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Flavonoids and phenylpropanoid derivatives from Campanula barbata
Phytochemistry 56 (2001) 631±636
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Flavonoids and phenylpropanoid derivatives from Campanula barbata Muriel Cuendet, Olivier Potterat, Kurt Hostettmann * Institut de Pharmacognosie et Phytochimie, Universite de Lausanne, BEP, CH-1015 Lausanne, Switzerland Received 4 April 2000; received in revised form 6 October 2000
Abstract Four new phenylpropanoid derivatives, barbatosides A±D, and a new catechin, barbato¯avan, were isolated from the whole plant of Campanula barbata L. (Campanulaceae) and identi®ed as wahlenbergioside-30 -O-glucoside, wahlenbergioside-30 -O-(2000 -(pmethoxycinnamoyl))-glucoside, wahlenbergioside-30 -O-(4000 -(trans-p-coumaroyl))-glucoside, wahlenbergioside-30 -O-(4000 -(cis-p-coumaroyl))-glucoside and 3-acetyl-5-methoxy-7,30 ,40 -trihydroxy-8-O-glucoside-¯avan-3-ol, respectively, by spectroscopic methods. In addition, four ¯avonols were isolated and identi®ed as kaempferol-3-O-glucoside, kaempferol-3-O-rutinoside, quercetin-3-O-glucoside and quercetin-3-O-rutinoside. Barbato¯avan demonstrated scavenging properties towards the DPPH radical. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Campanula barbata; Campanulaceae; Radical scavenging activity; Flavonoids; Phenylpropanoid derivatives; Barbatosides A±D; Barbato¯avan
1. Introduction In the course of our search for new antioxidants from higher plants, particular attention has been given to plants growing at high altitude (>2000 m). Such species have to face more intense UV radiation and may respond to the resulting oxidative stress by producing a greater diversity of antioxidant molecules. Campanula barbata L. (Campanulaceae) is a hairy plant of height 10±40 cm, growing in the alpine regions of central and boreal Europe. To our knowledge, no previous investigation has been done on its constituents. In a TLC autographic assay with the methanol extract of C. barbata whole plant, compounds were observed which reduced the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (Takao et al., 1994; Cuendet et al., 1997). 2. Results and discussion The methanolic extract of the whole plant was fractionated by a combination of CC on silicagel, medium-pressure * Corresponding author. Tel.: +41-21-692-45-61; fax: +41-21-69245-65. E-mail address: [email protected] (K. Hostettmann).
liquid chromatography (MPLC) and gel ®ltration on Sephadex LH-20 to provide compounds 1±9 (see Experimental). Compounds 1±4 were identi®ed as kaempferol-3-Oglucoside, kaempferol-3-O-rutinoside, quercetin-3-Oglucoside and quercetin-3-O-rutinoside by comparison of their spectroscopic data (1H - and 13C -NMR, UV, FAB-MS) with literature values (Markham et al., 1978). Compound 5, named barbatoside A, was obtained as a white amorphous powder. Its UV spectrum showed absorption at lmax 221 and 266 nm and suggested the presence of an aromatic system conjugated with an unsaturated side chain. Reaction with Godin reagent (Godin, 1954) gave a grey-blue colour. The ES±MS presented a signal at m/z 687, corresponding to [M+Na]+. A MS/MS experiment on this quasimolecular ion gave two fragments at m/z 525 [(M+Na)162]+ and 363 [(M+Na)-324]+ indicating the loss of two hexosyl groups. According to ES±MS, 13C and DEPT NMR data, the molecular formula was deduced to be C28H40O18. In the 1H NMR spectrum, the presence of a trans-propenyl alcohol was indicated by a pair of ole®nic signals at 6.55 (1H, d, J=16.0 Hz), 6.23 (1H, dt, J=16.0, 6.5 Hz), and a methylene doublet at 4.71 (2H, d, J=6.5 Hz). Two meta-coupled aromatic protons of the 3,4,5-trisubstituted phenyl residue
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00423-4
