Flavonoid glycosides from the aerial parts of Curcuma comosa

Article abstract of DOI:10.1016/j.phytol.2012.03.003

Four new flavonoid glycosides, curcucomosides A-D (1-4), three known flavonoid glycosides, 5-7, and four known diarylheptanoids, 8-11, were isolated from the ethanol extract of the aerial parts of Curcuma comosa. The structures of the new compounds were established as rhamnazin 3-O-α-l- arabinopyranoside (1), rhamnocitrin 3-O-α-l-arabinopyranoside (2), rhamnazin 3-O-α-l-rhamnopyranosyl-(1→2)-O-α-l-arabinopyranoside (3), and rhamnocitrin 3-O-α-l-rhamnopyranosyl-(1→2)-O-α-l- arabinopyranoside (4) by spectroscopic analysis and chemical reactions whereas those of the known compounds were identified by spectral comparison with those of the reported values.

Full text of DOI:10.1016/j.phytol.2012.03.003

Phytochemistry Letters 5 (2012) 361–366
Contents lists available at SciVerse ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Flavonoid glycosides from the aerial parts of Curcuma comosa
Ratchanaporn Chokchaisiri a, Phongsak Innok b, Apichart Suksamrarn a,
*
a Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand
b Department of Chemistry, Faculty of Science, Chandrakasem Rajabhat University, Bangkok 10900, Thailand
A R T I C L E I N F O
A B S T R A C T
Article history:
Four new flavonoid glycosides, curcucomosides A–D (1–4), three known flavonoid glycosides, 5–7, and
four known diarylheptanoids, 8–11, were isolated from the ethanol extract of the aerial parts of Curcuma
Received 3 January 2012
Received in revised form 12 February 2012
Accepted 3 March 2012
comosa. The structures of the new compounds were established as rhamnazin 3-O-
side (1), rhamnocitrin 3-O- -arabinopyranoside (2), rhamnazin 3-O-
-rhamnopyranosyl-(1!2)-O-
-arabinopyranoside (3), and rhamnocitrin 3-O- -arabinopyrano-
a-L-arabinopyrano-
a
-
L
a-L
Available online 17 March 2012
a
-
L
a
-
L
-rhamnopyranosyl-(1!2)-O-
a-L
side (4) by spectroscopic analysis and chemical reactions whereas those of the known compounds were
identified by spectral comparison with those of the reported values.
Keywords:
Curcuma comosa
Zingiberaceae
ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Flavonoid glycosides
Diarylheptanoids
1. Introduction
of four new flavonoid glycosides, and the isolation and
identification of three known flavonoid glycosides and four
Curcuma comosa Roxb. (Zingiberaceae), commonly known as
Waan Chak Modluk in Thai, has long been used in indigenous
medicine in Thailand. The rhizome of this plant species has
known as an anti-inflammatory agent and has been used widely
for the treatment of postpartum uterine bleeding and as an
aromatic stomachic. However, the folkloric medicinal property
of the aerial parts of this plant has not been reported. A number
of diarylheptanoids with nematocidal (Jurgens et al., 1994) and
estrogenic activity (Suksamrarn et al., 2008), and a phlorace-
tophenone glucoside with choleretic activity (Suksamrarn et al.,
1997) have been isolated. The n-hexane extract of the aerial
parts of C. comosa enhanced hemoglobin (Hb) F production
and labdane diterpenes with Hb F induction potency together
with a diarylheptanoid was isolated (Chokchaisiri et al., 2010).
Investigation of the ethanolic extract revealed the presence of
flavonoid glycosides and preliminary work indicated that
the sugar moiety was not the simple glucose residue. The
present work describes the isolation and structural elucidation
known diarylheptanoids from the ethanol extract of the aerial
parts of C. comosa.
2. Results and discussion
Column chromatography of the EtOH extract resulted in the
isolation of four new flavonoid glycosides, curcucomosides A–D
(1–4), three known flavonoid glycosides, 5–7, and four known
diarylheptanoids, 8–11. The known flavonoid glycosides were
identified as kaempferol 3-O-
2009), quercetin 3-O-arabinopyranoside (guaijaverin) (6) (Wang
and Lee, 1999) and kaempferol 3-O- -rhamnopyranosyl-
(1!2)-O- -arabinopyranoside (7) (Moon et al., 2010) by
a-L-arabinoside (5) (Zong et al.,
a
-L
a
-L
spectroscopic (1H and 13C NMR, and mass spectra) comparison
with the reported values. The isolated diarylheptanoids were
found to be 1-(4-hydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-one
(8), 1-(4-hydroxyphenyl)-7-phenyl-(4E,6E)-4,6-heptadien-3-one
(9), 1-(4-hydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol (10, 3:1
mixture of 3S and 3R enantiomers) and (3S)-1-(3,4-dihydrox-
yphenyl)-7-phenyl-(6E)-6-hepten-3-ol (11) by spectroscopic (1H
NMR and mass spectra) comparison with those reported
previously and TLC comparison with the authentic samples
(Suksamrarn et al., 2008).
