Chromones from the tubers of Eranthis cilicica and their antioxidant activity

Article abstract of DOI:10.1016/j.phytochem.2008.12.002

Chemical studies on the constituents of Eranthis cilicica led to isolation of ten chromone derivatives, two of which were previously known. Comprehensive spectroscopic analysis, including extensive 1D and 2D NMR data, and the results of enzymatic hydrolys

Full text of DOI:10.1016/j.phytochem.2008.12.002

Phytochemistry 70 (2009) 288–293
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Chromones from the tubers of Eranthis cilicica and their antioxidant activity
a
b
a,
Minpei Kuroda a, , Singo Uchida , Kazuki Watanabe , Yoshihiro Mimaki
*
*
a School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan
b Faculty of Pharmaceutical Science, Aomori University, 2-3-1, Koubata, Aomori, Aomori 030-0943, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 August 2008
Received in revised form 22 November 2008
Available online 21 January 2009
Chemical studies on the constituents of Eranthis cilicica led to isolation of ten chromone derivatives, two of
which were previously known. Comprehensive spectroscopic analysis, including extensive 1D and 2D NMR
data, and the resultsof enzymatic hydrolysis allowed the chemical structuresof the compounds tobe assigned
as 8,11-dihydro-5-hydroxy-2,9-dihydroxymethyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, 5,7-dihydroxy-8-
[(2E)-4-hydroxy-3-methylbut-2-enyl]-2-methyl-4H-1-benzopyran-4-one, 5,7-dihydroxy-2-hydroxymethyl-
Keywords:
8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-4H-1-benzopyran-4-one, 7-[(b-
8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-2-methyl-4H-1-benzopyran-4-one, 7-[(b-
hydroxy-2-hydroxymethyl-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-4H-1-benzopyran-4-one, 9-[(O-b-
glucopyranosyl-(1?6)-b- -glucopyranosyl)oxy]methyl-8,11-dihydro-5,9-dihydroxy-2-methyl-4H-pyrano-
[2,3-g][1]benzoxepin-4-one, 8,11-dihydro-5,9-dihydroxy-9-hydroxymethyl-2-methyl-4H-pyrano[2,3-g]-
[1]benzoxepin-4-one, and 7-[(O-b- -glucopyranosyl-(1?6)-b- -glucopyranosyl)oxy]methyl-4-hydroxy-
D-glucopyranosyl)oxy]-5-hydroxy-
Eranthis cilicica
Ranunculaceae
Chromones
Glycosides
Antioxidant activity
D-glucopyranosyl)oxy]-5-
D-
D
D
D
5H-furo[3,2-g][1]benzopyran-5-one, respectively. The isolated compounds were evaluated for their anti-
oxidant activity.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
The genus Eranthis belonging to the family Ranunculaceae is
composed of seven species, including Eranthis cilicica, E. hyemalis
and E. pinnatifida (Tsukamoto, 1989). Eranthis cilicica Schott et
Kotschy is indigenous to Turkey and Afghanistan. Previously, we
have reported the structural determination of two new bisdesmos-
idic triterpene glycosides, namely eranthisaponins A and B, along
with four known triterpene glycosides from E. cilicica tubers
(Watanabe et al., 2003). A literature survey showed that E. hyemalis
(Kopp et al., 1991; Junior, 1979) and E. pinnatifida (Wada et al.,
The dried tubers of E. cilicica (1.3 kg) were extracted with hot
MeOH, and the MeOH extract was passed through a porous-poly-
mer polystyrene resin (Diaion HP-20) column. The MeOH–H2O
(1:1, v/v) eluate fraction was subjected to column chromatography
over silica gel and octadecylsilanized (ODS) silica gel, as well as
preparative HPLC, giving compounds 1 (127 mg), 2 (172 mg), 3
(127 mg), 4 (42.4 mg), 5 (78.1 mg), 6 (25.3 mg), 7 (16.3 mg), 8
(297 mg), 9 (42.5 mg), and 10 (13.0 mg).
Compounds 1 and 2 were identified as 2,3-dihydro-7-hydroxy-
methyl-2-(1-hydroxy-1-methylethyl)-4-methoxy-5H-furo-[3,2-g]-
1974) contained
a variety of chromone derivatives, which
prompted us to make a phytochemical examination of E. cilicica tu-
bers with particular attention to chromones. As a result, eight new
chromones (3–10), along with two known chromones (1 and 2),
were isolated. This paper deals with their structural determination
on the basis of spectroscopic analysis, including extensive 1D and
2D NMR data, and the results of enzymatic hydrolysis. The antiox-
idant activity of the isolated compounds is also described.
