6-hydroxykaempferol and its glycosides from Carthamus tinctorius petals

Phytochemisrry, Vol. 31,No. 11,pp.400-4004,1992 Printedin GreatBritain.

6-HYDROXYKAEMPFEROL CARTHAMUS

0031 9422/92 $5.00 + 0.00 Q 1992PergamonPressLtd

AND ITS GLYCOSIDES TINCTORIUS PETALS

FROM

MASAO HATTORI, XIN-LI HUANG, QING-MING CHE, YUKIO KAWATA, YASUHIRO TEZUKA, TOHRU KIKUCHI and TSUNEO NAMBA

Research Institute for Wakan-Yaku (Traditional Sino-Japanese Medicines), Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan (Received in revised form 28 April 1992)

Key Word Index--Carthamus tinctorius; Compositae; petals; 6-hydroxykaempferol glycosides; flavonols. Abstract-From the dried petals of Carthamus tinctorius, five flavonoids, 6-hydroxykaempferol, 6-hydroxykaempferol 3-glucoside, 6-hydroxykaempferol3,6-diglucoside, 6-hydroxykaempferol3,6,7-triglucoside and 6-hydroxykaempferol 3-rutinoside-6-glucoside, were isolated along with 13 known compounds, and their structures determined by spectroscopic means including 2D NMR.

INTRODUCTION

The dried flower petals of safflower, Carthamus tinctorius L., have been used as an important crude drug in traditional Chinese medicine [l]. Among its chemical constituents carthamin [2-41, safflor yellows k[4] and B [a, safllomin A [6,7], safllomin C [S], isocarthamin [9] and isocarthamidin [9] have been reported. Isolation of adenosine as a platelet aggregation inhibitor from safflower was reported by Kutsuna et al. [lo]. In the present paper, we describe the isolation and characterization of flavonoids from the dried petals of C. tinctorius cultivated in China for medicinal use. RESULTS AND DISCUSSION

Repeated column chromatography of a methanolic extract of the dried petals led to isolation of five flavonoids l-5 along with known compounds, 3,4-dihydroxycinnamic acid, 4-hydroxycinnamic acid, apigenin, kaempferol, quercetin, (-)-eriodictyol, scutellarein, kaempferol3-glucoside, kaempferol3_rutinoside, quercetin 3,7_diglucoside, quercetin 3-glucoside, quercetin 3rutinoside and syringin. The known compounds were identified from their mass spectra, and comparison of their ‘H and 13CNMR spectra with authentic samples. The structures of new flavonoids were determined as fOllOW8.

Compound 1 showed a molecular ion peak at m/z 302. The ‘H NMR showed the presence of a p-hydroxyphenyl group (6, 6.92, 2H, d, J=8.6 Hz; 6a 8.04, 2H, d, J =8.6 Hz), an aromatic proton (6, 6.54, lH, s) and five hydroxy groups (6, 8.67, 9.20, 10.07, 10.47 and 12.25, exchangeable with deuterium). The hydroxy proton signal at &.,9.20 was long-range shift correlated with carbon signals at 6cl75.9 (C-4), 135.2 (C-3) and 146.8 (C-2) (Table I), the last signal being correlated with a proton signal at 6, 8.04 (H-2’, H-6’), indicating that 1 is a flavon-3-01 *Author to whom correspondence should be addressed. PMY31:11-v

1 : R,dls=Ra=H R2=Ra=H 3 : R,=Rp=Glc, RI=H 4: R,=R =R,=Glc 5 : R,=Gfc-Rhs, R,=Glc,