* Corresponding author. Tel.: +66 2 319 1900; fax: +66 2 319 0931.
E-mail addresses: asuksamrarn@yahoo.com, s_apichart@ru.ac.th
(A. Suksamrarn).
1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2012.03.003

362
R. Chokchaisiri et al. / Phytochemistry Letters 5 (2012) 361–366
by TLC comparison with standard pentose sugars (Ngounou et al.,
2000). The specific rotation of the sugar was positive as that of the
reported value of the -arabinose (Almeida et al., 1998; Arima and
Danno, 2002). The sugar resonance region in the 1H NMR
spectrum of 1 showed a signal at 6.23 (1H, d, J = 6.4 Hz)
L
d
assignable to the anomeric proton. The coupling constant
observed suggested a trans-diaxial relationship between H-100
and H-200, which indicated a
b-linkage of the sugar with the
flavonoid aglycone. The 1H NMR data (CDCl3, Section 3.9) of the
pentaacetate 1a also confirmed the structure of the sugar unit.
This was further confirmed by the NOE experiments on
compound 1 (Fig. 1). Thus, NOE correlations of H-100 with H-300
and H-500a, and H-500a with H-100 were observed (Halabalaki et al.,
2011). Similar NOE results on the acetate 1a were also obtained
(data not shown). The point of attachment of the glycosyl moiety
was established by the HMBC experiment.
correlation was observed between H-100
6.23) of the sugar
and C-3 ( 134.2) of the aglycone (Fig. 1). Compound 1 was thus
A long-range
(
d
d
elucidated as rhamnazin 3-O-a-L-arabinopyranoside and was
named curcucomoside A.
Compound 2 was obtained as pale yellow needles, ½a D29
ꢁ95.0 (c
ꢀ
0.74, pyridine). The UV spectrum showed maximum absorption
bands of a flavonoid system at lmax 349, 266, and 207 nm. The IR
spectrum indicated the presence of the hydroxyl (3209 cmꢁ1), and
carbonyl (1661 cmꢁ1) groups. The HRTOFMS (ES, negative ion
mode) at m/z 431.0980 [MꢁH]ꢁ was compatible with the
molecular formula C21H20O10. The 1H and 13C NMR spectral
(pyridine-d5, Table 1) features were similar to those of 1; the
significant difference was the absence of a methoxyl signal and the
presence of a p-substituted, instead of a 1,3,4-trisubstituted,
aromatic moiety at
(2H, d, J = 8.7 Hz, H-30 and H-50), which were correlated to the 13C
NMR signals at 131.1 and 115.4, respectively. Acid hydrolysis of 2
d
8.49 (2H, d, J = 8.7 Hz, H-20 and H-60) and 7.22
d
(Section 3.10) yielded rhamnocitrin or kaempferol-7-O-methyl
ether by 1H and 13C NMR, and ESMS spectra (data not shown) and
by comparison of the 1H NMR data with those reported previously
(Iwashina and Ootani, 1990). The sugar moiety of 2 was identified
as a
-arabinose by comparison of 1H and 13C NMR spectra of 2 with
those of compound 1. The structure of 2 was confirmed by 2D-NMR
and NOE experiments. On the basis of the above evidence, the
glycoside was characterized as rhamnocitrin 3-O-a-L-arabinopyr-
anoside and was named curcucomoside B.
Compound 3, ½a 3D0
ꢁ97.7 (c 0.90, MeOH), showed similar UV
ꢀ
spectrum as that of compound 1, thus suggesting structural
similarity of the aglycone part of these two compounds. The IR
data were also similar to those of compound 1. The HRTOFMS
(ES, negative ion mode) showed a pseudo-molecular ion peak at
m/z 607.1656 [MꢁH]ꢁ, which was used to establish the
Compound 1 was obtained as pale yellow needles, ½a D29
ꢁ87.3 (c
ꢀ
0.52, pyridine). The UV spectrum showed maximum absorption
molecular formula as C28H32O15.
