Isolation of 4 and 20–26 from S. dioica. The aerial part of S. dioica (320 g) was extracted (3×) with EtOH
(60%, 1:15) in an ultrasonic bath (100 W, 35 kHz) at 50°C (2 h). The obtained extract (86.4 g) was concentrated to dryness
under vacuum, suspended in H O (250 mL), and extracted with hexane, EtOAc, and BuOH. The EtOAc fraction (19.2 g) was
2
separated over polyamide for CC ( kg) with elution by H O and EtOH (20, 40, and 60%). The fraction eluted by 60% EtOH
2
was separated over Sephadex LH-20 (CC, 80 × 2 cm, MeOH–H O eluent, 90:10→0:100) and RP-SiO (CC, 40 × 2 cm,
2
2
H O–MeCN eluent, 70:30→20:80) and by prep. HPLC [isocratic mode (%B): 0–60 min, 65%] to give 4 (52 mg), divarioside
2
B (saponarin-2″-O-glucoside-6″″-O-feerulate, 46 mg, 22) [24], and saponarin-6″″-O-ferulate (17 mg, 23) [25]. The BuOH
fraction was separated analogously over polyamide, Sephadex LH-20, and RP-SiO and by prep. HPLC [gradient mode (%B):
2
0–30 min, 5–15%; 30–60 min, 15–40%; 60–80 min, 40–45%] to isolate 20 (9 mg), 21 (4 mg), isovitexin-7,2″-di-O-glucoside
(11 mg, 24) [26], isovitexin-7-O-xyloside-2″-O-glucoside (19 mg, 25) [4], and isovitexin-7-O-glucoside-2″-O-xyloside
(21 mg, 26) [4].
–1
Sileneside G (4). C H O . UV spectrum (ÌåÎÍ, λ , nm): 272, 327. IR spectrum (ν, cm ): 1652, 1717.
42 46 22
max
+
2
HR-ESI-MS, m/z: 903.4216 (calcd for C
H O , 903.7478). ESI-MS, m/z: 903 [M + H] ; MS [903]: 771
42 47 22
+
+
+
3
[(M + H) – C H O ] , 595 [(M + H) – C H O – C H O ] , 433 [(M + H) – C H O – C H O – C H O ] ; MS [433]:
5
8
4
5
8
4
10
8
3
5
8
4
10
8
3
6 10 5
+
+
343 [(M + H) – C H O – C H O – C H O – C H O ] , 315 [(M + H) – C H O – C H O – C H O – C H O – CO] ,
5
8
4
10
8
3
6
10
5
3
6
3
5
8
4
10
8
3
6
10
5
3 6 3
+
+
313 [(M + H) – C H O – C H O – C H O – C H O ] , 285 [(M + H) – C H O – C H O – C H O – C H O – CO] .
5
8
4
10
8
3
6
10
5
4
8
4
5
8
4
10
8
3
6
10
5
4 8 4
13
Table 2 lists the PMR spectrum (500 MHz, DMSO-d , δ, ppm). Table 3 lists the C NMR spectrum (125 MHz, DMSO-d , δ, ppm).
6
6
Total Hydrolysis. A weighed portion (5 mg) of compound was mixed with a mixture (5 mL) of H SO (30%) and
2
4
AcOH (30%) (1:1) thermostatted at 95°C for 10 h, neutralized with CaCO , and centrifuged. The supernatant was separated
3
over polyamide (5 g) with elution by H O (eluate 1) and MeOH (80%, eluate 2). Monosaccharides in eluate 1 were derivatized
2
with 3-methyl-1-phenyl-2-pyrazolin-5-one [31] and analyzed by anal. HPLC (conditions 1). Monosaccharides were assigned
to D- and L-series after reductive amination with L-tryptophan [32] using anal. HPLC (conditions 2). Non-carbohydrate
hydrolysis products (eluate 2) were analyzed by GC-MS [33] and NMR spectroscopy. Hydrolysis of 1 produced acacetin [34]
and D-glucose; of 2, apigenin [34], D-glucose, and L-arabinose; of 3, genkwanin [34], D-glucose, and L-arabinose; of 4,
apigenin, ferulic acid [27], D-glucose, and D-xylose.
Acid hydrolysis with TFA was performed in TFA (2 M) at 120°C. The hydrolysate was separated over polyamide
using the previously reported method [24]. The hydrolysis products of 1 contained isocytisoside (5) [6] and D-glucose;
of 2, schaftoside (17) [17] and D-glucose; of 3, genkwanin-6-C-β-D-glucopyranoside-8-C-α-L-arabinopyranoside (16) [3],
and D-glucose; of 4, isovitexin (15) [20], ferulic acid, D-glucose, and D-xylose.