2 : R,=Glc,

RpH

derivative. In addition, the aromatic proton signal was long-range shift correlated with quaternary carbon signals at 8, 148.9 and 103.4, assignable to C-9 and C-10, respectively, as well as with signals at 6, 128.5 (C-6) and 153.6 (C-7) in the HMBC spectrum. On the basis of these findings, 1 was determined as 6-hydroxykaempferol. This compound was isolated for the first time from a natural source as a free hydroxy form, although its 7-glucoside has been reported [l 11. Compound 2 in its FAB mass spectrum showed a quasi-molecular ion peak at m/z 465 [M-I- 11’. The ‘H NMR spectrum showed the presence of a p-hydroxyphenyl group and a singlet signal at 6,6.53, assignable to H-6 or H-8 in a flavonoid nucleus. The presence of a glucopyranosyl moiety was evident on the ‘H and 13CNMR spectral data (J1, 2=7.9 Hz for the anomeric proton; Table 2 for 13C NMR). In the HMBC spectrum, the proton signal at 6, 6.53 was shown to be shiftcorrelated with the C-6 (6, 129.1), C-7 (6, 153.6), C-9 (6, 149.0) and C-10 (6, 104.4) signals but not with the C-5 signal (6, 146.5), which enabled us to assign the proton signal to H-8. In addition, an anomeric proton signal at 6, 5.46 (Ho,,-1) was shift-correlated with the C-3 signal (6, 132.9), indicating that glucose is attached to the C-3 position. Furthermore, the long-range 13C-‘H shiftcorrelations of the signal at 6, 12.41 (OH-5) with C-5 (6, 146.5), C-6 and C-10 signals, and of the signal at &lo.56 (OH-7) with C-6, C-7 and C-8 signals, suggested the

4001

4002 Table

M. HAT-TORI et al. 1. 13C and

‘H chemical shifts and long-range correlated ‘H signals for 1 Long-range “C-‘H shift correlated proton signal

‘H

Position

13C

2 3 4 5 6 I 8 9 10 1’ 2 3’ 4

146.8 135.2 175.9 145.9 128.5 153.6 93.4 148.9 103.4 122.0 129.4 115.5 159.1

5’ 6’ OH-3 OH-5 OH-6 OH-7 OH-4

115.5 (d) 129.4 (d)

(s) (s) (s) (s) (s) (s) (6) (s) (s) (s) (d) (d) (s)

6.54 (s)

8.04 (d, J = 8.6 Hz) 6.92 (d, J = 8.6 Hz)

6.92 8.04 9.20 12.25 8.67 10.47 10.07

(d, J = 8.6 Hz) (d, J = 8.6 Hz) (s) (s) (s) (s) (s)

H-2’, H-6’, OH-3 OH-3 OH-3 OH-5 H-8,OH-7,OH-5 H-8 OH-7 H-8 H-8, OH-5 H-3’. H-5’ OH-4 H-3’, H-5’, H-6’. OH-4 OH’-4

H-2’.

‘H and ‘%NMR spectra were measured at 400 MHz and 100 MHz, respectively, in DMSO-d,. Assignments were performed by DEPT, ‘H-‘H COSY, ‘H-‘% COSY and HMBC experiments.

of a 5,6,7-trihydroxy system in the molecule. On acid hydrolysis, 2 gave 6-hydroxykaempferol (1) and glucose, which were identified by comparing their spectral data with those of authentic samples. On the basis of these findings, the structure of 2 was determined as 6-hydroxykaempferol 3-U-/I-D-glucopyranoside. Compound 3 in its FAB mass spectrum showed a quasi-molecular ion peak at m/z 627 [M+ 11’. The ‘H NMR spectral pattern was similar to that of 2 except for the signals due to the sugar moiety. On acid hydrolysis, 3 gave 6-hydroxykaempferol (1) and glucose. The presence of two anomeric proton signals at 6, 4.77 (J = 7.3 Hz) and 5.46 (J = 7.3 Hz) in the ‘H NMR spectrum, and the 13C NMR spectral data (Table 2) revealed that 3 was a 6-hydroxykaempferol diglucoside. No NOE was observed between the anomeric protons and the protons due to H-8 (S, 6.54) and H-3’, H-5’ (6, 6.88), suggesting that two glucose groups are not attached to C7 and C-4’. Further, a chelated hydroxy group was observed in the ‘HNMR spectrum (6, 12.78, OH-5) but no appreciable change in chemical shift of C-4 was observed between 2 and 3 in the 13CNMR spectrum. These findings led us to conclude that the structure of 3 is 6-hydroxykaempferol 3,6-di-0-/I-D-glucopyranoside. Compound 4 showed an ion peak at m/z 789 [M + 11’ in the FAB mass spectrum. The ‘H-‘H COSY spectrum was indicative of a 3,4’,5,6,7+entaoxygenated flavone derivative (6, 6.89, 2H, d, J=9.0 Hz, H-3’, H-5’; 6, 8.05, 2H, d, J=9.0 Hz, H-2’, H-6’; 6, 6.99, lH, s, H-8). In presence