The 1H NMR spectra in
bands of a flavonoid system at
l
max 355, 255, and 208 nm. The IR
pyridine-d5 showed more overlapping signals than those
recorded in CD3OD. The 1H and 13C NMR features (CD3OD, Table
1) of the aromatic region of 3 were very similar to those of
compound 1. The aglycone part of 3 was thus established as
rhamnazin. However, the spectral features of the sugar region of
3 were more complicated than those of 1. Acid hydrolysis of 3
yielded rhamnazin, together with two sugars identified as
arabinose and rhamnose (see Section 3.11). Determination of
the specific rotations of these two sugars revealed that they were
spectrum showed absorption bands for the hydroxyl (3287 cmꢁ1
)
and conjugated carbonyl (1661 cmꢁ1) groups. The molecular
formula C22H22O11 was deduced from the HRTOFMS (ES, negative
ion mode) spectrum, which showed an [MꢁH]ꢁ peak at m/z
461.1097. The 1H and 13C NMR assignments were based on the
DEPT and 2D-NMR (COSY, HMQC and HMBC) experiments. The
spectroscopic data indicated that this compound was a flavonoid
glycoside. The 1H and 13C NMR data (Table 1) revealed that the
aglycone part was rhamnazin or 7,30-dimethoxy-3,5,40-trihydrox-
yflavone structure by comparison of spectroscopic data with those
of the reported values (Agrawal, 1989; Subhadhirasakul et al.,
2003). The two methoxyl groups were confirmed to locate at C-7
and C-30, by HMBC and NOE experiments (see Fig. 1).
L
-arabinose and
The 1H NMR data of 3 revealed the presence of the
moiety as the characteristic anomeric signal (H-100) as a doublet
(J = 5.8 Hz) at
5.62. The H-200 and H-500a signals appeared as two
dd at
4.08 (J = 6.6, 5.8 Hz) and 3.41 (J = 9.5, 4.0 Hz). The H-300, H-400
and H-500b resonances appeared as partially overlapping signals at
3.80, 3.78 and 3.82, respectively. The evidence for the attachment
of the C-100 position of the arabinose unit to the C-3 position of the
L-rhamnose.
a
-arabinose
d
d
Acid hydrolysis of 1 (see Section 3.8) gave a flavonoid aglycone
and a sugar. The aglycone was identified as rhamnazin (Sub-
hadhirasakul et al., 2003). The sugar was identified as arabinose
d

Table 1
NMR data (100 MHz for 13C and 400 MHz for 1H,
d
ppm) of compounds 1, 2 (pyridine-d5), 3 and 4 (CD3OD).a
1
2
3
4
Position
dC
dH
HMBC
dC
dH
HMBC
dC
dH
HMBC
dC
dH
HMBC
2
156.8
134.2
178.0
156.3
91.6
–
156.8
134.5
178.1
156.3
91.5
–
158.6
135.1
179.4
158.2
93.0
–
158.9
135.3
179.5
158.3
93.0
–
3
–
–
–
–
4
–
–
–
–
5
–
–
–
–
6
6.66 (d, 1.7)
C-4, 5, 7, 8, 10
C-6, 7, 9, 10
6.63 (d, 1.9)
C-4, 5, 7, 8, 10
C-6, 7, 9, 10
6.57 (d, 2.0)
C-5, 7, 8, 10
C-6, 7, 9, 10
6.59 (br s)
–
C-5, 7, 8, 10
C-6, 7, 9, 10
7
165.0
97.7
–
165.0
97.6
–
167.2
98.9
–
167.2
98.9
8
6.57 (d, 1.7)
6.57 (d, 1.9)
6.29 (d, 2.0)
6.32 (br s)
–
9
161.5
105.4
122.1
113.4
147.3
150.6
115.5
121.0
102.9
72.0
–
161.5
105.3
120.9
131.1
115.4
161.0
115.4
131.1
102.9
71.8
–
