7
28
Vol. 59, No. 6
mon aglycone of compounds 2 and 3, pectolinarigenin, is fourth fraction, II-7-3-4 (124 mg), was purified by HPLC to furnish com-
pound 1 (10 mg) and compound 2 (21.1 mg). The fourth fraction, II-7-4
also known to have a preventing activity of hepatic injury
26)
(1.63 g), was subjected to silica gel (75 g) CC using an isocratic system of
CHCl –MeOH–H O (17 : 6 : 1), giving five fractions. The third fraction II-7-
caused by D-galactosamine via antioxidant mechanism.
Phenylethanoids were also reported to have radical scaveng-
3
2
4-3 (123.4 mg) was purified by HPLC to afford compound 9 (7 mg).
27)
ing acitivity.
The last fraction (III) eluted with 80% MeOH (5.0 g) was subjected to sil-
ica gel CC (200 g) (Fꢄ35 mm, Lꢄ60 cm), using CHCl –MeOH mixtures,
3
Experimental
yielding seven fractions. The third fraction, III-3 (500 mg), was purified by
General Experimental Procedures Optical rotation data were meas- RPCC, affording nine fractions. The third fraction, III-3-3 (13.4 mg), was
ured on a JASCO P-1030 polarimeter. IR and UV spectra were obtained on a
Horiba FT-710 Fourier transform infrared spectrophotometer and a JASCO
V-520 UV/Vis spectrophotometer, respectively. H- and C-NMR spectra
purified by HPLC to yield compound 10 (4.1 mg) and compound 11
(5.2 mg). The fourth fraction, III-3-4 (15.3 mg), was purified by HPLC to
give compound 12 (3.2 mg) and compound 13 (10.4 mg). The fifth fraction,
1
13
were recorded on a JEOL JNM a-400 spectrometer with tetramethylsilane III-3-5 (78.4 mg), was purified by HPLC to furnish compound 14 (5.7 mg)
as an internal standard. A circular dichroism (CD) spectrum was measured
on a JASCO J-720 spectropolarimeter. HR-ESI mass spectra were taken on
LTQ Orbitrap XL and QSTAR XL mass spectrometers. A highly porous
synthetic resin, Diaion HP-20, was purchased from Mitsubishi Chemical
Co., Ltd. (Tokyo, Japan). Silica gel column chromatography (CC) was per-
and compound 5 (4 mg). The sixth fraction, III-3-6 (31.5 mg), was purified
by HPLC to yield compound 15 (8 mg). The ninth fraction, III-3-9 (98 mg),
was subjected to DCCC, giving three fractions. The second fraction, III-3-9-
2 (16 mg), was purified by HPLC to afford compound 16 (2.3 mg). The
fourth fraction, III-4 (953 mg), was purified by RPCC, producing three frac-
formed on silica gel 60 [(E. Merck, Darmstardt, Germany) 70—230 mesh]. tions. The third fraction, III-4-3 (101 mg), was purified by HPLC to give
Reversed-phase [octadecylsilanized silica gel (ODS)] open CC (RPCC) was compound 17 (5.5 mg). The fifth fraction, III-5 (82.7 mg), was subjected to
performed on Cosmosil 75C -OPN (Nacalai Tesque, Kyoto, Japan) silica gel (50 g) CC using an isocratic system of CHCl –MeOH–H O
1
8
3
2
(
Fꢄ2 cm, Lꢄ40 cm, 10 g fractions being collected). The droplet counter-
(17 : 6 : 1), affording five fractions. The fifth fraction, III-5-5 (40 mg), was
purified by HPLC to furnish compound 3 (4.8 mg).
current chromatograph (DCCC) (Tokyo Rikakikai, Tokyo, Japan) was
equipped with 500 glass columns (Fꢄ2 mm, Lꢄ40 cm), and the lower and
Demethoxycentaureidin 7-O-b-D-Galacturonopyranoside (1): Pale yellow
2
5
ꢃ1
upper layers of
a
solvent mixture of CHCl –MeOH–H O–1-PrOH
amorphous powder. [a]D ꢃ32.9° (cꢄ1.00, DMSO); IR nmax (film) cm :
3
2
(9 : 12 : 8 : 2) were used as the mobile and stationary phases, respectively.
