Biflavonoids from flowers of Butea monosperma (Lam.) Taub.

Article abstract of DOI:10.3987/COM-11-12275

A new aurone glucoside (1) and three new biflavonoids (12 - 14), together with fourteen known compounds, were isolated from the flowers of Butea monosperma (Lam.) Taub. The structures of the new compounds were established by 1D, 2D NMR, MS and CD analyses. The isolated compounds were evaluated for their influenza A neuraminidase inhibitory activity and DPPH free-radical scavenging activity.

Full text of DOI:10.3987/COM-11-12275

HETEROCYCLES, Vol. 83, No. 9, 2011
2079
HETEROCYCLES, Vol. 83, No. 9, 2011, pp. 2079 - 2089. © The Japan Institute of Heterocyclic Chemistry
Received, 6th June, 2011, Accepted, 30th June, 2011, Published online, 7th July, 2011
DOI: 10.3987/COM-11-12275
BIFLAVONOIDS FROM FLOWERS OF BUTEA MONOSPERMA (LAM.)
TAUB.
Fakhruddin Ali Ahmed, Sang-Yong Kim, Shin-ichiro Kurimoto, Hisako
Sasaki, Hirofumi Shibata, Yoshiki Kashiwada,* and Yoshihisa Takaishi
Graduate School of Pharmaceutical Sciences, University of Tokushima, Shomachi
1
-78, Tokushima 770-8505, Japan
E-mail address: kasiwada@ph.tokushima-u.ac.jp
Abstract – A new aurone glucoside (1) and three new biflavonoids (12 - 14),
together with fourteen known compounds, were isolated from the flowers of
Butea monosperma (Lam.) Taub. The structures of the new compounds were
established by 1D, 2D NMR, MS and CD analyses. The isolated compounds were
evaluated for their influenza A neuraminidase inhibitory activity and DPPH
free-radical scavenging activity.
INTRODUCTION
Butea monosperma (Lam.) Taub. (Fabaceae), which is commonly known as ‘Flame of forest’ or ‘Palash’,
is a small to medium-sized deciduous tree widely distributed in tropical southern Asia up to an altitude of
4
,000 ft. This plant species has been used as a traditional herbal medicine in Ayurveda and Unani. It's
roots have been used for elephatiasis and several eyesight defects, while the bark decoction has been used
1
for the treatment of goiter, ulcer, dysentery, liver disorder and tumors. Flowers have been used as an
emmenagogue, diuretic, depurative and tonic, as well as for the treatment of leprosy, gout and skin
2
3
diseases, spermatorrhoea and leucorrhoea. The flowers of this plant are also used commercially as a
natural dyeing due to their bright orange-red color. Several biological activities for the flowers of B.
4
5
6
7
monosperma have been reported including antidiabetic, anticonvulsive, antiesterogenic, antioxidative,
8
9
10
hepatoprotective, chemopreventive and mast cell inactivation activities, and some of which validated
the traditional use of this plants. However, their active components have not been clarified. By contrast,
11
previous phytochemical analysis for the flowers of B. monosperma revealed the presence of flavone,
1
2-14
14
12-14
12,13,15
and aurones.
flavanones,
isoflavones, chalcones,
As part of our chemical studies of

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HETEROCYCLES, Vol. 83, No. 9, 2011
medicinal plants aimed at searching for biologically active compounds, we have examined the flowers of
Butea monosperma, which has resulted in the isolation of a new aurone glucoside and three new
biflavonoids together with fourteen known compounds. The influenza A neuraminidase inhibitory activity
and DPPH free-radical scavenging activity for the isolated compounds were also evaluated. The present
paper articulates the structure elucidation of new compounds and evaluation of biological activities for
the isolated compounds.