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appeared at 6.61 (2H, d, J=3.0 Hz). A three-proton singlet at 3.84 was characteristic of a methoxyl group placed on the aromatic nucleus. The remaining resonances, a methyl signal at 1.51 (3H, s), the four protons at 2.99 (1H, d, J=15.5 Hz), 2.91 (1H, d, J=15.5 Hz) and 2.85 (2H, d, J=6.5 Hz) corresponding to two methylenes, together with the carbonyl signal at 172.46 in the 13C NMR spectrum, demonstrated the presence of an ester derivative of a 3-hydroxyl-3methylglutarate moiety (HMG) (Mizutani et al., 1988). The diacid moiety was found to be bound to C-a through an ester linkage, by comparing the 1H NMR shifts of the methyleneoxy protons H2-a with those reported for esteri®ed derivatives (Mizutani et al., 1988; Yuda et al., 1990). Sugar moieties were identi®ed as two glucoses, by comparing 13C NMR data with that of the literature (Harborne and Mabry, 1982). This information
suggested that 5 was a ``tangshenoside type'' phenylpropanoid derivative (Mizutani et al., 1988; Yuda et al., 1990). Comparison of the spectral data of 5 with those of tangshenoside I (Mizutani et al., 1988) revealed that there was only one methoxyl group in compound 5 instead of two (in positions C-3 and C-5) in the case of tangshenoside I. A similar compound, without the glucosyl moiety in position 30 , is known as wahlenbergioside (Ma et al., 1997). A GHMBC experiment con®rmed the two glucose and methoxyl group positions. The stereochemistry of the HMG moiety was not established. Thus, compound 5, named barbatoside A, is wahlenbergioside-30 -O-glucoside. The reaction of 6 with Godin reagent gave a grey-blue colour and the ®rst part of its UV spectrum showed the same absorption peaks as that of compound 5. This suggested that 6 was also a ``tangshenoside type''
M. Cuendet et al. / Phytochemistry 56 (2001) 631±636
phenylpropanoid derivative. The ES±MS spectrum gave a signal at m/z 823 and another at m/z 483, corresponding to the pseudomolecular ion [M H] and to the loss of the trans-propenyl alcohol moiety linked to a 3,4,5-trisubstituted phenyl residue [(M H)-340] , respectively. In the 1H and 13C NMR spectra, all signals corresponding to compound 5 were present. However, the GHSQC spectrum showed that the C-2000 was shifted down®eld and thus this position was esteri®ed. The substituent at C-2000 was identi®ed as trans-p-methoxycoumaric acid. The trans con®guration was determined from the coupling constant Jb0 g0 . Treatment with b-glucosidase cleaved the glucose moiety located at HO-C-4 to provide 6a. The presence of a second glucose residue was demonstrated by acidic hydrolysis of 6a and by TLC comparison of the sugar with an authentic sample. Thus, compound 6, named barbatoside B, is wahlenbergioside-30 -O-(2000 -(p-methoxycinnamoyl))-glucoside. The UV spectrum of 7 was almost the same as that of 6. Moreover, this substance also had a grey-blue colour when sprayed on TLC with Godin's reagent. This suggested a structural relationship between 6 and compound 7. A molecular mass of 810 amu was deduced from the ES±MS spectrum which exhibited a quasimolecular ion [M+Na]+ at m/z 833. Moreover, the peak at m/z 493 corresponded to the loss of the trans-propenyl alcohol moiety linked to a 3,4,5-trisubstituted phenyl residue [(M+Na)-340]+. The 1H and 13C NMR spectra of 7 were very similar to those of 6, with the exception of the signal for the methoxyl group OCH3±C-400 , which was missing in 7. After enzymatic hydrolysis of 7 with b-glucosidase followed by acid hydrolysis, it was possible to conclude the presence of two glucosyl moieties, as in 6. However, the p-coumaroyl group was linked at position C-4 of the C-30 sugar because of the down®eld shift observed for this proton in the 1H NMR spectrum. Thus, compound 7, named barbatoside C, is wahlenbergioside-30 -O- (4000 (p-coumaroyl))- glucoside (trans con®guration). Compound 8 was separated from 7 by semipreparative HPLC. Compound 7 and 8 showed the same UV and MS spectra. However, in 8, the p-coumaroyl moiety was found to possess a cis con®guration, from the 1H NMR signals of H-b and H-g which appeared as two d with a coupling constant of 12.5 Hz. Thus, compound 8, named barbatoside D, is wahlenbergioside-30 -O-(4000 -(p-coumaroyl))-glucoside (cis con®guration). Phenylpropanoid derivatives with structures similar to compounds 5±8 have already been found in the genera Codonopsis, Lobelia and Wahlenbergia in the Campanulaceae family (Ishimaru et al., 1991; Lee and Ko, 1992; Ma et al., 1997). On the other hand, none possessed the p-coumaroyl moiety attached to the glucose at position C-30 , as in compounds 6±8. The APCI±MS spectrum of 9 showed a molecular mass of 524 amu. A fragment observed at m/z 481 [(M H)-42]
