Isolation of 1–21 from the BuOH Fraction of S. repens. The BuOH fraction from S. repens herb was obtained as
before [5] and then separated over polyamide (CC, 4 kg) with elution by H O (30 L) and EtOH (40%, 40 L; 80%, 20 L).
2
The fraction (154 g) obtained by elution with EtOH (40%) was separated over silica gel (CC, 40 × 20 cm, hexane–EtOAc
eluent, 90:10→50:50; EtOAc–Me CO, 100:0→50:50) and Sephadex LH-20 (CC, 90 × 2 cm, EtOH–H O eluent, 90:10→0:100)
2
2
and by preparative HPLC [gradient mode (%B): 0–60 min, 5–30%; 60–120 min, 30–65%] to isolate 2 (11 mg) [13], 3 (14 mg)
[
(
14], 4 (26 mg) [15], 5 (21 mg) [13], 6 (894 mg) [9], 7 (121 mg) [16], 8 (12 mg) [16], 9 (52 mg) {17], 10 (34 mg) [9], 11
27 mg) [18], 12 (9 mg) [19], 13 (19 mg) [20], and 14 (31 mg) [21]. The fraction (58 g) eluted over polyamide by EtOH (80%)
was separated by CC over silica gel (30 × 10 cm, hexane–EtOAc eluent, 90:10→50:50) and Sephadex LH-20 (90 × 2 cm,
EtOH–H O eluent, 90:10→50:50) and by preparative HPLC [gradient mode (%B): 0–60 min, 5–80%] to afford 1 (24 mg),
2
1
5 (11 mg) [22], 16 (18 mg) [18], 17 (21 mg) [23], 18 (31 mg) [24], 19 (25 mg) [25], 20 (26 mg) [26], and 21 (29 mg) [27].
Silenerepin (1). C H O . UV spectrum (60% EtOH, λ , nm): 274, 333; +AlCl 281, 353; +AlCl /HCl 281,
2
9
34 15
max
3
3
–
3
6
5
46; +NaOAc 274, 332; +NaOAc/H BO 273, 332. HR-ESI-MS, m/z 621.2307 [M – H] (calcd 621.5540); (+)ESI-MS, m/z:
3 3
+
+
–
–
–
23 [M + H] , 645 [M + Na] ; (–)ESI-MS, m/z: 621 [M – H] , 621→531 [(M – C H O ) – H] , 501 [(M – C H O ) – H] ,
3
6
3
4 8 4
–
–
–
–
31→441 [(M – C H O ) – H] , 411 [(M – C H O ) – H] , 501→411 [(M – C H O ) – H] , 381 [(M – C H O ) – H] .
3
6
3
4
8
4
3
6
3
4 8 4
1
3
Table 1 lists the PMR and C NMR spectra.
Hydrolysis of 1 in HCl–AcOH. A weighed portion of 1 (5 mg) was suspended in HCl–AcOH–H O (5 mL, 10:4:5),
2
heated at 110°C for 5 h, and neutralized by KOH (10%). The resulting solution was placed on polyamide (10 g) and eluted by
H O (100 mL, eluate 1) and EtOH (80%, 200 mL, eluate 2). Both eluates were concentrated to dryness. Eluate 1 was worked
2
up with 3-methyl-1-phenyl-2-pyrazolin-5-one [29] and analyzed by analytical HPLC (conditions 1). Monosaccharides were
not detected. Eluate 2 was analyzed by analytical HPLC (conditions 2). Compound 1 (t 16.53 min) was observed.
R
Oxidative Hydrolysis of 1 by FeCl . A weighed portion of 1 (5 mg) was mixed with FeCl (5%) in HCl (5%,
3
3
1
0 mL), heated at 90°C for 3 h, placed on polyamide (10 g), and worked up as above. Derivatization of eluate 1 with
L-tryptophan [30] detected D-glucose (t 12.52 min); eluate 2, 5-hydroxy-7,4′-dimethoxyflavone (t 26.41 min), which was
R
R
identified by UV spectroscopy and mass spectrometry and comparison with a known sample (≥98%, No. CFN98819, Wuhan
ChemFaces Biochemical Co., Ltd., Wuhan, Hubei, PRC).
Analytical HPLC. Conditions 1: mobile phase NH CH COO (100 mM, pH 4.5) (A) and MeCN (B); gradient mode
4
3
(
(
(
%B): 0–20 min, 20–26%; flow rate 150 μL/min; column temperature 35°C; UV detector at λ 250 nm. The retention time
t , min) of reference standard D-glucose was 12.52; L-glucose, 12.87. Conditions 2: mobile phase HCOOH (0.1%) in H O
A) and HCOOH (0.1%) in MeCN (B); gradient mode (%B): 0–10 min, 5–25%; 10–25 min, 25–50%; 25–35 min, 50–100%;
R
2
flow rate 200 μL/min; column temperature 30°C; UV detector at λ 330 nm. The retention time (t , min) of reference standard
R
vicenin-2 (7) was 14.11; silenerepin (1), 16.51; apigenin, 22.28; and 5-hydroxy-7,4′-dimethoxyflavone, 26.40.
ACKNOWLEDGMENT
The work was sponsored by FASO Russia (AAAA-AA17-117011810037-0).
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