0.3 mL/min, capillary potential +30 kV (flavonoids) and –25 kV (ecdysteroids), field potential 3.0 kV, mass scan range
(m/z) 100–1,000. NMR spectra were recorded on a VXR 500S NMR spectrometer (Varian, Palo Alto, CA, USA).
Preparative (prep.) HPLC used a Summit liquid chromatograph (Dionex, Sunnyvale, CA, USA), LiChrospher RP-18 column
(250 × 10 mm, ∅ 10 μm, Supelco, Bellefonte, PA, USA), mobile phase H O (A) and MeCN (B) at flow rate (ν) 1 mL/min,
2
column temperature 30°C, and UV detector at λ = 250 and 330 nm. Analytical (anal.) HPLC used a MilichromA-02 microcolumn
liquid chromatograph (EcoNova, Novosibirsk, Russia) and ProntoSIL-120-5-C18AQ column (2 × 75 mm, ∅ 5 μm, Metrohm
AG, Herisau, Switzerland).
Extraction and Fractionation. Plant raw material was extracted and the Me CO fraction was produced as described
2
earlier [1]. The Me CO fraction (162 g) was separated by CC over polyamide (800 g) with elution by H O and then
2
2
EtOH–H O mixtures (20:80→40:60→60:40→80:20) and NH OH solution (0.5%) in EtOH (90%). This produced subfractions
2
4
A-2 (4 g), A-3 (32 g), A-4 (5 g), A-5 (2 g), and A-6 (29 g), respectively. Subfraction A-2 was chromatographed over polyamide
(CC, 1.5 × 30 cm, H O–EtOH eluent, 100:0→10:90) and Sephadex LH-20 (CC, 2 × 40 cm, EtOH–H O eluent, 90:10→0:100)
2
2
to isolate five compounds that were identified as lucenin-2 (9 mg, 9) [4], orientin (11 mg, 10) [5], isoorientin (15 mg, 11) [5],
vitexin (7 mg, 12) [6], and isovitexin (8 mg, 13) [6].
Subfraction A-3 was separated using CC over polyamide (2 × 40 cm, H O–EtOH eluent, 100:0→10:90), Sephadex
2
LH-20 (2 × 50 cm, EtOH–H O eluent, 90:10→0:100), SiO (2 × 40 cm, hexane–EtOAc eluent, 100:0→60:40), and RP-SiO
2
2
2
(1 × 30 cm, H O–MeCN eluent, 100:0→50:50) and prep. HPLC [gradient mode (%B): 0–90 min, 2–25%] to isolate
2
6,8-dihydroxyluteolin-7-O-glucoside (zeravschanoside, 5 mg, 14) [7], 6-hydroxyluteolin-7-O-rutinoside (11 mg, 15) [8],
6-hydroxyluteolin-7-O-glucoside (26 mg, 16) [9], nepetin-7-O-rutinoside (22 mg, 17) [10], nepetin-7-O-glucoside (nepitrin,
34 mg, 18) [11], luteolin-7-O-rutinoside (scolimoside, 14 mg, 19) [12], luteolin-7-O-glucoside (cynaroside, 16 mg, 20) [12],
6-hydroxykaempferol-7-O-glucoside (8 mg, 27) [18], 6-methoxykaempferol-7-O-glucoside (6 mg, 28) [19], 6-hydroxyquercetin-
7-O-glucoside (quercetagitrin, 7 mg, 29) [20], and 6-methoxyquercetin-7-O-glucoside (patulitrin, 9 mg, 30) [20].
Subfraction A-4 was chromatographed over SiO (1 × 45 cm, hexane–EtOAc eluent, 100:0→60:40), RP-SiO
2
2
(1 × 30 cm, H O–MeCN eluent, 100:0→30:70), and SiO (1 × 20 cm, EtOAc–Me CO eluent, 100:0→70:30) and by prep.
2
2
2
TLC (SiO , mobile phase CHCl –MeOH, 5:1→2:1) to afford 3 (12 mg), 6 (10 mg), apigenin-7-O-glucoside (cosmosiin,
2
3
8 mg, 21) [13], nepetin-4′-O-glucoside (22 mg, 22) [14], luteolin-4′-O-glucoside (8 mg, 23) [15], and luteolin-3′-O-glucoside
(5 mg, 24) [15].
Subfraction A-5 was separated over SiO (1 × 50 cm, hexane–EtOAc eluent, 100:0→80:20) and by prep. HPLC
2
[gradient mode (%B): 0–60 min, 50–100%] to give nine compounds including 6-hydroxyluteolin (5 mg, 36) [26], nepetin
(18 mg, 37) [26], 5,6,7,3′-tetrahydroxy-4′-methoxyflavone (5 mg, 38) [21], nodifloretin (5,6,7,4′-tetrahydroxy-3′-
methoxyflavone, 7 mg, 39) [27], luteolin (9 mg, 40) [26], hispidulin (3 mg, 41) [26], diosmetin (2 mg, 42) [26], chrysoeriol
(4 mg, 43) [26], and apigenin (2 mg, 44) [26].
Subfraction A-6 was separated over RP-SiO (2 × 50 cm, H O–MeCN eluent, 100:0→0:100) and SiO (1 × 45 cm,
2
2
2
EtOAc–Me CO, 100:0→50:50) and by prep. HPLC [gradient mode (%B): 0–20 min, 10–35%; 20–40 min, 35–40%;
2
40–60 min, 40–68%; 60–80 min, 68–100%] and prep. TLC [SiO , mobile phase EtOAc–1,2-C H Cl –AcOH–HCOOH
2
2
4
2
(85%)–H O, 10:2.5:1:1:0.8] to isolate 1 (14 mg), 2 (10 mg), 4 (21 mg), 5 (18 mg), 7 (10 mg), 8 (11 mg), luteolin-7-O-
2
glucuronide (19 mg, 25) [16], apigenin-7-O-glucuronide (33 mg, 26) [17], 6-hydroxyluteolin-7-O-(6″-O-caffeoyl)glucoside
(spicosideA, 15 mg, 31) [21], luteolin-7-O-(6″-O-caffeoyl)glucoside (18 mg, 32) [22], luteolin-7-O-(6″-O-cinnamoyl)glucoside
(24 mg, 33) [23], 6-hydroxykaempferol-7-O-(6″-O-caffeoyl)glucoside (11 mg, 34) [24], 6-hydroxyquercetin-7-O-(6″-O-
caffeoyl)glucoside (12 mg, 35) [25], 20-hydroxyecdysone-2-O-cinnamate (18 mg, 45) [28], and polypodine-2-O-cinnamate
(6 mg, 46) [28].
–1
Rhaunoside A (1). C H O . UV spectrum (ÌåÎÍ, λ , nm): 254, 288, 335. IR spectrum (ν, cm ): 3375, 1687,
30 26 15
max
–
2
3
1
1648, 1622, 1563. ESI-MS, m/z: 625 [M – H] ; [MS ] 625→463, 301; [MS ] 463→301. Í NMR spectrum (500 MHz, ÌåÎÍ-d ,
4
13
δ, ppm), see Table 1a, C NMR spectrum (125 MHz, ÌåÎÍ-d , δ, ppm), see Table 2a.
4
–1
Rhaunoside B (2). C H O . UV spectrum (ÌåÎÍ, λ , nm): 253, 283, 347. IR spectrum (ν, cm ): 3381, 1680,
30 26 13
max
–
2
3
1
1645, 1620, 1561. ESI-MS, m/z: 593 [M – H] ; [MS ] 593→463, 301; [MS ] 463→301. Í NMR spectrum (500 MHz,
13
ÌåÎÍ-d , δ, ppm), see Table 1a, C NMR spectrum (125 MHz, ÌåÎÍ-d , δ, ppm), see Table 2a.
4
4
–1
Rhaunoside C (3). C H O . UV spectrum (ÌåÎÍ, λ , nm): 287, 335. IR spectrum (ν, cm ): 3381, 1679,
21 20 12
–
max
2
1
1615. ESI-MS, m/z: 463 [M – H] ; [MS ] 463→301. Í NMR spectrum (500 MHz, ÌåÎÍ-d , δ, ppm), see Table 1a,
4
13
C NMR spectrum (125 MHz, ÌåÎÍ-d , δ, ppm), see Table 2a.
4
262