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corresponded to the loss of an acetyl moiety. The loss of a hexosyl group led to the peak at m/z 361 [(M±H)162] . The elimination of both the sugar and the acetyl moieties explained the detected fragment at m/z 319 [(M H)-204] . From the 1H and 13C NMR data, the molecular formula was established to be C24H28O13. The 1H NMR spectrum showed three signals in the low®eld region at 6.75 (1H, d, J=8.0 Hz), 6.80 (1H, dd, J=8.0, 1.5 Hz) and 7.06 (1H, d, J=1.5 Hz) indicative of a 3,4-dihydroxyphenyl moiety attached at C-2. Other signals at 6.16 (1H, s), 5.38 (1H, br. s), 5.01 (1H, s), 2.96 (1H, dd, J=17.5, 4.5 Hz), 2.84 (1H, dd, J=17.5, 2.0 Hz) corresponded to a chromane substituted in positions 3, 5, 7 and 8 (Adinol® et al., 1989; Sakar et al., 1993). The remaining signals were attributed to a methoxyl group at 3.79 (3H, s), to a methyl at 1.87 (3H, s), and to a glucosyl moiety. The 13C NMR spectrum allowed con®rmation of the catechin skeleton (Bennini et al., 1993). A signal at 172.01 for the carbonyl group of the acetyl moiety, together with a signal for the methyl group at 20.77 was observed. The linkage position of the acetyl moiety was at C-3 because of the down®eld shift of this carbon compared with an unsubstituted C-3 skeleton (Nonaka et al., 1983). A GHMBC experiment placed the methoxyl group in position C-5 and showed the glucosyl moiety to be linked to the hydroxyl at C-8. Shifts obtained in the 1H and 13C NMR spectra for positions C-2 and C-3 indicated that the stereochemistry of 9 was that of epicatechin (Shen et al., 1993; Harborne, 1994). Thus, compound 9, named barbato¯avan, is 3-acetyl-5-methoxy-7,30 ,40 -trihydroxy-8-O-glucoside-¯avan-3-ol. Compounds 5±9 were tested in a spectrophotometric assay for free radical scavenging activity, using DPPH. Quercetin and BHT [2,6-di(tert-butyl)-4-methylphenol] were used as reference compounds. Wahlenbergioside derivatives 5±8 were inactive. On the other hand, 9 had an activity similar to that of quercetin (Table 3). Antioxidant properties of catechins have been the subject of several studies due to their contribution to the antioxidant properties of black and green teas (Sera®ni et al., 1994). Green tea beverages have demonstrated protective eects against cardio-vasucular and liver diseases (Imai and Nakachi, 1995). Catechins were also found useful as food preservatives. These compounds prevent the formation of toxic oxygen species and peroxides in food (Nakayama et al., 1994). 3. Experimental 3.1. General TLC: Silica gel 60 F254 sheets (Merck); CHCl3± MeOH±H2O (65:35:5). Open column chromatography (CC): Sephadex LH-20 (Pharmacia) and silica gel
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Table 1 1 H NMR spectral data of compounds 5, 6, 7 and 8 (measured in CD3OD). Attributions are supported by GHSQC and GHMBC dataa Assignment
5
H-2 H-6 OCH3 H2-a H-b H-g Ha-20 Hb-20 Ha-40 Hb-40 H-60 H-200 H-300 H-500 H-600 OCH3 H-b0 H-g0 H-1000 H-2000 H-3000 H-4000 H-5000 Ha-6000 Hb-6000 H-10000 H-20000 H-30000 H-40000 H-50000 Ha-60000 Hb-60000
6.61 6.61 3.84 4.71 6.23 6.55 2.99 2.85 2.91 2.85 1.51
a
4.60 3.17 3.28 3.28 3.39 3.82 3.63 4.68 3.49 3.45 3.43 3.28 3.82 3.73
6 (1H, (1H, (3H, (2H, (1H, (1H, (1H, (1H, (1H, (1H, (3H,
(1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, J=3.0) d, J=3.0) s) d, J=6.5) dt, J=16.0, 6.5) d, J=16.0) d, J=15.5) d, J=6.5) d, J=15.5) d, J=6.5) s) ± ± ± ± ± ± ± d, J=8.0) dd, J=9.0, 8.0) m) m) m) dd, J=12.0, 2.5) dd, J=12.0, 4.5) d, J=8.0) m) m) m) m) d, J=12.0) dd, J=12.0, 4.5)
