2
P. Wang et al. / Fitoterapia 103 (2015) 1–8
2
2
. Experimental
column to remove flavanoids and tannins, and the 60% methanol
eluate (240 mg) was further chromatographed over an RP MPLC
(50% methanol) and a preparative HPLC (65% methanol) to give
.1. General
8
(6.1 mg). Part of Fr. B4 (1.0 g) was also treated by RP polyamide
UV and IR spectra were recorded on a Shimadzu UV-2450
and the 60% methanol eluate was further chromatographed over
an RP MPLC (45% methanol) and a preparative HPLC (60%
methanol) to give 9 (16.0 mg) and 10 (9.3 mg). Fr. C (32 g) was
chromatographed over a silica gel column (200–300 mesh) and
eluted with a gradient of petroleum ether/acetone (10:1, 5:1,
2:1, and 1:1, v/v) to give four subfractions (Fr. C1–4). Part of
Fr. C2 (1.0 g) was treated by RP polyamide and Sephadex LH-20
to remove flavanoids and tannins. The purified Fr. C2 (220 mg)
was further chromatographed on RP MPLC (45% methanol) to
afford 7 (51.0 mg) and 13 (2.1 mg). Part of Fr. C3 (1.0 g) was
treated like Fr. C2 to remove flavanoids and tannins, and further
chromatographed on RP MPLC (40% methanol) and preparative
HPLC (50% methanol) to afford 11 (9.2 mg) and 12 (5.4 mg).
After removal of flavanoids and tannins, part of Fr. C4 (1.0 g) was
purified by RP MPLC and preparative HPLC to give 14 (4.2 mg).
Fr. D had almost one peak in the HPLC chromatogram, and part
of Fr. D (50.0 mg) was purified by RP MPLC (55% methanol) and
preparative HPLC (65% methanol) to afford 15 (27.4 mg).
spectrometer and a Bruker Tensor 27 spectrometer, respec-
tively. Optical rotations were measured on a JASCO P-1020
polarimeter. CD spectra were obtained on a JASCO 810
spectropolarimeter. NMR spectra were recorded on a Bruker
1
13
AV-500 NMR instrument at 500 MHz ( H) and 125 MHz ( C)
in CD OD, pyridine-d or DMSO-d . HRESIMS experiments
3
6
6
were performed on an Agilent UPLC-Q-TOF (6520B). Silica
gel (100–200 mesh and 200–300 mesh, Qingdao Marine
Chemical Co., Ltd., China), polyamide (100–200 mesh, Sijia
Corp., Zhejiang, China.), ODS (40–63 μm, FuJi, Japan), and
Sephadex LH-20 (Pharmacia, Sweden) were used for column
chromatography. Preparative HPLC was carried out on a
Shimadzu LC-6A instrument with an SPD-10A detector and a
shim-pack RP-C18 column (20 × 200 mm, 10 μm). RP-MPLC
was carried out on a Quiksep system (H & E Co., Ltd., Beijing,
China). Analytical HPLC was performed on an Agilent 1200
series instrument using a DAD detector and a shim-pack
VP-ODS column (250 × 4.6 mm, 5 μm). L-cysteine methyl ester
and o-tolylisothiocyanate were purchased from TCI (Tokyo,
Japan). D-glucose was purchased from Sinopharm Chemical
Reagent Co., Ltd. (Shanghai, China). Human hepatoma cell line
HepG2, human breast carcinoma cell line MCF-7, and human
osteosarcoma cell line U2-OS were purchased from the Cell
Bank of Shanghai Institute of Biochemistry and Cell Biology,
Chinese Academy of Sciences (Shanghai, China). HepG2 and
MCF-7 cell lines were cultured in Dulbecco's modified Eagle
medium (DMEM, Gibco Invitrogen Corp., Carlsbad, CA, USA).
