J. He et al. / Carbohydrate Research 346 (2011) 1903–1908
1907
1D and 2D NMR spectra were run on INOVA 500 MHz or BRUKER
AV500 spectrometer. HRESIMS were performed on a Finnigan LTQ
FT mass spectrometer. The ESI mass spectra were recorded on an
Agilent 1100 series LC/MSD TOF from Agilent Technologies. Column
chromatography was performed with Macroporous resin (Diaion
HP-20, Mitsubishi Chemical Corp. Tokyo, Japan), Sephadex LH-20
(Pharmacia Fine Chemicals, Uppsala, Sweden). Preparative HPLC
was carried out on a Shimadzu LC-6AD instrument with an
SPD-20A detector, using a YMC-Pack ODS-A column (250 mm ꢀ
1160, 1074, 1039, 896 cmꢁ1; 1H NMR see Table 1; 13C NMR see Ta-
ble 3; (+)-HRESIMS m/z 403.1364 (calcd for 19H24O8Na,
C
403.1363).(2E)-10R-Tetradecaene-4,6-diyne-1,10,14-triol-1-O-b-D-
glucopyranoside (4): ½a D20
ꢂ
ꢁ8.5 (c 0.07, MeOH); IR
mmax 3405, 2932,
2234, 2142, 1724, 1605, 1496, 1453, 1365, 1076, 1041, 751, 701,
633 cmꢁ1; 1H NMR see Table 1; 13C NMR see Table 3; (+)-HRESIMS
m/z 421.1834 (calcd for C20H30O8Na, 421.1833).(2E,8E)-12R-
Tetradecadiene-4,6-diyne-1,12,14-triol-1-O-b-D-glucopyranoside
(5): ½a 2D0
ꢁ31.1 (c 0.13, MeOH); IR mmax 3297, 2924, 2882, 2204,
ꢂ
20 mm, 5
1200 series system (Agilent Technologies, Waldbronn, Germany)
m; Grace
l
m). HPLC-DAD analysis was performed using an Agilent
2132, 1615, 1515, 1441, 1426, 1363, 1256, 1161, 1068, 1036,
951, 836 cmꢁ1; 1H NMR see Table 2; 13C NMR see Table 3; (+)-HRE-
SIMS m/z 419.1685 (calcd for C20H28O8Na, 419.1676).(2Z,8Z)-12R-
with an Apollo C18 column (250 mm ꢀ 4.6 mm, 5
l
Davison). Precoated silica gel GF-254 plates (Yantai Jiangyou Silica
Tetradecadiene-4,6-diyne-1,12,14-triol-1-O-b-
D
-glucopyranoside
Gel Exploitation Company) were used for analytical TLC.
(6): ½a 2D0
ꢂ
+5.3 (c 0.07, MeOH); IR mmax 3352, 2928, 2203, 2130, 1657,
1603, 1409, 1366, 1280, 1159, 1072, 1048, 896, 739, 614 cmꢁ1 1H
;
3.2. Plant material
NMR see Table 2; 13C NMR see Table 3; (+)-HRESIMS m/z 419.1673
(calcd for C20H28O8Na, 419.1676).(2Z,8E)-12R-Tetradecadiene-4,6-
The florets of C. tinctorius L. were collected from Changji Hui
tribe autonomous prefecture of the Xinjiang Uygur Autonomous
Region of China in July 2005. The plant was identified by Professor
Lin Ma (Institute of Materia Medica, Peking Union Medical College,
and Chinese Academy of Medical Science, China). A voucher spec-
imen has been deposited in the Herbarium of the Department of
Medicinal Plants, Institute of Materia Medica, Chinese Academy
of Medical Sciences, Beijing 100050, People’s Republic of China.
diyne-1,12,14-triol-1-O-b-
D
-glucopyranoside (7):
½
a 2D0
ꢂ
ꢁ2.8 (c
0.08, MeOH); IR mmax 3333, 2928, 2889, 2204, 2130, 1616, 1567,
1419, 1338, 1158, 1076, 1045, 1022, 955, 613 cmꢁ1
;
1H NMR see
Table 2; 13C NMR see Table 3; (+)-HRESIMS m/z 419.1682 (calcd
for C20H28O8Na, 419.1676).(2E,8Z)-12R-Tetradecadiene-4,6-diyne-
1,12,14-triol-1-O-b-
MeOH); IR mmax 3367, 2930, 2205, 2129, 1603, 1417, 1365, 1309,
1160, 1076, 1045, 956, 623 cmꢁ1 1H NMR see Table 2; 13C NMR
D
-glucopyranoside (8):
½
a 2D0
ꢂ
ꢁ11.2 (c 0.11,
;
see Table 3; (+)-HRESIMS m/z 419.1673 (calcd for C20H28O8Na,
3.3. Extraction and isolation
419.1676).
The florets of C. tinctorius L. (7.0 kg) were extracted with H2O
under refluxed condition (3 ꢀ 2 h). The H2O extracts were then
concentrated under reduced pressure to give a residue (1.5 kg).