6.56 6.54 3.83 4.60 6.14 6.42 2.86 2.86 2.86 2.86 1.52 7.50 6.90 6.90 7.50 3.80 6.39 7.65 4.87 4.79 3.59
7 (1H, (1H, (3H, (2H, (1H, (1H, (1H, (1H, (1H, (1H, (3H, (1H, (1H, (1H, (1H, (3H, (1H, (1H, (1H, (1H, (1H,
d, J=2.0) d, J=2.0) s) d, J=6.0) dt, J=16.0, 6.0) d, J=16.0) m) m) m) m) s) d, J=8.5) d, J=8.5) d, J=8.5) d, J=8.5) s) d, J=16.0) d, J=16.0) d, J=7.5) t, J=7.5) t, J=9.0)
3.85 (1H, dd, J=12.0, 2.0) 3.67 (1H, dd, J=12.0, 4.5) 4.68 (1H, d, J=7.5)
3.80 (1H, dd, J=12.0, 2.5) 3.72 (1H, dd, J=12.0, 4.5)
6.63 6.61 3.85 4.73 6.26 6.57 2.89 2.89 2.98 2.98 1.50 7.47 6.81 6.81 7.47 ± 6.36 7.66 4.68
8 (1H, (1H, (3H, (2H, (1H, (1H, (1H, (1H, (1H, (1H, (3H, (1H, (1H, (1H, (1H,
d, J=1.5) d, J=1.5) s) d, J=6.5) dt, J=16.0, 6.5) d, J=16.0) m) m) m) m) s) d, J=9.0) d, J=9.0) d, J=9.0) d, J=9.0)
(1H, d, J=16.0) (1H, d, J=16.0) (1H, d, J=8.0)
6.63 6.61 3.85 4.73 6.26 6.57 2.89 2.89 2.98 2.98 1.50 7.67 6.75 6.75 7.67 ± 5.79 6.90 4.68
(1H, (1H, (3H, (2H, (1H, (1H, (1H, (1H, (1H, (1H, (3H, (1H, (1H, (1H, (1H,
d, J=1.5) d, J=1.5) s) d, J=6.5) dt, J=16.0, 6.5) d, J=16.0) m) m) m) m) s) d, J=9.0) d, J=9.0) d, J=9.0) d, J=9.0)
(1H, d, J=12.5) (1H, d, J=12.5) (1H, d, J=8.0)
3.65 (1H, t, J=9.5) 4.81 (1H, m)
3.65 (1H, t, J=9.5) 4.81 (1H, m)
3.58 (1H, d, J=12.0)
3.58 (1H, d, J=12.0)
4.68 (1H, d, J=8.0)
4.68 (1H, d, J=8.0)
3.42 (1H, t, J=9.0)
3.42 (1H, t, J=9.0)
3.79 (1H, dd, J=12.0, 2.5) 3.72 (1H, dd, J=12.0, 4.5)
3.79 (1H, dd, J=12.0, 2.5) 3.72 (1H, dd, J=12.0, 4.5)
Proton coupling constants (J in Hz), signal multiplicities and integrations are in parentheses.
(63±200 mm, Merck). Medium-pressure liquid chromatography (MPLC): home-packed LiChroprep-RP-18 column (15±25 mm, 46036 mm i.d., Merck). Anal. HPLC: Hewlett Packard 1050 instrument equipped with a photodiode array detector; Nucleosil RP-18 column (7 mm; 2504 mm i.d., Macherey±Nagel); MeCN±H2O (5:95!50:50 in 30 min, 0.05% CF3COOH, 1 ml/min). UV: Shimadzu UV-160A and Perkin-Elmer Lambda-3 spectrophotometers. Mp: Mettler FP-80/82 hot-stage apparatus; uncorrected. [a]20 D : Perkin-Elmer 241 polarimeter. 1H and 13C NMR: Varian Inova 500 spectrometer; in ppm rel. to TMS; J in Hz; carbon multiplicities from DEPT experiments. FAB±MS: Finnigan-MAT TSQ-700 triple-stage quadrupole instrument. ES±MS, APCI±MS: MAT-LCQ ion trap instrument. 3.2. Plant material The whole plant of Campanula barbata L. was collected in La Creusaz above Les Marecottes, Valais, Switzerland, in July 1996. A voucher specimen (No. 96169) has been deposited at the Institute of Pharmacognosy and Phytochemistry, University of Lausanne, Switzerland.
3.3. Extraction and isolation The dried whole plant (430 g) was ground and extracted at room temp. successively with CH2Cl2 (31500 ml) and MeOH (31500 ml) to yield 14 and 72 g of extract, respectively. A portion (50 g) of the MeOH extract was subjected to CC on silica gel eluted with CHCl3±MeOH (9:1±1:1) to provide 15 fractions (1±15). Fraction 4 contained pure kaempferol-3-O-glucoside (1; 45 mg). Fraction 6 was puri®ed by gel ®ltration on Sephadex LH-20 MeOH±H2O (1:1) to yield 9 (68 mg). Fraction 7 was also separated by ®ltration on Sephadex LH-20 MeOH to give kaempferol-3-O-rutinoside (2; 140 mg) and quercetin-3-O-glucoside (3; 65 mg). Two successive ®ltrations on Sephadex LH-20 (MeOH and MeOH±H2O 1:1) were made on fraction 8 to obtain fractions 8c1 7 and 8d1 6. 8c6 and 8d4 were then mixed and separated by semi-preparative HPLC (RP-18, MeCN±H2O 27:73, ¯ow rate 4 ml/min) aording 7 (3.5 mg) and 8 (1.5 mg). 6 (15 mg) was obtained after separation of fraction 10 by MPLC (RP-18, MeCN± H2O 15:85). Fraction 12 was separated by MPLC (RP18, MeOH±H2O 20:80) to yield 13 fractions. Fractions
M. Cuendet et al. / Phytochemistry 56 (2001) 631±636
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12e and 12i contained pure compounds 5 (120 mg) and 4 (quercetin-3-O-rutinoside; 180 mg), respectively.
