Phenolic glycosides from Kaempferia parviflora

Article abstract of DOI:10.1016/j.phytochem.2008.09.001

Three phenolic glycosides were isolated together with two known flavonol glycosides from the H₂O-soluble fraction of rhizomes of Kaempferia parviflora. Their structures were determined to be rel-(5aS,10bS)-5a,10b-dihydro-1,3,5a,9-tetrahydroxy-8

Full text of DOI:10.1016/j.phytochem.2008.09.001

Phytochemistry 69 (2008) 2743–2748
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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Phenolic glycosides from Kaempferia parviflora
Toshiaki Azuma a, Yasuo Tanaka b, Hiroe Kikuzaki a,
*
a Division of Food and Health Sciences, Graduate School of Human Life Science, Osaka City University, 3-3-138, Sugimoto, Sumiyoshi, Osaka, Japan
b Taiyo Corporation, 1-6-27, Higashiawaji, Higashiyodogawa, Osaka, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 May 2008
Received in revised form 29 August 2008
Available online 14 October 2008
Three phenolic glycosides were isolated together with two known flavonol glycosides from the H2O-sol-
uble fraction of rhizomes of Kaempferia parviflora. Their structures were determined to be rel-(5aS,10bS)-
5a,10b-dihydro-1,3,5a,9-tetrahydroxy-8-methoxy-6H-benz[b]indeno[1,2-d]furan-6-one 5a-O-[
pyranosyl-(1 ? 6)-b- -glucopyranoside] (1), its rel-5aS,10bR isomer (2), and (2R,3S,4S)-3-O-[
pyranosyl-(1 ? 6)-b- ? O ? 3,4 ? 4)-(5aS, 10bS)-5a,
-glucopyranosyl]-30-O-methyl-ent-epicatechin-(2
10b-dihydro-1,3,5a,9-tetrahydroxy-8-methoxy-6H-benz[b]indeno[1,2-d]furan-6-one 5a-O-[ -rhamno-
pyranosyl-(1 ? 6)-b- -glucopyranoside] (3). The structures were elucidated on the basis of analyses of
chemical and spectroscopic evidence.
a-L-rhamno-
D
a-L-rhamno-
D
a
a
Keywords:
Kaempferia parviflora
Zingiberaceae
a
-L
D
Phenolic glycoside
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
anones,
a
chalcone,
a
diarylheptanoid, b-sitosteryl myristate,
methyl linolate, three glyceroglycolipids and a sphingoglycolipid.
The H2O-soluble fraction was subjected to successive column chro-
matography using Sephadex LH-20 and ODS to give three new com-
pounds (1–3) (see Fig. 1) and two known flavonol glycosides. The
structures of the known compounds were identified by comparison
of their spectroscopic data with those in the literature as 5-hydro-
xy-3,7-dimethoxyflavone (Jaipetch et al., 1983), 5-hydroxy-3,7,40-
trimethoxyflavone (Dong et al., 1999), 3,5,7-trimethoxyflavone (Jo-
seph-Nathan et al., 1981), 3,5,7,40-tetramethoxyflavone (Joseph-
Nathan et al., 1981), 5,7-dimethoxyflavone (Jaipetch et al., 1983;
Joseph-Nathan et al., 1981), 5,7,40-trimethoxyflavone (Jaipetch
et al., 1983; Joseph-Nathan et al., 1981), 5-hydroxy-7,40-dimeth-
oxyflavone (Gonzalez et al., 1989), 5-hydroxy-7-methoxyflavone
(Asakawa, 1971), 5-hydroxy-3,7,30,40-tetramethoxyflavone (Vidari
et al., 1971), 5,30-dihydroxy-3,7,40-trimethoxyflavone (Dong et al.,
1999; Wang et al., 1989), 3,5,7,30,40-pentamethoxyflavone (Dong
et al., 1999; Joseph-Nathan et al., 1981), 5-hydroxy-7,30,40-trimeth-
oxyflavone (Ahond et al., 1990), 40-hydroxy-5,7-dimethoxyflavone
(Kao et al., 2004), 5,7,30,40-tetramethoxyflavone (Joseph-Nathan
et al., 1981), (2S)-5-hydroxy-7-methoxyflavanone (Gonzalez et al.,
1989; Su et al., 2003), (2S)-5,7-dimethoxyflavanone (Bick et al.,
1972), (E)-20-hydroxy-40,60-dimethoxychalcone (Jhoo et al., 2006),
(1E,6E)-1,7-diphenyl-1,6-heptadiene-3,5-dione (Matthes et al.,
1980), b-sitosteryl myristate (Parmar et al., 1998), methyl 9Z,12Z-
Kaempferia parviflora Wall., which has a black rhizome, belongs
to the family Zingiberaceae. In the northeast of Thailand, the rhi-
zomes of K. parviflora have been used for the treatment of colic dis-
order as well as peptic and duodenal ulcers (Yenjai et al., 2004).
Recently, various bioactivities of extracts of K. parviflora and/or
methoxyflavones isolated from this plant, have been reported.
These include: gastroprotective effects in rats (Rujjanawate et al.,
2005); effects on P-glycoprotein function (Patanasethanont et al.,
2006); inhibition of viral proteases (Sookkongwaree et al., 2006)
and promotion of nitric oxide production (Wattanapitayakul
et al., 2007). Some methylated flavonoids have also previously
been isolated from K. parviflora (Jaipetch et al., 1983; Yenjai
et al., 2004). However, the polar components of this plant have
not been identified. In the course of our study on phytochemical
constituents of the Zingiberaceae plant (Kikuzaki et al., 2001;
Kikuzaki and Tesaki, 2002; Akiyama et al., 2006), the structure elu-
cidation of three new compounds (1–3) isolated from the H2O-sol-
uble fraction of an extract of rhizomes of this plant is reported.
2. Results and discussion
Freeze–dried rhizomes of K. parviflora were successively ex-
tracted with CH2Cl2 and 70% aqueous acetone. After the evapora-
tion of the acetone, the aqueous residue was extracted with
EtOAc. The CH2Cl2 extract and EtOAc-soluble fraction were sepa-
rated by column chromatography using silica gel, Sephadex LH-20
and Chromatorex ODS, respectively, to obtain 14 flavones, two flav-
octadecadienoate, 1-O-(9Z,12Z-octadecadienoyl)-3-O-[
pyranosyl-(1 ? 6)-b-galactopyranosyl]glycerol (Yoshikawa et al.,
1992, 1994), 1-O-hexadecanoyl-3-O-[ -galactopyranosyl-(1 ? 6)-
b-galactopyranosyl]glycerol (Murakami et al., 1994), 1-O-hexa-
decanoyl-2-O-(9Z,12Z-octadecadienoyl)-3-O-[ -galactopyranosyl-
(1 ? 6)-b-galactopyranosyl]glycerol (Jung et al., 1996; Murakami
et al., 1991), 1-O-b-glucopyranosyl-(8Z)-2-(2-hydroxytetra-
a-galacto-
a
a
* Corresponding author. Tel.: +81 6 6605 2812; fax: +81 6 6605 3086.
E-mail address: kikuzaki@life.osaka-cu.ac.jp (H. Kikuzaki).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.09.001

2744
T. Azuma et al. / Phytochemistry 69 (2008) 2743–2748
cosanoylamino)-8-octadecene-1,3,4-triol (Kang et al., 2001;
Tuntiwachwuttikul et al., 2004), quercetin 3-O-[ -rhamnopyrano-
syl-(1 ? 6)-b-glucopyranoside] and isorhamnetin 3-O-[ -rhamno-
a
a
pyranosyl-(1 ? 6)-b-glucopyranoside] (Beck and Haberlein, 1999).
Compound 1 was isolated as a brownish amorphous solid with a
ꢀ
ꢁ
negative optical rotation
½
a 2D5
ꢀ
ꢁ 131:3 . In the FABMS, a molecu-
lar ion peak was observed at m/z 625 [M + H]+. The molecular for-
mula was established as C28H32O16 by HRFABMS measurement. In
the 1H NMR spectrum, doublets for meta-coupled protons [d5.85
(1H, d, J = 2.0 Hz, H-8) and 5.98 (1H, d, J = 2.0 Hz, H-6)] indicated
the presence of a 1,2,3,5-tetrasubstituted phenyl moiety. Signals
for two additional aromatic protons [d7.22 (1H, s, H-20) and 7.34
(1H, d, J = 1.0 Hz, H-50)] and a methine proton at d5.17 (1H, br s,
H-4) were observed. Furthermore, methoxy protons [d3.89 (3H,
s)], which showed a NOESY correlation with H-20, were observed.
The 13C NMR spectrum showed the signals of one carbonyl carbon
[d196.1 (C-2)] and one acetal carbon [d112.6 (C-3)]. In the HMBC
experiment with 1, correlations were observed between the fol-
lowing protons and carbons (H-4 and C-2, 3, 5, 9, 10, 10, 60; H-20
and C-2, 10, 30, 40, 60; H-50 and C-4, 10, 30, 40, Fig. 2), indicating that
the aglycone of 1 was 5a,10b-dihydro-1,3,5a,9-tetrahydroxy-8-
methoxy-6H-benz[b]indeno[1,2-d]furan-6-one. In the 1H NMR
spectrum for 1, the signals of two anomeric protons at d4.54 and
4.66 showed the presence of two sugar moieties. The sugars were
Fig. 3. Key NOESY correlations for compounds 1 and 2.
tyl derivative of 1, and three phenolic acetyl methyl signals [d2.26
(3H, s), 2.35 (3H, s) and 2.43 (3H, s)] were observed in the 1H NMR
spectrum. This result confirmed the presence of three phenolic hy-
droxy group in 1. In the NOESY experiment with 1, the correlation
between H-4 and the anomeric proton of glucose indicated a cis
relationship between C-3 and H-4 (Fig. 3). Thus, compound 1
was determined to be rel-(5aS,10bS)-5a,10b-dihydro-1,3,5a,9-tet-
rahydroxy-8-methoxy-6H-benz[b]indeno[1,2-d]furan-6-one 5a-O-
[a-
L-rhamnopyranosyl-(1 ? 6)-b-
D-glucopyranoside].
To
our
knowledge, this is the first report of a 6H-benz[b]indeno[1,2-d]fur-
an-6-one-type compound as a natural product.
Compound 2 was obtained as a brownish amorphous solid.
The HRFABMS gave an [M + H]+ peak at m/z 625.1762 correspond-
ing to the same molecular formula (C28H32O16) as 1. The spectro-
scopic data of 2 were very similar to those of 1 except for the
determined to be b-glucopyranose and
on observed coupling constants as well as comparison to the car-
bon chemical shifts of isorhamnetin 3-O-[ -rhamnopyranosyl-
(1 ? 6)-b-glucopyranoside] (Beck and Haberlein, 1999). In addi-
tion, acid hydrolysis of 1 afforded -glucose and -rhamnose whose
structures were confirmed by co-HPLC analysis of their 1-[(S)-N-
acetyl- -methylbenzylamino]-1-deoxyalditol acetate derivatives
with the same derivatives of standard sugars. The anomeric proton
of glucose (d4.54) was correlated to C-3 of the aglycone and H-1 of
rhamnose (d4.66) to C-6 of glucose (d67.0), which indicated that 6-
a-rhamnopyranose based
ꢀ
ꢁ
a
optical rotation
½
a 2D5
ꢀ
þ 138:3 and the proton and carbon signals
at the 4-position of aglycone and the 1- and 5-positions of the
glucose moiety. Its structure was elucidated to be the same as 1
by HMQC, HMBC, and NOESY experiments. The absence of a cor-
relation in the NOESY experiment between H-4 (d4.82) and the
anomeric proton (d4.90) of glucose confirmed the trans stereo-
chemistry of 2 (Fig. 3). As with 1, acid hydrolysis of 2 yielded
D
L
a
D
-glucose and
tified as rel-(5aS,10bR)-5a,10b-dihydro-1,3,5a,9-tetrahydroxy-8-
methoxy-6H-benz[b]indeno[1,2-d]furan-6-one 5a-O-[ -rhamno-
pyranosyl-(1 ? 6)-b- -glucopyranoside].
L-rhamnose. Therefore, the structure of 2 was iden-
a-L-rhamnopyranosyl b-D-glucopyranose was attached to C-3.
Compound 1 was acetylated by Ac2O in pyridine to give a nonaace-
a-L
D
Compound 3 was obtained as a brownish amorphous solid.
An [M + H]+ peak at m/z 1233 was observed in the FABMS exper-
iment, and the molecular formula C56H64O31 was established by
HRFABMS measurement. The 13C and 1H NMR spectra of 3
showed many similarities to those of 1. A key difference in the
1H NMR spectrum was the presence of the singlet at d6.17 in-
stead of meta-coupled signals of the 6- and 8-positions (d5.85
and 5.98) in 1. Additionally, five aromatic protons [d5.80 (1H,
d, J = 2.2 Hz, H-8), 5.81 (1H, d, J = 2.2 Hz, H-6), 6.84 (1H, d,
J = 8.5 Hz, H-50), 7.17 (1H, dd, J = 8.5, 2.0 Hz, H-60) and 7.26
(1H, d, J = 2.0 Hz, H-20)] were observed in the 1H NMR spectrum
of 3, suggesting the presence of a 1,2,4-trisubstituted phenyl and
1,2,3,5-tetrasubstituted phenyl moieties. Two methine protons at
d4.50 (1H, d, J = 3.7 Hz, H-3) and 4.90 (1H, d, J = 3.7 Hz, H-4) and
an acetal carbon [d100.0 (C-2)] were also observed. Long-range
correlations in the HMBC spectrum were observed between the
following protons and carbons: H-4 and C-2, 3, 5, 9, 10; H-20
and C-2; H-60 and C-2. In addition, methoxy protons (d3.89,
3H, s) were correlated to H-20 in the NOESY spectrum. These re-
sults and comparison with the NMR spectroscopic data of A-
linked proanthocyanidin (Lou et al., 1999; Hatano et al., 2002)
suggest that the 30-O-methylepicatechin moiety was connected
to 1 as in proanthocyanidin type-A. In the HMBC spectrum, both
H-4 and a proton at d6.17 were correlated with a carbon at
d155.1, and H-4 and H-400 (d5.33) with a carbon at d158.5, sug-
gesting that C-4 was attached to C-600 or 800 and C-2 linked C-
500 or 700 through an ether linkage. Acetylation of 3 gave a hepta-
decaacetyl derivative (3a) having five phenolic acetyl groups
[d2.20 (3H, s), 2.31 (3H, s), 2.32 (3H, s), 2.42 (3H, s) and 2.59
Fig. 1. Structures of compounds 1–3.
Fig. 2. Key HMBC correlations for compound 1.