Table

shift

2. 13C NMR spectral

data for 2-5

C

2

3

4

5

2 3 4 5 6 7 8 9 10 1’ 2 3’ 4 5 6

156.2 132.9 177.6 146.5 129.1 153.6 93.5 149.0 104.4 121.2 130.9 115.1 159.8 115.1 130.9

157.3 132.8 177.6 152.3 128.2 156.4 93.9 152.0 104.3 120.9 130.8 115.0 159.9 115.0 130.8

157.1 133.2 177.8 152.3 128.9 156.0 94.4 151.6 106.2 120.8 131.0 115.2 160.1 115.2 131.0

157.6 132.9 177.6 152.2 128.4 156.9 94.1 152.2 104.1 120.9 130.8 115.1 159.9 115.1 130.8

3-Glucose G-l G-2 G-3 G-4 G-5 G-6

moiety 101.1 74.3 76.5 69.9 77.5 60.9

100.8 74.2” 76.3b 69.9’ 77.4d 60.7

100.8 74.3’ 76.4’ 69.9# 77.5” 60.9’

101.3 73.9 75.7 69.9 76.2 66.9

6-Glucose G-l’ G-2 G-3’ G-4 G-5’ G-6

moiety 104.2 73.9” 76.2b 69.6’ 17.2d 60.7

103.4 73.4= 76.4’ 69.88 77.2h 60.8’

104.4 74.2 76.4 69.6 77.2 60.7

7-Glucose G-l” G-2” G-3” G-4” G-5” G-6”

moiety

Rhamnose R-l R-2 R-3 R-4 R-5 R-6

moiety

loo.7 74.2’ 75.9’ 69.78 77.2” 60.7’

100.8 70.3 70.6 71.8 68.2 17.7

Measured in DMSO-d,. Values with identical superscripts a-i within a column may be interchanged. Other signals due to sugar carbons were assigned on the basis of the ‘H-‘H COSY, ‘H-l% COSY, HMBC, relayed COSY, and double-relayed COSY experiments.

addition, the ‘%NMR spectrum (Table 2) showed the presence of three glucopyranosyl groups which were directly linked to the flavonoid ring. In the HMBC spectrum, appreciable shift correlations were observed between the signal at 6c 156.0 (C-7) and an anomeric proton signal at 6, 5.04; between the signal at 6, 128.9 (C-6) and an anomeric proton signal at 6 4.87; between the signal at 6c133.2 (C-3) and an anomeric proton signal