[1]-benzopyran-5-one (Sasaki et al., 1982) and 9-[(O-b-
pyranosyl-(1?6)-b- -glucopyranosyl)oxy]methyl-8,11-dihydro-5-
hydroxy-2-methyl-4H-pyrano[2,3-g][1]benzoxepin-4-one [eran-
thin 9-b- -glucopyranosyl-(1?6)-b- -glucopyranoside] (Kopp
et al., 1991), respectively.
Compound 3 was obtained as a yellow amorphous solid. Its
molecular formula was determined to be C15H14O6 on the basis of
the HRESI-TOFMS data, showing an accurate [M + H]+ ion at
m/z 291.0877. The UV spectrum of 3 had absorption maxima at
324 and 258 nm. The IR spectrum was consistent with the presence
of hydroxy groups (3272 cmÀ1) and a conjugated carbonyl group
(1652 cmÀ1). The 1H NMR spectrum of 3 (DMSO–d6) displayed sig-
nals for a chelated proton of a hydroxy group at d 12.76 (s), an aro-
matic proton at d 6.40 (s), two olefinic protons at d 6.32 (s) and 5.92
(t-like, J = 5.7 Hz), three oxymethylene groups at d 4.69 (2H, s), 4.47
D-gluco-
D
D
D
* Corresponding authors. Tel.: +81 426 76 4573x5; fax: +81 426 76 4579.
E-mail addresses: kurodam@ps.toyaku.ac.jp (M. Kuroda), mimakiy@ps.toyaku.
ac.jp (Y. Mimaki).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.12.002

M. Kuroda et al. / Phytochemistry 70 (2009) 288–293
289
(2H, s), and 3.85 (2H, s), and a deshielded methylene group at d 3.58
(2H, d, J = 5.7 Hz). These NMR spectroscopic data were closely re-
lated to those of the aglycone moiety of 2. However, the methyl sin-
glet signal at d 2.44 (C(2)–Me) observed in the 1H NMR spectrum of 2
was absent from that of 3 and was replaced by the oxymethylene
resonace at d 4.47. In the HMQC spectrum of 3, the oxymethylene
proton signal was associated with the one-bond coupled carbon
resonance at d 60.6. These data indicated that the methyl group at-
tached to C-2 of the chromone skeleton in 2 is modified to a
hydroxymethyl group in 3. This was supported by long-range corre-
lations between the oxymethylene signal at d 4.47 and the C-2 (d
172.0) and C-3 (d 106.5) carbon resonances. Thus, structure 3 was
identified as 8,11-dihydro-5-hydroxy-2,9-dihydroxymethyl-4H-
pyrano[2,3-g][1]benzoxepin-4-one.
293.1037 [M + H]+), which was higher than that of 4 by one
oxygen atom. Comparison of the 1H and 13C NMR spectra of 5
with those of
4 completed the following information: The
methyl group located at C-2 position of 4, of which the 1H
and 13C NMR signals were observed at dH 2.36 and dC 20.8,
was missing in 5. Instead, resonances for an oxymethylene
group showed up at dH 4.42 (s) and dC 60.6. Moreover, the
HMBC spectrum of 5 featured cross-peaks from the oxymethyl-
ene protons to the C-2 (d 171.4) and C-3 (d 105.9) carbons,
which implied the locus of
a hydroxymethyl group at C-2.
Therefore, was established as 5,7-dihydroxy-2-hydroxy-
5
methyl-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-4H-1-benzopyran-
4-one.
Compound 6 was obtained as an amorphous yellow solid
with a molecular formula C21H26O10, as determined by HRESI-
TOFMS (m/z 439.1637 [M + H]+). As in the case of 4, the 1H
NMR spectrum exhibited signals for a chelated proton of a hy-
droxy group at d 12.79 (s), an aromatic proton at d 6.58 (s), two
olefinic protons at d 6.24 (s) and 5.40 (t-like, J = 7.3 Hz), two
methyl groups attached to double bonds at d 2.39 and 1.75
(each 3H, s), an oxymethylene group at d 3.76 (2H, s), and a
deshielded methylene group at d 3.55 (dd, J = 14.1, 7.3 Hz) and
3.36 (dd, J = 14.1, 7.3 Hz). In addition, a resonance due to an
anomeric proton of a hexopyranosyl group could be observed
at d 4.97 (d, J = 7.3 Hz). Enzymatic hydrolysis of 6 with narin-
Compound
4 was shown to have the molecular formula
C15H16O5 on the basis of the HRESI-TOFMS (m/z 277.1064