162.8
106.6
123.0
114.3
148.6
150.8
116.1
123.7
101.1
77.5
–
162.9
106.6
122.6
132.2
116.3
161.6
116.3
132.2
102.2
77.2
10
10
20
30
40
50
60
100
200
300
400
500
–
–
–
–
–
–
–
–
8.54 (br d, 0.8)
C-2, 10, 30, 40, 60
8.49 (d, 8.7)
C-2, 40, 60
C-10, 40, 50
7.96 (d, 1.7)
–
C-2, 10, 30, 40, 60
8.06 (d, 8.7)
6.90 (d, 8.7)
–
C-2, 40, 60
C-10, 20, 40
–
7.22 (d, 8.7)
–
–
–
7.30 (d, 8.4)
C-30, 40, 60
C-2, 20, 40
C-3, 300, 500
C-100, 300, 400
C-100, 200
C-200, 300
C-100, 300, 400
7.22 (d, 8.7)
C-10, 30, 40
C-2, 20, 40
C-3, 300, 500
C-100, 300, 400
C-100, 200
C-200
C-100, 300, 400
6.91 (d, 8.4)
7.59 (dd, 8.4, 1.7)
5.62 (d, 5.8)
4.08 (dd, 6.6, 5.8)
3.80b
3.78b
H-500a: 3.41 (dd, 9.5,
4.0); H-500b: 3.82b
C-10, 20, 30, 40, 60
C-2, 20, 40
C-3, 300, 500
C-100, 300, 400, 1000
C-200
C-300
C-100, 300, 400
6.90 (d, 8.7)
8.06 (d, 8.7)
5.53 (d, 5.0)
4.09 (dd, 6.2, 5.0)
3.81b
3.74b
H-500a: 3.32c;
H-500b: 3.77b
C-10, 40, 60
C-2, 20, 40
C-3, 200, 300, 500
C-100, 300, 400, 1000
C-200
C-300
C-300, 400
7.90 (dd, 8.4, 0.8)
8.49 (d, 8.7)
6.23 (d, 6.4)
6.18 (d, 5.7)
4.80 (dd, 7.1, 6.4)
4.87 (dd, 6.4, 5.7)
73.2
4.34b
4.35b
73.0
4.39b
4.41b
73.3
72.7
67.7
67.2
68.9
68.3
66.1
H-500a: 3.80 (br d,
11.8); H-500b: 4.31
65.4
H-500a: 3.79 (br d,
11.4); H-500b: 4.31
65.9
65.1
(dd, 11.8, 2.7)
(dd, 11.4, 3.9)
1000
–
–
–
–
–
–
102.4
72.3
72.4
73.9
70.0
17.5
56.4
57.0
–
5.08 (br s)
3.93b
C-200, 2000, 5000
C-3000, 4000
C-2000, 4000, 5000
C-2000, 5000
C-3000, 4000
C-4000, 5000
C-7
101.0
72.2
72.4
74.0
70.1
17.7
56.4
–
5.08 (br s)
3.89b
C-200, 3000, 5000
C-3000, 4000
C-2000, 4000, 5000
C-3000, 5000
C-3000, 4000
C-4000, 5000
C-7
2000
–
–
3000
–
–
–
–
3.69 (dd, 9.5, 3.1)
3.32 (t, 9.5)
3.90b
3.69 (dd, 9.3, 3.2)
3.35 (t, 9.3)
3.86b
4000
–
–
–
–
5000
–
–
–
–
6000
–
–
–
–
0.98 (d, 6.1)
3.86 (s)
1.07 (d, 6.1)
3.87 (s)
–
7-OCH3
30-OCH3
5-OH
55.1
55.5
–
3.74 (s)
4.00 (s)
13.22 (s)
C-7
C-30
55.1
–
3.74 (s)
–
C-7
3.99 (s)
C-30
–
13.20 (s)
–
–
–
a
Assignments were based on DEPT, HMQC, and HMBC experiments.
Partially overlapping signal.
b
c
Obscured signal.

364
R. Chokchaisiri et al. / Phytochemistry Letters 5 (2012) 361–366
since the 1H and 13C NMR data of the sugar moieties of these two
compounds were almost identical. On the basis of the above
evidence, compound 4 was characterized as rhamnocitrin 3-O-
OMe
a-L-
OH
rhamnopyranosyl-(1!2)-O-
a-
L-arabinopyranoside
and
was
named curcucomoside D.
MeO
O
O
Compounds 1–7 have been evaluated for cytotoxic activities
against human epidermoid carcinoma (KB), human breast cancer
(MCF7) and human small cell lung cancer (NCI-H187) cells, and
antiplasmodial activity against Plasmodium falciparum. These
compounds have also been evaluated for the enhancement of
fetal hemoglobin production. However, none of them showed
these activities. The diarylheptanoids 8–11 have been reported to
exhibit estrogenic activity (Suksamrarn et al., 2008).