RESULTS AND DISCUSSION
The flowers of B. monosperma were extracted with MeOH at room temperature. The MeOH extract was
partitioned between EtOAc and water. The EtOAc-soluble fraction was further partitioned between
hexane and 90% MeOH. Repeated chromatography of the 90% MeOH-soluble fraction over Sephadex
LH-20, MCI gel CHP20P, YMC ODS-A, silica gel and purification by HPLC to afford a new aurone
glucoside (1) and three new biflavonoids (12 - 14), together with fourteen known compounds. The known
1
6
17
13
18
compounds were identified as sulfurein (2), butein (3), monospermoside (4), isobutrin (5),
1
9
20
13
21
22
coreopsin (6), butin (7), isomonospermoside (8), formononetin (9), aformosin (10), kaempferide
2
3
24
25
26
(11),
umbelliferon,
7,8-dihydroxychromone,
methyl
3,4,5-trihydroxybenzoate
and
2
7
2
,4-dihydroxybenzoic acid by comparison of their physical and spectral data with those reported in the
literature. 7,8-Dihydroxychromone was isolated for the first time as a natural product.
–
Compound 1 gave an [M–H] ion peak at m/z 431.0978 in the HRESIMS, indicating the molecular
1
formula C21
H
20
O
10. The H NMR spectrum of 1 revealed, together with an olefinic proton signal [!
H
7.05
6.93 (1H, dd, J = 8.4, 1.8 Hz), 7.13 (1H, d, J = 1.8 Hz),
and 7.83 (1H, d, J = 8.4 Hz); 7.24 (1H, d, J = 8.3 Hz), 7.67 (1H, dd, J = 8.3, 1.8 Hz), and 8.28 (1H, d, J =
(
s)], two sets of ABX-type aromatic signals [!
H
1
.8 Hz)], suggesting the presence of two 1,2,4-trisubstituted aromatic rings. It also showed an anomeric
1
3
H
proton signal at ! 5.62 (d, J = 7.8 Hz) suggestive of a glycosidic nature of 1. The C-NMR spectrum
2
3
indicated the presence of 21 carbons, including 15 sp carbons and six sp carbons arising from a hexosyl
moiety, whose chemical shifts were in good agreement with the occurrence of a glucosyl moiety. These
1
3
spectral data were closely related to those of sulfurein (2), but differed the C chemical shifts for the
aromatic signals, suggesting the position of the glucosyl moiety was different. The 2D-NMR analyses
indicated the aglycone moiety of 1 to be sulphuretin, which was confirmed by acid hydrolysis of 1 with
2
8
5
% HCl, which afforded an aglycone, identical with (Z)-6,3',4'-trihydroxyaurone, together with glucose.
The HMBC correlation of the anomeric proton signal with C-3' indicated the location of the glucosyl
moiety to be at C-3'. The !-linkage of the glucosyl moiety was concluded from the coupling constant
value (7.8 Hz) of the anomeric proton signal. Thus, the structure of 1 was assigned as sulphuretin
3
'-O-!-glucopyranoside.

HETEROCYCLES, Vol. 83, No. 9, 2011
2081
OH
OR
OH
R O
2
OH
O
OH
R O
1
O
O
HO
O
O
OR2
OR1
R1
R2
R1
R2
Glc
H
R
H
3
4
5
6
H
H
1
2
H
7
8
Glc
Glc
H
H
Glc
Glc
Glc
Glc
HO
O
O
OMe
HO
O
O
R
OMe
OH
R
H
OH
9
0
11
1
OMe
OH
6'
OH
'
OH
6
'
OH
4
2'
8
H
4'
4'
H
8
H
H
HO
9
O
4
8
HO
9
O
OH
HO
9
O
4
2
2
'
OH
O
2
6'
2
2'
H
"
6
2"
10
OH
OH
6
4
OH
6
H
10
1
0
H
!
1
0"
4"'
H
H
O
6"
O
"
4
"
O
6"'
O
6"'
6"
7
2
"'
4"
2
"
HO
8"
OH
HO
O
O
9"
OH
2"'
8
"
H
2" 10"
2
"'
5"
9
"
HO 4"'
6
"'
HO 4"'
OH
O
12
13
14
Figure 1. The structures of flavonoids isolated from the flowers of B. monosperma
–
An [M–H] ion peak at m/z 541.1115 (calcd for 541.1135) was observed in the HRESIMS of compound
1
1
2, indicating the molecular formula C30
aromatic signals [!
.1 Hz), 6.99 (dd, J = 8.1, 2.0 Hz) and 7.43 (d, J = 2.0 Hz)] and a pair of doublets [!