terminal glucose was obtained in the butanolic fraction.
3.4. Reduction of 2,2-diphenyl-1-picrylhydrazyl (2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl; DPPH) radical
3.6. Acid hydrolysis of 2, 4, 6 and 7
Thirty microlitres of a soln. containing the compound to be tested and 200 ml of MeOH were added to 50 ml of a 0.02% MeOH soln. of DPPH. Absorbance at 517 nm was determined after 30 min, and the percentage of activity was calculated. 3.5. Enzymatic hydrolysis of 3, 6 and 7 One milligramme of compound with 1 mg of b-glucosidase (Sigma G-0395) in 2 ml of buer (NaOAc 0.5 M), adjusted to pH 5 with acetic acid, was left at room temp. for 14 h; the reaction mixt. was partitioned successively with AcOEt and n-BuOH. The compound without the
Two milligrammes of pure compound (2 and 4), or the residue obtained in the butanolic extract after enzymatic hydrolysis (6 and 7), in 10 ml 2 N HCl aqueous solution was heated at 100 C for 3 h. The reaction mixture was diluted with water (5 ml) and successively extracted with AcOEt and BuOH. A TLC with organic fractions was run with reference substances to identify the aglycone. The aqueous phase, containing free sugars, was neutralised with NaHCO3 to pH 6, then dried. The residue was checked for sugars by TLC (comparison with authentic samples) using AcOEt±MeOH±H2O±HOAc (65:15:15:20) and anisidine phthalate as the detection reagent. The presence of glucose (2, 4, 6 and 7) and rhamnose (2 and 4) was con®rmed. 3.7. Wahlenbergioside-30 -O-glucoside (barbatoside A; 5)
Table 2 13 C NMR spectral data of compounds 5, 6, and 7 (measured in CD3OD). Attributions are supported by GHSQC and GHMBC data Carbon no.
5
6
7
1 2 3 4 5 6 OCH3 a b g 20 30 40 50 60 100 200 300 400 500 600 OCH3 a0 b0 g0 1000 2000 3000 4000 5000 6000 10000 20000 30000 40000 50000 60000
135.05 108.86 154.20 135.12 151.84 103.48 56.75 66.06 124.17 134.89 44.33 77.59 44.33 172.46 24.80 ± ± ± ± ± ± ± ± ± ± 98.22 75.04 77.65 71.43 77.89 62.75 106.69 75.28 77.51 70.78 78.24 62.05
135.11 108.92 154.22 135.17 151.88 103.44 56.75 65.89 124.47 134.32 44.59 77.82 44.59 171.86 24.19 128.39 130.98 115.43 163.11 115.43 130.98 55.88 167.93 116.47 146.23 96.57 75.18 76.39 71.62 77.88 62.71 106.79 75.35 77.68 70.83 78.33 62.08
135.12 108.91 154.28 135.21 151.94 103.48 56.75 66.09 124.19 134.99 44.28 77.68 44.28 172.45 24.85 127.15 131.26 116.85 161.44 116.85 131.26 ± 168.58 114.83 147.21 98.36 75.24 75.72 72.45 75.97 62.52 106.77 75.35 77.68 70.85 78.34 62.09
