J. Hu et al.
Materials and methods
petroleum ether (1 L 9 3), chloroform (1 L 9 3), and
ethyl acetate (1 L 9 3) gradually to afford three fractions
(51.0, 81.0, and 143.0 g respectively). The ethyl acetate
fraction was further fractionated through a silica gel col-
umn (200–300 mesh, 10 9 80 cm) eluting with a gradient
of CHCl3–MeOH (100:1, 50:1, 30:1, 20:1, 15:1, 10:1, 5:1,
2:1, 1:1, each 10 L) to afford 9 fractions. Fraction 3 (5.6 g)
was applied to an ODS MPLC column and eluted with
MeOH/H2O (10:90, 20:80, 30:70, 40:60, 50:50, 60:40,
80:20, 90:10, each1 L) to yield 3 subfractions. Subfraction
2 (830 mg) was purified by a preparative RP-HPLC using
25 % methanol as mobile phase to obtain 2 (112 mg).
Fraction 4 (6.1 g) was applied to an ODS column and
eluted with MeOH/H2O (10:90, 20:80, 30:70, 40:60, 50:50,
60:40, 70:30, 90:10, each 500 mL), followed by a Sepha-
dex LH-20 column eluting with MeOH/H2O (50:50, 3 L) to
obtain 3 (138 mg). Fraction 5 (4.9 g) was applied to an
ODS column using MeOH/H2O (10:90, 20:80, 30:70,
40:60, 50:50, 60:40, 70:30, 90:10, each 1 L) as mobile
phase to yield 4 subfractions. Subfraction 2 (898 mg) was
purified by a preparative RP-HPLC eluting with 22 %
methanol to get 1 (151 mg).
General experimental procedures
Optical rotations were taken on a Perkin-Elmer 341 polar-
imeter. IR spectra were recorded on Nicolet Magna FT-IR
750 spectrophotometer using KBr disks. NMR spectra were
recorded on Bruker AM-300, AM-400, and INVOR-600
NMR spectrometers. The chemical shift (d) values are given
in ppm with TMS as internal standard, and coupling con-
stants (J) are in Hz. FAB-MS spectra were recorded on a
Finnigan MAT TSQ-700. EI-MS and HR-EI-MS spectra
were recorded on a Finnigan MAT-95 mass spectrometer
(San Jose, CA, USA). Column chromatographic separations
were carried out using silica gel (200–300 mesh and H60,
Qingdao Haiyang Chemical Group Corporation, China),
MCI gel CHP20P (75–150 lm, Mitsubishi Chemical
Industries, Japan), and Sephadex LH-20 (Pharmacia Biotech
AB, Uppsala, Sweden) as packing material. TLC was carried
out on precoated silica gel GF254 plates (Yantai Chemical
Industrials), and the TLC spots were viewed at 254 nm and
visualized using 5 % sulfuric acid in alcohol containing
10 mg/mL vanillin. Analytical HPLC was performed on a
Waters 2690 instrument with a 996 PAD (photodiode array
detector) coupled with an Alltech ELSD 2000 detector.
Semipreparative and preparative HPLC was performed on a
Varian SD1 instrument with a 320 single-wave detector.
Their chromatographic separations were carried out on C-18
columns (250 9 10 mm, 5 lm, Waters; 220 9 25 mm,
10 lm, Merck, respectively), usinga gradient solvent system
comprised of H2O and MeOH, with a flow rate of 3.0 and
15.0 mL/min, respectively. All cell lines were purchased
from Cell Bank of Shanghai Institute of Biochemistry & Cell
Biology, Chinese Academy of Sciences. Other reagents were
purchased from Shanghai Sangon Biological Engineering
Technology & Services CO., Lt.
Acid hydrolysis and sugar analysis of compounds 1–3
A solution of compound 1 (2 or 3) (about 10.0 mg) in
1 M HCl (dioxane-H2O, 1:1, 1 mL) was heated at 95 °C for
2 h based on a previous reference (Zhang et al. 2008). After
cooling, the reaction mixture was neutralized by passage
through an Amberlite IRA-93ZU (Organo, Tokyo, Japan)
column and subjected to silica gel chromatography using a
gradient mixture of CHCl3–MeOH (19:1; 9:1; 1:1) to give an
aglycone fraction and a sugar fraction (3.0 mg). The agly-
cone fraction was purified by silica gel CC eluting with
hexane-Me2CO (4:1) to give an aglycone 1a (2.9 mg) [2a
(3.3 mg) or 3a (3.1 mg)]. HPLC analysis of the sugar frac-
tion under the following conditions showed the presence of
D-glucose and L-arabinose. Column: Capcell Pak NH2 UG80
(4.6 mm i.d. 9 250 mm, 5 lm, Shiseido); detector: Shodex
OR-2 (Showa-Denko, Tokyo, Japan); solvent: MeCN–H2O
(17:3); flow rate: 1.0 mL/min. Rt (min): 8.27 (L-arabinose,
positive optical rotation); 13.39 (D-glucose, positive optical
rotation). All chemical reagents and standard sugars were
purchased from Sigma-Aldrich Corporation.
Plant materials
The dried roots of S. officinalis were collected in the suburb
of Qujing, Yunnan province of China, in October of 2012
and identified by one of the authors (X. Mao). A voucher
specimen (SO20121001) was deposited in the Herbarium
of the College of Biological Resources and Environment
Science, Qujing Normal University, Qujing, Yunnan
province, China.
3b-[(a-L-arabinopyranosyl)oxy]-19a,23-dihydroxyolean-
12-en-28-oic acid 28-[6-O-acetyl-b-D-glucopyranosyl]
ester (1): White amorphous powder; [a]2D3.3 = ?23.8
(c 0.64, MeOH); IR (KBr) mmax 3450, 2943, 1731, 1459,
Extraction and isolation
1389, 1073, 860, 781 cm-1
;
1H NMR (pydirine-d5,
The air-dried roots of S. officinalis (5 kg) were ground into
powder and were extracted thrice with 80 % EtOH. After
evaporation of the EtOH, the aqueous brownish syrup (1 L)
was suspended in H2O (l L) and then partitioned with
600 MHz) data see Table 1, 13C NMR (pydirine-d5,
150 MHz) data see Table 2; FAB-MS (pos.) m/z: 847
[M ? Na]?; HR-ESI–MS (pos.) m/z: 847.4451
([M ? Na]?, C43H68O15Na; calc. 847.4456).
123