3330, 2930, 1700, 1652, 1614, 1512, 1458, 1360, 1269, 1140, 1020, 953;
Five-gram fractions were collected and numbered according to their order of
elution with the mobile phase. HPLC was performed on an ODS column
[
UV lmax (MeOH) nm (log e): 235 (3.88), 348 (3.86); ꢂNaOMe 238 (3.90),
375 (3.80); ꢂAlCl3 230 (3.85), 378 (3.94); ꢂAlCl /HCl 230 (3.89), 378
3
1
Inertsil ODS-3; GL Science, Tokyo, Japan; (Fꢄ6 mm, Lꢄ25 cm, flow rate: (3.85); ꢂNaOAc 238 (3.88), 375 (3.90); H-NMR (400 MHz, DMSO-d6)
1
3
1
6
.0 ml/min), using a refractive index and/or UV detector. Precoated silica gel
0 F254 plates (E. Merck; 0.25 mm in thickness) were used for TLC analyses,
and C-NMR (100 MHz, DMSO-d ): Table 1; HR-ESI-MS (positive-ion
6
ꢂ
mode) m/z: 529.0944 [MꢂNa] (Calcd for C H O Na: 529.0952).
23
22 13
with visualization by spraying with a 10% H SO solution in ethanol and
then heating to around 150 °C on a hotplate.
Pectolinarigenin 7-O-a-L-Rhamnopyranosyl-(1ꢅ→4ꢀ)-b-D-glucopyra-
2
4
25
noside (2): Pale yellow amorphous powder. [a] ꢃ59.5° (cꢄ1.40, DMSO);
D
ꢃ1
Plant Material The leaves of R. patula were collected in March 2007 IR nmax (film) cm : 3350, 2921, 1652, 1608, 1571, 1509, 1460, 1419, 1357,
from the Orman Botanical Garden in Giza, Egypt. A voucher specimen of
the plant has been deposited in the Herbarium of Faculty of Pharmacy,
Minia University, Egypt (Minia-07-Mar-RP).
Extraction and Isolation Powdered air-dried leaves (2.80 kg) of R. pat-
ula was exhaustively extracted with 70% methanol (5 lꢆ5) and then concen-
trated under reduced pressure to yield a viscous gummy material. This
residue (330 g) was dissolved in 250 ml of water and defatted with n-hexane
1294, 1262, 1181, 1052, 834; UV lmax (MeOH) nm (log e): 240 (3.83), 355
(3.86); ꢂNaOMe 242 (3.82), 380 (3.89); ꢂAlCl 243 (3.89), 390 (3.93);
3
1
ꢂAlCl /HCl 245 (3.82), 390 (3.97); ꢂNaOAc 240 (3.85), 355 (3.87); H-
3
1
3
NMR (400 MHz, DMSO-d ) and C-NMR (100 MHz, DMSO-d ): Table 1;
6 6
ꢃ
HR-TOF-ESI-MS (negative-ion mode) m/z: 621.1836 [MꢃH] (Calcd for
C H O : 621.1825).
2
9
33 15
Pectolinarigenin 7-O-a-L-rhamnopyranosyl-(1ꢅ→4ꢀ)-b-D-glucuronopyra-
2
5
(
1 lꢆ5). The aqueous layer was evaporated to remove a trace amount of or-
ganic solvent, and then extracted with EtOAc and 1-BuOH, successively
1 lꢆ5 each). The EtOAc and 1-BuOH fractions were concentrated under re-
noside (3): Pale yellow amorphous powder. [a] ꢃ42.3° (cꢄ0.48, DMSO);
D
ꢃ1
IR nmax (film) cm : 3390, 2950, 1712, 1655, 1607, 1510, 1462, 1357, 1258,
(
1183, 1106, 1073, 1034, 831; UV lmax (MeOH) nm (log e): 225 (4.18), 345
duced pressure to give 14 g and 137 g of residues, respectively. The remain-
ing aqueous layer was concentrated to furnish a water-soluble fraction
(4.21); ꢂNaOMe 225 (4.23), 375 (4.21); ꢂAlCl 225 (4.29), 370 (4.25);
3
1
ꢂAlCl /HCl 227 (4.20), 370 (4.26); ꢂNaOAc 238 (4.21), 375 (4.14); H-
3
1
3
(60 g).
NMR (400 MHz, DMSO-d ) and C-NMR (100 MHz, DMSO-d ): Table 1;
6 6
ꢂ
The EtOAc fraction (14 g) was subjected to silica gel CC (520 g) (Fꢄ
HR-ESI-MS (positive-ion mode) m/z: 659.1577 [MꢂNa] (Calcd for
5
5 mm, Lꢄ60 cm). The column was eluted initially with CHCl (5 l), and C H O Na: 659.1583).
3
29 32 16
25
then with a CHCl –MeOH gradient mixture, 500 ml fractions being col-
Byzantionoside B 6ꢁ-O-Sulfate (4): Off-white amorphous powder. [a]
D
ꢃ1
3
lected. Similar fractions were combined, affording 16 fractions. The second
fraction was precipitated with MeOH to provide 6 (35 mg).
