Kaempferol 3-O-rhamnosyl-(1→4)-[rhamnosyl-(1→6)-galactoside] (F1)

HPLC (tR): 24.7 min. The authentic sample used for qualitative HPLC analysis was isolated from leaves of soybean cultivars (Murai et al., 2013). UV λmax nm (MeOH): 266, 348. +NaOMe: 274, 325, 394 (inc.). +AlCl3: 274, 304, 354, 396. +AlCl3/HCl: 275, 303, 350, 391. +NaOAc: 274, 312, 382. +NaOAc/H3BO3: 267, 297sh, 352. Kaempferol, rhamnose and galactose were obtained by acid hydrolysis. LC-ESI-MS: m/z 763 [M+Na]+, 739 [M-H]- (kaempferol + 1 mol galactose and 2 mol rhamnose), m/z 595 [M-146+H] + (kaempferol + each 1 mol rhamnose and galactose), m/z 449 [M-292+H] + (kaempferol + 1 mol galactose), m/z 287 [M-454+H] + (kaempferol). 1H NMR (600 MHz, pyridine-d5): δ 13.44 (1H, s, 5-OH), 8.58 (2H, d, J = 8.8 Hz, H-2’, 6’), 7.29 (2H, d, J = 8.8 Hz, H-3’, 5’), 6.80 (1H, d, J = 2.0 Hz, H-6), 6.75 (1H, d, J = 1.9 Hz, H-8), 6.47 (1H, d, J = 7.8 Hz, galactosyl H-1), 6.35 (1H, s, 4’’-rhamnosyl H-1), 5.15 (1H, s, 6’’-rhamnosyl H-1), 1.56 (3H, d, J = 6.2 Hz, 6’’-rhamnosyl CH3), 1.44 (3H, d, J = 5.6 Hz, 4’’-rhamnosyl CH3). 13C NMR (150 MHz, pyridine-d5): (kaempferol) δ 157.3 (C-2), 134.1 (C-3), 178.6 (C-4), 162.9 (C-5), 99.7 (C-6), 165.7 (C-7), 94.5 (C-8), 157.5 (C-9), 105.4 (C-10), 122.1 (C-1’), 131.8 (C-2’, 6’), 116.2 (C-3’, 5’), 161.5 (C-4’); (galactose) δ 100.7 (C-1), 69.9 (C-2), 74.7 (C-3), 76.6 (C-4), 75.8 (C-5), 66.1 (C-6); (4’’-rhamnose) δ 102.5 (C-1), 72.5 (C-2), 72.8 (C-3), 74.1 (C-4), 70.0 (C-5), 18.5 (C-6); (6’’-rhamnose) δ 101.8 (C-1), 72.0 (C-2), 72.7 (C-3), 73.8 (C-4), 69.6 (C-5), 18.3 (C-6). Sugar-sugar linkage of F1 was also determined by 2D NMR.

Kaempferol 3-O-rhamnosyl-(1→2)-[rhamnosyl-(1→6)-glucoside] (F2)