[M + H]+) data. The UV spectrum of 4 had absorption maxima
at 299 and 258 nm. The IR spectrum showed absorption bands
of hydroxy groups at 3228 cmÀ1 and a conjugated carbonyl
group at 1658 cmÀ1. The 1H NMR spectrum exhibited signals
for a chelated proton of a hydroxy group at d 12.79 (s), an aro-
matic proton at d 6.27 (s), two olefinic protons at d 6.15 (s) and
5.37 (t-like, J = 7.4 Hz), two methyl groups attached to double
bonds at d 2.36 and 1.73 (each 3H, s), an oxymethylene group
at d 3.76 (2H, s), and a deshielded methylene group at d 3.33
(2H, d, J = 7.4 Hz). The above spectral features were essential
analogous to those of the aglycone moiety of 2. However, the
signal due to the H2-8 oxymethylene group in the oxepin moi-
ety, which was observed at d 4.74 (2H, s) in the 1H NMR spec-
trum of 2, could not be detected in 4. In the HMBC spectrum of
4, the olefinic proton at d 5.37 (H-2’) showed long-range corre-
lations with the hydroxymethyl carbon at d 67.1 (C-4’) and the
methyl carbon at d 14.4 (C-5’), whereas the deshielded methy-
lene protons at d 3.33 (H2-1’) were correlated with the aromatic
carbons of the chromone nucleus at d 162.4 (C-7), 106.5 (C-8),
and 155.9 (C-8a), together with the quaternary olefinic carbon
at d 136.2 (C-3’) (Fig. 1). The H2-1’ methylene protons showed
proton spin-coupling correlations with the H-2’ olefinic proton
in the 1H–1H COSY spectrum. Furthermore, the aromatic proton
at d 6.29 (H-6) and a chelated proton of the C-5 hydroxy group
at d 12.70 showed long-range correlations with the aromatic
carbon at d 159.5 (C-5). These data implied that the oxepin ring
is opened and that a 4’-hydroxy-3’-methylbut-2’-enyl group is
located at the C-8 position in 4. In the PHNOESY spectrum of
4, an NOE correlation between the protons of H-2’ and H2-4’
ginase gave 4 and D-glucose. Identification of D-glucose, includ-
ing its absolute configuration, was carried out by direct HPLC
analysis of the hydrolysate. In the HMBC spectrum of 6, the
anomeric proton showed a long-range correlation with the oxy-
genated aromatic carbon at d 160.6, which exhibited long-range
correlations with the H-6 (d 6.58) and H2-1’ (d 3.55 and 3.36)
protons and was assigned to C-7 of the aglycone (Fig. 2). Thus,
structure 6 was defined as 7-[(b-D-glucopyranosyl)oxy]-5-hydro-
xy-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-2-methyl-4H-1-benz-
opyran-4-one.
Compound 7 had the molecular formula C21H26O11 on the
basis of HRESI-TOFMS (m/z 455.1572 [M + H]+), which differed
from 6 by containing one more oxygen atom. When the 1H
NMR spectrum of 7 was compared with that of 6, the signal
for the C(2)–methyl group, which was observed at d 2.39 in 6,
disappeared in 7, and a resonance for a hydroxymethyl group
was detected at d 4.44 (2H, s). These 1H NMR spectroscopic
data, along with the hydroxymethyl carbon signal at d 59.5,
allowed the structure of 7 to be assigned as 7-[(b-D-glucopyran-
osyl)oxy]-5-hydroxy-2-hydroxymethyl-8-[(2E)-4-hydroxy-3-meth-
ylbut-2-enyl]-4H-1-benzopyran-4-one.
provided evidence for the (2’E)-geometry. Thus, structure
4
was established as 5,7-dihydroxy-8-[(2E)-4-hydroxy-3-methyl-
but-2-enyl]-2-methyl-4H-1-benzopyran-4-one.
Compound 5, obtained as an amorphous yellow solid, was
analyzed for C15H16O6 on the basis of HRESI-TOFMS (m/z
Compound 8 was obtained as an amorphous yellow solid. Its
molecular formula was determined as C27H34O16 by HRESI-TOF-
MS, showing an [M + H]+ peak at m/z 615.1920. The spectro-
scopic properties of 8 were similar to those of 2 and were
Fig. 1. HMBC correlations of 4.
Fig. 2. HMBC correlations of 6.

290
M. Kuroda et al. / Phytochemistry 70 (2009) 288–293
Fig. 3. HMBC correlations of 8.