H
22
O
10. The H NMR spectrum showed two sets of ABX-type
H
8.14 (d, J = 8.7 Hz), 6.82 (dd, J = 8.7, 2.2 Hz) and 6.66 (d, J = 2.2 Hz); 7.31 (d, J =
6.61 and 3.46 (each
d, J = 11.9 Hz)], while the C NMR spectrum of 12 revealed 15 carbon signals, including a carbonyl
8
H
1
3
2
2
carbon (! 191.6), six sp quaternary carbon (! 114.2, 128.7, 146.8, 147.7, 163.7, and 165.8), six sp
C
C
3
methine (!
C
103.1, 111.0, 116.1, 116.1, 119.6, and 129.2), an oxygen-bearing sp methine (!
C
84.9) and
3
sp methine (!
C
51.3). Taking its molecular formula into account, compound 12 was indicated to be a
symmetrical biflavanone with a C-3/C-3" linkage. The positions of hydroxyl groups were assigned to be
at C-7/C-7˝, C-3´/C-3˝´ and C-4´/C-4˝´ from the HMBC correlations of H-5/H-5" with C-4/C-4", and of
H-2'/H-2"' and H-6'/H-6"' with C-2/C-2". The trans configuration of H-2/H-3 (H-2˝/H-3˝) was elucidated
from their J values (11.9 Hz). The absolute stereochemistry was assigned as 2S,3R by the CD spectral

2082
HETEROCYCLES, Vol. 83, No. 9, 2011
analyses, in which the first positive ["# CD +11.9 (334 nm)] and the second negative ["# CD –17.5 (304
2
9
nm)] Cotton effects were similar to those of the related compound. Therefore, the structure of 12 was
elucidated as shown.
–
The molecular formula of compound 13 was assigned as C30
H
20
O
10 by HRESIMS (m/z 539.0940 [M–H] ),
1
suggesting that 13 was also a biflavonoid. The H NMR spectrum, together with signals closely correlated
with those of 12, showed ABX-type aromatic signals [!
.75 (1H, d, J = 8.6 Hz)] and three singlet signals [! 7.42, 7.52 and 8.29 (each s)] in the aromatic region.
The C-NMR spectrum of 13 displayed the presence of 30 carbons, in which 15 signals were closely
H
6.60 (1H, brs), 6.87 (1H, brd, J = 8.6 Hz) and
7
H
1
3
1
3
correlated with those found in 12. The rest of 15 C resonances were closely correlated with those of
sulphuretin, except for the signals of the B-ring moiety, suggesting that 13 is an auronoflavanone-type
1
1
biflavonoid. Detailed interpretation of the H- H COSY, HSQC, and HMBC data indicated that 13 is a
biflavonoid, consisting from a butin and a sulphuretin units. The linkage of these units was elucidate to be
C-3–C-6"' from the HMBC correlation of H-3 with C-6"' and of H-5"' with C-3. The Z-configuration of
1
3
30
the aurone moiety was assigned from the C resonance of C-10". By contrast, the 2,3-trans
configuration of the butin unit was assigned from the J-value of H-2 (J = 11.0 Hz). In addition, the CD
spectrum of 13 showed positive ("# +2.2 at 332 nm) and negative ("# –1.3 at 304 nm) Cotton effects,
3
1
which were similar to those seen in 3-aryl-flavanone derivatives with a 2S,3R-configuration.
Accordingly, the structure of 13 was elucidated as shown.