Amorphous white powder. Mp 115±118 C. [a]20 24 D (MeOH, c 0.3). UV lmax (MeOH) nm (log "): 221 (4.38), 266 (4.06). ES±MS (positive ion mode) m/z 687.3 [(M+H)+Na]+, 525.1 [(M+Na)-162]+, 363.1 [(M+Na)324]+. 1H and 13C NMR : Tables 1 and 2. 3.8. Wahlenbergioside- 30 -O-(2000 -(p-methoxycinnamoyl))glucoside (barbatoside B; 6) Amorphous white powder. Mp 124±125 C. [a]20 40 D (MeOH, c0.1). UV lmax (MeOH) nm (log "): 222 (4.69), 271 (4.43), 309 (4.33). ES±MS (negative ion mode) m/z 823.1 [M-H] , 483 [(M H)-340] . 1H and 13C NMR: Tables 1 and 2. 3.9. Wahlenbergioside-30 -O-(4000 -p-coumaroyl)-glucoside (trans) (barbatoside C; 7) Amorphous white powder. Mp 121±122 C. UV lmax (MeOH) nm (log "): 222 (5.71), 266 (4.48), 314 (4.32). ES±MS (positive ion mode) m/z 833.0 [(M+H)+Na]+, 493 [(M+Na)-340]+. 1H and 13C NMR: Tables 1 and 2. Table 3 Percentage of DPPH radical reduction in a spectrophotometric assay caused by compound 9 at dierent concentrations. Measurement at 517 nm, determination after 30 min Concentration (mM)
Quercetin
BHT
9
80.0 40.0 20.0 10.0 5.0 2.5
97.1 96.5 94.9 53.7 25.8 15.3
41.3 29.5 14.9 8.1 3.3 3.3
95.9 89.7 49.9 26.5 16.2 8.8
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3.10. Wahlenbergioside-30 -O-(4000 -p-coumaroyl)glucoside (cis) (barbatoside D; 8) Amorphous white powder. 1H NMR: Table 1. 3.11. 3-Acetyl-5-methoxyl-7,30 ,40 -trihydroxyl-8-O-glucoside-¯avan-3-ol (barbato¯avan; 9) Amorphous cream powder. Mp 143±145 C. [a]20 78 D (MeOH, c 0.2). UV lmax (MeOH) nm (log "): 230 (sh) (4.02), 281 (3.54). APCI±MS (negative ion mode) m/z 523.1 [M H] , 481.3 [(M-H)-42] , 361.4 [(M-H)-162] , 319.5 [(M H)-204] . 1H NMR (CD3OD) : 7.06 (1H, d, J=1.5 Hz, H-20 ); 6.80 (1H, dd, J=8.0, 1.5 Hz, H-60 ); 6.75 (1H, d, J=8.0 Hz, H-50 ); 6.16 (1H, s, H-6); 5.38 (1H, br. s, H-3); 5.01 (1H, s, H-2); 4.68 (1H, d, J=7.5 Hz, H-100 ); 3.79 (3H, s, OCH3); 3.76 (2H, m, H-600 ); 3.39±3.48 (3H, non resolved, H-200 , H-300 , H-400 ); 3.24 (1H, m, H-500 ); 2.96 (1H, dd, J=17.5, 4.5 Hz, Ha-4); 2.84 (1H, dd, J=17.5, 2.0 Hz, Hb-4); 1.87 (3H, s, CH3). 13 C NMR (CD3OD): 172.01 (CO); 153.33 (C-7); 152.55 (C-5); 149.43 (C-9); 146.01 (C-40 ); 145.98 (C-30 ); 130.92 (C-10 ); 128.68 (C-8); 119.04 (C-60 ); 115.98 (C-50 ); 115.01 (C-20 ); 106.66 (C-100 ); 101.16 (C-10); 93.79 (C-6); 78.38 (C-2); 78.00 (C-500 ); 77.66 (C-300 ); 75.75 (C-200 ); 71.13 (C400 ); 69.33 (C-3); 62.51 (C-600 ); 56.83 (OCH3); 26.62 (C-4); 20.77 (CH3). Acknowledgements Financial support was provided by the Swiss National Science Foundation. References Adinol®, M., Aquila, T., Barone, G., Lanzetta, R., Parrilli, M., 1989. Homoiso¯avanones from Bellevalia romana. Phytochemistry 28, 3244±3246. Bennini, B., Chulia, A.J., Kaouadji, M., Delage, C., 1993. (2R,3R)Dihydro¯avonol aglycone and glycosides from Erica cinerea. Phytochemistry 33, 1233±1236.
Cuendet, M., Hostettmann, K., Dyatmiko, W., Potterat, O., 1997. Iridoid glucosides with free radical scavenging properties from Fagraea blumei. Helvetica Chimica Acta 80, 1144±1152. Godin, P., 1954. A new spray reagent for paper chromatography of polyols and cetoses. Nature 174, 134. Harborne, J.B., 1994. The Flavonoids: Advances in Research Since 1986. Chapman and Hall, London. Harborne, J.B., Mabry, T.J., 1982. The Flavonoids: Advances in Research. Chapman and Hall, London. Imai, K., Nakachi, K., 1995. Cross sectional study of eects of drinking green tea on cardiovascular and liver diseases. British Medical Journal 310, 693±696. Ishimaru, L., Yonemitsu, H., Shimomura, K., 1991. Lobetyolin and lobetyol from hairy root culture of Lobelia in¯ata. Phytochemistry 30, 2255±2257. Lee, I.R., Ko, J.H., 1992. Isolation of triterpenoids and phenylpropanoids from Codonopsis ussuriensis. Archives of Pharmaceutical Research 15, 289±291. Ma, W.G., Tan, R.X., Fuzzati, N., Li, Q.S., Wolfender, J.-L., Hostettmann, K., 1997. Wahlenbergioside, a phenylpropanoid glucoside from Wahlenbergia marginata. Phytochemistry 45, 411±415. Markham, K.R., Ternai, B., Stanley, R., Geiger, H., Mabry, T.J., 1978. Carbon-13 NMR studies of ¯avonoids Ð III., Naturally occurring ¯avonoid glycosides and their acylated derivatives. Tetrahedron 34, 1389±1397. Mizutani, K., Yuda, M., Tanaka, O., Saruwatari, Y.I., Jia, M.R., Ling, Y.K., 1988. Tangshenosides I and II from Chuan-Dangshen, the root of Codonopsis tangshen Oliv. Chemical and Pharmaceutical Bulletin 36, 2726±2729. Nakayama, T., Kawagishi, S., Oosawa, T., 1994. Quercetin, kaempferol, catechin and taxifolin as antioxidants for food preservation. CA 122:54685 JP 06,248,267. Nonaka, G.-I., Kawahara, O., Nishioka, I., 1983. Tannins and related compounds. XV. A new class of dimeric ¯avan-3-ol gallates, theasinensins A and B, and proanthocyanidin gallates from green tea leaf. Chemical and Pharmaceutical Bulletin 31, 3906±3914. Sakar, M.K., Petereit, F., Nahrstedt, A., 1993. Two phloroglucinol glucosides, ¯avan gallates and ¯avonol glycosides from Sedum sediforme ¯owers. Phytochemistry 33, 171±174. Sera®ni, M., Ghiselli, A., Ferro-Luzzi, A., 1994. Red wine, tea, and antioxidants. Lancet 344, 626. Shen, C.-C., Chang, Y.-S., Ho, L.-K., 1993. Nuclear magnetic resonance studies of 5,7-dihydroxy¯avonoids. Phytochemistry 34, 843±845. Takao, T., Kitatani, F., Watanabe, N., Yagi, A., Sakata, K., 1994. A simple screening method for antioxidants and isolation of several antioxidants produced by marine bacteria from ®sh and shell®sh. Bioscience, Biotechnology, Biochemistry 58, 1780±1783. Yuda, M., Ohtani, K., Mizutani, K., Kasai, R., Tanaka, O., Jia, M.R., 1990. Neolignan glycosides from roots of Codonopsis tangshen. Phytochemistry 29, 1989±1993.
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Flavonoids and phenylpropanoid derivatives from Campanula barbata Muriel Cuendet, Olivier Potterat, Kurt Hostettmann * Institut de Pharmacognosie et Phytochimie, Universite de Lausanne, BEP, CH-1015 Lausanne, Switzerland Received 4 April 2000; received in revised form 6 October 2000
Abstract Four new phenylpropanoid derivatives, barbatosides A±D, and a new catechin, barbato¯avan, were isolated from the whole plant of Campanula barbata L. (Campanulaceae) and identi®ed as wahlenbergioside-30 -O-glucoside, wahlenbergioside-30 -O-(2000 -(pmethoxycinnamoyl))-glucoside, wahlenbergioside-30 -O-(4000 -(trans-p-coumaroyl))-glucoside, wahlenbergioside-30 -O-(4000 -(cis-p-coumaroyl))-glucoside and 3-acetyl-5-methoxy-7,30 ,40 -trihydroxy-8-O-glucoside-¯avan-3-ol, respectively, by spectroscopic methods. In addition, four ¯avonols were isolated and identi®ed as kaempferol-3-O-glucoside, kaempferol-3-O-rutinoside, quercetin-3-O-glucoside and quercetin-3-O-rutinoside. Barbato¯avan demonstrated scavenging properties towards the DPPH radical. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Campanula barbata; Campanulaceae; Radical scavenging activity; Flavonoids; Phenylpropanoid derivatives; Barbatosides A±D; Barbato¯avan
1. Introduction In the course of our search for new antioxidants from higher plants, particular attention has been given to plants growing at high altitude (>2000 m). Such species have to face more intense UV radiation and may respond to the resulting oxidative stress by producing a greater diversity of antioxidant molecules. Campanula barbata L. (Campanulaceae) is a hairy plant of height 10±40 cm, growing in the alpine regions of central and boreal Europe. To our knowledge, no previous investigation has been done on its constituents. In a TLC autographic assay with the methanol extract of C. barbata whole plant, compounds were observed which reduced the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (Takao et al., 1994; Cuendet et al., 1997). 2. Results and discussion The methanolic extract of the whole plant was fractionated by a combination of CC on silicagel, medium-pressure * Corresponding author. Tel.: +41-21-692-45-61; fax: +41-21-69245-65. E-mail address: [email protected] (K. Hostettmann).
