Journal of Asian Natural Products Research
481
3. Experimental
concentrated under reduced pressure to
yield an extract (1.6 kg). The alcohol
extract was partitioned successively with
CHCl3, EtOAc, and n-BuOH. The n-
BuOH-soluble extract (175 g) was sub-
jected to silica gel column chromatog-
raphy with gradient elution [CHCl3–
MeOH 20:1 (2 l), CHCl3 –MeOH 9:1
(2 l), CHCl3–MeOH 4:1 (2 l), CHCl3–
MeOH–H2O 7:3:0.5 (2 l), CHCl3 –
MeOH–H2O 6:4:0.5 (2 l), MeOH (2 l)] to
give 11 fractions (Fr1–11). Compounds 1
(16 mg) and 2 (20 mg) from Fr8 (9.2 g)
were purified by repeated column chro-
matography over silica gel column chro-
matography with gradient elution
[CHCl3 –MeOH–H2O 7:3:0.5], ODS
(MeOH–H2O, 65:35), and Sephadex LH-
20 (MeOH). Further fractionation of Fr6
by silica gel column chromatography with
gradient elution [CHCl3 –MeOH–H2O
3.1 General experimental procedures
Optical rotations were measured with a
Perkin-Elmer 241 MC polarimeter (Perkin-
Elmer, Foster City, CA, USA). UV spectra
were recorded with a UV-2401 spec-
trometer (Shimadzu Corp., Kyoto, Japan).
IR spectra were obtained on a Nicolet 5700
IR spectrometer (Thermo corp., West Palm
Beach, FL, USA). NMR spectra were
recorded on a VARIAN INOVA 500 (1H,
500 MHz; 13C, 125 MHz) spectrometer
(Varian, Palo Alto, CA, USA). ESI-MS
was carried out with Angilent 1100 LC/
MSD (Angilent Technologies Ltd, Santa
Clara, CA, USA). For column chromatog-
raphy, silica gel (200–300 mesh; Qingdao
Marine Chemical Inc., Qingdao, China),
ODS (40–60 mm; YMC Co. Ltd, Kyoto,
Japan), and Sephadex LH-20 (Pharmacia
Biotech AB, Uppsala, Sweden) were used.
The analytical HPLC was carried out on
Angilent 1200 LC with DAD, and the
preparative HPLC was carried out on
Shimadzu LC-20A (Shimadzu Corp.,
Kyoto, Japan) with YMC-Pack ODS
column (20 mm £ 250 mm, 10 mm; YMC
Co. Ltd).
7:3:0.5
(4 l),
CHCl3 –MeOH–H2O
6:4:0.5 (4 l)] gave six subfractions (Fr10 –
60). Compounds 3 (15 mg), 4 (460 mg), 5
(24 mg), and 6 (17 mg) from Fr60 (11.4 g)
were isolated by repeated column chro-
matography over silica gel with CHCl3–
MeOH–H2O 6:4:0.5, ODS (MeOH–H2O,
60:40), and Sephadex LH-20 (MeOH).
Compound 7 (21 mg) from Fr50 (4.9 g) was
separated by repeated column chromatog-
raphy over silica gel with CHCl3 –
MeOH–H2O 7:3:0.5, Sephadex LH-20
(MeOH), and preparative HPLC
(MeOH–H2O, 65:35; flow rate: 7 ml/min;
tR: 20.03 min; detection wavelengths:
210 nm). Compound 8 (12 mg) from Fr5
(6.4 g) was isolated by repeated normal
phase silica gel with CHCl3–MeOH–H2O
6:4:0.5, Sephadex LH-20 (MeOH), and
preparative HPLC (MeOH–H2O, 60:40;
flow rate: 7 ml/min; tR: 26.80 min; detec-
tion wavelengths: 210 nm).
3.2 Plant material
The seeds of M. charantia were purchased
from Anguo of Hebei Province in 2011, and
identified by Associate Professor Lin Ma at
Institute of Materia Medica, Chinese Acad-
emy of Medical Sciences and Peking Union
Medical College. A voucher specimen
(No. ID-S-2547) has been deposited at our
laboratory in the Institute of Materia Medica,
Chinese Academy of Medical Sciences and
Peking Union Medical College.
3.3 Extraction and isolation
The seeds of M. charantia (15.0 kg) were
defatted three times by petroleum ether
(90 l each time), then extracted three times
under reflux in 95% ethanol (90 l each
time), and the combined solution was
3.3.1 Compound 1
25
White powder. ½aꢀD þ 100:0 (c ¼ 1.0,
MeOH); UV (MeOH) lmax nm (log 1):
233 (0.41), 327 (0.57); IR (KBr) nmax
: