The BuOH fraction (3.5 g) was separated over polyamide, Sephadex LH-20, and RP-SiO as above and by prep.
2
HPLC [gradient mode (%B): 0–20 min, 5–23%; 20–45 min, 23–34%; 45–90 min, 34–52%] to isolate 1 (195 mg), 2 (173 mg),
isorhamnetin-3-O-(2″-O-rhamnosyl-6″-O-glycosyl)-glucoside (14 mg, 10) [5], kaempferol-3-O-neohesperidoside-7-O-glucoside
(
22 mg, 11) [4], quercetin-3-O-(2″-O-rhamnosyl-6″-O-glucosyl)-glucoside (11 mg, 12) [9], quercetin-3-O-neohesperidoside-
7
-O-glucoside (56 mg, 13) [10], and quercetin-3-O-gentiobioside-7-O-glucuronide (12 mg, 14) [7].
Quercetin-3-O-neohesperidoside-7-O-glucuronide (1). C H O . UV spectrum (70% ÌåÎÍ, λmax, nm): 255,
3
3 38 22
2
67 sh, 352; +NaOMe 270, 395; + NaOAc 264, 329 sh, 397; +AlCl 272, 297 sh, 437; +AlCl /HCl 268, 295 sh, 360 sh, 403.
3
3
+
+
HR-ESI-MS, m/z 787.4271 (calcd for C H O , 787.5990 [M + H] ). ESI-MS, m/z (%): 787 [M + H] (2), 641
3
3 39 22
+
+
+
[
(M + H) – C H O ] (40), 479 [(M + H) – C H O – C H O ] (100), 303 [(M + H) – C H O – C H O – C H O ]
6 10 4 6 10 4 6 10 5 6 10 4 6 10 5 6 8 6
1
3
(
4). Table 1 gives the PMR spectrum (500 MHz, MeOH-d , 298 K, δ, ppm) and C NMR spectrum (125 MHz, MeOH-d4,
4
2
98 K, δ, ppm).
Kaempferol-3-O-neohesperidoside-7-O-glucuronide (2). C H O . UV spectrum (70% ÌåÎÍ, λmax, nm): 265,
3
3 38 21
3
47; +NaOMe 242, 270, 345 sh, 385; + NaOAc 265, 379; +AlCl 272, 301 sh, 352, 401; +AlCl /HCl 272, 300 sh, 348, 401.
3
3
+
+
HR-ESI-MS, m/z: 771.5742 (calcd for C H O , 771.6000 [M + H] ). ESI-MS, m/z (%): 771 [M + H] (20), 625
3
3 39 21
+
+
+
[
(M + H) – C H O ] (22), 463 [(M + H) – C H O – C H O ] (100), 287 [(M + H) – C H O – C H O – C H O ]
6 10 4 6 10 4 6 10 5 6 10 4 6 10 5 6 8 6
1
3
(
5). Table 1 gives the PMR spectrum (500 MHz, MeOH-d , 298 K, δ, ppm) and C NMR spectrum (125 MHz, MeOH-d4,
4
2
98 K, δ, ppm).
Total hydrolysis was performed in TFA (2 M) followed by separation of the reaction mixture over polyamide [16]
and analysis of the hydrolysis products by GC-MS (aglycons) [17], derivatization with 3-methyl-1-phenyl-2-pyrazolin-5-one
and reductive amination with L-tryptophan [18] (monosaccharides of the D/L-series) [19]. The hydrolysis products of 1
included quercetin [20], D-glucose, L-rhamnose, and D-glucuronic acid; of 2, kaempferol [20], D-glucose, L-rhamnose, and
D-glucuronic acid.
Hydrolysis with β-glucosidase and α-rhamnosidase was conducted as described earlier [16]. Hydrolysis by
β-glucosidase did not change 1 and 2. The products from hydrolysis of 1 by α-rhamnosidase consisted of quercetin-3-O-
glucoside-7-O-glucuronide (8) [7]; of 2, kaempferol-3-O-glucoside-7-O-glucuronide (5) [7], which were identified using UV
and NMR spectroscopic and mass spectrometric data.
Quercetin-3-O-glucoside-7-O-glucuronide (8). C H O . UV spectrum (70% ÌåÎÍ, λmax, nm): 256, 268 sh,
2
7 28 18
3
51; +NaOMe 271, 396; + NaOAc 263, 330 sh, 399; +AlCl 271, 298 sh, 435; +AlCl /HCl 270, 296 sh, 361 sh, 401.