HPLC (tR): 25.1 min. The authentic sample used for qualitative HPLC analysis was isolated from flowers of tulip cultivars (Murai et al., unpublished data). UV λmax nm (MeOH): 266, 285sh, 345. +NaOMe: 274, 325, 392 (inc.). +AlCl3: 275, 305, 352, 390. +AlCl3/HCl: 275, 303, 348, 390sh. +NaOAc: 274, 309, 379. +NaOAc/H3BO3: 267, 286sh, 348. Kaempferol, rhamnose and glucose were obtained by acid hydrolysis. LC-ESI-MS: m/z 763 [M+Na]+, 739 [M-H]- (kaempferol + 1 mol galactose and 2 mol rhamnose), m/z 595 [M-146+H] + (kaempferol + each 1 mol rhamnose and galactose), m/z 449 [M-292+H] + (kaempferol + 1 mol galactose), m/z 287 [M-454+H] + (kaempferol). 1H NMR (600 MHz, pyridine-d5): δ 13.40 (1H, s, 5-OH), 8.52 (2H, d, J = 8.8 Hz, H-2’, 6’), 7.38 (2H, d, J = 8.8 Hz, H-3’, 5’), 6.74 (1H, d, J = 2.0 Hz, H-8), 6.71 (1H, d, J = 2.1 Hz, H-6), 6.46 (1H, s, 2’’-rhamnosyl H-1), 6.40 (1H, d, J = 7.9 Hz, glucosyl H-1), 5.23 (1H, s, 6’’-rhamnosyl H-1), 1.63 (3H, d, J = 6.2 Hz, 2’’-rhamnosyl CH3), 1.44 (3H, d, J = 5.7 Hz, 6’’-rhamnosyl CH3). 13C NMR (150 MHz, pyridine-d5): (kaempferol) δ 157.6 (C-2), 134.1 (C-3), 178.5 (C-4), 162.9 (C-5), 99.6 (C-6), 165.7 (C-7), 94.5 (C-8), 158.0 (C-9), 105.5 (C-10), 122.4 (C-1’), 131.8 (C-2’, 6’), 116.2 (C-3’, 5’), 161.5 (C-4’); (glucose) δ 100.6 (C-1), 77.2 (C-2), 79.1 (C-3), 71.7 (C-4), 78.4 (C-5), 68.0 (C-6); (2’’-rhamnose) δ 102.5 (C-1), 72.5 (C-2), 72.7 (C-3), 74.2 (C-4), 70.0 (C-5), 18.4 (C-6); (6’’-rhamnose) δ 102.4 (C-1), 72.0 (C-2), 72.6 (C-3), 73.8 (C-4), 69.6 (C-5), 18.5 (C-6). Sugar-sugar linkage of F2 was also determined by 2D NMR.

Kaempferol 3-O-rhamnosyl-(1→6)-galactoside (F3)

HPLC (tR): 27.7 min. The authentic sample used for qualitative HPLC analysis was isolated from leaves of soybean cultivars (Murai et al., 2013). UV λmax nm (MeOH): 266, 348. +NaOMe: 275, 323, 399 (inc.). +AlCl3: 274, 304, 354, 393. +AlCl3/HCl: 273, 302, 348, 389. +NaOAc: 274, 315, 394. +NaOAc/H3BO3: 266, 301, 354. Kaempferol, rhamnose and galactose were obtained by acid hydrolysis. LC-ESI-MS: m/z 471 [M+Na]+, 447 [M-H]- (kaempferol + 1 mol glucose), m/z 287 [M-162+H]+ (kaempferol).

Kaempferol 3-O-rhamnosyl-(1→6)-glucoside (F4)

HPLC (tR): 28.8 min. The authentic sample used for qualitative HPLC analysis was isolated from leaves of soybean cultivars (Murai et al., 2013). UV λmax nm (MeOH): 266, 348. +NaOMe: 275, 323, 399 (inc.). +AlCl3: 274, 304, 354, 393. +AlCl3/HCl: 273, 302, 348, 389. +NaOAc: 274, 315, 394. +NaOAc/H3BO3: 266, 301, 354. Kaempferol, rhamnose and glucose were obtained by acid hydrolysis. LC-ESI-MS: m/z 471 [M+Na]+, 447 [M-H]- (kaempferol + 1 mol glucose), m/z 287 [M-162+H]+ (kaempferol).

Kaempferol 3-O-glucoside (F5)

HPLC (tR): 30.0 min. The authentic sample used for qualitative HPLC analysis was isolated from fronds of Cyrtomium species (Iwashina et al., 2006). UV λmax nm (MeOH): 266, 297sh, 348. +NaOMe: 275, 324, 398 (inc.). +AlCl3: 274, 303, 348, 393. +AlCl3/HCl: 274, 302, 346, 392. +NaOAc: 275, 312, 389. +NaOAc/H3BO3: 267, 296sh, 354. Kaempferol and glucose were obtained by acid hydrolysis. LC-ESI-MS: m/z 471 [M+Na]+, 447 [M-H]- (kaempferol + 1 mol glucose), m/z 287 [M-162+H]+ (kaempferol).