OH
O
HO
H
H
O
In summary, four new and three known flavonoid glycosides
were isolated from the aerial parts of C. comosa. The aglycone
moieties are the flavonols rhamnazin, rhamnocitrin, kaempferol
and quercetin, whereas the glycoside parts are arabinopyranoside
and rhamnopyranosyl arabinopyranoside. Four known diarylhep-
tanoids were also isolated from this plant species.
H
HO
OH
H
H
H
Fig. 1. Selected HMBC (one-headed arrow) and NOE (two-headed arrow)
correlations of compound 1.
3. Experimental
3.1. General
flavonoid was in the same analogy to that of the compound 1 case.
Melting points were determined with an electrothermal
melting point apparatus and are uncorrected. Optical rotations
were measured on a JASCO-1020 polarimeter. IR spectra were
obtained using a Perkin Elmer FT-IR Spectrum BX spectrophotom-
eter. 1H and 13C NMR spectra were recorded on a Bruker AVANCE
400 FT-NMR spectrometer, operating at 400 MHz (1H) and
100 MHz (13C). ESMS and ESTOFMS spectra were measured with
a Finnigan LC-Q and a Bruker micrOTOF mass spectrometer. Unless
indicated otherwise, column chromatography was carried out
using Merck silica gel 60 (<0.063 mm) and Pharmacia Sephadex
LH-20. Merck Lichrolut RP-18 (0.040–0.060 mm) was used for
reversed phase column chromatography. For TLC, Merck precoated
silica gel 60 F254 plates were used. Spots on TLC were detected
under UV light and by spraying with anisaldehyde-H2SO4 reagent
followed by heating.
Thus, a long-range correlation between H-100
(
d
5.62) of the sugar
135.1) of the aglycone was observed. Another sugar
moiety was suggested to be rhamnose from the characteristic
signals of H-1000 and H-6000 at
5.08 (br s) and 0.98 (d, J = 6.1 Hz). The
H-3000 and H-4000 signals were located at
3.69 (dd, J = 9.5, 3.1 Hz)
and 3.32 (t, J = 9.5 Hz). The remaining proton resonances, H-2000 and
H-5000, were two partially overlapping signals at
3.93 and 3.90,
and C-3 (
d
d
d
d
respectively. The spectroscopic data together with the 2-D and
NOE experiments have indicated that this second sugar moiety is
a
-L
-rhamnopyranosyl unit. The point of attachment of the two
glycosyl units was established by the HMBC experiment. A long-
range correlation was observed between H-1000
5.08) of
rhamnose and C-200 77.5) of arabinose, and between H-200
4.08) of arabinose and C-1000
102.4) of rhamnose. Furthermore, a
(
d
(
d
(d
(
d
4.8 ppm downfield shift of the C-200 resonance of the arabinose unit
comparing with the C-200 resonance of the unsubstituted arabinose
in compound 6 in the same solvent (data not shown) confirmed
that the rhamnosyl unit was linked to C-200 position of the
arabinose moiety. The observed NOE enhancements between H-200
and H-1000 (65%), and H-1000 and H-200 (44%) further confirmed the
linkage positions of these two sugars (Ye et al., 1999). The 1H and
13C NMR data of 3 were similar to those of 7, which has recently
been isolated from Draba nemorosa (Moon et al., 2010). The
significant differences were the absence of two methoxyl signals of
3.2. Plant material
The aerial parts of C. comosa were collected from Kampaeng-
saen district, Nakhon Pathom province, Thailand, in January, 2008.
A voucher specimen (Apichart Suksamrarn, no. 052) of this plant
species is deposited at the Faculty of Science, Ramkhamhaeng
University.
3.3. Extraction and isolation
the latter glycoside. Compound
3
was thus established as
-arabinopyra-
rhamnazin 3-O-
a
-
L
-rhamnopyranosyl-(1!2)-O-
a
-L
The air-dried aerial parts of C. comosa (2.20 kg) were milled and
macerated successively with n-hexane and EtOH to yield, after
evaporation of the solvents under reduced pressure, the hexane
(171.0 g) and EtOH (193.2 g) extracts, respectively.
noside and was named curcucomoside C.