The residue was dissolved in H2O again, then chromatographed
over macroporous adsorbent resin (HP-20) column. After eluting
with H2O, the adsorbed constituents were eluted with 10% ethanol,
30% ethanol, and 50% ethanol, respectively. The 50% ethanol part
(110 g) was chromatographed over Sephadex LH-20 eluting with
H2O–MeOH (from 100:0 to 0:100) to give 126 fractions. Fr. 29
was further purified by reversed-phase preparative HPLC with
MeOH–H2O (45:55) as mobile phase to give 5 (35 mg), 9 (7 mg),
10 (50 mg), and 11 (5 mg). Fr. 43 was further purified by re-
versed-phase preparative HPLC with MeOH–H2O (40:60) as mobile
phase to give 6 (15 mg), 7 (16 mg), and 8 (11 mg). Fr. 20 and Fr. 21
were mixed together on the basis of HPLC-DAD analysis and sub-
jected to chromatography over Sephadex LH-20 with MeOH–H2O
(30:70) as mobile phase to yield 16 subfractions. Subfraction 4
was further purified by reversed-phase preparative HPLC with
MeOH–H2O (35:65) as mobile phase to afford 1 (31 mg) and 4
(25 mg). Subfraction 10 was further purified by reversed-phase
preparative HPLC with MeOH–H2O (40:60) as mobile phase to af-
ford 2 (9 mg) and 3 (12 mg).
3.5. Acid hydrolysis of 1
A solution of compound 1 (2 mg) in MeOH (1 mL) was treated
with 5% HCl (1 mL) at 85 °C for 4 h. After evaporation of MeOH,
the reaction mixtures were extracted with EtOAc (3 ꢀ 2 mL); Then
D-glucuronate and glucose were determined by the measurement
of optical rotation (gave positive optical rotation). The solvent sys-
tems CHCl3–MeOH–AcOH–H2O (14:6:2:1)16 were used for TLC
identification of glucuronate (Rf = 0.18) and glucose (Rf = 0.22).
3.6. Enzymatic hydrolysis of 2–8
Compound 5 (9.0 mg) was dissolved in 0.1 M acetate buffer
(3.0 mL). The reaction mixture was added to b-glucosidase
(9.0 mg, SIGMA, EC 3.2.1.21) and left at 38 °C until 5 was com-
pletely hydrolyzed (ca. 1 h). The reaction was monitored by
HPLC-DAD analysis on a C-18 column using MeOH-0.2% AcOH
(45:55) as mobile phase. The solution was then transferred to a li-
quid–liquid extractor and extracted with CHCl3. The aqueous frac-
tion was subjected to silica gel TLC [CHCl3–MeOH–H2O (7: 3: 1)] to
show the presence of D-glucose (Rf = 0.16). The organic layer was
evaporated in vacuo and then purified by reversed-phase prepara-
tive HPLC, using MeOH–H2O (45:55) as mobile phase to give 5a
(6 mg), which was identified by comparing with compound 9 on
HPLC-DAD analysis. The hydrolysis of 2–4 and 6–8 and sugar
identification were performed according to the procedure
described for 5.
3.4. Identification
(8Z)-Decaene-4,6-diyn-1-ol-1-O-b-
D
-glucuronyl-(100-20)-b-
ꢁ29.2 (c 0.07, MeOH); IR mmax 3336,
D-
glucopyranoside (1): ½a D20
ꢂ
2926, 2232, 1723, 1656, 1610, 1423, 1366, 1253, 1171, 1064,
719, 615 cmꢁ1
;
1H NMR see Table 1; 13C NMR see Table 3; (+)-
3.7. Determination of absolute configuration of 3–9
ESIMS m/z 509 [M+Na]+; (ꢁ)-ESIMS m/z 485 [MꢁH]ꢁ; (+)-HRESIMS
m/z 509.1629 (calcd for C22H30O12Na, 509.1630).(2E,8Z)-Decadi-
Following the reported procedure,11 the solid compound 9
(0.85 mg) was dissolved in a dry solution of the stock [Rh2(O-
COCF3)4] complex (3.50 mg) in chloroform (1 mL). The observed
sign of the diagnostic band at 350 nm in the ICD was correlated
to the absolute configuration of the chiral C-12 in 9. To analyze
3–8, firstly, enzymatic hydrolyzing of 3–8 affords their aglycones
3a–8a. Then the absolute configuration identification of 3–8 was
performed according to the procedure described for 9.
ene-4,6-diyne-1-ol-1-O-b-
0.07, MeOH); IR mmax 3385, 2925, 2881, 2206, 2130, 1575, 1418,
1366, 1159, 1074, 1045, 952, 615 cmꢁ1 1H NMR see Table 1; 13C
NMR see Table 3; (+)-HRESIMS m/z 331.1158 (calcd for
16H20O6Na, 331.1152).(2E,8E,10E)-12R-Tridecatriene-4,6-diyne-
1,12,13-triol-1-O-b- -glucopyranoside (3):
ꢁ22.8 (c 0.07,
MeOH); IR mmax 3337, 2928, 2880, 2200, 1628, 1589, 1410, 1362,
D-glucopyranoside (2):
½
a 2D0
ꢂ
ꢁ7.4 (c
;
C
D
½ ꢂ
a 2D0