separated over polyamide (CC, 10 ꢅ 40 cm) with elution by H O (fraction E-01, 30.7 g), EtOH (40%, E-02, 10.7 g), EtOH
2
(90%, E-03, 2.4 g), NH (0.1% in 90% EtOH, E-04, 26.1 g), and NH (0.5% in 90% EtOH, E-05, 46.7 g). Fraction E-02 (10 g)
3
3
was separated using flash chromatography over SiO (CC, 6 ꢅ 50 cm) and a hexane–EtOAc gradient (100:0ꢄ0:100) to
2
produce subfractions E-02/01–E-02/10. Subfractions E-02/04, E-02/05–E-02/06, E-02/08, and E-02/09 were rechromatographed
over RP-SiO (CC, 4 ꢅ 40 cm, H O–MeCN eluent, 100:0ꢄ0:100) to isolate four compounds that were identified as quercetagetrin
2
2
(quercetagetin-7-O-glucoside, 36 mg, 7), patulitrin (patuletin-7-O-glucoside, 12 mg, 8) [15], quercimeritrin (quercetin-7-O-
glucoside, 10 mg, 6) [16], and spinacetin-7-O-glucoside (18 mg, 11). CC of fraction E-03 (2 g) over RP-SiO (4 ꢅ 40 cm,
2
H O–MeCN eluent, 100:0ꢄ0:100) and SiO (5 ꢅ 30 cm, hexane–Me CO eluent, 100:0ꢄ80:20) isolated nepetin-7-O-glucoside
2
2
2
(eupafolin-7-O-glucoside, 11 mg, 2) [3] and jaceosidin-7-O-glucoside (16 mg, 3) [2]. Fraction E-04 (22 g) was separated by
CC over RP-SiO (4 ꢅ 60 cm, H O–MeCN eluent, 100:0ꢄ0:100) and Sephadex LH-20 (4 ꢅ 60 cm, MeOH–H O eluent,
2
2
2
100:0ꢄ0:100) and by preparative HPLC (conditions 1) to produce five compounds including quercetagetin-7-O-(6ꢀ-O-
caffeoyl)glucoside (27 mg, 9) [15], tinctoside [patuletin-7-O-(6ꢀ-O-caffeoyl)glucoside, 39 mg, 10] [17], gnaphaloside A
[jaceosidin-7-O-(6ꢀ-O-caffeoyl)glucoside, 26 mg, 4] [2, 3], gnaphaloside B [nepetin-7-O-(6ꢀ-O-caffeoyl)glucoside, 79 mg, 5]
[2], and 1 (39 mg). Fraction E-05 was chromatographed over Sephadex LH-20 (4 ꢅ 80 cm, MeOH–H O eluent, 100:0ꢄ0:100)
2
and by preparative HPLC (conditions 2) to produce 11 compounds that were identified as caffeic acid (10 mg, 12);
3-O- (102 mg, 13), 4-O- (24 mg, 14), 1,3-di-O- (16 mg, 15), 1,5-di-O- (27 mg, 16), 3.4-di-O- (14 mg, 17), 3,5-di-O- (114 mg,
18), 4,5-di-O- (29 mg, 19), 1,3,5-tri-O- (11 mg, 20), 3,4,5-tri-O- (57 mg, 21) caffeoylquinic acids [5, 18]; and leontopodic acid
(8 mg, 22) [19].
The compositions of another two samples of the aerial part of G. uliginosum were studied analogously. Sample B-02
(2.5 kg) yielded 1 (29 mg), 2 (10 mg), 4 (31 mg), 5 (85 mg), 8 (14 mg), 10 (35 mg), 12 (19 mg), 13 (86 mg), 14 (22 mg), 15
(15 mg), 16 (26 mg), 17 (14 mg), 18 (127 mg), 19 (26 mg), 20 (9 mg), 21 (61 mg), 22 (10 mg), cosmosiin (apigenin-7-O-
glucoside, 7 mg, 23) [20], plantaginin (scutellarein-7-O-glucoside, 11 mg, 24) [21], and isoquercitrin (quercetin-3-O-glucoside,
5 mg, 25) [22]. Sample C-03 (0.9 kg) produced 4 (18 mg), 5 (10 mg), 6 (5 mg), 12 (5 mg), 13 (22 mg), 15 (10 mg), 16 (9 mg),
17 (5 mg), 18 (29 mg), 19 (11 mg), 20 (4 mg), 21 (9 mg), 22 (4 mg), and 24 (9 mg).
–
Gnaphaloside C (1). C H O . HR-ESI-MS, m/z 669.601 [M – H] ; calcd 669.578. ESI-MS/MS, m/z: 669
32 30 16
–
–
–
–
–
[M – H] , 507 [M – H – caffeoyl] , 345 [M – H– caffeoyl-glucose] , 323 [caffeoyl-glucose – H] , 179 [caffeic acid – H] .