–
A bioflavonoid nature of 14 was revealed by the HRESIMS, which gave an [M–H] ion peak at m/z
1
5
41.1135 (calcd for 541.1135), indicating the molecular formula C H O . The H NMR spectrum
3
0
22 10
revealed, along with three sets of ABX-type aromatic signals and two aromatic signals, four methine
signals [! 5.47 (d, J = 13.0 Hz), 4.03 (d, J = 11.0 Hz), 3.94 (brt, J = 11.0 Hz) and 3.57 (dd, J = 13.0, 11.0
H
OH
OH
OH
OH
HO
O
O
HO
O
O
OH
OH
OH
OH
HO
O
O
1
-1
HO
OH
14
H H COSY
O
HMBC
13
Figure 2. Selected 2D NMR correlations for compounds 13 and 14

HETEROCYCLES, Vol. 83, No. 9, 2011
2083
1
3
2
Hz)]. The C NMR spectrum exhibited the presence of two carbonyl carbons, six sp quaternary carbons,
2
3
3
six sp methine carbons, one-oxygen-bearing sp methine, and three sp methines (Table 1). Detailed
1
1
analyses of the H- H COSY, HSQC, and HMBC data indicated the presence of a butin unit and a
,4,2',4'-tetrahydroxy-dihydrochalcone unit in 14. In addition, the HMBC correlations of H-3 with C-$,
3
C-!, and CO, and of H-" with C-1' and C-6' indicated the connectivity of these units at C-3–C-# and
C-6'–C-!. The 2,3-trans configuration of the butin unit was elucidated from the J-value of H-2 (J = 11.0
Hz). The coupling pattern and J-values of H-# (brt, J = 11.0 Hz), together with the NOE correlation of
H-# with H-2, indicated trans configurations of H-3/H-# and H-#/H-". Compound 14 was considered to
1
13
Table 1. H and C NMR spectroscopic data for compounds 12-14
a
a
b
c
1
2
13
14
1
c
13
d
1
c
13
d
1
13
d
Position
H
C
H
C
H
C
2
3
4
5
6
7
8
9
6.61 (d, 11.9)
3.46 (d, 11.9)
84.9
51.3
6.00 (d, 11.0)
5.15 (d, 11.0)
85.3
54.5
5.47 (d, 13.0)
3.57 (dd, 13.0, 11.0)
79.6
51.9
191.6
129.2
111.0
165.8
103.1
163.7
114.2
128.7
116.1
146.8
147.7
116.1
119.6
191.1
129.4
111.1
165.8
103.1
163.5
114.2
129.6
115.3
146.4
146.5
115.5
119.2
193.3
129.9
111.8
166.5
103.7
165.8
114.7
126.3
112.9
145.0
145.4
116.8
131.9
136.1
117.2
145.4
145.0
116.2
121.9
8.14 (d, 8.7)
8.18 (d, 8.7)
7.54 (d, 8.6)
6.82 (dd, 8.7, 2.2)
6.87 (brd, 8.7)
6.45 (brd, 8.6)
6.66 (d, 2.2)
7.43 (d, 2.0)
6.78 (brs)
7.52 (bs)
6.46 (d, 1.9)
1
0
1
´
´
´
´
´
´
˝
˝
˝
˝
˝
˝
˝
˝
˝
2
7.14 (s)
3
4
5
6
1
2
3
4
5
6
7
8
9
7.31 (d, 8.1)
7.07 (d, 8.0)
6.23 (s)
6.99 (dd, 8.1, 2.0)
7.12 (brd, 8.0)
146.9
181.7
125.4
112.7
167.6
98.6
6.44 (d, 1.7)
7.75 (d, 8.6)
6.87 (brd, 8.6)
6.52 (d, 8.1)
6.34 (dd, 8.1, 1.7)
6.60 (brs)
166.9
113.7
108.6
130.6
118.2
148.8
145.7
118.2
123.3
1
0˝
7.52 (brs)
8.29 (s)
1
˝´
˝´
˝´
˝´
˝´
˝´
$
!
115.9
165.4
103.0
165.8
108.3
134.4
47.6
2
3
4
5
6
6.13 (d, 2.3)
7.42 (s)
6.04 (dd, 8.9, 2.3)
7.21 (d, 8.9)
3.94 (brt, 11.0)
4.03 (d, 11.0)
51.3
CO
207.7
a
b
c
d
Measured in pyridine-d
5
. Measured in CD
3
OD. % ppm (mult., J in Hz), 500 MHz. % ppm, 125 MHz.

2084
HETEROCYCLES, Vol. 83, No. 9, 2011
be racemic from its small optical rotation value and CD spectrum, in which obvious Cotton effect was not
observed. Consequently, the structure of 14 was characterized as shown.
Compounds 13 and 14 were an auronoflavanone-type and a chalconoflavanone-type biflavonoid,
respectively, with new types of linkage.