The 1-BuOH fraction (137 g) was fractionated by CC on a highly porous
ꢂ10.5° (cꢄ1.13, MeOH); IR nmax (film) cm : 3420, 2963, 1650, 1377,
1257, 1220, 1069, 1012; UV l (MeOH) nm (log e): 236 (3.31), 280
max
1
13
(3.02); H-NMR (400 MHz, CD OD) and C-NMR (100 MHz, CD OD):
3
3
-
5
synthetic resin, Diaion HP-20 (Fꢄ40 mm, Lꢄ55 cm). The column was Table 2; CD De (nm): ꢂ0.43 (320), ꢂ1.21 (240) (cꢄ2.4ꢆ10 M, MeOH);
ꢂ
eluted initially with H O (3 l) and then with a MeOH–H O stepwise gradient
HR-ESI-MS (positive-ion mode) m/z: 497.1424 [MꢃHꢂ2Na] (Calcd for
2
2
of increasing MeOH content, i.e., 20% (2 l), 50% (2 l), 80% (2 l), and 100%
MeOH (3 l). The effluent was collected in fractions (500 ml each). Similar
fractions were combined to provide three fractions. The first fraction (I)
eluted with 20% MeOH (4.33 g) was subjected to silica gel CC (160 g)
C H O Na S: 497.1428).
1
9
31 10
2
(Z)-Hex-3-en-1-ol O-b-D-Xylopyranosyl-(1ꢀ→2ꢁ)-b-D-glucopyranoside
2
5
(5): White amorphous powder. [a] ꢂ34.2° (cꢄ0.26, MeOH); IR nmax
D
ꢃ1
1
(film) cm : 3390, 2927, 1075, 1040; H-NMR (400 MHz, DMSO-d ) and
6
1
3
(
Fꢄ30 mm, Lꢄ50 cm), using CHCl –MeOH gradients, yielding six frac-
C-NMR (100 MHz, DMSO-d ): Table 2. HR-ESI-MS (positive-ion mode)
6
ꢂ
3
tions. The fourth fraction, I-4 (316 mg), was purified by DCCC to give three
fractions. The second fraction, I-4-2 (256 mg), was purified by HPLC to pro-
duce compound 7 (6.56 mg) and compound 8 (2.48 mg). The fifth fraction, I-
m/z: 417.1730 [MꢂNa] (Calcd for C H O Na: 417.1731).
17 30 10
Analysis of the Sugar Moiety About 1 mg each of compounds 1—5
was hydrolyzed with 1 M HCl (1.0 ml) at 80 °C for 2 h. The reaction mixtures
were neutralized with Amberlite MB-3 and then partitioned with an equal
amount of EtOAc (1.0 ml), the resulting water layers being analyzed for their
sugar components. The sugars were identified by HPLC on an amino col-
umn [Shodex Asahipak NH P-50 4E (4.6 mmꢆ250 mm), CH CN–H O
5
(1.13 g), was purified by RPCC, affording four fractions. The fourth frac-
tion, I-5-4 (219.9 mg), was subjected to DCCC to give three fractions. The
second fraction, I-5-4-2 (108.2 mg), was purified by HPLC to furnish com-
pound 4 (34.6 mg).
2
3
2
The second fraction (II) eluted with 50% MeOH (6.36 g) on Diaion HP-
(4 : 1), 1 ml/min], using chiral detector (JASCO OR-2090plus), in compari-
son with authentic sugars (D-galacturonolactone, D-glucuronolactone, D-xy-
lose, D-glucose and L-rhamnose). Compound 1 gave a peak for D-galactur-
onolactone at the retention time of 11.5 min. Compound 2 gave peaks for L-
rhamnose and D-glucose at the retention times of 6.2 min and 11.4 min, re-
spectively. Compound 3 gave peaks for L-rhamnose and D-glucuronolactone
at the retention times of 6.2 min and 12.2 min, respectively. Compound 4
2
0 CC was chromatographed on silica gel (350 g), (Fꢄ40 mm, Lꢄ55 cm),
using a CHCl –MeOH gradient system, 200 ml fractions being collected.
3
Similar fractions were combined to yield seven fractions. The seventh frac-
tion, II-7 (4.65 g), was purified by RPCC, producing five fractions. The third
fraction, II-7-3 (1.58 g), was subjected to silica gel (75 g) CC with isocratic
elution with CHCl –MeOH–H O (17 : 6 : 1), providing seven fractions. The
3
2