suggestive of a chromone diglucoside related to 2. However, dif-
ferences between 8 and 2 were observed in the NMR signals
arising from the oxepin moiety, which is connected through
C-6a and C-11a. On the basis of the 1H NMR spectroscopic data,
the oxepin moiety of 8 was composed of two oxymethylene
groups [d 4.34 (d, J = 11.6 Hz, H-8a) and 3.93 (d, J = 11.6 Hz,
H-8b); d 3.82 (d, J = 10.6 Hz, H-12a) and 3.46 (d, J = 10.6 Hz,
H-12b)] and a pair of olefinic protons [d 6.80 (d, J = 12.4 Hz,
H-11); d 5.87 (d, J = 12.4 Hz, H-10)]. Furthermore, the 13C NMR
spectrum showed signal due to a quaternary carbon bearing a
hydroxy group at d 73.5 (C-9). In the HMBC spectrum, long-
range correlations were observed between H-11 and C-6a (d
164.3)/C-11b (d 155.0)/C-9 (d 73.5)/C-10 (d 132.7), and between
H-10 and C-8 (d 74.2)/C-9 (d 73.5)/C-11a (d 105.8)/C-12 (d 73.0)
(Fig. 3). Thus, the oxepin moiety with a double bond at C-
10(11), and a hydroxy and an oxymethylene group at C-9 was
shown to be attached to the chromone ring through C-6a and
C-11a. A linkage of a b-D-glucopyranosyl-(1?6)-b-D-glucopyran-
osyl group to C-12 as in 2 was ascertained by HMBC correla-
tions between the H-1 proton of the terminal glucosyl moiety
at d 4.22 and C-6 of the inner glucosyl moiety at d 68.5, and
between the H-1 proton of the inner glucosyl at d 4.24 and
C-12 at d 73.0. On the basis of the above evidence, structure
8 was determined as 9-[(O-b-D-glucopyranosyl-(1?6)-b-D-gluco-
pyranosyl)oxy]methyl-8,11-dihydro-5,9-dihydroxy-2-methyl-4H-
pyrano[2,3-g][1]benzoxepin-4-one.
Compound 9 had the molecular formula C15H14O6 on the basis
of HRESI-TOFMS (m/z 291.0877 [M + H]+). The 1H and 13C NMR
spectra of 9 strongly suggested that it is identical to the aglycone
of 8. Enzymatic hydrolysis of 8 with naringinase resulted in the
production of 9 and D-glucose. The structure 9 was elucidated as
8,11-dihydro-5,9-dihydroxy-9-hydroxymethyl-2-methyl-4H-pyr-
ano-[2,3-g][1]benzoxepin-4-one.
The antioxidative property of the isolated compounds was
examined against superoxide anion using a chemiluminescence as-
say. Although the chromone glycosides 2, 6, 7, 9, and 10 did not
show any apparent superoxide anion scavenging activity even at
a sample concentration of 1000
mone derivatives 1, 3, and 5 were slightly active with IC50 values
of 179, 198, and 274 g/ml, respectively. In 1, 3, and 5, the 2-
lg/ml, the 2-hydroxymethylchro-
Compound 10, isolated as an amorphous yellow solid,
showed an [M + H]+ ion at m/z 577.1525 in HRESI-TOFMS,
l
hydroxymethyl group may contribute to the appearance of their
antioxidant activity. Epigallocatechin gallate used as a positive
control had an IC50 value of 3.0 lg/ml.
corresponding to the empirical molecular formula C24H28O15
.
Analysis of the 1H and 13C NMR spectra of 10 implied that
it was a chromone diglycoside whose sugar sequence is the
same as that of
spectrum of the aglycone moiety of 10 with that of 5,
the AX pattern signals at 8.05 and 7.12 (each d,
J = 2.2 Hz), assignable to H-2 and H-3 of furan ring, ap-
2
and 8. On comparison of the 1H NMR
3. Concluding remarks
d
a
Eight new chromone derivatives (3–10), along with two known
ones, were isolated from the tubers of E. cilicica, and the structures
of the new chromones were determined by spectroscopic analysis,
including extensive 1D and 2D NMR data, and the results of enzy-
matic hydrolysis. Chromones are a group of natural products with
the 4H-1-benzopyran-4-one skeleton and are considered to be dis-
tributed in the relatively limited species of higher plants belonging
to the family Ranunculaceae, Umbelliferae, Leguminosae, and
Cneoraceae on the basis of the facts reported up to the present
(Harborne et al., 1999). In plants of the family Ranunculaceae,
chromones have been identified only in genus Eranthis (Kopp
peared in 10 instead of the signals for the (2E)-4-hydroxy-
3-methylbut-2-enyl unit in 5. Enzymatic hydrolysis of 10
with naringinase liberated 4-hydroxy-7-hydroxylmethyl-5H-
furo[3,2-g][1]benzopyran-5-one (10a, norkhellol) (Cao et al.,
2005) and
D-glucose. The diglycoside O-b-D-glucopyranosyl-
(1?6)-b- -glucopyranosyl group was ascertained to be linked
D
to C-7 of the aglycone by analysis of the HMBC spectrum
of 10. Thus, the structure of 10 was characterized as 7-
[(O-b-
D-glucopyranosyl-(1?6)-b-D-glucopyranosyl)oxy]methyl-4-
hydroxy-5H-furo[3,2-g][1]benzopyran-5-one.

M. Kuroda et al. / Phytochemistry 70 (2009) 288–293
291
et al., 1991; Junior, 1979; Wada et al., 1974) and Cimicifuga (Cao
4.4. Compound 3
et al., 2005; Kondo and Takemoto, 1972). This is the first compre-
hensive report on chromone constitusion of E. cilicica.
