Molecules 2017, 22, 133
5 of 7
After evaporation of the ethanol in vacuo, the aqueous part (400 g) was subjected to a macroporous
resin column (HP2MGL, 12 L) eluted with water and 20% EtOH. The latter portion (200 g, A)
was subjected to column chromatography on silica gel (200–300 mesh, 10 cm
×
100 cm, 2.8 kg)
using a stepwise gradient elution of CHCl –MeOH–H O (80:20:2–70:30:5, v/v/v) to afford thirteen
3
2
fractions (A1–A13). The last portion A13 (27 g) was further chromatographed over a sephadex LH-20
column (5 cm 130 cm, 600 g) using 50% EtOH as a mobile phase to give five subfractions (A13a–A13e).
Fraction A13b (9.5 g) was separated by reversed-phase silica MPLC with 10%–40% aqueous MeOH
25 mL/min, 6 h) to afford forty parts (A13b1-40). Fraction A13b21 (500 mg) was performed repeatedly
on an RP-18 column (ODS, 50 m, YMC) with MeOH–H O (14:86, v/v) as the mobile phase to give
(5 mg) and (4 mg). Fraction A13b31 (170 mg) was followed by repeated separation on an RP-18
column (ODS, 50 m, YMC) with MeOH–H O (14:86, v/v) as the mobile phase yielded (14 mg) and
(13 mg), using phenyl column (50
get 1 (14 mg).
×
(
µ
2
4
5
µ
2
2
3
µ
m, ZORBAX) with MeOH–H O (9:91, v/v) as the mobile phase to
2
Conferyl alcohol-4-O-
amorphous solid. [α]D
β
-D-glucopyranoside-9-O-
β
-D-apiofuranosyl(1
→
6)-
β
-D-glucopyranoside (
1): white,
20
−
117.4 (c 0.13, MeOH). UV (MeOH) λmax (log
ε
−
): 211 (4.56), 260 (4.35),
1
1
2
5
98 (3.90) nm; IR (microscope) νmax 3382, 2921, 1512, 1266, 1076, 636 cm
00 MHz) and 13C-NMR (DMSO-d , 125 MHz), see Table 1. HRESIMS m/z 659.2171 [M + Na]
.
H-NMR (DMSO-d ,
6
+
6
(
calcd. for C H NaO , 659.2158).
27
40
17
Umbelliferone 7-O-
amorphous solid. [α]D
α
-D-glucopyranosyl(1
→
3)-[
β
-D-apiofuranosyl(1
→
6)]-
β
-D-glucopyranoside (
2): white,
20
−
35.4 (c 0.17, MeOH). UV (MeOH) λmax (log ε): 204 (4.57), 291 (4.12),
−
1
1
3
6
18 (4.26) nm; IR (microscope) νmax 3382, 2925, 1708, 1617, 1072, 1026 cm . H-NMR (DMSO-d ,
00 MHz) and 13C-NMR (DMSO-d , 150 MHz), see Table 1. HRESIMS m/z 641.1685 [M + Na]
6
+
6
(
calcd. for C H NaO , 641.1688).
26
34
17
Umbelliferone 7-O-
amorphous solid; [α]D
α
-D-glucopyranosyl(1
→
4)-[
β
-D-apiofuranosyl(1
→
6)]-
β
-D-glucopyranoside (
3): white,
20
−
50.6 (c 0.15, MeOH). UV (MeOH) λmax (log ε): 204 (4.64), 291 (4.12), 318
−
1 1
(
4.25) nm; IR (microscope) νmax 3359, 2923, 1708, 1618, 1026 cm . H-NMR (DMSO-d , 600 MHz)
6
+
and 13C-NMR (DMSO-d , 150 MHz), see Table 1. HRESIMS m/z 641.1699 [M + Na] (calcd. for
6
C H NaO , 641.1688).
26
34
17
3
.4. Acid Hydrolysis and Sugar Analysis
Each compound (2 mg) was dissolved in 2 mL 2 M HCl and refluxed at 80 C for 5 h. The reaction
mixture was extracted with EtOAc (3 1 mL), and the aqueous layer was evaporated to give a mixture
◦
×
of monosaccharides. The residue was further dissolved in anhydrous pyridine (1 mL) followed by
the addition of 2 mg L-cysteine methyl ester hydrochloride (J&K Scientific Ltd., Beijing, China; 99%).
◦
After heating at 60 C for 2 h, the solvent was eliminated under N , and 0.2 mL trimethylsilylimidazole
(
2
◦
J&K Scientific Ltd., 99%) was added. Then, the mixture was heated at 60 C for another 2 h and
partitioned with n-hexane and water (2 mL each). The organic layer was analyzed by GC. The authentic
samples were treated with the same method, D-glucose and D-apiose in a ratio of 2:1 were detected
from 1 to 3.
3
.5. Hepatoprotective Activity Assay
Human HepG2 hepatoma cells were cultured in DMEM medium supplemented with 10% fetal
◦
calf serum, 100 U/mL penicillin, and 100
µ
g/mL streptomycin at 37 C in a humidified atmosphere
of 5% CO + 95% air. The cells were then passaged by treatment with 0.25% trypsin in 0.02% EDTA.
2
The MTT assay was used to assess the cytotoxicity of test samples. The cells were seeded in 96-well
◦
multiplates. After an overnight incubation at 37 C with 5% CO , 50
µM test samples and APAP
2
(
final concentration of 8 mM) were added into the wells and incubated for another 48 h. Then, 100
of 0.5 mg/mL MTT was added to each well after the withdrawal of the culture medium and incubated
for an additional 4 h. The resulting formazan was dissolved in 150 L of DMSO after aspiration of the
µL
µ