X. Zhu et al. / Fitoterapia 82 (2011) 632–636
633
were recorded with Bruker AM-400 and DRX-500 instru-
ments with TMS as internal standard. EI–MS was measured
with VG AutoSpec 3000 spectrometers. ESI–MS and HR–ESI–
MS were measured with API QSTAR Pulsarimass spectro-
meters. Silica gel (200–300 mesh, Qingdao Marine Chemical
Inc., China) and Sephadex LH-20 (Amersham Biosciences,
Sweden) were used as stationary phase for column chroma-
tography. Silica gel (GF254, Qingdao Marine Chemical Inc.,
China) was used for stationary phase for TLC.
kemia type cell line K562, hepatocellular carcinoma HepG2
cell line, carcinomic human alveolar basal epithelial cells
A549, and human mammary carcinoma MDA435 cell line by
MTT assay. Cell lines were maintained in RPMI-1640 medium
(Hyclone) containing 10% heat inactivated fetal bovine serum
(Hyclone), 100 μg/ml penicillin sodium salt and 100 μg/ml
streptomycin sulphate, and kept in humidified incubator at
5% CO2 at 37 °C. After 80% confluence was reached cells were
harvested and seeded in a 96-well plate with 6000 cells per
well to which tested compounds of concentration at 100 μM
were added and incubated for 48 h followed by MTT assay at
the wave length of 490 nm.
2.2. Plant material
Aerial parts of E. rosea were collected in May, 2004 in
Xishuangbanna, Yunnan province, People's Republic of China.
The plant was identified by Dr. Li Rong, Kunming Institute of
Botany, Chinese Academy of Sciences (CAS). A sample (Kun
No.20040501) has been deposited in Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming, Yunnan, PR
China.
2.5. Acidic hydrolysis
Compounds 1 and 2 (each 5 mg) were dissolved in a
mixture of CH3OH (1.0 ml) and 2 M HCl (1.0 ml) and
hydrolyzed for 2 h by refluxing in a boiling water bath. The
hydrolysate was partitioned between water and EtOAc after it
was cooled to room temperature and diluted two-fold with
distilled water and then the aqueous layer was neutralized
and concentrated in vacuo to obtain a residue which was
identified by TLC compared with standard samples with
solvent A: petrol ether/CH3COCH3 (3:2), solvent B: CH2Cl2/
C2H5OH (9:1). Glucose was identified by TLC compared with
authentic sample with solvent CHCl3/CH3OH/H2O (4:3:1).
Ecdysantheroside A (1=14β, 20-dihydroxy-18-oic (18→
20) lactone-5-pregnene 3-O-β-D-cymaropyranoside).
2.3. Extraction and isolation
The dried powdered stems and leaves (11 kg) of E. rosea
were extracted three times for 2 h under reflux with 60, 50, and
40 L EtOH, successively. Then the combined extracts were
concentrated under reduced pressure to yield a dark residue
which was suspended in water and partitioned with petroleum
ether, EtOAc and n-BuOH, respectively. The EtOAc part (79 g)
was subjected to silica gel column chromatography (CC, Ø×L,
15×150 cm) eluting with a gradient mixture of CHCl3–MeOH
(100:0, 95:1, 90:10, 80:20, 70:30v/v) to give seven fractions
(Fr.1–Fr.7). And Fr. 2 (10 g) was further isolated by silica gel
column chromatography (CC) (Ø×L, 20×60 cm) eluting with a
gradient mixture of CHCl3–MeOH (95:1–90:10v/v) to obtain
twelve subfractions named as Fr2.1–Fr2.12. Furthermore Fr2.3
(1.6 g) was chromatographed on silica gel eluted with
petroleumether–Me2CO (9:1→7:3, v/v) followed by Sephadex
LH-20 column chromatography (CC) (Ø×L, 1×150 cm)
(MeOH) to afford compound 4 (21 mg), 5 (11 mg), and 6
(16 mg). And Fr2.6 (1.2 g) was chromatographed on silica gel
eluted with CHCl3–MeOH (10:1, 8:1, 7:3) and further purified
on sephadex-LH-20 eluted with CHCl3–MeOH (1:1, v/v) and
then on reverse phase (RP-18) column chromatography (CC)
(Ø×L, 1×60 cm) eluted with CH3OH: H2O 1: 1 to afford 1
(87 mg), 7 (12 mg) and 8 (6 mg). Moreover, F6 (22 g) was
further isolated by silica gel column chromatography (CC)
(Ø×L, 20×100 cm) eluting with a gradient mixture of CHCl3–
MeOH (95:5–80:20v/v) to give ten subfractions named as
F6.1–Fr6.10 respectively. And Fr6.6 (1.2 g) was further chro-
matographed on silica gel eluted with CHCl3–MeOH (10:1, 8:1,
and 7:3) followed by purifying on Sephadex LH-20 column
chromatography (CC) (Ø×L, 1×150 cm) eluted with CHCl3–
MeOH 1:1 and then on reverse phase (RP-18) column
chromatography (CC) (Ø×L, 1×60 cm) eluted with CH3OH:
H2O 1:1 to afford 2 (127 mg).
White solid, molecular formula, [α]2D5=+22.4 (c 0.57,
MeOH). Negative FAB-MS m/z: 489 [M–H]−, negative
HRFABMS m/z: 489.2863 [M–H]− calc. for C28H41O7 489.2852.
UV: 217, 241 nm; IR bands (KBr) 3441 (OH), 2930, 2856, 1744
(C=O), 1640 (C=C), 1453, 1088 cm−1
spectral data see Table 1.
;
1H and 13C NMR
Ecdysantheroside B (2=14β, 20-dihydroxy-18-oic (18→
20) lactone-5-pregnene 3-O-β-D-glucopyranosyl-(1→6)-β-D-
glucopyranosyl-1→4)-α-L-cymaropyranosyl-(1→4)-β-D-
cymaropyranosyl-(1→4)-β-D-cymaropyranoside).
White solid, molecular formula, [α]2D5=−21.7 (c 0.58,
CH3OH). Negative FAB-MS m/z: 1101 [M–H]−, negative
HRFABMS m/z: 1101.5474 [M–H]− calc. for C54H85O23
1101.5481. UV: 224, 249 nm; IR bands (KBr) 3441 (OH),
2933, 2968, 1746 (C=O), 1633 (C=C), 1452, 1368, 1088 cm−1
1H and 13C NMR spectral data see Table 2.
;
3. Results and discussion
Ecdysantheroside A (1) was found to possess a molecular
formula of C28H41O7 based on the negative FAB-MS (m/z 489
[M–H]−), negative HR-FAB-MS (m/z=489.2863 [M–H]−
,
calcd.: 489.2852) and the NMR spectroscopic data (Table 1).
IR spectrum showed the absorption bands due to hydroxyl
(3441 cm− 1), carbonyl (1744 cm− 1) and olefinic-bond
(1640 cm−1) groups. The 13C NMR spectrum of compound
1 displayed 28 carbon signals including one methoxy, 21
carbons of which were assigned to the aglycon part and 6 to
the glycoside. This can be further identified by acid hydrolysis
of 1 that led to the isolation of the aglycone unit, identified as
3β, 14β, 20-trihydroxy-18oic (18→20) lactone 5-pregnene
(3) [3] and a monosaccharide, cymarose, identified by
comparison with a standard sample (Fig. 1). The 1H NMR
2.4. Cytotoxicity assay
The cytotoxicity assay was performed against human
acute lymphoblastic T-cell leukemia (CEM) cells, human
umbilical vein endothelial cells (HUVEC), human erythroleu-