JoURnAL oF ASIAn nATURAL PRoDUCTS ReSeARCH
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3.3. Extraction and isolation
e air-dried aerial parts of C. japonicum (12.5 kg) were extracted three times with 95%
EtOH (18 L × 3) under reflux to afford a dark gum (1.4 kg). e combined gum was succes-
sively diluted in H2O and partitioned with petroleum ether, ethyl acetate, and n-butanol [18],
yielding petroleum ether fraction (99.6 g), ethyl acetate fraction (210.4 g), and n-butanol
fraction (294.5 g). According to the screening results of C. japonicum, it was found that the
n-butanol fraction exhibited potential cytotoxic activity.
e n-butanol fraction was subjected to the macroporous absorption resin (Diaion-101)
column, which was eluted with 15, 30, 50, and 95% ethanol to obtain four fractions: A
(45.2 g), B (87.4 g), C (44.7 g), and D (21.6 g). e purification of A fraction was per-
formed on the Sephadex LH-20 column with a gradient system of MeOH/H2O (MeOH
in H2O, 90, 95, and 100%) to give three sub-fractions: A1 (10.5 g), A2 (12.8 g), A3 (8.2 g).
e separation of A2 was dealt with silica gel (100–200 mesh or 200–300 mesh) column
eluted with petroleum ether–acetone (5:1 → 3:1 → 1:1, v/v) to give three sub-fractions: A2a
(1.87 g), A2b (3.65 g), A2c (1.26 g). e A2b fraction was subjected to silica gel (200–300 mesh)
column eluted with petroleum ether–acetone (3:1, v/v) and Sephadex LH-20 column with
MeOH/H2O (MeOH in H2O, 95%), successively, and yielded 5 (10.20 mg, purity > 97.3%
by HPLC), 6 (8.34 mg, purity > 96.5% by HPLC), 8 (9.65 mg, purity > 98.3% by HPLC).
In the same way, B fraction was subjected to Sephadex LH-20 column with a gradient
system of MeOH/H2O (MeOH in H2O, 85, 90, 95, and 100%) to give four sub-fractions:
B1 (7.52 g), B2 (13.08 g), B3 (11.37 g), B4 (8.41 g). e sub-fraction B3 was chromatographed
over silica gel (100–200 mesh) column eluted with petroleum ether–acetone (6:1→4:1→2:1,
v/v) to obtain three sub-fractions: B3a (2.30 g), B3b (4.52 g), B3c (1.89 g). e B3b fraction
was subjected to Sephadex LH-20 with MeOH/H2O (MeOH in H2O, 95%) and silica gel
(200–300 mesh) column eluted with petroleum ether–acetone (4:1, v/v), successively, and
yielded 1 (6.89 mg, purity > 97.0% by HPLC), 2 (8.32 mg, purity > 98.3% by HPLC),
3 (7.20 mg, purity > 97.7% by HPLC), 7 (10.25 mg, purity > 96.6% by HPLC), and 10
(12.10 mg, purity > 97.4% by HPLC). Similarly, C fraction was also performed on Sephadex
LH-20 with a gradient system of MeOH/H2O (MeOH in H2O, 90, 95, and 100%) and sil-
ica gel (100–200 mesh and 200–300 mesh) column eluting with petroleum ether–acetone
(8:1 → 5:1 → 3:1 → 2:1, v/v), successively, and yielded 4 (9.56 mg, purity > 97.4% by HPLC),
9 (12.58 mg, purity > 96.7% by HPLC), 11 (10.21 mg, purity > 96.9% by HPLC), and 12
(9.52 mg, purity > 97.8% by HPLC).
3.3.1. 1-[3-(4-Hydroxyphenoxyl)-1-propenyl]-3,5-dimethoxyphene-4-O-β-D-
glucopyranoside (1)
Pale yellow powder;[ꢀ]D20 + 31.52 (c 0.30, MeOH); UV (MeOH) λmax: 205, 330 nm; IR νmax
:
3428, 1612, and 1582 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz)
spectral data see Table 2; HR-ESI-MS: m/z 487.1562 [M + Na]+ (calcd for C23H28O10Na,
487.1580).
3.3.2. 1-[3-(3,4,5-Trimethoxyphenoxyl)-1-propenyl]-3-methoxyphene-4-O-β-D-
glucopyranoside (2)
Pale yellow powder;[ꢀ]D20 + 41.27 (c 0.30, MeOH); UV (MeOH) λmax: 205, 331 nm; IR νmax
:
3422, 1611, and 1576 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz)