W. Disadee et al. / Tetrahedron 67 (2011) 4221e4226
4225
ꢀ
(
9:1); (II) EtOAceMeOHeH
2
O (40:10:1); (III) EtOAceMeOHeH
2
O
Table 2; negative HRMS (APCI-TOF): [MþCl] , found 849.2259.
46ClO21 requires 849.2226.
(
70:30:3); (IV) EtOAceMeOHeH
2
O (6:4:1); (V) 10e80% aqueous
36
C H
MeOH; (VI) 5% aqueous MeCN; and (VII) 20% aqueous MeCN. The
spraying reagent used for TLC was 10% H SO in 50% EtOH.
2
4
4.3.7. Acid hydrolysis of sesuvioside A (1). A solution of sesuvioside
A (149 mg) in 1,4-dioxane (0.5 mL) and 2 M HCl (4.5 mL) was heated
ꢄ
4
.2. Plant material
The aerial portion of S. portulacastrum (L.) L. was collected from
at 80 C for 4 h. After cooling, H
2
O (5 mL) was added and neu-
tralized with 2 M KOH. The reaction was extracted with EtOAc
(30 mLꢁ3) and the combined organic part was concentrated in
vacuo to yield an aglycone 1a (69.5 mg) after re-crystallization from
southern coastal area in November 2007, Pattani province, Thai-
land. The identification of the plant was done by Mr. Nopporn
Nontapa of Department of Pharmaceutical Botany and Pharma-
cognosy, Faculty of Pharmaceutical Sciences, Khon Kaen University.
A voucher specimen (TK-PSKKU-0062) is on file in the Herbarium of
the Faculty of Pharmaceutical Sciences, Khon Kaen University.
0
MeOH. The structure of 1a was identified to be 3,5,4 -trihydroxy-
6,7-dimethoxyflavone (eupalitin) by NMR spectral analysis.
The aqueous layer was concentrated to dryness, providing the
sugar fraction. This fraction was subjected to a silica gel column and
eluted with increasing polarity mixtures of EtOAceMeOH as sol-
vent system to give
and -galactose (23.9 mg, [
2
6
L
-rhamnose (25.2 mg, [
a
]
D
þ5.3; H
2
O, c 1.26)
2
7
4
.3. Extraction and isolation
D
a
]
D
þ65.7; H
2
O, c 1.20) in comparison
with authentic samples. Aglycones of sesuvioside BeF, compounds
3a and 5a, were also obtained in an analogous manner.
The aerial portion of S. portulacastrum (2.4 kg) was macerated
three times with MeOH (12 L for each extraction) at room tem-
perature. The MeOH extract was concentrated in vacuo to dryness.
0
4.3.8. 3,5,4 -Trihydroxy-6,7-dimethoxyflavone (eupalitin, 1a). Yellow
1
This residue (556.0 g) was suspended in H
Et O (each 1.0 L, three times). The aqueous soluble fraction (345.4 g)
was subjected to a Diaion HP-20 column, and eluted with H O,
MeOH, and (CH CO, successively. The fraction eluted with MeOH
2
O, and partitioned with
amorphous powder; H NMR (DMSO-d
6
):
d
H
12.42 (1H, br s, 5-OH),
0
0
0
0
2
8.08 (2H, d, J¼8.9 Hz, H-2 /6 ), 6.93 (2H, d, J¼8.9 Hz, H-3 /5 ), 6.83
13
2
(1H, s, H-8), 3.90 (3H, s, 7-OMe), 3.73 (3H, s, 6-OMe); C NMR
0
3
)
2
(DMSO-d
6
):
d
C
176.1 (C-4), 159.3 (C-4 ), 158.5 (C-7), 151.5 (C-5), 151.0
0 0 0
(
(
(
29.7 g) was subjected to a silica gel column using solvent systems I
4.0 L), II (4.0 L), III (6.0 L), and IV (12.0 L) affording six fractions
fractions AeF), monitored by TLC. Fraction B (3.2 g), which showed
(C-9), 147.3 (C-2), 135.8 (C-3), 131.2 (C-6), 129.6 (C-2 /6 ), 121.6 (C-1 ),
115.4 (C-3 /5 ), 104.3 (C-10), 91.1 (C-8), 60.1 (6-OMe), 56.4 (7-OMe).
0
0
0
0
the presence of the one major compound, was applied to an RP-18
column using solvent system V to give ecdysterone (1.8 g) by pre-
cipitation. Fraction C (4.7 g) was subjected to an RP-18 column
using solvent system V, providing five fractions (C-1 to C-5). Frac-
tion C-3 was purified by preparative HPLC-ODS (column A) with
solvent system V to afford compounds 1 (1.3 g), 3 (56 mg), and 5
4.3.9. 3,5,3 ,4 -Tetrahydroxy-6,7-dimethoxyflavone (eupatolitin, 3a)-
1
. Yellow amorphous powder; H NMR (DMSO-d
6
):
d
H
12.44 (1H, br s,
0
0
5-OH), 7.72 (1H, d, J¼1.9 Hz, H-2 ), 7.57 (1H, dd, J¼8.5, 1.9 Hz, H-6 ),
0
6.89 (1H, d, J¼8.5 Hz, H-5 ), 6.81 (1H, s, H-8), 3.90 (3H, s, 7-OMe), 3.72
13
(3H, s, 6-OMe); C NMR (DMSO-d
6
):
d
C
176.1 (C-4), 158.5 (C-7), 151.5
0
0
(C-5), 151.1 (C-9), 147.8 (C-4 ), 147.4 (C-2), 145.1 (C-3 ), 135.8 (C-3),
131.2 (C-6), 121.9 (C-6 ), 120.0 (C-1 ), 115.6 (C-2 ), 115.3 (C-5 ), 104.3
(C-10), 91.1 (C-8), 60.1 (6-OMe), 56.4 (7-OMe).