Compound 4, ½a 3D0
ꢁ155.2 (c 0.18, MeOH), exhibited UV
ꢀ
absorption maxima very similar to those of compound 2. The IR
absorption bands of this compound were also similar to other
isolated flavonoid glycosides. The molecular formula C27H30O14
was deduced from the HRTOFMS spectrum (ES, negative ion
mode), which show an [MꢁH]ꢁ peak at m/z 557.1556. The 1H and
13C NMR spectra of 4 were similar to those of 3. The significant
differences were the absence of a methoxyl signal and the 1,3,4-
trisubstituted aromatic system, and the presence of p-disubstitut-
The EtOH extract (190.0 g) was fractionated by column
chromatography (Merck silica gel 60 PF254, 520 g), using a gradient
solvent system of n-hexane, n-hexane–EtOAc, EtOAc, EtOAc–
MeOH and MeOH with increasing amounts of the more polar
solvent. The eluates were examined by TLC and 5 combined
fractions (E1–E5) were obtained. Fraction E1 (29.5 g) was
subjected to column chromatography on silica gel using n-
hexane–EtOAc (100:2) to afford 5 subfractions. Subfraction 3
(1.01 g) was subjected to column chromatography twice, using
CH2Cl2–MeOH (100:1) to give compound 8 (Suksamrarn et al.,
2008) (15.2 mg) as colorless viscous oil. Subfraction 4 was
chromatographed, eluting with CH2Cl2–MeOH and MeOH in
ed aromatic protons at
(2H, d, J = 8.7 Hz, H-30, H-50) which were correlated to the 13C-NMR
signals at 132.2 and 116.3, respectively, by the HMQC
d
8.06 (2H, d, J = 8.7 Hz, H-20, H-60) and 6.90
d
experiments. The aglycone part of 4 was thus concluded as
rhamnocitrin. The sugar residue of 4 was the same as that of 3,

R. Chokchaisiri et al. / Phytochemistry Letters 5 (2012) 361–366
365
increasing proportion of the more polar solvent and followed by
Sephadex LH-20, eluted with MeOH, to yield compound
(KBr) 3491, 1654, 1594, 1497, 1431, 1344, 1289, 1212, 1159, 1126,
1080, 1039 cmꢁ1; 1H and 13C NMR data, see Table 1; HRTOFMS (ES,
negative ion mode) m/z 607.1656 [MꢁH]ꢁ (calc. for C28H32O15–H,
607.1663).
9
(Suksamrarn et al., 2008) (28.1 mg) as prisms, m.p. 129–130 8C
(EtOAc–n-hexane), lit. 129–130 8C (Suksamrarn et al., 2008).
Fraction E2 (15.2 g) was chromatographed, eluting under isocratic
condition of CH2Cl2–MeOH (100:2) followed by Sephadex LH-20,
eluted with MeOH, to yield compound 10 (Suksamrarn et al., 1994,
3.7. Rhamnocitrin 3-O-
a
-L
-rhamnopyranosyl-(1!2)-O-
a-L-
arabinopyranoside (4)
2008) (67.2 mg), ½a D26
ꢁ1.73 (c 1.48, EtOH). Compound 10 was
ꢀ
shown to be a 3:1 mixture of 3S and 3R enantiomers by the
modified Mosher’s method (Dale and Mosher, 1973; Ohtani et al.,
1991) in the same manner as described in the previous work
(Suksamrarn et al., 2008). Fraction E3 (42.1 g) was subjected to
column chromatography twice, using CH2Cl2–MeOH (100:3) to
furnish compound 11 (Suksamrarn et al., 2008) (111.4 mg), m.p.
99–100 8C (from CH2Cl2–n-hexane), lit. 100–101 8C (Suksamrarn
Pale yellow amorphous solid; ½a D30
ꢁ155.2 (c 0.18, MeOH); UV
ꢀ
(MeOH) lmax (log e) 350 (4.30), 266 (4.36), 207 (4.56) nm; IR nmax
(KBr) 3491, 1657, 1594, 1513, 1497, 1447, 1431, 1345, 1289, 1212,
1159, 1127, 1078 cmꢁ1 1H and 13C NMR data, see Table 1;
;
HRTOFMS (ES, negative ion mode) m/z 577.1556 [MꢁH]ꢁ (calc. for
C27H30O14–H, 577.1557).
et al., 2008), ½a D28
ꢀ
ꢁ5.2 (c 0.41, EtOH). The absolute stereochemistry
3.8. Acid hydrolysis of 1
of 11 was determined to be 3S by the modified Mosher’s method.
Fraction E4 (63.2 g) was subjected to column chromatography
eluted with solvents of increasing polarity, CH2Cl2, CH2Cl2–MeOH
and MeOH, to give 6 subfractions. Subfraction 3 (178.3 mg) upon
standing yellow solid separated out, which was rechromato-
graphed eluting under isocratic condition with CH2Cl2–MeOH
(100:5) to furnish compound 1 (13.1 mg) and compound 2
(23.4 mg). The mother liquor was evaporated and the residue
(97.8 mg) was chromatographed on a Sephadex LH-20 column
eluting with MeOH, followed by repeated column chromatography
eluting with CH2Cl2–MeOH (100:8) to yield compound 5 (Zong
et al., 2009) (15.4 mg) as yellow amorphous solid, m.p. 241–242 8C
(from MeOH), lit. 251–253 8C (MeOH) (Abou-Shoer et al., 1993);
A mixture of 1 (10 mg) in EtOH (10 ml) and 5% HCl (10 ml) was
heated at 80 8C for 3 h. The mixture was neutralized with 5% aq.
K2CO3 and EtOH was evaporated under reduced pressure. The
mixture was extracted with EtOAc (3 ꢂ 15 ml) to give the
aglycone (1.5 mg) which was identified as 7,30-di-O-methyl-
quercetin or rhammazin by comparison of spectroscopic data
with those reported in the literature (Subhadhirasakul et al.,
2003). Most of the water was removed by azeotropic distillation
with n-BuOH under reduced pressure. The sugar in the
concentrated water-soluble portion was compared with stan-
dard sugars on a TLC plate, with n-BuOH–EtOAc–iso-PrOH–
AcOH–H2O (7:20:12:7:6) as developing solvent system (Ngou-
nou et al., 2000) and the spots were detected with the
anisaldehyde reagent. The Rf value of arabinose, lyxose, ribose
and xylose was 0.38, 0.46, 0.45, and 0.47, respectively. The sugar
component was identified as arabinose. The inorganic salt was
removed from the concentrated aq. solution by repeated
½
a 2D9
ꢀ
ꢁ79.7 (c 0.20, MeOH), lit. ½aꢀD ꢁ49 (c 0.1, MeOH) (Abou-Shoer
et al., 1993). Subfraction 4 was separated on Sephadex LH-20
column, eluting with MeOH, followed by silica column chroma-
tography, eluting with CH2Cl2–MeOH (100:6), to afford compound
6 (Wang and Lee, 1999) (15.4 mg) as yellow needles, m.p. 249–
250 8C (from MeOH), lit. 250–251 8C (MeOH) (Wang and Lee,
precipitation with MeOH to yield
a
-arabinose (1.86 mg), ½a D30
ꢀ
1999); ½a 2D9
ꢀ
ꢁ120.4 (c 0.51, pyridine), lit. ½a D23
ꢀ
ꢁ97.2 (c 0.72,
+70.9 (c = 0.186, H2O), lit. ½a D30
ꢀ
+103 (Arima and Danno, 2002).
pyridine) (Wang and Lee, 1999). Subfraction 5 was chromato-
graphed on Sephadex LH-20 twice, eluted with MeOH and then
subjected to repeated reversed phase RP-18 using MeOH–H2O
Positive optical rotation indicated that this sugar is
(Arima and Danno, 2002).
L-arabinose
(30:70) to afford compounds
4
(11.6 mg) and
3
(24.8 mg).
3.9. Acetylation of 1
Subfraction 6 was purified by Sephadex LH-20 using MeOH to
give compound 7 (4.4 mg).
Compound 1 (2.0 mg) was dissolved in pyridine (1 ml) and
Ac2O (8 drops) was added. The mixture was stirred at ambient
temperature for 6 h. Water (10 ml) was added and the mixture
was extracted with CH2Cl2 (3 ꢂ 15 ml). The combined CH2Cl2
layer was washed with water and evaporated under reduced
pressure to dryness. The crude mixture was purified by Sephadex
3.4. Rhamnazin 3-O-a-L-arabinopyranoside (1)
Pale yellow needles; m.p. 240–241 8C (MeOH); ½a D29
ꢁ87.3 (c
ꢀ
0.52, pyridine); UV (MeOH) lmax (log e) 355 (4.30), 255 (4.38), 208
(4.58) nm; IR
n
max (KBr) 3287, 1661, 1606, 1590, 1520, 1498, 1340,
LH-20, eluting with MeOH, to give rhamnazin 3-O-
pyranoside 5,40,200,300,400-pentaacetate (1a) (2.4 mg), 1H NMR
(CDCl3, 400 MHz)
7.94 (1H, d, J = 1.1 Hz, H-20), 7.59 (1H, dd,
a-L-arabino-
1292, 1208, 1093, 1073, 1007 cmꢁ1
;
1H and 13C NMR data, see
Table 1; HRTOFMS (ES, negative ion mode) m/z 461.1097 [MꢁH]ꢁ
d
(calc. for C22H22O11–H, 461.1084).