Kaempferol 3-O-rhamnosyl-(1→2)-galactoside (F6)

HPLC (tR): 26.4 min. UV λmax nm (MeOH): 266, 287, 346. +NaOMe: 275, 323, 393 (inc.). +AlCl3: 273, 304, 352, 393. +AlCl3/HCl: 274, 301, 346, 390. +NaOAc: 273, 305, 382. +NaOAc/H3BO3: 266, 285sh, 350. Kaempferol, rhamnose and galactose were obtained by acid hydrolysis. LC-ESI-MS: m/z 617 [M+Na]+, 593 [M-H]- (kaempferol + each 1 mol galactose and rhamnose), m/z 449 [M-146+H]+ (kaempferol + 1 mol galactose), m/z 287 [M-308+H]+ (kaempferol). 1H NMR (600 MHz, pyridine-d5): δ 13.41 (1H, s, 5-OH), 8.57 (2H, d, J = 8.8 Hz, H-2’, 6’), 7.29 (2H, d, J = 8.8 Hz, H-3’, 5’), 6.71 (1H, d, J = 2.0 Hz, H-6), 6.71 (1H, d, J = 1.9 Hz, H-8), 6.59 (1H, d, J = 7.7 Hz, galactosyl H-1), 6.33 (1H, s, 2’’-rhamnosyl H-1), 1.52 (3H, d, J = 6.2 Hz, 2’’-rhamnosyl CH3). 13C NMR (150 MHz, pyridine-d5): (kaempferol) δ 157.0 (C-2), 134.2 (C-3), 178.6 (C-4), 162.9 (C-5), 99.7 (C-6), 165.7 (C-7), 94.4 (C-8), 157.4 (C-9), 105.4 (C-10), 122.2 (C-1’), 131.8 (C-2’, 6’), 116.2 (C-3’, 5’), 161.5 (C-4’); (galactose) δ 100.6 (C-1), 77.2 (C-2), 76.0 (C-3), 70.3 (C-4), 76.9 (C-5), 61.6 (C-6); (2’’-rhamnose) δ 102.5 (C-1), 72.7 (C-2), 72.9 (C-3), 74.2 (C-4), 69.3 (C-5), 18.3 (C-6). Sugar-sugar linkage of F6 was also determined by 2D NMR.

Kaempferol 3-O-rhamnosyl-(1→2)-glucoside (F7)

HPLC (tR): 26.9 min. The authentic sample used for qualitative HPLC analysis was isolated from flowers of carnation cultivars (Iwashina et al., 2010). UV λmax nm (MeOH): 266, 287, 346. +NaOMe: 275, 323, 393 (inc.). +AlCl3: 273, 304, 352, 393. +AlCl3/HCl: 274, 301, 346, 390. +NaOAc: 273, 305, 382. +NaOAc/H3BO3: 266, 285sh, 350. Kaempferol, rhamnose and glucose were obtained by acid hydrolysis. LC-ESI-MS: m/z 617 [M+Na]+, 593 [M-H]- (kaempferol + each 1 mol glucose and rhamnose), m/z 449 [M-146+H]+ (kaempferol + 1 mol glucose), m/z 287 [M-308+H]+ (kaempferol).

Kaempferol 3-O-galactoside (F8)

HPLC (tR): 28.9 min. The authentic sample used for qualitative HPLC analysis was isolated from fronds of Cyrtomium species (Iwashina et al., 2006). UV λmax nm (MeOH): 266, 297sh, 348. +NaOMe: 275, 323, 398 (inc.). +AlCl3: 274, 304, 353, 392. +AlCl3/HCl: 275, 302, 346, 392. +NaOAc: 274, 311, 390. +NaOAc/H3BO3: 266, 296sh, 352. Kaempferol and galactose were obtained by acid hydrolysis. LC-ESI-MS: m/z 471 [M+Na]+, 447 [M-H]- (kaempferol + 1 mol galactose), m/z 287 [M-162+H]+ (kaempferol).