6
X.-Y. Wang et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
over ODS CC using a MeOH-H2O (1:4–2:1) as eluent to give four sub-fractions
(Fr. 9.1.5.1–Fr. 9.1.5.4). Compound 9 (9 mg) and 10 (34 mg) were obtained
from Fr. 9.1.5.3 and Fr. 9.1.5.4, respectively, by purification using
semipreparative HPLC [MeOH-H2O (64:36), 7.5 mL/min, tR 27.7 min for 9;
MeOH–H2O (80:20), 8 mL/min, tR 16.6 min for 10]. Fr. 9.2 (9.5 g) was subjected
to ODS CC using a stepwise gradient [MeOH–H2O (1:4–4:1)] to afford eight
fractions (Fr. 9.2.1–Fr. 9.2.8). Fr. 9.2.2 (0.8 g), Fr. 9.2.3 (2.0 g) and Fr. 9.2.4
(1.2 g) were submitted to gel permeation chromatography on Sephadex LH-20
in MeOH to remove the pigments and carbohydrates. Fr. 9.2.2 was further
purified by semipreparative HPLC [MeOH-H2O (56:44), 6.5 mL/min] to give
decreased the resultant cytotoxic activity. However, the cytotoxic
activity of such saponins was very sensitive to their precise func-
tionalization, and more extensive studies are needed before a clear
structure-activity relationship can be reached. In addition, it is
worthy of mention that saponins 1–4, and 9 based on SA or OA
as the aglycone exhibited selective cytotoxicity against the
U87MG cells. To the best of our knowledge and after examination
of the literature data, bisdesmosidic oleanane-type saponins were
rarely cytotoxic.41–43 Works are in progress to elucidate the possi-
ble mechanism of action of this class of saponins.
compound
1 [12 mg, tR 12.3 min] and compound 2 [12 mg, tR 17.4 min].
Compounds 11 and 12 were obtained from Fr. 9.2.3 by semipreparative HPLC
[MeOH–H2O (59:41), 7.2 mL/min]: compound 11 [24 mg, tR 19.8 min] and
compound 12 [45 mg, tR 38.3 min]. Fr. 9.2.4 was purified by semipreparative
HPLC to yield compound
3 [45 mg, MeOH–H2O (65:35), 7.0 mL/min, tR
Acknowledgements
21.5 min]. Fr. 9.3 (8.3 g) was separated by ODS CC eluting with a gradient of
MeOH–H2O (1:10–3:1) to afford six fractions (Fr. 9.3.1–Fr. 9.3.6). Fr. 9.3.2
(1.2 g) and Fr. 9.3.3 (2.0 g) were further purified by semipreparative HPLC after
CC over Sephadex LH-20 (MeOH), to give compound 4 [23 mg, MeOH–H2O
(54:46), 6.0 mL/min, tR 22.7 min from Fr. 9.3.2] and compound 5 [55 mg,
MeOH-H2O (55:45), 6.0 mL/min, tR 31.4 min from Fr. 9.3.3].
This work was financially supported by the National Natural
Science Foundation of China (Nos. 30873402 and 81274029) and
the Administration of Traditional Chinese Medicine of Shaanxi
Province, PR China (No. zy36). The authors would like to thank
Prof. Ji-Tao Wang of Shaanxi University of Chinese Medicine for
identifying the plant materials and Mr. Min-Chang Wang of Nucle-
ar Magnetic Resonance Center, Xi’an Modern Chemistry Research
Institute, for the NMR measurements.
15. Compound 1: White amorphous powder; ½a D20
ꢂ
+9.1 (c 0.15, MeOH); for 1H and
13C NMR spectroscopic data, see Tables 1 and 2; key HMBC and NOESY
correlations, see Figure 2; ESIMS (pos. ion mode) m/z 1259 [M+Na]+; HRESIMS
(pos. ion mode) m/z 1259.6031 [M+Na]+ (calcd for C59H96NaO27, 1259.6037).
Supplementary data
Supplementary data associated with this article can be found, in
21. Acid Hydrolysis and GC Analysis: Compounds 1–5 (each 4 mg) in 5 mL of 1 M HCl
(dioxane–H2O 1: 1, v/v) were heated at 95 °C for 6 h, respectively. The reaction
mixture was evaporated in vacuo and the residue was extracted with CHCl3
three times. The aqueous phase was concentrated and dissolved in pyridine
(5 mL) and 1-(trimethylsilyl)-imidazole (0.5 mL) at room temperature for
30 min. The reaction mixture was dried with a stream of N2. The residue was
partitioned between CHCl3 and H2O. The organic layer was subjected to GC
References and notes
analysis using an L-Chirasil-Val column. The sugar units were identified by
comparing the retention times of the corresponding trimethylsilylated
derivatives with those of the authentic samples prepared in the same
manner.20 Retention times for authentic samples after being derivatized
were 9.02 and 9.93 min (
and 10.55 min ( -rhamnose), 9.66 and 10.49 min (
12.29 min ( -xylose), 11.25 and 12.32 min ( -xylose), 14.90 min (
and 15.08 min ( -glucose), respectively. -arabinose, -rhamnose and
were identified in a 1:1:3 ratio for 1, while the sugar moieties of 2–5 were
identified as -arabinose, -rhamnose, -xylose and -glucose in the ratio of
1:2:1:2 for 2, 1:2:1:3 for 3 and 4, and 1:2:1:4 for 5.