UV spectrum (EtÎÍ, ꢈ , nm): 254, 270 sh, 331, 370; +AlCl 265, 287 sh, 335, 427; +AlCl /HCl 263, 288 sh, 334, 425;
max
3
3
1
+NaOAc 255, 333, 376; +NaOAc/H BO 255, 330, 370; +NaOMe 269, 338 sh, 397. Í NMR spectrum (500 MHz,
ÌåÎÍ-d , ꢃ, ppm) see Table 1, C NMR spectrum (125 MHz, ÌåÎÍ-d , ꢃ, ppm) see Table 1.
3
3
13
4
4
Acid Hydrolysis of 1. Compound 1 (5 mg) was dissolved in TFA (10 mL, 5%) and heated to 110°C (2 h).
The hydrolysate was concentrated in vacuo. The residue was dissolved in MeOH. The resulting hydrolysate was
chromatographed over polyamide (CC, 30 g) with elution by H O (100 mL, eluate 1), EtOH (90%, 250 mL, eluate 2), and NH
2
3
(0.5% in 90% EtOH, 150 mL, eluate 3). HPLC of eluate 1 detected glucose (conditions 3, t 14.10 min); of eluate 3, caffeic
R
acid (conditions 4, t 7.22 min). Eluate 2 was separated by preparative HPLC (conditions 1) to isolate spinacetin (2 mg) that
R
13
was identified using MS and C NMR spectroscopy.
–
13
Spinacetin. C H O . HR-ESI-MS, m/z345.295 [M – H] ; calcd 345.288. C NMR spectrum (125 MHz, ÌåÎÍ-d ,
17 14
8
4
ꢃ, ppm, DEPT): 55.9 (CH , 3ꢂ-OCH ), 60.1 (CH , 6-OCH ), 92.4 (ÑH, C-8), 103.9 (C, C-10), 114.0 (CH, C-5ꢂ), 114.8 (CH,
3
3
3
3
C-2ꢂ), 120.9 (CH, C-6ꢂ), 121.3 (C, C-1ꢂ), 130.5 (Ñ, Ñ-6), 135.8 (Ñ, Ñ-3), 145.4 (C, C-3ꢂ), 147.3 (C, C-4ꢂ), 148.2 (Ñ, Ñ-5), 148.9
(Ñ, Ñ-2), 154.7 (Ñ, Ñ-7), 156.4 (C, C-9), 178.0 (Ñ, Ñ-4) [23].
Alkaline Hydrolysis of 1. Compound 1 (5 mg) was dissolved in NaOH solution (1 mL, 0.4%) and heated at 50°C
(30 min). The hydrolysate was neutralized with HCl (0.4%) and chromatographed over polyamide (CC, 40 g) with elution by
H O (150 mL, eluate 1), EtOH (60%, 300 mL, eluate 2), and NH (0.5% in 90% EtOH, 200 mL, eluate 3). HPLC of eluate
2
3
3 detected caffeic acid (conditions 4, t 7.22 min). Eluate 2 was separated by preparative HPLC (conditions 1) to produce
R
13
spinacetin-7-O-ꢁ-D-glucopyranoside (2.5 mg) that was identified using UV, MS, PMR , and C NMR spectroscopy.
–
Spinacetin-7-O-ꢁ-D-glucopyranoside (11). C H O . HR-ESI-MS, m/z 507.422 [M – H] ; calcd 507.432.
23 24 13
–
–
–
ESI-MS/MS, m/z: 507 [M – H] , 345 [M – H – glucose] , 330 [M – H – glucose – CH ] . UV spectrum (EtÎÍ, ꢈ , nm): 255,
3
max
270 sh, 369; +AlCl 264, 285 sh, 425; +AlCl /HCl 265, 287 sh, 424; +NaOAc 256, 375; +NaOAc/H BO 255, 371; +NaOMe
3
3
3
3
1
13
270, 395. Í NMR spectrum (500 MHz, ÌåÎÍ-d , ꢃ, ppm) and C NMR spectrum (125 MHz, ÌåÎÍ-d , ꢃ, ppm) see Table 1.
4
4
HPLC. Conditions 1: preparative HPLC, mobile phase H O (A) and MeCN (B), gradient mode (%B) 0–90 min
2
40–100%, flow rate 2 mL/min, column temperature 30°C, UV detector at 270 nm. Conditions 2: preparative HPLC, mobile
phase H O (A) and MeCN (B), gradient mode (%B) 0–100 min 0–70%, flow rate 2 mL/min, column temperature 30°C,
2
1089