0
0
0
0
(
77 mg). Fraction E (5.5 g) was repeatedly separated on an RP-18
column using solvent system V, giving seven fractions (E-1 to E-
). Fraction E-7 was further purified by preparative HPLC-ODS
7
0
0
(
(
column B) with solvent system VII to afford compounds 2
236 mg), 4 (58 mg), and 6 (192 mg).
4.3.10. 3,5,4 -Trihydroxy-6,7,3 -trimethoxyflavone (5a). Yellow amor-
phous powder; H NMR (DMSO-d
1
6
):
d
H
12.43 (1H, br s, 5-OH), 7.79
0
0
(
1H, d, J¼1.9 Hz, H-2 ), 7.75 (1H, dd, J¼8.5, 1.9 Hz, H-6 ), 6.94 (1H, d,
2
7
0
0
4
.3.1. Sesuvioside A (1). Yellow amorphous powder, [
a
]
D
ꢀ27.0
J¼8.5 Hz, H-5 ), 6.90 (1H, s, H-8), 3.92 (3H, s, 7-OMe), 3.86 (3H, s, 3 -
1
13
13
(
H
2
O, c 1.14); H NMR (DMSO-d
6
): Table 1 and C NMR (DMSO-d
Table 2; negative HRMS (APCI-TOF): [MþCl] , found 673.1531.
6
):
OMe), 3.74 (3H, s, 6-OMe); C NMR (DMSO-d
6
):
d
C
176.1 (C-4), 158.6
ꢀ
0
0
(C-7),151.6(C-5),151.0(C-9),149.0(C-4 ),147.4(C-2),147.1(C-3 ),136.0
(C-3), 131.3 (C-6), 121.9 (C-6 ), 121.9 (C-1 ), 115.5 (C-5 ), 111.7 (C-2 ),
04.3 (C-10), 91.3 (C-8), 60.1 (6-OMe), 56.4 (7-OMe), 55.9 (3 -OMe).
0
0
0
0
C
29
H34ClO16 requires 673.1541.
0
1
2
6
4
.3.2. Sesuvioside B (2). Yellow amorphous powder, [
a
]
D
ꢀ40.6
1
13
(
H
2
O, c 1.26); H NMR (DMSO-d
6
): Table 1 and C NMR (DMSO-d
Table 2; negative HRMS (APCI-TOF): [MþCl] , found 819.2116.
6
):
4.4. Assay for radical scavenging activity
ꢀ
C
35
H44ClO20 requires 819.2120.
4.4.1. Scavenging of diphenyl-picrylhydrazyl (DPPH) radicals. This
assay was performed using a previously reported method with
2
6
15,16
4
.3.3. Sesuvioside C (3). Yellow amorphous powder, [
a
]
D
ꢀ18.7
some minor modifications.
ples (in DMSO, 5
The mixtures containing test sam-
M, 195 L)
1
13
(
H
2
O, c 1.06); H NMR (DMSO-d
6
): Table 1 and C NMR (DMSO-d
Table 2; negative HRMS (APCI-TOF): [MþCl] , found 689.1473.
6
):
m
L) and DPPH ethanolic solution (100
m
m
ꢀ
were allowed to react in a 96-well microplate. The plate was in-
ꢄ
C
29
H34ClO17 requires 689.1490.
cubated at 37 C for 30 min. Reduction of the DPPH free radical was
measured by the absorbance at 515 nm using a UVevis microplate
reader. L-Ascorbic acid (10 mM) was used as a positive control. The
scavenging activity was expressed in terms of the concentration of
samples, which scavenged free radical by 50% (SC50).
2
6
4
.3.4. Sesuvioside D (4). Yellow amorphous powder, [
a
]
D
ꢀ31.0
1
13
(
H
2
O, c 1.00); H NMR (DMSO-d
6
): Table 1 and C NMR (DMSO-d
Table 2; negative HRMS (APCI-TOF): [MþCl] , found 835.2084.
6
):
ꢀ
C
35
H44ClO21 requires 835.2069.
4
.4.2. Measurement of oxygen radical absorbance capacity (OR-
2
7
4
.3.5. Sesuvioside E (5). Yellow amorphous powder, [
a
]
D
ꢀ44.4
AC). The peroxyl radical absorbance capacity of test samples was
determined using a previously reported method.
1
13
16,17
(
MeOH, c 0.22); H NMR (DMSO-d
6
): Table 1 and C NMR (DMSO-
): Table 2; negative HRMS (APCI-TOF): [MþCl] , found 703.1641.
The reaction
L) in
ꢀ
ꢀ5
d
C
6
mixture containing fluorescein solution (7ꢁ10 mM, 175
m
30
H36ClO17 requires 703.1647.
phosphate buffer (75 mM, pH 7.4) was added to either test sample
10 L) or DMSO (10 L) (as a blank) diluted in phosphate buffer and
(
m
m
ꢄ
2
6
4
.3.6. Sesuvioside F (6). Yellow amorphous powder, [
a
]
D
ꢀ28.5
pre-incubated at 37 C for 10 min. The reaction was initiated by
addition of 2,2 -azobis-(2-amidinopropane) dihydrochloride
1
13
0
(
MeOH, c 1.16); H NMR (DMSO-d
6
): Table 1 and C NMR (DMSO-d ):
6