Journal of Natural Medicines
extracts were evaporated to dryness under vacuum to obtain
a petroleum ether-soluble portion (fraction A, 34.6 g) and an
80% ethanol-soluble portion (320.7 g). Fraction A (33.0 g)
was chromatographed on an MCI gel column, eluting with
EtOH-H2O (0:100–95:5, v/v, successively) to yield seven-
teen fractions (Fr. A1-Fr. A17). Pale-yellow needle crystals
(6, 128 mg) were obtained from Fr. A4.
Experimental
General experimental procedures
The optical rotations and CD spectra were experimented by
the Autopol® VI Automatic Polarimeter (Rudolph Research
Analytical, Hackettstown, NJ, USA) and JASCO J-810 Cir-
cular Dichroism Spectropolarimeter (JASCO, Easton, MD,
USA), respectively. The UV spectra was measured on Shi-
madzu UV-2700 UV–Visible Spectrophotometer (Shimadzu
Corporation, Kyoto, Japan). The IR data were measured on
VERTEX 70v FTIR Spectrometer (Bruker Optik GmbH.,
Ettlingen, Germany). The NMR spectra were recorded
with Bruker Avance 400/600 NMR spectrometer (Bruker-
Biospin, Billerica, MA, USA) and calibrated based on the
solvent peak used. HRESIMS reports were obtained from
a SYNAPT G2-Si HDMS (Waters Corp., Manchester, UK)
with an electrospray ion source (Waters, Milford, MA) con-
nected to a lock-mass apparatus that performed the real-time
calibration correction. Analytical HPLC was performed on a
Waters 2535 Series machine equipped with an Xbridge C18
column (4.6×250 mm, 5 μm), and preparative HPLC was
performed on a semi-preparative Xbridge Prep C18 OBD
column (19×250 mm, 5 μm). Column chromatography was
performed on CHP20P MCI gel (75–150 μm, Mitsubishi
Chemical Corporation, Japan), silica gel (200–300 mesh,
Qingdao Haiyang Chemical Co., Ltd.), Sephadex LH-20
(GE Healthcare Bio-Sciences AB, Sweden), and reversed-
phase C18 silica gel (50 μm, YMC, Kyoto, Japan). Analytical
and preparative TLC was performed on precoated GF254
plates (0.25 or 0.5 mm thickness, Qingdao Haiyang Chemi-
cal Co. Ltd.). Detection was performed by spraying the
plates with 10% sulfuric acid followed by heating.
Fraction B (300.0 g) was chromatographed on an MCI
gel column, eluting with EtOH-H2O (0: 100 to 95:5, v/v,
successively) to yield twenty-two fractions (Fr. B1-Fr. B22)
based on the TLC profles. Fr. B1 (180.0 g) was chroma-
tographed on the MCI gel column again, and eluted in a
step gradient of EtOH-H2O (0:100–95:5, v/v) to aford eight
subfractions (Fr. B1a-B1h). Subfraction Fr. B1c was further
separated on an MCI gel column using the same method as
for Fr. B1 to give six subfractions (Fr. B1c1-Fr. B1c6), and
Fr. B1c2 was purifed by semi-preparative HPLC (MeCN-
H2O, 5:95 → 30:70, 10 mL/min) to aford compounds 8
(15 mg), 3 (6 mg), and 9 (13 mg). Subfraction Fr. B1c5 was
chromatographed on a Sephadex LH-20 column in MeOH
and further purifed by semi-preparative HPLC (MeCN-
H2O, 5:95 → 30:70, 10 mL/min), yielding 2 (8.1 mg), 4
(4.1 mg), and 13 (18 mg). 15.1 mg of Fr. B2 was purifed by
semi-preparative HPLC (MeCN-H2O, 5:95→30:70, 10 mL/
min) to give 1 (8.3 mg). 20.0 mg of Fr. B3 was purifed by
semi-preparative HPLC (MeCN-H2O, 5:95→30:70, 10 mL/
min) to aford 7 (5.6 mg, tR = 17.6 min), and 10 (4.9 mg,
tR = 14.0 min). 43 mg of Fr. B4 was purifed by semi-pre-
parative HPLC (MeCN-H2O, 20:80→80:20, 10 mL/min),
yielding 11 (20.6 mg), and 12 (8.4 mg).
Spectroscopic data (UV and IR, ms)
Crocusatin M (1): Colorless oil. 1H NMR (CD3OD,
400 MHz) and 13C NMR (CD3OD, 100 MHz) spectro-
scopic data, see Table 1; IR νmax 3321, 2928, 1672, 1063,
1019 cm−1; UV λmax (MeOH) (log ε): 216 (2.57), 246
(2.90) nm; HRESIMS m/z 185.1174 [M+H]+ (calcd. for
C10H17O3, 185.1178). [α]19.4D +6.25 (c 0.096, MeOH); CD
(MeOH) (log Δε):198 (+3.20), 230 (− 2.89), 263 (+1.88),
281(− 1.38), 341(+2.27) nm;
Plant material
The safron raw materials were collected at the Chongming
Island in Shanghai city and were friendly donated by the Saf-
fron Division of the Shanghai Traditional Chinese Medicine
Co., LTD. (Shanghai, China) in August 2016. The scientifc
name was identifed by one of the authors (Wenwei Fu). A
voucher specimen (TCMNDD20160801) was deposited at
the Engineering Research Centre of Shanghai Colleges for
TCM New Drug Discovery (Shanghai University of Tradi-
tional Chinese Medicine).
1
Crocusatin N (2): Colorless solid. H NMR (CD3OD,
600 MHz) and 13C NMR (CD3OD, 150 MHz) spectro-
scopic data, see Table 1; IR νmax 3344, 2911, 1672, 1608,
1020 cm−1; UV λmax (MeOH) (log ε): 215 (2.07), 252
(2.73) nm; HRESIMS m/z 345.1554 [M–H]− (calculated for
C16H25O8); [α]19.7D − 37.26 (c 0.229, MeOH); CD (MeOH)
(log Δε): 190 (− 3.18), 210 (+ 2.76), 253 (− 2.87), 283
(+1.83) nm.
Extraction and isolation
1
Crocusatin O (3): Colorless solid. H NMR (CD3OD,
400 MHz) and 13C NMR (CD3OD, 100 MHz) spectro-
scopic data, see Table 1; IR νmax 3340, 2467, 1657, 1590,
1375, 1070, 1034, 973 cm−1;UV λmax (MeOH) (log ε):
Air-dried and powdered stigmas of the plant (930 g) were
percolated successively with petroleum ether (20 L) and
80% EtOH (40 L) at room temperature. The combined
1 3