Influenza A neuraminidase inhibitory activity and DHPP free-radical scavenging activity for the isolated
compounds were evaluated. In an influenza A neuraminidase inhibitory assay, compound 3 displayed
relatively potent inhibitory activity with an IC value of 5.4 µgmL. Compounds 1, 4, and 6 showed
5
0
moderate influenza A neuraminidase inhibitory activity with IC50 values ranging from 28.5 to 34.9 µg/mL.
By contrast, compound 14 showed potent activity in a DPPH free-radical scavenging assay with an IC50
value of 1.7 µg/mL. Compounds 3, 7, 13 and 7,8-dihydroxychromone also showed relatively potent
activity with IC50 values ranging from 3.5 to 9.8 µg/mL.
Table 2. Influenza A neuraminidase inhibitory activity and DPPH free-radical scavenging
activity of compounds isolated from the flowers of B. monosperma
Anti-influenza neuraminidase
DPPH free-radical scavenging
a
a
activity IC50 (µg/mL)
activity IC50 (µg/mL)
1
2
3
4
5
6
7
8
9
32.0 ± 2.7
> 100
> 50
14.9 ± 0.4
5.4 ± 0.3
18.9 ± 0.3
> 50
5.4 ±0.3
28.5±2.8
> 100
34.9 ±4.2
89.9±4.3
> 100
26.2 ± 0.3
3.5 ± 0.2
> 50
> 10
> 50
1
0
2
3
4
> 10
> 50
1
1
1
67.8 ±2.9
> 100
12.5 ± 0.4
9.8± 0.2
1.7 ± 0.1
7.0 ± 0.1
> 100
7
,8-dihydroxychromone
> 100
oseltamivir carboxylate
ascorbic acid
0.002 ±0.0002
7.3 ± 0.4
a
Data are mean ± SE from three or four experiments.
Owing to the high fluorescence intensity from the sample, assay was performed at the highest concentration of 10
b
µg/mL.

HETEROCYCLES, Vol. 83, No. 9, 2011
2085
EXPERIMENTAL
General experimental procedures
Optical rotations were measured on a JASCO P-2200 polarimeter. NMR spectra were recorded on a
1
13
Bruker AVANCE-500 instrument ( H NMR: 500 MHz, C NMR: 125 MHz) using TMS as an internal
standard. HRESIMS were obtained on a Waters LCT Premier. Column chromatography was performed
with silica gel 60N (63-210 µm, Kanto Kagaku), Sephadex LH-20 (25 – 100 µm, GE Health Care), Toyo
pearl HW-40C (TOSOH Corporation), MCI gel CHP20P (75-150 µm; Mitsubishi Chemical), YMC-pack
ODS-A (S-50 µm, YMC Co., Ltd). HPLC was performed on a JASCO apparatus consisting of a PU-980
prep pump, UV-970UV/VIS (at the wavelength of 280nm) and RI-930 refractive index detectors, and a
Mightysil RP-18 GP (250 & 20 mm; 5 µm, Kanto Chemical) or a COSMOSIL 'NAP (5 µm, (20"250
mm, nacalai tesque) column. TLC was conducted on precoated silica gel 60 F254 (0.20 mm, Merck) and
spots were detected by UV illumination and by spraying 10% sulfuric acid reagent followed by heating.
Plant Material
The flowers of Butea monosperma (Lam.) Taub. were collected in July, 2007 at the Jahangirnagar
University campus, Savar, Dhaka. Identification was carried out by Mr. Sardar Nasir Uddin (Bangladesh
National Herbarium, Dhaka, Bangladesh). A voucher specimen (DACB acc. no. 34193) was deposited at
the Graduate School of Pharmaceutical Sciences, University of Tokushima.
Extraction and Isolation
The dried flowers of B. monosperma (2.6 kg) were extracted three times with MeOH (5 L x 3) at room
temperature. The MeOH extract was concentrated under reduced pressure to give a residue (450 g), which
2
was partitioned with EtOAc and H O. The EtOAc layer, after removal of the solvent by evaporation, was
further partitioned with n-hexane and 90% aqueous MeOH to give a hexane-soluble fraction (51.6 g) and
a 90% MeOH-soluble fraction (93.8 g). The 90% MeOH-soluble fraction was subjected to
chromatography over Sephadex LH-20 with H
fractions (frs. 1.1–1.12). Fr. 1.3 (30.7 g) was subjected to MCI gel CHP20P [H
furnish 12 fractions (frs. 1.3.1–1.3.12). Fr. 1.3.4 (2.8 g) was further fractionated by YMC ODS-A
O–MeOH (0:1#1:0)] chromatography into nine fractions (frs. 1.3.4.1–1.3.4.9). Sephadex LH-20
chromatography [H O–MeOH (1:0#1:4)] of fr. 1.3.4.4 (392 mg) followed by crystallization gave
,8-dihydroxychromone (12 mg) and methyl 3,4,5-trihydroxybenzoate (25 mg). 2,4-Dihydroxybenzoic
acid (10 mg) was obtained by repeated chromatography of fr. 1.3.4.7 (163 mg) over Sephadex LH-20
2
O containing increasing amounts of MeOH to give twelve
2
O–MeOH (1:0#0:1)] to
[H
2
2
7
[H
2
O–MeOH (1:0#4:1)] and silica gel [CHCl
3 2
–MeOH–H O (10:1:0.1)]. Chromatography of fr. 1.3.4.6
(
1.5 g) with an ODS-A column [H O–MeOH (4:1#0:1)] followed by silica gel chromatography
2

2086
HETEROCYCLES, Vol. 83, No. 9, 2011
[CHCl
3
–MeOH–H
2
O (10:1:0.1)] gave compound 6 (8 mg). Sephadex LH-20 chromatography
2
[H O–MeOH (1:0#0:1)] of fr. 1.3.4.8 (163 mg) afforded seven fractions (frs. 1.3.4.8.1–1.3.4.8.7). Fr.
1
.3.4.8.2 was repeatedly separated by ODS-A [H O–MeOH (1:0#3:7)] and silica gel
2
[CHCl
3
–MeOH–H
2
O (10:1:0.1)] chromatography to furnish compounds 5 (12 mg) and 8 (8.5 mg). Fr.
1
.3.4.8.5 was purified by silica gel chromatography [CHCl
compound 2 (14 mg). Purification of fr. 1.3.4.9 by silica gel chromatography [CHCl
afforded compound 4 (6 mg). Fr 1.3.5 (14 g) was subjected to ODS-A chromatography [H
1:0#0:1)] to give eight fractions (fr. 1.3.5.1–1.3.5.8). Chromatography of fr. 1.3.5.5 (10 g) over
Sephadex LH-20 with EtOH yielded five fractions (frs. 1.3.5.5.1–1.3.5.5.5). Purification of fr. 1.3.5.5.2
65.5 mg) with a silica gel [CHCl –MeOH (8:2)] column gave umbelliferone (35.5 mg). Fr. 1.5 (15 g)
3
–MeOH–H
2
O (10:1:0.1)] to furnished
–MeOH (10:1)]
O–MeOH
3
2
(
(
3
was rechromatographed over MCI gel CHP20P [H
2
O–MeOH (7:3#0:1)] to furnish ten fractions
frs.1.5.1–1.5.10). Fraction 1.5.4 (700 mg) was further fractionated by ODS-A chromatography
O–MeOH (1:0#0:1)] into further seven fractions (frs. 1.5.4.1–1.5.4.7). Sephadex LH-20
O–MeOH (1:0#1:1)] of fr. 1.5.4.7 (185 mg), followed by crystallization (from
EtOAc) gave compound 1 (7.9 mg). Fr. 1.5.5 (8.5 g) was chromatographed over ODS-A [H O–MeOH