T. Azuma et al. / Phytochemistry 69 (2008) 2743–2748
2745
(3H, s)]. In the HMBC spectrum of 3a, a proton at d6.56 and H-400
(d5.05) were correlated with a carbon at d145.7 (C-500). The up-
through addition of water on C-2 position of malvidin followed
by oxidation (Es-Safi et al., 2008). Therefore, we conducted the fol-
lowing experiment to ascertain whether or not compounds 1–3
were artifacts derived in the extraction and purification process.
Immediately on extraction of freeze–dried rhizomes of K. parviflora
with 3% TFA in 70% aqueous MeOH, HPLC analysis of the extract
was performed using an acidic eluate to detect 1–3 (tR 6.8, 5.7
and 12.1 min, respectively) together with some compounds having
absorption maximum at 520 nm which were expected to be antho-
cyanins (tR of 7.7, 8.9, 15.8 and 18.8 min). This finding indicated
that compounds 1–3 were not artifacts of the extraction and puri-
fication process of the rhizomes of K. parviflora. Further study on
structure determination of anthocyanins are now in progress.
field shift of this carbon signal, compared with that of
3
(d154.0) indicated that the C-500 position was acetylated (Ewing,
1979) and the signal at d6.56 was assignable to H-600. Thus, the
2- and 4-positions of the methylepicatechin moiety were con-
nected to 700-O and C-800, respectively. The presence of two 6-
a-
L-rhamnopyranosyl-D-glucopyranose moieties was confirmed by
the 1H and 13C NMR spectroscopic data of 3 and acid hydrolysis
of 3. In addition, complete assignments of protons of two disac-
charide units were achieved by TOCSY experiment. In the HMBC
spectrum, cross-peaks were observed from two anomeric protons
of glucose moieties at d4.53 and 4.70 to C-3 (d70.1) and C-300
(d112.8) carbons, respectively. The relative stereochemistry of 3
was determined on the basis of the coupling constants in the
1H NMR spectrum and the NOESY experiment. With regard to
the methylcatechin part, the 2,3-trans form was identified by
comparison of the coupling constant of H-3 (3.7 Hz) with that
in the literature (Lou et al., 1999). The NOESY correlation
between H-400 and the glucose anomeric proton (d4.70) indicated
a cis relationship with the group attached to C-300. Furthermore, a
cross-peak between H-6 and H-2000 was observed. The upfield
shifts of H-2000 (d6.99) and the OCH3 at C-3000 (d3.50), compared
with those of 1, were considered to be caused by shielding of
the A-ring in the catechin moiety (Fig. 4). A negative Cotton
effect at short wavelength ([h]213–157,000) in the CD spectrum
of 3 was consistent with the 4S absolute configuration (Hatano
et al., 2002; Barrett et al., 1979; Kolodziej et al., 1991).
These results indicated the absolute configuration of 3 to be
2R, 3S, 4S, 300S, and 400 S. Thus, compound 3 was determined to
3. Conclusions
Three new phenolic glycosides (1–3) having a rare skeleton,
were isolated from the rhizomes of K. parviflora. A feature of these
compounds was the 6H-benz[b]indeno[1,2-d]furan-6-one sub-
structure. A further characteristic of 3 was that 30-O-methylepi-
catechin unit was connected to the benz[b]indeno[1,2-d]furan
skeleton through both C–C bond and ether linkage as A type
proanthocyanidins.
4. Experimental
4.1. General experimental procedures
Optical rotations were recorded on a JASCO P1030 polarimeter
(Tokyo, Japan). The CD spectrum was recorded on a JASCO J-820
spectropolarimeter. UV spectra were measured on a Shimadzu
UV-2500PC UV–Vis spectrophotometer. 1H, 13C, and 2D NMR spec-
tra were obtained on a Varian Unity 500 spectrometer (500 MHz,
Varian Inc., Palo Alto, CA) using TMS as an internal standard. FAB
and HRFABMS were measured using glycerol as the matrix on a
JEOL JMS-700T mass spectrometer. HPLC analysis was carried out
with a JASCO PU-980 Intelligent Pump equipped with a JASCO
MD-910 Multiwavelength Detector. The column for HPLC was a
be (2R,3S,4S)-3-O-[
syl]-30-O-methyl-ent-epicatechin-(2
5a,10b-dihydro-1,3,5a,9-tetrahydroxy-8-methoxy-6H-benz[b]indeno-
a-L-rhamnopyranosyl-(1 ? 6)-b-D-glucopyrano-
a
? O ? 3,4 ? 4)-(5aS,10bS)-
a
[1,2-d]furan-6-one 5a-O-[a-L-rhamnopyranosyl-(1 ? 6)-b-D-gluco-
pyranoside].
Recently, Es-Safi et al. reported that compounds having the
same skeleton as 1 and 2 were formed as artifacts from malvidin
3-O-glucoside incubated in an ethanolic solution at 40 °C for sev-
eral days. They proposed that such compounds might be formed
Mightysil Si 60 (5
lm, 250 ꢂ 4.6 mm, Kanto Chemical). Si gel 60
Fig. 4. Key HMBC and NOESY correlations for compound 3.