3 3
+
+
HR-ESI-MS, m/z: 641.0231 (calcd for C H O , 641.4674 [M + H] ). ESI-MS, m/z (%): 641 [M + H] (12), 479
2
7 29 18
+
+
1
[
(M + H) – C H O ] (100), 303 [(M + H) – C H O – C H O ] (18). Í NMR spectrum (500 MHz, MeOH-d , 298 K, δ,
6 10 5 6 10 5 6 8 6 4
ppm, J/Hz)*: 6.45 (1H, d, J = 2.1, H-6), 6.78 (1H, d, J = 2.1, H-8), 7.91 (1H, d, J = 2.1, H-2′), 6.89 (1H, d, J = 9.0, H-5′), 7.50
1H, dd, J = 2.1, 9.0, H-6′), 12.53 (1H, br.s, 5-OH)**, 9.55 (1H, br.s, 3′-OH)**, 9.70 (1H, br.s, 4′-OH)**; 3-Î-β-D-glucopyranose
5.31 (1H, d, J = 7.2, H-1′′), 3.70 (1H, m, H-2′′), 3.55 (1H, m, H-3′′), 3.48 (1H, m, H-4′′), 3.61 (1H, m, H-5′′), 3.90 (1H, dd,
J = 2.1, 12.0, H -6′′), 3.79 (1H, m, H -6′′); 7-Î-β-D-glucuronopyranose – 5.10 (1H, d, J = 7.5, H-1′′′), 3.52 (1H, m, H-2′′′),
(
–
A
B
1
3
3
.38 (1H, m, H-3′′′), 3.45 (1H, m, H-4′′′), 4.08 (1H, d, J = 9.0, H-5′′′). C NMR spectrum (125 MHz, MeOH-d , 298 K, δ,
4
ppm): 157.6 (C-2), 133.3 (C-3), 177.5 (C-4), 160.1 (C-5), 99.3 (C-6), 162.9 (C-7), 94.6 (C-8), 156.2 (C-9), 105.7 (C-10),
20.4 (C-1′), 115.2 (C-2′), 144.0 (C-3′), 148.7 (C-4′), 116.1 (C-5′), 121.4 (C-6′); 3-Î-β-D-glucopyranose – 102.6 (C-1′′), 74.2
1
(
(
C-2′′), 78.1 (C-3′′), 70.4 (C-4′′), 77.7 (C-5′′), 60.5 (C-6′′); 7-Î-β-D-glucuronopyranose – 99.7 (C-1′′′), 73.2 (C-2′′′), 75.6
C-3′′′), 71.4 (C-4′′′), 75.2 (C-5′′′), 171.1 (C-6′′′).
Kaempferol-3-O-glucoside-7-O-glucuronide (5). C H O . UV spectrum (70% ÌåÎÍ, λmax, nm): 266, 345;
2
7 28 17
+
NaOMe 241, 272, 341 sh, 381; + NaOAc 266, 375; +AlCl 272, 300 sh, 351, 400; +AlCl /HCl 272, 301 sh, 347, 400.
3 3
+
+
HR-ESI-MS, m/z: 625.0967 (calcd for C H O , 625.4684 [M + H] ). ESI-MS, m/z (%): 625 [M + H] (33), 463
2
7 29 17
+
+
[
(M + H) – C H O ] (100), 287 [(M + H) – C H O – C H O ] (12).
6 10 5 6 10 5 6 8 6
Oxidation by H O . A weighed portion (5 mg) of a compound was suspended in a mixture (10 mL) of H O
2 2
2
2
(
40%)–NH (25%)–H O (75:14:11), incubated at 25°C for 48 h, treated with catalase (100 U, 1.11.1.6, ≥10,000 U/mg,
3
2
®
+
No. C40; Sigma-Aldrich), and passed after 2 h through Amberlyst 15 (H -form, Sigma-Aldrich) that was eluted with H O
2
(
100 mL). The aqueous eluate was concentrated and analyzed by HPLC on a Milichrom A-02 chromatograph (EcoNova,
Novosibirsk, Russia) equipped with a Separon SGX NH column (2 × 75 mm, ∅ 5 μm; Tessek Ltd., Prague, Czech Rep.) in
2
isocratic mode (MeCN–H O, 75:25, flow rate 100 μL/min) at column temperature 30°C with UV detection (λ 190 nm).
2
_
_____
∗
Compound 8 was previously characterized only from hydrolysis results and UV spectroscopy [7], PMR and 13C NMR
spectroscopic data are given for the first time; data obtained from DMSO-d solutions.
*
*
6
1
038