U2-OS cell lines were cultured in modified RPMI-1640 medium
The n-butanol extract (320 g) was dissolved by appropriate
quantity of 40% methanol and adsorbed over a macroporous
adsorption resin column. Then the column was eluted by a
gradient of EtOH/H
give four fractions Fr. 1–4. Part of Fr. 3 (20.0 g) was subjected
to a silica gel column and eluted with CH Cl /MeOH system
2
O (10:90, 20:80, 30:70 and 50:50, v/v) to
2
2
(10:1, 5:1, 2:1 and 1:1, v/v) to give four subfractions (Fr. 3.1–
3.4). Fr. 3.2 (3.32 g) was chromatographed over an ODS column
and eluted with a gradient MeOH/H O system (20:80, 30:70,
2
40:60, 50:50 and 60:40, v/v) to yield Fr. 3.2.1–Fr. 3.2.5. Fr. 3.1.3
was subjected to an RP polyamide column to remove flavanoids
and tannins, and the 60% methanol eluate (520 mg) was
further purified by Sephadex LH-20 and preparative HPLC to
afford 17 (112 mg), 1 (13.1 mg), 2 (5.4 mg) and 3 (8.8 mg). Part
of Fr. 3.3 (2 g) was subjected to an ODS column and eluted with
a gradient methanol/water system (20:80, 30:70, 40:60, 50:50
and 60:40, v/v) to yield Fr. 3.3.1–Fr. 3.3.5. Fr. 3.3.1 (120 mg) was
further purified by Sephadex LH-20 and preparative HPLC to
give 4 (9.1 mg) and 5 (11.2 mg). Fr. 3.3.4 (131 mg) was purified
by Sephadex LH-20, RP MPLC and preparative HPLC to yield
20 (36.1 mg) and 21 (14.5 mg). Part of Fr. 3.4 (2 g) was
chromatographed on ODS, Sephadex LH-20 and preparative
HPLC to give 6 (6.8 mg), 18 (420 mg) and 19 (15.2 mg). Part of
Fr. 4 (1 g) was subjected to RP MPLC and preparative HPLC
successively to afford 15 (133 mg) and 16 (12.1 mg).
(
1
Gibco Invitrogen Corp.). The cells were all supplemented with
0% fetal bovine serum (Sijiqing, Hangzhou, China), 100 U/mL
penicillin and 100 μg/mL streptomycin at 37 °C with 5% CO
2
.
2
.2. Plant material
The fruits of C. acuminata Decne were collected in November
012 from the campus of China Pharmaceutical University,
2
Jiangsu Province, China, and were authenticated by Professor
Mian Zhang, Department of Pharmacognosy, China Pharmaceu-
tical University. A voucher specimen (No. XS201211) was
deposited in the Department of Natural Medicinal Chemistry,
China Pharmaceutical University.
2
.3. Extraction and isolation
2
.3.1. Camptothecoside (1)
The air-dried and powdered fruits of C. acuminata (8 kg)
were extracted by 95% ethanol (3 × 2 h) under reflux. After
removal of the solvent under reduced pressure, the residue
960 g) was suspended in water and extracted by petroleum
ether, chloroform and n-butanol, respectively. The chloroform
extract (102 g) was subjected to a silica gel column (100–
2 2
00 mesh) and eluted with CH Cl /MeOH system to give four
fractions Fr. A-D (50:1, 20:1, 10:1 and 5:1, v/v). Fr. B (26 g) was
further subjected to a silica gel column (200–300 mesh), and
eluted with a gradient of petroleum ether/acetone (20:1, 10:1,
Pale yellow amorphous powder; [α]25
D
−50.6 (c 0.10,
MeOH); UV (MeOH) λmax (log ε) 218 (4.52), 254 (4.38), 290
(4.02), 366 (4.21) nm; CD (MeOH) 261 (Δε +0.72), 238
(Δε −6.56), 222 (Δε +11.9), 204 (Δε −25.9) nm; IR (KBr)
(
−
1 1
ν
max 3340, 1654, 1596, 1504, 1185, 1066, 891, 825 cm
(DMSO-d , 125 MHz) data,
, 500 MHz) and 13C NMR (DMSO-d
see Table 1; HRESIMS m/z: 495.1759 [M + H] (calcd. for
, 495.1762).
; H
6
6
+
2
26 27 2 8
C H N O
2.3.2. Camptothecin 11-O-β-D-glucopyranoside (2)
Pale yellow amorphous powder; [α]25
−18.8 (c 0.08,
MeOH); UV (MeOH) λmax (log ε) 219 (4.55), 255 (4.39), 288
5
:1, 2:1, and 1:1, v/v), to yield 5 subfractions (Fr. B1–5). Part of
Fr. B3 (1.0 g) was subjected to a reversed-phase polyamide
D