6-Hydroxykaempferol glycosides from Carthnmus tinctor’ius at 6u5.47. Furthermore, a significant NOE was observed between the anomeric proton at 6,504 and H-8. On the basis of these findings and its ‘%NMR spectral data (Table 2), the structure of 4 was concluded to be 6-hydroxykaempferol 3,6,7-tri-O-/I-D-glucopyranoside. Compound 5 showed an ion peak at m/z 771 [M - l]in the FAB mass spectrum. The ‘H NMR spectrum of 5 in DMSO-d, showed the presence of a 4-hydroxyphenyl group and a singlet signal at 6u6.51 (lH, s), assignable to H-8 in a flavonoid nucleus as in 2-4. In addition, characteristic signals at 6, 0.99 (d, J = 6.1 Hz, H,,-6) and 4.38 (br s, H,,,-1), suggested the presence of a rhamnose unit, and two doublet signals at 6, 4.78 and 5.30 (J = 7.7 Hz and 7.4 Hz, respectively) were assigned as anomeric protons of two other sugar units. On acid hydrolysis, 5 gave 6-hydroxykaempferol (I), rhamnose and glucose. The 13CNMR spectrum showed the presence of 6-0-a-~rhamnosyl-D-glucose (rutinose) and glucose moieties which are linked to the 5avonoid ring (Table 2), their chemical shifts being assigned in comparison with those of the sugar moieties of kaempferol 3-rutinoside, quercetin 3-rutinoside and Z-4, and further, by ‘H-‘H COSY and r3C-rH COSY experiments On the basis of the relayed- and double-relayed COSY and ‘H-‘H COSY spectra measured in pyridine-d,, all proton signals for HGlc-l’, Horc-2, Ho,c-3’7 Ho,c-4, Ho,,-5’ and Ho,,-6 in one glucose moiety were assigned as 6, 5.61, 4.19, 4.29, 4.31, 4.07 and 4.3914.52, since the chemical shift value of C-6’ (6c62.3 in pyridine-d,), which correlated with proton signals at 6n4.39 and 4.52 (Ho,,-6 x 2) in the “C-‘H COSY spectrum, indicated that the primary hydroxyl group (OH-6’) of this sugar was not linked with rhamnose, but free in form [12]. In the HMBC spectrum in pyridine-d, (or in DMSOd6) signals at 6, 6.02 and 5.61 (6, 5.30 and 4.78 in DMSO-d,), assignable to anomeric protons of rutinose and glucose residues were shown to shift-correlate with signals at 6, 134.8 and 130.1 (6, 132.9 and 128.4 in DMSO-d,, C-3 and C-6), respectively, indicating that rutinose and glucose are linked at the C-3 and C-6 positions, respectively. These findings led us to conclude the structure of 5 to be 6-hydroxykaempferol 3-O-/3rutinoside-6-0-fi-D-glucopyranoside. We have previously reported that a methanolic extract of the dried petals of C. tinctorius stimulates the beating amplitude of cultured myocardial cells prepared from mouse embryonic heart though the extract does not affect the beating rate of the cells [13]. Of the compounds isolated in the present experiments, scutellarein, 4-hydroxycinnamic acid, 3,4_dihydroxycinnamic acid, 6hydroxykaempferol 3-glucoside (2) and quercetin 3,7-diglucoside significantly increased the beating amplitude [ 131. In addition, 6-hydroxykaempferol 3glucoside (2) appreciably inhibited Na+-K’ ATPase activity, which may result in inducing a positive inotropic effect [13]. Furthermore, we have recently found that these flavonoids inhibit 3a-hydroxysteroid dehydrogenase [ 141, thus suggesting that they may be associated with the anti-inflammatory effect of the C. tinctorius petals [l5].

EXPEUIMENTAL Mps are uncorr. ‘H and ‘%NMR: 400/270 and 100/22.5 MHz, respectively. EIMS: 70 eV, FABMS: BkV; Xe and glycerol as a neutral gas and a matrix, respectively.

4003

Chromatography. Wakogel C-200 was used for CC and Merck Kieselgel60 F,,, plates (0.25 mm thickness), were used for TLC. Plant material. Dried petals of C. tinctorius cultivated in Sichuang province of China, were obtained from Tochimoto Tenkaido Co. (Osaka, Japan) and a voucher specimen is deposited at the Museum of Materia Medica of Toyama Medical and Pharmaceutical University. Extraction Mdfractionation. Dried petals (6 kg) of C. tinctorius were extracted with MeOH (40 1x 3) for 48 hr at room temp. A MeOH extract (1140 g) was suspended in Hz0 (2 1)and successively extracted with hexane (2 1x lo), Et,0 (2 1x lo), EtOAc (2 1 x 10)and BuOH (2 1 x 10). The Et@-soluble and BuOH-soluble (226 9) fractions were further fractionated by CC on silica gel (column size, 55 x 4.5 cm) and Sephadex LH-20 (column size, 74 x 8 cm), followed by prep. TLC. Scutellarein (50 mg), 6-hydroxykaempferol (98 mg), apigenin (88 mg), kaempferol (22 mg), astragalin (198 mg), (-)-eriodictyol (9 mg), quercetin (74 mg), 4-hydroxycinnamic acid (211 mg) and 3,4-dihydroxycinnamic acid (308 mg) were isolated from the EtzO-soluble fraction, while the new flavonol glycosides 2 (307 mg), 3 (333 mg), 4 (58 mg) and 5 (62 mg), kaempferol 3-rutinoside (131 mg), quercetin 3-glucoside (131 mg), quercetin 3,7-diglucoside (152 mg), quercetin 3rutinoside (83 mg) and syringin (204 mg) were isolated from the BuOH-soluble fraction. 6-Hydroxykaempferol (1). Yellowprisms from MeOH, mp 280-282”. EI-MS m/z: 302 [Ml’. IR e; cn- i: 3400, 1640. 6-Hydroxykaempferol 3-glucoside (2). Yellow needles from MeOH, mp 263265”. FARMS (positive ion) m/z: 465 [M + l] +, 303 [aglycone]+. IR VIA cm-‘: 3402,1625. ‘H NMR (400 MHz, DMSO-d,): flavonoid moiety: 6, 6.53 (lH, s, H-8), 6.88 (2H, d, J=9.2 Hz, H-3’, H-S), 8.03 (2H, d, J=9.2 Hz, H-2, H-6’), 8.79 (lH, br s, OH-6), 10.17 (lH, s, OH-4’), 10.56 (lH, s, OH-7), 12.41 (lH, s, OH-5); sugar moiety: 6, 3.08 (Ho,,-5), 3.09 (Ho,,-4), 3.17 (Ho,,-2), 3.21 (Ho,,-3), 3.33 (Hole-6), 3.57 (lH, d, J=11.5 Hz, Ho,,-6), 5.46 (lH, d, J=7.9 Hz, Ho,,- 1). The assignments were performed on the basis of the ‘H-‘H COSY, ‘“C-‘H COSY and HMBC data. 6-Hydroxykaempferol 3,6_diglucoside (3). Yellow prisms from MeOH, mp 183-186”. FAB-MS (positive ion) m/z: 627 [M + l]‘, 303 [aglycone] +. IR vi:; cm - i: 3400,163O. ‘H NMR (400 MHz, pyridine-d,): 6, 3.95-4.6 (sugar protons), 5.55 (lH, d, J=7.6 Hz, Ho,,-1 ), 6.21 (lH, d, J=7.0Hz, Ho,,- 1), 6.73 (lH, s, H-8), 7.21 (2H, d, J = 8.9 Hz, H-3’, H-S), 8.41 (2H, d, J =8.9 Hz, H-2’, H-6’). ‘H NMR (270 MHz, DMSO-d,): 6n 3.0-3.7 (sugar protons), 4.77 (lH, d, J = 7.3 Hz, Hols- l’), 5.46 (lH, d, J=7.3 Hz, Ho,,-1), 6.54 (lH, s, H-8), 6.88 (lH, d, 3=8.8 Hz, H-3’, 5’), 8.04 (lH, d, J=8.8 Hz, H-2, H-6),10.18 (1H s, OH), 12.78 (lH, s, OH-5). 6-Hydroxykaempferol 3,6,7_triglucoside (4). Yellow needles from MeOH, mp 202-205”. FAB-MS (positive ion) m/z: 789 465 [M-2Glc+l]+. IR CM+ll+, 627 [M-Glc+l]+, v;: cm-‘: 3400, 1620. ‘H NMR (400 MHz, DMSO-d,): 6, 3.0-5.5(sugar protons),4.87(1H,d,J=7.3 Hz,Ho,,-13,5.04(1H, d, J=7.7 Hz, Ho,,-l”), 5.47 (lH, d, J=7.3 Hz, Ho,,-1), 6.89 (2H, d, J=9.0 Hz, H-3’, H-5’), 6.99(1H, s, H-8), 8.05 (lH, d, J=9.0 Hz, H-2, H-6’), 10.26 (lH, br s, OH-4’), 12.65 (lH, br s, OH-5). 6-Hydroxykaempfmol3-rutinoside-glucoside (5). Yellow plates from MeOH; mp 205-207”. FABMS (negative ion) m/z: 771 3402, 1620. ‘HNMR (400 MHz, [M-l]-. IR vE;cn-‘: DMSO-d.J: 6nO.99 (3H, d, J =6.1 Hz, H,,-6), 3.c3.7 (sugar-H), 4.37 (lH, d, J=4.37 Hz, OH), 4.38 (lH, br s, H,-l), 4.50 (lH, d, J=5.4Hz, OH), 4.78 (lH, d, J=7.7Hz, Ho,,-1’), 4.96 (lH, d, 5=4.7Hz, OH), 5.04 (lH, d, 5=6.1 Hz, OH), 5.06 (lH, d,J=5.1 Hz,OH),5.30(1H,d,J=7.4Hz,Ho,,-1),5.33(1H,d,J =4.7 Hz, OH), 6.51 (lH, s, H-8), 6.87 (2H, d, J=8.9 Hz, H-3’, H5’), 7.98 (2H, d, J=8.9 Hz, H-2’, H-6’), 12.70 (lH, s, OH-S). ‘aCNMR (lOOMHz, pyridine-d,): 6c161.7 (C-2), 134.8 (C-3),