D
-arabinose), 9.10 and 10.06 min (
-rhamnose), 11.12 and
-glucose),
-glucose
L-arabinose), 9.71
D
L
D
L
D
L
L
L
D
L
L
D
D
23. Compound 2: White amorphous powder; ½a D22
ꢂ
ꢀ9.1 (c 0.18, MeOH); for 1H and
13C NMR spectroscopic data, see Tables 1 and 2; key HMBC and NOESY
correlations, see Fig. S1 in Supplementary data; ESIMS (pos. ion mode) m/z
1375 [M+Na]+; HRESIMS (pos. ion mode) m/z 1375.6514 [M+Na]+ (calcd for
C
64H104NaO30, 1375.6510).
24. Compound 3: White amorphous powder; [
a
] ½a 2D2
ꢂ
ꢀ12.6 (c 0.31, MeOH); for 1
H
and 13C NMR spectroscopic data, see Tables 1 and 2; key HMBC and NOESY
correlations, see Fig. S2 in -Supplementary data; ESIMS (pos. ion mode) m/z
1521 [M+Na]+, 1051 [1521–146–162–162]+, 949 [1521–132–146–162–132]+,
773 [1051–132–146]+; HRESIMS (pos. ion mode) m/z 1521.7081 [M+Na]+
(calcd for C70H114NaO34, 1521.7089).
14. Extraction and isolation: Anemone taipaiensis was collected on Taibai Mountain,
Shaanxi Province, China, in August 2009, and identified by Prof. Ji-Tao Wang
(Department of Pharmacognosy, School of Pharmacy, Shaanxi University of
Chinese Medicine). A voucher specimen (No. 090918) has been deposited in
the Herbarium of Shaanxi University of Chinese Medicine. The air-dried
rhizomes of A. taipaiensis (5 kg) were powdered and extracted three times
(each for 2 h) with 70% EtOH (5 L) under reflux. The extract was evaporated in
vacuo to yield a residue (650 g) which was suspended in water (8 L) and
partitioned successively with petroleum ether (8 L ꢁ 2) and n-BuOH (8 L ꢁ 3).
The n-BuOH extract (110 g) was separated by silica gel CC using a stepwise
gradient of CHCl3–MeOH–H2O (10:1:0.05–6:4:0.8) to give nine fractions (Fr. 1–
Fr. 9). Fr. 4 (7.6 g) was applied to silica gel CC and eluted with a CHCl3–MeOH–
H2O gradient (10:1:0.1–8:2:0.2) to afford eight sub-fractions (Fr. 4.1–Fr. 4.8).
Fr. 4.1 (1.2 g) was purified by semipreparative HPLC to give compound 6
26. Compound 4: White amorphous powder; [
a
ꢂ
+10.6 (c 0.22, MeOH); for 1
H
and 13C NMR spectroscopic data, see Tables 1 and 2; key HMBC and NOESY
correlations, see Fig. S3 in Supplementary data; ESIMS (pos. ion mode) m/z
1537 [M+Na]+, 1067 [1537–146–162–162]+, 789 [1067–132–146]+, 595
[132+146+162+132+Na]+; HRESIMS (pos. ion mode) m/z 1537.7042 [M+Na]+
(calcd for C70H114NaO35, 1537.7038).
27. Compound 5: White amorphous powder; ½a D22
ꢂ
ꢀ15.8 (c 0.25, MeOH); for 1H and
13C NMR spectroscopic data, see Tables 1 and 2; key HMBC and NOESY
correlations, see Fig. S4 in Supplementary data; ESIMS (pos. ion mode) m/z
1699 [M+Na]+, 1537 [1699–162]+, 1405 [1537–132]+, 1097 [1405–146–162]+,
1299 [1699–146–162–162]+; HRESIMS (pos. ion mode) m/z 1699.7562
[M+Na]+ (calcd for C76H124NaO40, 1699.7567).
[20 mg, MeOH–H2O (90:10), 8 mL/min, tR 12.5 min]. Fr.
9 (29.2 g) was
chromatographed on silica gel CC with a stepwise gradient of CHCl3–MeOH–
H2O gradient (8:2:0.2–6:4:0.5) to yield five fractions (Fr. 9.1–Fr. 9.5). Fr. 9.1
(8.0 g) was chromatographed on silica gel CC with a stepwise gradient of
CHCl3-n-BuOH (6:1–1:1) to yield five sub-fractions (Fr. 9.1.1–Fr. 9.1.5). Fr. 9.1.1
(1.5 g) and Fr. 9.1.4 (1.5 g) were submitted to gel permeation chromatography
on Sephadex LH-20 in MeOH to remove the pigments and carbohydrates, and
further purified by semipreparative HPLC to give compound 7 [8 mg, MeOH–
H2O (78:22), 8 mL/min, tR 24.0 min] and compound 8 [19 mg, MeOH–H2O
(87:13), 8 mL/min, tR 23.2 min], respectively. Fr. 9.1.5 (1.0 g) was fractionated