2746
T. Azuma et al. / Phytochemistry 69 (2008) 2743–2748
Table 1
(70–230 mesh, Merck), Sephadex LH-20 (Pharmacia) and Chromat-
orex ODS DM1020T (100-200 mesh, Fuji Silysia Chemical) were
used for column chromatography.
NMR spectroscopic data (500 MHz, CD3OD) for compounds 1 and 2a
Position
1
2
dH (m, J in Hz)
dC
dH (m, J in Hz)
dC
4.2. Plant material
2
3
4
5
6
7
8
9
196.1
112.6
49.7
161.7
97.7
196.0
113.3
51.3
161.6
97.4
5.17 (br s)
4.82 (br s)
Rhizomes of K. parviflora from Thailand were supplied by Taiyo
Co., Osaka, Japan. (Lot. No. CP000135820LA).
5.98 (d, 2.0)
5.85 (d, 2.0)
5.94 (d, 2.0)
5.86 (d, 2.0)
160.8
90.1
160.8
90.8
4.3. Extraction and isolation
156.0
105.5
124.7
106.2
150.8
158.5
113.7
152.4
99.2
74.7
77.5
70.8
77.0
155.9
105.4
125.9
106.3
150.7
157.6
113.7
150.5
100.3
74.8
10
Freeze–dried rhizomes (1960 g) of K. parviflora were extracted
with CH2Cl2 at room temperature to afford a crack of extract
(131.8 g). The residue was further extracted with aqueous ace-
tone (3:7, v/v). After filtration, the acetone was evaporated under
reduced pressure and an aqueous extract was obtained. The lat-
ter was partitioned between EtOAc and H2O to give the EtOAc-
soluble (31.8 g) and H2O-soluble fractions (112.6 g). The CH2Cl2
extract (62.5 g) was subjected to silica gel CC using benzene-ace-
tone to give 42 fractions. Fractions 6, 11, 14, 18, 22, 25, 28, 32
and 36 were recrystallized to obtain 5-hydroxy-3,7-dimethoxyf-
lavone (384 mg), 5-hydroxy-3,7,40-trimethoxyflavone (106 mg),
5-hydroxy-7,40-dimethoxyflavone (858 mg), 5-hydroxy-3,7,30,40-
tetramethoxyflavone (15 mg), 5,30-dihydroxy-3,7,40-trimethoxyf-
lavone (29 mg), 3,5,7-trimethoxyflavone (146 mg), 3,5,7,40-tetra-
methoxyflavone (21 mg), 5,7-dimethoxyflavone (7 mg) and
10
20
7.22 (s)
7.22 (s)
30
40
50
7.34 (d, 1.0)
7.33 (d, 0.7)
60
Glc-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6a
Glc-6b
Rha-1
Rha-2
Rha-3
Rha-4
Rha-5
Rha-6
OCH3
4.54 (d, 7.6)
3.29 (m)
3.30 (m)
4.90 (m)
3.20 (dd, 9.3, 7.8)
3.35 (m)
3.27 (m)
3.27 (m)
3.86 (br d, 10.7)
3.51 (br d, 10.7)
4.70 (d, 1.7)
3.90 (dd, 3.4, 1.7)
3.72 (dd, 9.5, 3.4)
3.35 (dd, 9.8, 9.5)
3.61 (dq, 9.8, 6.1)
1.21 (d, 6.1)
3.89 (s)
77.9
71.3
77.3
67.4
3.37 (dd, 9.8, 9.0)
3.03 (ddd, 9.8, 4.6, 2.0)
3.90 (dd, 10.7, 2.0)
3.51 (dd, 10.7, 4.6)
4.66 (d, 1.7)
3.92 (dd, 3.4, 1.7)
3.69 (dd, 9.8, 3.4)
3.35 (dd, 9.8, 9.5)
3.64 (dq, 9.5, 6.3)
1.26 (d, 6.3)
67.0
101.8
72.1
72.3
74.1
69.7
18.0
56.5
102.0
72.2
72.3
74.2
69.7
18.0
56.6
5,7,40-trimethoxyflavone (2023 mg), respectively. Fractions
1
3.89 (s)
and 2 were purified by using silica gel CC (n-hexane-acetone,
99:1) to yield b-sitosteryl myristate (6 mg). Fractions 3 and 4
were separated by silica gel CC (n-hexane-acetone, 97:3) to yield
(2S)-5-hydroxy-7-methoxyflavanone (8 mg), (E)-20-hydroxy-40,60-
dimethoxychalcone (3 mg) and (1E,6E)-1,7,diphenyl-1,6-heptadi-
ene-3,5-dione (5 mg). Fraction 7 was re-applied to a silica gel
column (benzene) to give 5-hydroxy-7-methoxyflavone (11 mg).