8,11-Dihydro-5-hydroxy-2,9-dihydroxymethyl-4H-pyrano[2,3-
g][1]benzoxepin-4-one (3); amorphous yellow solid; HRESI-TOFMS
(positive-mode) m/z: 291.0877 [M + H]+ (calculated for C15H15O6,
4. Experimental
291.0869); UV kmax (log e) nm: 324 (3.68), 258 (4.33); IR mmax (film)
cmÀ1: 3272 (OH), 1652 (C@O); 1H NMR (DMSO–d6): d 12.76 (1H, s,
C(5)–OH), 6.40 (1H, s, H-6), 6.32 (1H, s, H-3), 5.92 (1H, t-like,
J = 5.7 Hz, H-10), 4.69 (2H, s, H-8), 4.47 (2H, s, C(2)–CH2OH), 3.85
(2H, s, H-12), 3.58 (2H, d, J = 5.7 Hz, H-11); for 13C NMR (DMSO–
d6) spectroscopic data, see Table 1.
4.1. General
Optical rotations were measured using a JASCO DIP-360 (Tokyo,
Japan) automatic digital polarimater. IR spectra were recorded on a
JASCO FT-IR 620 spectrophotometer. NMR spectra were recorded
on a Bruker DRX-500 spectrometer (500 MHz for 1H NMR, Kar-
lsruhe, Germany) using standard Bruker pulse programs. Chemical
shifts are given as d-value with reference to tetramethylsilane
(TMS) as internal standard. HRESI-TOFMS data were obtained on
a Waters-Micromass LCT mass spectrometer (Manchester, UK).
Diaion HP-20 (Mitsubishi-Chemical, Tokyo, Japan), silica gel (Fuji-
Silysia Chemical, Aichi, Japan), and ODS silica gel (Nacalai Tesque,
Kyoto, Japan) were used for column chromatography (CC). TLC
was carried out on precoated Kieselgel 60 F254 (0.25 mm, Merck,
Darmstadt, Germany) and RP-18 F254 S (0.25 mm thick, Merck)
plates, and spots were visualized by spraying with 10% H2SO4 fol-
lowed by heating. HPLC was performed by using a system com-
prised of a CCPM pump (Tosoh, Tokyo, Japan), a CCP PX-8010
controller (Tosoh), an RI-8010 detector (Tosoh) or a Shodex OR-2
detector (Showa Denko, Tokyo, Japan), and a Rheodyne injection
port. A Capcell Pak C18 UG120 column (10 mm i.d. Â 250 mm,
4.5. Compound 4
5,7-Dihydroxy-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-2-methyl-
4H-1-benzopyran-4-one (4); amorphous yellow solid; HRESI-TOF-
MS (positive-mode) m/z: 277.1064 [M + H]+ (calculated for
C15H17O5, 277.1076); UV kmax (log e) nm: 299 (3.80), 258 (4.39);
IR mmax (film) cmÀ1: 3228 (OH), 1658 (C@O); 1H NMR (DMSO–
d6): d 12.79 (1H, s, C(5)–OH), 6.27 (1H, s, H-6), 6.15 (1H, s, H-3),
5.37 (1H, t-like, J = 7.4 Hz, H-2’), 3.76 (2H, s, H-4’), 3.33 (2H, d,
J = 7.4 Hz, H-1’), 2.36 (3H, s, C(2)–Me), 1.73 (3H, s, Me-5’); for 13C
NMR (DMSO–d6) spectroscopic data, see Table 1.
4.6. Compound 5
5,7-Dihydroxy-2-hydroxy-methyl-8-[(2E)-4-hydroxy-3-meth-
ylbut-2-enyl]-4H-1-benzopyran-4-one (5); amorphous yellow
solid; HRESI-TOFMS (positive-mode) m/z: 293.1037 [M + H]+
5 lm, Shiseido, Tokyo, Japan) was used for preparative HPLC. The
following reagents were obtained from the indicated companies:
xanthine oxidase (Sigma, St. Luis, MO, USA); hypoxantnine (Wako,
Osaka, Japan); MPEC (2-methyl-6-p-methoxyphenylethynylimi-
dazopyrazinone) (ATTO, Tokyo, Japan). All other chemicals used
were of biochemical reagent grade.
(calculated for C15H17O6, 293.1025); UV kmax (log
e) nm: 301
(3.72), 258 (4.30); IR mmax (film) cmÀ1: 3276 (OH), 1669 (C@O);
1H NMR (DMSO–d6): d 12.78 (1H, s, C(5)–OH), 6.29 (1H, s, H-6),
6.22 (1H, s, H-3), 5.37 (1H, t-like, J = 7.2 Hz, H-2’), 4.42 (2H, s,
C(2)–Me), 3.75 (2H, s, H-4’), 3.34 (2H, m, H-1’), 1.72 (3H, s, Me-5’);
for 13C NMR (DMSO–d6) spectroscopic data, see Table 1.