3:2#0:1)] to give eight fractions (frs. 1.5.5.1–1.5.5.8). Crystallization of fraction 1.5.5.4 (from EtOAc)
gave compound 7 (28 mg). Fr. 1.5.5.6 (1.6 g) was further chromatographed over Sephadex LH-20
(
[
H
2
chromatography [H
2
2
(
[H
2
O–MeOH (1:0#0:1)] to give six fractions (frs. 1.5.5.6.1–1.5.5.6.6). ODS-A chromatography
O–MeOH (3:2#0:1)] of fr. 1.5.5.6.6 (50 mg) followed by crystallization (from H O) gave
O–MeOH
1:1#0:1)] to give six fractions (frs. 1.5.8.1–1.5.8.6). Fr. 1.5.8.6, consisted mainly of a mixture of
compounds 9 and 10, which was separated by HPLC on Mightysil RP-18 [MeOH–1% AcOH (1:2)] and
pure samples were crystallized from H O in each case; 9 (9.2 mg) and 10 (11.4 mg). Fr. 1.10 (5.3 g) was
chromatographed over MCI gel CHP20P [H O–MeOH (1:0#0:1)] to afford eight further fractions
1.10.1–1.10.8). Fr. 1.10.4 (1.0 g) was separated by ODS-A chromatography [H O–MeOH (1:1#1:4)]
to give three fractions (1.10.4.1–1.10.4.3). Further fractionation of fr. 1.10.4.2 (700 mg) by Sephadex
LH-20 [H O–MeOH (1:0#0:1)] afforded seven fractions (1.10.4.2.1–1.10.4.2.6). Fr. 1.10.4.2.6 was
crystallized from H O giving compound 12 (39 mg). Sephadex LH-20 chromatography [H O–MeOH
4:1#0:1)]) of fr.1.10.4.2.4 (50 mg) followed by HPLC on Mightysil RP-18 [MeOH-1% AcOH (2:3)]
gave compound 13 (6.1 mg). Fr. 1.10.4.3ꢀ170 mgꢁwas chromatographed over Sephadex LH-20
O–MeOH (4:1#0:1)] and then purified by ODS HPLC (Mightysil RP18) [MeOH–1%AcOH (2:3)]
to yield compound 14 (19.7 mg). Fr. 1.10.7 (1.6 g) was repeatedly chromatographed over ODS-A
O–MeOH (7:3#0:1)] and silica gel [CHCl –MeOH–H O (10:1:0.1)] to furnish compound 11 (19
mg).
[H
2
2
compound 3 (14.5 mg). Fr. 1.5.8 (500 mg) was fractionated by ODS-A chromatography [H
2
(
2
2
(
2
2
2
2
(
[H
2
[H
2
3
2

HETEROCYCLES, Vol. 83, No. 9, 2011
2087
2
0
Sulphuretin 3'-O- !! -glucopyranoside (1): Yellowish brown amorphous powder; [$]
–16.5 (c 0.11,
D
–
1 1
MeOH); IR (KBr) )max 3400, 1628, 1513 cm ; H NMR (500 MHz, pyridine-d ): % 4.13 (1H, m, H-5˝),
5,
4
.24 (1H, m, H-2˝), 4.32 (1H, m, H-3˝), 4.35 (1H, m, H-4˝), 4.42 (1H, dd, 12.0, 4.8 Hz, H-6˝ ), 4.55 (1H,
dd, 12.0, 2.2 Hz, H-6˝), 5.62 (1H, d, J = 7.8 Hz, H-1˝), 6.93 (1H, dd, J = 8.4, 1.8 Hz, H-5), 7.05 (1H, s,
H-10), 7.13 (1H, d, J = 1.8 Hz, H-7), 7.24 (1H, d, J = 8.3 Hz, H-5´), 7.67 (1H, dd, J = 8.3, 1.8 Hz, H-6´),
1
3
7
.83 (1H, d, J = 8.4 Hz, H-4), 8.28 (1H, d, J = 1.8 Hz, H-2´); C NMR: (125 MHz, pyridine-d ): % 61.4
5,
(C-6˝), 70.3 (C-4˝), 74.1 (C-2˝), 77.6 (C-3˝), 78.3 (C-5˝), 98.8 (C-7), 103.5 (C-1˝), 111.0 (C-10), 112.9
(C-5), 113.4 (C-9), 116.9 (C-5´), 120.7 (C-2´), 124.0 (C-1´), 125.2 (C-4), 127.6 (C-6´), 146.1 (C-3´),
–
1
46.4 (C-2), 150.5 (C-4´), 166.9 (C-8), 168.0 (C-6), 181.5 (C-3); HRESIMS: m/z 431.0941[M–H] (calcd
19
for C21H O10, 431.0978).
Acid hydrolysis of 1: Compound 1 (2 mg) was hydrolyzed with 10% HCl for 3 h at reflux. The reaction
mixture was diluted with H O, and extracted with EtOAc. The EtOAc-soluble fraction was purified by
2
ODS on HPLC to afford an aglycone (0.8 mg), which was identified as (Z)-6,3',4'-trihydroxyaurone by
spectral comparison. The aqueous layer was neutralized with Amberlite IRA-400 resin and was directly
f 2
analyzed by TLC to detect glucose [R : 0.35, n-BuOH/Pyridine/H O (6:4:3), Avicel SF cellulose].
2
0
Compound 12: Pale yellow granules; mp 222-228 °C; [$]
+92.3 (c 0.18, MeOH); IR (KBr) )max
D
–
1
1
13
-5
3
3
380, 1649, 1604, 1531, 1462 cm ; H and C NMR data: Table 1; CD (MeOH; 1.84 " 10 M, "#) *max
–
34 (+11.9), 304 (–17.5) ; HRESIMS: m/z 541.1115 [M–H] (calcd for C30
H
21
O
10, 541.1135).
2
0
Compound 13: Pale orange amorphous powder; [$] +5.3 (c 0.07, MeOH); IR (KBr) )max 3430, 1630,
D
–
1
1
13
-5
1
500 cm ; H and C NMR: see Table 1; CD (MeOH; 1.84 " 10 M, "#) * 332 (+2.2), 304 (-1.3);
max
–
HRESIMS: m/z 539.0940 [M–H] (calcd for C30
H
19
O10, 539.0978).
2
0
Compound 14: Yellow granules; mp 220-225 °C; [$] +5.6 (c 0.1, MeOH); IR (KBr) )max 3445, 1665,
D
–
1
1
13
–
1
5
21 10
605, 1517 cm ; H and C NMR: see Table 1; HRESIMS: m/z 541.1135 [M–H] (calcd for C30H O ,
41.1135).
Viruses and cells
Madin-Darby canine kidney (MDCK) cells were routinely passaged in the DMEM supplemented with
0% FBS and 100 µg/mL kanamycin. Influenza A/PR/8/34 (H1N1) was purchased from the American
1
Type Culture Collection (ATCC; Manassas, VA, USA). Virus-infected cells were maintained in DMEM
supplemented with 0.5% bovine serum albumin. Influenza viruses were propagated in MDCK cells.

2088
HETEROCYCLES, Vol. 83, No. 9, 2011
Influenza A neuraminidase inhibition assay
A modified fluorometric assay using the fluorogenic substrate 2'-(4-methylumbellyferyl)-$-D-N-acetyl-
neuraminic acid (MUNANA, Sigma) was used to determine the neuraminidase activity (NA) on the type
3
2
A (H1N1) viruses. All compounds were dissolved in DMSO and diluted to the corresponding
5
concentrations in PBS. A 96-well plate containing a mixture of the diluted virus suspension (1"10 PFU,
5
0 µL) and the different concentration of compound solution (50 µL) was incubated on ice for 1h.
MUNANA substrate solution [0.2 mM in 0.1 M acetate buffer (pH 4.6), 25 mL] was added and the
mixture was incubated for 30 min at 37 ˚C. The enzymatic reaction was quenched by adding 100 µL of
glycine-NaOH buffer solution (pH 10.7). The fluorescence intensity of the product
(4-methylumbellyferone) was measured in a spectrophotometer with excitation and emission wavelengths
of 360 and 440 nm, respectively. Oseltamivir carboxylate was used as a positive control. The drug
concentrations required to inhibit 50% of the NA (IC50) were determined by plotting the percent
3
3
inhibition of NA as a function of the drug concentrations.
Determination of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical
3
4
This assay was performed using a previously reported method with some minor modifications. The
mixtures containing different concentration of test samples (in EtOH 100 µL) and DPPH ethanolic
solution (60 µM, 100 µL) were allowed to react in a 96-well microplate. The plate was vigorously vortex
mixed at room temperature for 30 min. Reduction of the DPPH free radical was measured by the
absorbance at 540 nm using a UV-vis microplate reader. L-Ascorbic acid was used as a positive control.
The scavenging activity was expressed in terms of the concentration of samples, which scavenged free
radical by 50% (IC50).
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