Fractions 19 and 20 were separated by silica gel (benzene-
MeOH, 99:1) and ODS (MeOH–H2O, 4:1) CC to yield 5-hydro-
xy-7,30,40-trimethoxyflavone (106 mg) and (2S)-5,7-dimethoxy-
flavanone (3 mg). 3,5,7,30,40-Pentamethoxyflavone (132 mg) was
obtained from fraction 32 by CC using silica gel (CH2Cl2-acetone,
19:1). The EtOAc-soluble fraction (27.9 g) was fractionated by
silica gel CC using benzene-acetone to give 29 fractions. Fraction
22 was purified by silica gel CC (CH2Cl2-acetone, 19:1) to afford
40-hydroxy-5,7-dimethoxyflavone (8 mg) and 5,7,30,40-tetrameth-
oxyflavone (11 mg). Fraction 24 was purified by silica gel CC
(CH2Cl2-acetone, 19:1) to give 1-O-b-glucopyranosyl-(8Z)-2-(2-
a
Chemical shifts are shown in d values (ppm) relative to the solvent peak.
graphed on ODS gel (MeOH–H2O, 45:55) to yield quercetin
3-O-[ -rhamnopyranosyl-(1 ? 6)-b-glucopyranoside] (6 mg).
a
4.4. Compound 1
MeOH
max
Brownish amorphous solid; ½
aꢀ
ꢁ 131:3ꢃ (c 1.0, MeOH); UV
MeOH
max
k
nm (log e
) 236 (4.3), 283 (4.0), 326 (4.0); for 1H and 13C
NMR spectroscopic data, see Table 1; FABMS m/z 625 [M + H]+,
317; HRFABMS m/z 625.1769 [M + H]+ (calcd for C28H33O16
,
625.1768).
4.5. Acetylation of compound 1
hydroxytetracosanoylamino)-8-octadecene-1,3,4-triol
Fraction 26 was subjected to be Sephadex LH-20 (MeOH) and sil-
ica gel (EtOAc–MeOH–H2O, 17:2:1) CC to yield 1-O-hexadeca-
(14 mg).
To a solution of compound 1 (3.0 mg) in pyridine (0.5 ml) was
added an excess of Ac2O (0.5 ml) and the mixture was allowed to
stand overnight at room temperature. The reaction mixture was
partitioned between H2O and EtOAc. The EtOAc layer was washed
with satd. NaCl aq. and evaporated to give compound 1a (4.1 mg).
Compound 1a: pale yellow amorphous solid; 1H NMR (500 MHz,
CDCl3) d1.16 (3H, d, J = 6.1 Hz, Rha-6), 1.97, 2.01, 2.037, 2.040,
2.09, 2.11, 2.26, 2.35, 2.43 (each 3H, s, COCH3), 3.55 (1H, m, Glc-
6b), 3.66 (2H, m, Glc-5, 6a), 3.87 (1H, m, Rha-5), 3.87 (3H, s,
OCH3), 4.74 (1H, br s, Rha-1), 4.93 (1H, br s, H-4), 5.00–5.26 (7H,
m, Glc-1,2,3,4, Rha-2,3,4), 6.63 (1H, dd, J = 2.0, 0.5 Hz, H-8), 6.66
(1H, d, J = 2.0 Hz, H-6), 7.33 (1H, s, H-20), 7.34 (1H, d, J = 0.7 Hz, H-
50); 13C NMR (125 MHz, CDCl3) d17.4 (Rha-6), 20.7–21.3 (COCH3),
49.9 (C-4), 56.4 (OCH3), 66.5 (Rha-5), 66.9 (Glc-6), 69.0–72.6 (Glc-
2,3,4, Rha-2,3,4), 73.0 (Glc-5), 95.7 (Glc-1), 97.9 (Rha-1), 102.6 (C-
8), 107.0 (C-20), 109.9 (C-6), 110.8 (C-3), 116.3 (C-10), 120.7 (C-
50), 131.4 (C-10), 143.4 (C-60), 146.6 (C-5), 147.7 (C-40), 152.1 (C-7),
152.6 (C-30), 159.3 (C-9), 167.9 (COCH3), 168.0 (COCH3), 168.7
(COCH3), 169.5 (COCH3), 169.6 (COCH3), 169.8 (COCH3), 169.9
(COCH3), 170.2 (COCH3), 170.3 (COCH3), 191.6 (C-2).
noyl-3-O-[
(9 mg) and 1-O-hexadecanoyl-2-O-(9Z,12Z-octadecadienoyl)-3-O-
-galactopyranosyl-(1 ? 6)-b-galactopyranosyl]-glycerol (67 mg).
a-galactopyranosyl-(1 ? 6)-b-galactopyranosyl]-glycerol
[a
Fraction 27 was separated by CC using Sephadex LH-20 (MeOH)
and ODS gel (MeOH–H2O, 9:1) to obtain methyl 9Z,12Z-octade-
cadienoate (12 mg) and 1-O-(9Z,12Z-octadecadienoyl)-3-O-[
galactopyranosyl-(1 ? 6)-b-galactopyranosyl]-glycerol (4 mg).