4.2. Plant material
Eranthis cilicica was purchased from a nursery in Heiwaen,
Japan, in October 2000 and was identified by Dr. Yutaka Sashida,
emeritus professor of the Tokyo University of Pharmacy and Life
Sciences. A voucher specimen has been deposited in our laboratory
(voucher No. 00-7-EC, Laboratory of Medicinal Pharmacognosy).
4.7. Compound 6
7-[(b-
D-Glucopyranosyl)oxy]-5-hydroxy-8-[(2E)-4-hydroxy-3-
methylbut-2-enyl]-2-methyl-4H-1-benzopyran-4-one (6); amor-
25
phous yellow solid; [
(positive-mode) m/z: 439.1637 [M + H]+ (calculated for C21H27O10
439.1640); UV kmax (log ) nm: 325 (3.66), 254 (4.36); IR max (film)
a
]
À38.3° (c 0.05; MeOH); HRESI-TOFMS
D
,
e
m
4.3. Extraction and isolation
cmÀ1: 3375 (OH), 1657 (C@O); 1H NMR (DMSO–d6): d 12.79 (1H, s,
C(5)–OH), 6.58 (1H, s, H-6), 6.24 (1H, s, H-3), 5.40 (1H, t-like,
J = 7.3 Hz, H-2’), 4.97 (1H, d, J = 7.3 Hz, H-1’’), 3.76 (2H, s, H-4’),
3.71 (1H, br d, J = 11.9 Hz, H-6’’a), 3.55 (1H, dd, J = 14.1, 7.3 Hz, H-
1’a), 3.46 (1H, dd, J = 11.9, 5.7 Hz, H-6’’b), 3.36 (1H, dd, J = 14.1,
7.3 Hz, H-1’b), 2.39 (3H, s, C(2)–Me), 1.75 (3H, s, H-5’); for 13C
NMR (DMSO–d6) spectroscopic data, see Table 1.
The dry tubers of E. cilicica (1.3 kg) were extracted with MeOH
(10 l  2) under reflux for 2 h. Following removal of MeOH, the res-
idue (135 g) suspended in MeOH–H2O (3:7, v/v) was applied to a
Diaion HP-20 column [MeOH–H2O (3:7, v/v), MeOH–H2O (1:1,
v/v), MeOH–H2O (4:1, v/v), MeOH, EtOH, and EtOAc (each 3 l)].
The MeOH–H2O (1:1, v/v) eluate portion (35 g) was subjected to
silica gel CC eluted with CHCl3–MeOH–H2O (20:10:1) and MeOH
alone, to give five fractions (I–V). Fraction II was purified by ODS
silica gel CC eluted with MeOH–H2O (11:9) into three subfractions
(II-1–II-3). Fraction II-1 was applied to a silica gel column, this
being eluted with CHCl3–MeOH (49:1; 19:1) to yield 4 (42.4 mg).
Fraction II-2 was subjected to silica gel CC eluted with CHCl3–
MeOH (49:1) to give 3 (127 mg) and 8 (297 mg). Fraction II-3
was applied to a silica gel column eluted with CHCl3–MeOH
(14:1; 9:1) to yield 1 (127 mg) and 5 (78.1 mg), and 6 with a few
impurities, which was further purified by preparative HPLC using
MeOH–H2O (1:1) to furnish 6 (25.3 mg). Fraction III was subjected
to ODS silica gel CC eluted with MeOH–H2O (11:9; 1:1) and silica
gel CC with CHCl3–MeOH–H2O (20:10:1) to yield 7 (16.3 mg).
Compounds 2 (172 mg), 9 (42.5 mg), and 10 (13.0 mg) were iso-
lated from fraction IV via preparative HPLC using MeCN–H2O (1:4).
4.8. Enzymatic hydrolysis of 6
Compound 6 (9.4 mg) was dissolved in AcOH–NaOAc buffer (pH
5.0, 5 ml) with naringinase (Sigma, EC 232-962-4, b-glucosidase
activity: 69 units/g) (15.0 mg) and incubated at room temperature
for 14 h. The crude reaction mixture was applied to a silica gel col-
umn eluted with CHCl3–MeOH–H2O (7:4:1) to yield 4 (3.9 mg) and
a sugar fraction (1.5 mg). The sugar fraction was analyzed by HPLC
under the following conditions: column, Shodex Sugar SC1011
(8.0 mm i.d. Â 300 mm, 5
rate, 1.0 ml/min; column temperature, 80 °C; detection, RI and
OR. Identification of -glucose was carried out by comparison of
its retention time and optical rotation with those of authentic sam-
ples. Rt (min): 7.61 ( -glucose, positive polarity).
lm, Showa Denko); solvent, H2O; flow
D
D

292
M. Kuroda et al. / Phytochemistry 70 (2009) 288–293
Table 1
13C NMR spectroscopic data for 3–10 in DMSO–d6.