J = 8.4, 1.1 Hz, H-60), 7.12 (1H, d, J = 8.4 Hz, H-50), 6.78 (1H, d,
J = 1.8 Hz, H-6), 6.58 (1H, d, J = 1.8 Hz, H-8), 5.51 (1H, d, J = 7.2 Hz,
H-100), 5.35 (1H, dd, J = 9.6, 7.2 Hz, H-200), 5.21 (1H, m, W1/2 = 7 Hz,
H-400), 5.13 (1H, dd, J = 9.6, 3.3 Hz, H-300), 3.79 (1H, dd, J = 13.0,
2.4 Hz, H-500b), 3.51 (1H, br d, J = 13.0 Hz, H-500a), 3.96 (3H, s, 30-
OCH3), 3.88 (3H, s, 7-OCH3), 2.43 (3H, s, 5-OAc), 2.32 (3H, s, 40-
OAc), 2.09 (3H, s, OAc), 2.06 (3H, s, OAc), 2.01 (3H, s, OAc); ES-MS
m/z: 673 [M+H]+.
3.5. Rhamnocitrin 3-O-a-L-arabinopyranoside (2)
Pale yellow needles; m.p. 242–243 8C (MeOH); ½a D29
ꢁ95.0 (c
ꢀ
0.74, pyridine); UV (MeOH) lmax (log e) 349 (4.25), 266 (4.31), 207
(4.46) nm; IR
nmax (KBr) 3209, 1661, 1601, 1586, 1498, 1343, 1288,
1212, 1182, 1079, 1010 cmꢁ1
;
1H and 13C NMR data, see Table 1;
HRTOFMS (ES, negative ion mode) m/z 431.0980 [MꢁH]ꢁ (calc. for
C20H21O10–H, 431.0978).
3.10. Acid hydrolysis of 2
3.6. Rhamnazin 3-O-
a
-
L
-rhamnopyranosyl-(1!2)-O-
a
-
L
-
Compound 2 (10 mg) was subjected to hydrolysis in the same
manner employed for compound 1 to yield rhamnocitrin or
kaempferol 7-O-methyl ether (2.1 mg). The identity of the
flavonoid was confirmed by comparison of spectroscopic data
with the reported values (Iwashina and Ootani, 1990).
arabinopyranoside (3)
Pale yellow amorphous solid; ½a D30
ꢁ97.7 (c 0.90, MeOH); UV
ꢀ
(MeOH) lmax (log e) 353 (4.23), 255 (4.32), 207 (4.52) nm; IR nmax

366
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ꢁ9.0
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Acknowledgments
This work was supported by the Strategic Basic Research Grant
of The Thailand Research Fund (TRF). RC is grateful for a
scholarship from the Royal Golden Jubilee Ph.D. Program of TRF.
Partial support from the Center of Excellence for Innovation in
Chemistry (PERCH-CIC), Office of the Higher Education Commis-
sion, Ministry of Education is gratefully acknowledged.
Suksamrarn, A., Eiamong, S., Piyachaturawat, P., Charoenpiboonsin, J., 1994. Phe-
nolic diaryheptanoids from Curcuma xanthorriza. Phytochemistry 36,
1505–1508.
Appendix A. Supplementary data
Suksamrarn, A., Ponglikitmongkol, M., Wongkrajang, K., Chindaduang, A., Kittida-
nairak, S., Jankam, A., Yingyongnarongkul, B., Kittipanumat, N., Chokchaisiri, R.,
Khetkam, P., Piyachaturawat, P., 2008. Diarylheptanoids, new phytoestrogens
from the rhizomes of Curcuma comosa: isolation, chemical modification and
estrogenic activity evaluation. Bioorg. Med. Chem. 16, 6891–6902.
Wang, P.H., Lee, S.S., 1999. Polar chemical constituents from Phoebe formosana. J.
Chin. Chem. Soc. 46, 215–219.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytol.2012.03.003.
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