Hydrolysis by β-glucosidase used β-glucosidase from Amygdalus (3.2.1.21, 30 U/mg, No. G4511; Sigma-Aldrich)
as described earlier [24]. The hydrolysis products were analyzed by HPLC, mass spectrometry, and NMR spectroscopy [24].
The hydrolysis products of 4 were 6-O-feruloylglucose [28] and isovitexin-2″-O-xyloside [29].
Hydrolysis of 4 by β-Xylosidase. A weighed portion of 4 (10 mg) was dissolved in DMSO (150 μL). The volume
was adjusted to 5 mL using sodium succinate solution (50 mM, pH 5.3). The mixture was treated with β-xylosidase (2 U) from
Selenomonas ruminantium (3.2.1.37, 115 U/mg; Megazyme Ltd., Bray, Ireland). The reaction mixture was incubated at 40°C
for 10 h, heated at 95°C (15 min), and centrifuged (6,000 rpm, 15 min). The hydrolysate was separated over polyamide (5 g)
with elution by H O (50 mL) and EtOH (60%, 100 mL). The EtOH eluate afforded saponarin-6″′-O-ferulate (4 mg, 23), which
2
13
was identified using C NMR spectroscopy and mass spectrometry [25].
Alkaline hydrolysis of 4 with NaOH used the previously reported method [24]. The hydrolysate of 4 contained
ferulic acid [27] and saponarin-2″-O-β-D-xylopyranoside (4a) according to GC-MS [33] and NMR spectroscopy.
Saponarin-2″-O-β-D-xylopyranoside (4a), C H O . UV spectrum (ÌåÎÍ, λ , nm): 271, 334. HR-ESI-MS,
32 38 19
max
+
2
+
m/z 727.7202 (calcd for C H O , 727.5924). ESI-MS, m/z: 727 [M + H] ; MS [727]: 595 [(M + H) – C H O ] , 433
32 39 19
5 8 4
+
3
+
[(M + H) – C H O – C H O ] ; MS [433]: 343 [(M + H) – C H O – C H O – C H O ] , 315 [(M + H) – C H O – C H O
5
8
4
6
10
5
5
8
4
6
10
5
3
6
3
5
8
4
6 10 5
+
+
+
– C H O – CO] , 313 [(M + H) – C H O – C H O – C H O ] , 285 [(M + H) – C H O – C H O – C H O – CO] .
3
6
3
5
8
4
6
10
5
4
8
4
5
8
4
6
10
5
4 8 4
1
Í NMR spectrum (500 MHz, DMSO-d , δ, ppm, J/Hz): apigenin – 6.74 (1H, s, H-3), 6.51 (1H, s, H-8), 7.90 (2H, d, J = 8.1,
6
H-2′, 6′), 6.99 (2H, d, J = 8.1, H-3′, 5′), 13.38 (1H, br.s, 5-OH), 10.29 (1H, br.s, 4′-OH); 6-Ñ-β-D-glucopyranose – 4.87 (1H,
d, J = 9.0, H-1′′), 4.41 (1H, m, H-2′′), 3.59 (1H, m, H-3′′), 3.17–3.25 (2H, m, H-4′′, 5′′), 3.79 (1H, dd, J = 3.0, 11.4, H -6′′),
A
3.37 (1H, m, H -6′′); 2′′-Î-β-D-xylopyranose – 4.96 (1H, d, J = 7.0, H-1′′′), 3.10 (1H, m, H-2′′′), 3.27–3.35 (2H, m, H-3′′′,
B
4′′′), 3.62 (1H, m, H -5′′′), 2.93 (1H, m, H -5′′′); 7-Î-β-D-glucopyranose – 5.09 (1H, d, J = 7.1, H-1′′′′), 3.39–3.57 (4H, d,
A
B
13
H-2′′′′–5′′′), 3.92 (1H, dd, J = 3.1, 11.6, H -6′′′′), 3.65 (1H, m, H -6′′′′). C NMR spectrum (125 MHz, DMSO-d , δ, ppm):
A
B
6
apigenin – 164.1 (C-2), 103.6 (C-3), 182.4 (C-4), 160.1 (C-5), 110.4 (C-6), 162.3 (C-7), 94.7 (C-8), 156.4 (C-9), 105.9 (C-10),
1032