The H2O-soluble fraction (35.5 g) was subjected to Sephadex
LH-20 CC using H2O, H2O–MeOH (1:1, v/v), and MeOH to yield
20 fractions. Fractions 11–15, eluted by MeOH–H2O (1:1, v/v)
were rechromatographed separately. Fraction 11 (650 mg) was
subjected to ODS CC (MeOH–H2O, 3:7) to yield compounds 1
(68 mg) and 2 (72 mg). Fraction 12 (340 mg) was purified by
a-
ODS gel (MeOH–H2O, 3:7) to give isorhamnetin 3-O-[
pyranosyl-(1 ? 6)-b-glucopyranoside] (5 mg). Compound
(55 mg) was obtained from fraction 13 (290 mg) by CC using
ODS gel (MeOH–H2O, 3:7). Fraction 15 (105 mg) was rechromato-
a-rhamno-
3

T. Azuma et al. / Phytochemistry 69 (2008) 2743–2748
2747
Table 2
NMR spectroscopic data (500 MHz) for compounds 3 and 3a
4.6. Compound 2
Brownish amorphous solid: ½a D25
ꢀ
þ 138:3ꢃ (c 1.0, MeOH); UV
Position
3a
3ab
MeOH
max
k
nm (log e
) 236 (4.3), 280 (4.0), 322 (3.9); for 1H and 13C
dH (m, J in Hz)
dC
dH (m, J in Hz)
dC
NMR spectroscopic data see Table 1; FABMS m/z 625 [M + H]+,
Aglycone
2
3
4
317; HRFABMS m/z 625.1762 [M + H]+ (calcd for C28H33O16
,
100.0
70.1
24.9
98.0
70.0
26.2
625.1768).
4.50 (d, 3.7)
4.90 (d, 3.7)
4.44 (d, 3.2)
4.56 (m)
5
6
7
8
156.4
97.3
158.5
95.3
147.6
109.7
150.1
107.1
152.8
114.1
136.2
112.1
150.6
140.5
122.3
119.6
191.4
111.4
49.6
145.7
104.5
153.0
103.0
157.0
112.3
131.3
107.2
152.5
147.7
120.5
144.2
56.2
4.7. Compound 3
5.81 (d, 2.2)
5.80 (d, 2.2)
6.53 (d, 2.2)
6.57 (d, 2.2)
Brownish amorphous solid; ½a D25
ꢀ
ꢁ 131:3ꢃ (c 1.0, MeOH); CD
(MeOH)
[h]205
+45,900,
[h]213 ꢁ 157,000,
[h]246 + 89,500,
9
154.1
105.3
131.8
112.4
148.1
147.9
115.5
121.3
196.0
112.8
49.4
[h]282 ꢁ 13,000, [h]292 ꢁ 5720, [h]322 ꢁ 23,700; UV kMmaexOH nm (log
10
e
) 234 (4.6), 281 (4.1), 324 (3.9); for 1H and 13C NMR spectroscopic
10
data see Table 2; FABMS m/z 1233 [M + H]+, 617; HRFABMS (+NaI)
m/z 1255.3334 [M + Na]+ (calcd for C56H64O31Na, 1255.3328).
20
7.26 (d, 2.0)
7.32 (d, 2.0)
30
40
500
6.84 (d, 8.5)
7.17 (dd, 8.5, 2.0)
7.09 (d, 8.3)
7.23 (dd, 8.3, 2.0)
4.8. Acetylation of compound 3
600
200
300
400
5
Compound 3 (3.0 mg) was acetylated in the same way as com-
pound 1 to give compound 3a (4.1 mg). Compound 3a: pale yellow
solid; for 1H and 13C NMR spectroscopic data see Table 2.
5.33 (d, 0.7)
6.17 (s)
5.05 (d, 0.7)
6.56 (s)
154.0
97.5
600
700
800
900
1000
1000
2000
3000
4000
5000
6000
30-OCH3
155.1
100.1
158.5
107.2
124.6
106.4
150.5
158.5
113.6
152.3
56.7
4.9. Acid hydrolysis of compounds 1, 2 and 3
Solutions of compounds 1, 2 and 3 (1.0 mg, each) in 2 M TFA
(1 ml) were heated at 90 °C for 3 h. After cooling, MeOH was
repeatedly added to and evaporated from the reaction mixture un-
til the acid was completely removed. The residue was dissolved in
6.99 (br s)
7.32 (br s)
7.32 (br s)
7.32 (br s)
H2O (1 ml), to which a solution of (S)-a-methylbenzylamine (5 mg)
and NaBH3CN (8 mg) in EtOH (1 ml) were added. After being al-
lowed to stand overnight at room temperature, glacial AcOH
(0.2 ml) was added to the reaction mixture. After evaporation,
the residue was acetylated with Ac2O (0.3 ml) in pyridine
(0.3 ml) and the mixture was allowed to stand overnight at room
temperature. After evaporation, H2O (1 ml) was added and the
solution was passed through a Sep-Pak C18 cartridge and washed
with H2O, CH3CN–H2O (1:4, v/v) and CH3CN–H2O (1:1, v/v) (each
5 ml), successively. The CH3CN–H2O (1:1, v/v) eluate was analyzed
3.89 (s)
3.50 (br s)
3.92 (s)
3.83 (s)
3
000-OCH3
56.1
56.3
C-3 sugar
Glc-1
Glc-2
Glc-3
Glc-4
4.53 (d, 7.6)
2.95 (dd, 9.3, 7.6)
3.55 (m)
3.12 (dd, 9.3, 9.0)
3.45 (m)
3.91 (m)
3.45 (m)
4.65 (d, 1.7)
3.72 (dd, 3.4, 1.7)
3.58 (m)
99.6
75.2
77.2
71.7
76.9
68.1
4.56 (m)
4.65 (m)
5.14 (m)
4.79 (m)
3.56 (m)
3.52 (m)
95.0
68.6–73.0
68.6–73.0
68.6–73.0
72.4
Glc-5
Glc-6a
Glc-6b
Rha-1
Rha-2
Rha-3
Rha-4
Rha-5
Rha-6
67.1
3.32 (m)
and the 1-[(S)-N-acetyl-a-methylbenzylamino]-1-deoxyalditol
102.2
72.1
72.3
73.9
69.8
17.9
4.62 (m)c
98.01f
acetate derivatives were identified by co-HPLC analysis (column,
Mightysil Si 60, 250 ꢂ 4.6 mm; solvent, n-hexane-EtOH, 9:1 v/v;
flow rate, 1.0 ml/min; detection, UV absorbance at 200–300 nm)
with derivatives of the standard sugars prepared under the same
conditions (Oshima and Kumanotani, 1981; Shao et al., 2007).
5.02–5.21 (m)
5.02–5.21 (m)
5.02–5.21 (m)
3.76 (m)d
1.17 (d, 6.1)e
68.6–73.0
68.6–73.0
68.6–73.0
66.5g
3.28 (m)
3.55 (m)
1.17 (d, 6.1)
17.3h
C-300 sugar
Glc-1
Glc-2
Glc-3
Glc-4
The derivatives of D-glucose and L-rhamnose were detected with
tR of 19.9 min and 13.8 min, respectively.
4.70 (d, 7.6)
3.33 (m)
3.66 (m)
3.35 (m)
3.29 (m)
3.92 (m)
3.51 (m)
4.66 (d, 1.7)
3.94 (dd, 3.4, 1.7)
3.71 (dd, 9.8, 3.4)
3.35 (m)
99.4
75.4
76.4
71.1
76.5
67.1
5.34 (d, 8.1)
5.02 (m)
5.37 (dd, 9.5, 9.3)
5.19 (m)
3.66 (m)
3.69 (m)
97.3
68.6–73.0
68.6–73.0
68.6–73.0
72.9
4.10. Extraction and HPLC analysis of compounds 1, 2 and 3
Glc-5
Glc-6a
Glc-6b
Rha-1
Rha-2
Rha-3
Rha-4
Rha-5
Rha-6
67.7
Freeze–dried rhizomes (100 mg) of K. parviflora were extracted
with 3% TFA (1 ml) in MeOH–H2O (3:7, v/v) (20 min ꢂ 2) at room
temperature. After filtration, MeOH was evaporated under reduced
pressure. An aqueous residue was washed with EtOAc and imme-
3.63 (m)
101.8
72.1
72.3
74.1
69.7
18.0
4.76 (d, 1.5)c
5.02–5.21 (m)
5.02–5.21 (m)
5.02–5.21 (m)
3.91 (m)d
98.05f
68.6–73.0
68.6–73.0
68.6–73.0
66.6g
diately analysed by HPLC (column, Mightysil RP-18 GP (5 lm,
250 ꢂ 4.6 mm, Kanto Chemical); solvent, 0.3% TFA in CH3CN-0.3%
TFA in H2O, 3:17; flow rate, 1.0 ml/min; detection, UV absorbance
at 200–600 nm). Compounds 1–3 were detected with tR of 6.8, 5.7
and 12.1 min, respectively.
3.64 (m)
1.23 (d, 6.3)
1.18 (d, 6.1)e
17.5h
a
Recorded in CD3OD. Chemical shifts are shown in d values (ppm) relative
to the solvent peak.
b
Recorded in CDCl3. Phenolic acetyl methyl protons were detected at
d2.20, 2.31, 2.32, 2.42 and 2.59 (each, 3H, s), glycosyl acetyl methyl protons
were detected at d1.949 (3H, s), 1.954 (6H, s), 1.96, 1.99, 2.02, 2.04, 2.050,
2.053, 2.07, 2.12 and 2.17 (each, 3H, s), acetyl methyl carbons were detected
in the range of d20.5–21.3 and acetyl carbonyl carbons were detected in the
range of d168.0–170.2.
Acknowledgement
We are grateful to Ms. Tomomi Maekawa for measurement of
the NMR spectra and to Ms. Tomomi Shimonaka and Ms. Rika
Miyake for recording MS.
c–h
Assignments with the same superscript may be interchanged.

2748
T. Azuma et al. / Phytochemistry 69 (2008) 2743–2748
Lou, H., Yamazaki, Y., Sasaki, T., Uchida, M., Tanaka, H., Oka, S., 1999. A-type
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