Position
3
8
9
Position
4
5
6
7
Position
10
2
3
4
4a
5
6
6a
8
9
10
11
11a
11b
12
172.0
106.5
183.5
107.3
160.0
104.6
165.1
70.8
140.4
123.1
21.6
111.4
154.1
63.8
168.7
108.6
182.6
106.1
160.3
102.5
164.3
74.2
171.4
105.5
182.5
105.4
159.5
98.4
165.0
75.1
66.2
134.7
117.5
106.7
155.6
66.2
2
3
4
4a
5
6
7
8
8a
1’
2’
3’
4’
5’
168.3
108.5
182.9
104.2
159.9
99.1
162.4
106.5
155.9
21.5
171.4
105.9
183.0
104.7
160.0
99.2
162.6
106.6
155.6
21.4
168.3
108.1
182.5
105.0
159.5
98.3
160.6
108.1
154.5
21.0
171.4
105.5
182.5
105.4
159.5
98.4
160.8
108.2
154.1
20.9
2
3
3a
4
4a
5
6
7
147.3
104.9
113.4
159.2
106.3
184.7
107.2
168.7
155.2
92.0
73.5
8a
9
9a
132.7
117.3
105.8
155.0
73.0
121.7
136.2
67.1
121.7
136.2
67.1
121.1
135.6
66.5
121.0
135.7
66.5
154.5
66.3
C(2)–CH2O–
14.4
14.4
13.8
13.7
C(2)–CH2–
60.6
20.1
20.8
C(2)–CH2–
20.8
60.6
20.2
59.5
1’
2’
3’
4’
5’
6’
1’’
2’’
3’’
4’’
5’’
6’’
103.9
73.7
76.5
70.9
76.0
68.5
103.4
73.7
76.9
70.0
1’’
2’’
3’’
4’’
5’’
6’’
100.7
73.5
76.7
69.8
77.3
100.7
73.5
76.7
69.8
77.3
1’
2’
3’
4’
5’
6’
1’’
2’’
3’’
4’’
5’’
6’’
104.4
74.2
77.6
70.9
76.7
69.5
103.3
74.4
77.4
70.9
77.7
61.9
60.8
60.8
77.0
61.1
25
4.9. Compound 7
[a]
–2.6° (c 0.10; MeOH); HRESI-TOFMS (positive-mode) m/z:
D
291.0877 [M + H]+ (calculated for C15H14O6, 291.0869); UV kmax
7-[(b-
D
-Glucopyranosyl)oxy]-5-hydroxy-2-hydroxymethyl-8-
(log e
) nm: 334 (3.51), 257 (4.68); IR mmax (film) cmÀ1: 3387
[(2E)-4-hydroxy-3-methylbut-2-enyl]-4H-1-benzopyran-4-one (7),
(OH), 1660 (C@O); 1H NMR (DMSO–d6): d 13.00 (1H, s, C(5)–OH),
6.74 (1H, d, J = 12.4 Hz, H-11), 6.39 (1H, s, H-6), 6.31 (1H, s, H-3),
5.85 (1H, d, J = 12.4 Hz, H-10), 4.29 (1H, d, J = 11.5 Hz, H-8a), 3.89
(1H, d, J = 11.5 Hz, H-8b), 3.41 (2H, s, H-12), 2.44 (3H, s, C(2)–Me);
for 13C NMR (DMSO–d6) spectroscopic data, see Table 1.
25
amorphous yellow solid; [
MS (positive-mode) m/z: 455.1572 [M + H]+ (calculated for
C21H27O11, 455.1553); UV kmax (log ) nm: 327 (3.59), 255 (4.30);
a]
À38.3° (c 0.05; MeOH); HRESI-TOF-
D
e
IR mmax (film) cmÀ1: 3375 (OH), 1659 (C@O); 1H NMR (DMSO–
d6): d 12.74 (1H, s, C(5)–OH), 6.60 (1H, s, H-6), 6.29 (1H, s, H-3),
5.39 (1H, t-like, J = 7.4 Hz, H-2’), 4.98 (1H, d, J = 7.5 Hz, H-1’’),
4.44 (2H, s, C(2)–CH2OH), 3.75 (2H, s, H-4’), 3.70 (1H, br d,
J = 11.5 Hz, H-6’’a), 3.54 (1H, dd, J = 14.2, 7.4 Hz, H-1’a), 3.47 (1H,
dd, J = 11.5, 5.0 Hz, H-6’’b), 3.35 (1H, dd, J = 14.2, 7.4 Hz, H-1’b),
1.74 (3H, s, H-5’); for 13C NMR (DMSO–d6) spectroscopic data, see
Table 1.