M. HATTORIet al.

4004

178.9 (C-4), 153.5 (C-5), 130.1 (C-6), 158.8 (C-7), 94.4 (C-8), 153.8 (C-9) 105.6 (C-lo), 122.0 (C-l’) 131.9 (C-2’) 116.1 (C-3’), 158.1 (C4’) 116.1 (C-5’), 131.9 (C-6’), 104.3 (G-l), 76.0 (G-2), 78.1 (G-3), 71.4 (G-4), 77.5 (G-5), 68.4 (G-6), 107.1 (G-l’) 75.5 (G-2’), 78.5 (G3’) 71.2 (G-4’), 79.2 (G-5’), 62.3 (G-6’), 102.5 (R-l), 72.1 (R-2), 72.6 (R-3), 73.9 (R-4), 69.6 (R-5). 18.9 (R-6). Scutellarein. EIMS m/z: 286 [Ml’, ‘HNMR (270MHz, DMSO-de): 6,, 6.58 (lH, s. H-3), 6.74 (lH, s, H-8), 6.93 (2H, d, J =8.8 Hz, H-3’, H-5’), 7.92 (2H, d, J=8.8 Hz, H-2’, H-6’) 8.73, 10.31, 10.46, 12.81 (each lH, OH-4’. OH-5, OH-6, OH-7). i3C NMR (22.5 MHz, DMSO-d,): 6,163.5 (s, C-2). 102.2 (d, C-3), 181.9 (s, C-4), 147.0 (s, C-5), 129.1 (s, C-6), 153.2 (s. C-7), 93.8 (d, C-81, 149.6 (s, C-9). 104.0 (s, C-lo), 121.4 (s, C-l’), 128.2 (d, C-2’, C-6’), 115.9 (d, C-3’, C-5’). 160.9 (s. C-4).

REFERENCES 1. Dictionary House of 2. Obara, H. 3. Onodera, 1327. 4. Takahashi,

of Chinese Materia Medica (1985) Publishing Science and Technology of Shang Hai, p. 992. and Onodera, J. (1979) Chetn. Letters 201. J., Saito, T. and Obara, H. (1979) Chem. Letters Y., Miyasaka,

N., Tasaka,

S., Miura, I., Urano,

S.,

Ikura, M., Hikichi, K., Matsumoto, T. and Wada, M. (1982) Tetrahedron Letters 23, 5163 5. Takahashi, Y., Saito. K., Yanagiya, M., Ikura, M., Hikichi, K., Matsumoto, T. and Wada, M. (1984) Tetrahedron Letters 25, 2471. 6. Onodera. J., Obara, H., Osone, M., Maruyama, Y. and Sato, S. (1981) Chem. Letters 433. 7. Onodera, J., Sato, S. and Obara, H. (1981) Chem. Letters 887. 8. Onodera, J., Obara, H., Hirose, R., Matsuba, S., Saito, N., Sato. S. and Suzuki, M. (1989) Chem. Letters 1571. 9. Seshadri, T. R. and Thakur. R. S. (1960) Curr. Sci. India 29,54. 10. Kutsuna, H., Fujii, S.. Kitamura, K., Komatsu, K. and Nakano, M. (1988) Yakugaku Zasshi 108, 1101. 11. Bacon. J. D.. Urbatsch. L. E., Bragg, L. H., Mabry, T. J., Neuman. P. and Jackson, D. W. (1978) Phytochemistry 17, 1939. 12. Mttsuhashi. H., Tanaka, O., Nozoe, S. and Nagai, M. (1992) Chemistry of Organic Natural Products. Fourth Edition, p. 83. Nankodo Co., Tokyo. 13. Huang, S., Hattori, M. and Namba, T. (1992) Shoyakugaku Zasshi 46 (in press). 14. Akao, T., Che. Q., Hattori, M., Namba, T. and Kobashi, K. (1992) Shoyakugaku Zasshi 46 (in press). 15. Kasahara, Y., Kumaki, K. and Katagiri, S. (1991) Shoyakugaku Zasshr 45, 306.