4.12. Enzymatic hydrolysis of 8
Compound 8 (20.0 mg) was subjected to enzymatic hydroly-
sis with naringinase as described for 6 to give an aglycone 9
(6.4 mg) and a sugar fraction. HPLC analysis of the sugar frac-
tion under the same conditions as in the case of that of 6
showed the presence of
sitive polarity).
D-glucose. Rt (min): 7.73 (D-glucose, po-
4.10. Compound 8
9-[(O-b-D-Glucopyranosyl-(1?6)-b-D-glucopyranosyl)oxy]methyl-
4.13. Compound 10
8,11-dihydro-5,9-dihydroxy-2-methyl-4H-pyrano[2,3-g][1]benzoxe-
25
pin-4-one (8); amorphous solid; [
HRESI-TOFMS (positive-mode) m/z: 615.1920 [M + H]+ (calculated
for C27H35O16, 615.1925); UV kmax (log ) nm: 338 (3.57), 258
a
]
À5.57° (c 0.10; MeOH);
7-[(O-b-
D-Glucopyranosyl-(1?6)-b-D-glucopyranosyl)oxy]methyl-
D
4-hydroxy-5H-furo[3,2-g][1]benzopyran-5-one (10); amorphous
25
e
solid; [
m/z: 557.1525 [M + H]+ (calculated for C24H29O15, 557.1506); UV
kmax (log
) nm: 340 (3.45), 252 (4.49); IR mmax (film) cmÀ1: 3376
a
]
À41.8° (c 0.10; MeOH); HRESI-TOFMS (positive-mode)
D
(4.54); IR mmax (film) 3376 (OH), 1660 (C@O); 1H NMR (DMSO–
d6): d 13.01 (1H, s, C(5)–OH), 6.80 (1H, d, J = 12.4 Hz, H-11), 6.43
(1H, s, H-6), 6.27 (1H, s, H-3), 5.87 (1H, d, J = 12.4 Hz, H-10), 4.34
(1H, d, J = 11.6 Hz, H-8a), 4.24 (1H, d, J = 7.4 Hz, H-1’), 4.22 (1H, d,
J = 7.5 Hz, H-1’’), 3.97 (1H, br d, J = 10.3 Hz, H-6’a), 3.93 (1H, d,
J = 11.6 Hz, H-8b), 3.82 (1H, d, J = 10.6 Hz, H-12a), 3.63 (1H, dd,
J = 11.4, 5.2 Hz, H-6’’a), 3.57 (1H, dd, J = 11.5, 6.3 Hz, H-6’b), 3.46
(1H, d, J = 10.6 Hz, H-12b), 3.40 (1H, dd, J = 11.4, 5.8 Hz, H-6’’b),
2.44 (3H, s, C(2)–Me); for 13C NMR (DMSO–d6) spectroscopic data,
see Table 1.
e
(OH), 1659 (C@O); 1H NMR (DMSO–d6): d 13.65 (1H, s, C(5)–OH),
8.05 (1H, d, J = 2.2 Hz, H-2), 7.39 (1H, s, H-8), 7.12 (1H, d,
J = 2.2 Hz, H-3), 6.65 (1H, s, H-6), 4.77 and 4.68 (each 1H, d,
J = 15.7 Hz, C(2)–CH2O–), 4.34 (1H, d, J = 7.8 Hz, H-1’’), 4.25 (1H, d,
J = 7.8 Hz, H-1’), 4.02 (1H, br d, J = 11.5 Hz, H-6’a), 3.66 (1H, dd,
J = 11.6, 4.9 Hz, H-6’’a), 3.57 (1H, dd, J = 11.5, 7.1 Hz, H-6’b), 3.42
(1H, d, J = 11.6, 5.8 Hz, H-6’’b); for 13C NMR (DMSO–d6) spectro-
scopic data, see Table 1.
4.11. Compound 9
4.14. Enzymatic hydrolysis of 10
8,11-Dihydro-5,9-dihydroxy-9-hydroxymethyl-2-methyl-4H-
pyrano-[2,3-g][1]benzoxepin-4-one (9); amorphous yellow solid;
Compound 10 (6.5 mg) was subjected to enzymatic hydrolysis
with naringinase as described for 6 to give norkhellol (10a,

M. Kuroda et al. / Phytochemistry 70 (2009) 288–293
293
1.8 mg) and sugar fractions (3.0 mg). HPLC analysis of the sugar
fraction under the same conditions as in the case of that of 6
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ml, which was then stirred at 30 °C for 20 s. The intensity of
chemiluminescence of the mixture was automatically deter-
mined at 30 °C for 30 s using an AB-2200-R Luminescencer-
PSN (ATTO). The superoxide anion radical scavenging activity
was expressed as the sample concentration (lg/ml) necessary
to give a 50% reduction in the sample intensity of lumines-
cence (IC50).