liquid chromatography (MPLC) and gel ®ltration on Sephadex LH-20 to provide compounds 1±9 (see Experimental). Compounds 1±4 were identi®ed as kaempferol-3-Oglucoside, kaempferol-3-O-rutinoside, quercetin-3-Oglucoside and quercetin-3-O-rutinoside by comparison of their spectroscopic data (1H - and 13C -NMR, UV, FAB-MS) with literature values (Markham et al., 1978). Compound 5, named barbatoside A, was obtained as a white amorphous powder. Its UV spectrum showed absorption at lmax 221 and 266 nm and suggested the presence of an aromatic system conjugated with an unsaturated side chain. Reaction with Godin reagent (Godin, 1954) gave a grey-blue colour. The ES±MS presented a signal at m/z 687, corresponding to [M+Na]+. A MS/MS experiment on this quasimolecular ion gave two fragments at m/z 525 [(M+Na)162]+ and 363 [(M+Na)-324]+ indicating the loss of two hexosyl groups. According to ES±MS, 13C and DEPT NMR data, the molecular formula was deduced to be C28H40O18. In the 1H NMR spectrum, the presence of a trans-propenyl alcohol was indicated by a pair of ole®nic signals at 6.55 (1H, d, J=16.0 Hz), 6.23 (1H, dt, J=16.0, 6.5 Hz), and a methylene doublet at 4.71 (2H, d, J=6.5 Hz). Two meta-coupled aromatic protons of the 3,4,5-trisubstituted phenyl residue
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00423-4
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appeared at 6.61 (2H, d, J=3.0 Hz). A three-proton singlet at 3.84 was characteristic of a methoxyl group placed on the aromatic nucleus. The remaining resonances, a methyl signal at 1.51 (3H, s), the four protons at 2.99 (1H, d, J=15.5 Hz), 2.91 (1H, d, J=15.5 Hz) and 2.85 (2H, d, J=6.5 Hz) corresponding to two methylenes, together with the carbonyl signal at 172.46 in the 13C NMR spectrum, demonstrated the presence of an ester derivative of a 3-hydroxyl-3methylglutarate moiety (HMG) (Mizutani et al., 1988). The diacid moiety was found to be bound to C-a through an ester linkage, by comparing the 1H NMR shifts of the methyleneoxy protons H2-a with those reported for esteri®ed derivatives (Mizutani et al., 1988; Yuda et al., 1990). Sugar moieties were identi®ed as two glucoses, by comparing 13C NMR data with that of the literature (Harborne and Mabry, 1982). This information
suggested that 5 was a ``tangshenoside type'' phenylpropanoid derivative (Mizutani et al., 1988; Yuda et al., 1990). Comparison of the spectral data of 5 with those of tangshenoside I (Mizutani et al., 1988) revealed that there was only one methoxyl group in compound 5 instead of two (in positions C-3 and C-5) in the case of tangshenoside I. A similar compound, without the glucosyl moiety in position 30 , is known as wahlenbergioside (Ma et al., 1997). A GHMBC experiment con®rmed the two glucose and methoxyl group positions. The stereochemistry of the HMG moiety was not established. Thus, compound 5, named barbatoside A, is wahlenbergioside-30 -O-glucoside. The reaction of 6 with Godin reagent gave a grey-blue colour and the ®rst part of its UV spectrum showed the same absorption peaks as that of compound 5. This suggested that 6 was also a ``tangshenoside type''
M. Cuendet et al. / Phytochemistry 56 (2001) 631±636
phenylpropanoid derivative. The ES±MS spectrum gave a signal at m/z 823 and another at m/z 483, corresponding to the pseudomolecular ion [M H] and to the loss of the trans-propenyl alcohol moiety linked to a 3,4,5-trisubstituted phenyl residue [(M H)-340] , respectively. In the 1H and 13C NMR spectra, all signals corresponding to compound 5 were present. However, the GHSQC spectrum showed that the C-2000 was shifted down®eld and thus this position was esteri®ed. The substituent at C-2000 was identi®ed as trans-p-methoxycoumaric acid. The trans con®guration was determined from the coupling constant Jb0 g0 . Treatment with b-glucosidase cleaved the glucose moiety located at HO-C-4 to provide 6a. The presence of a second glucose residue was demonstrated by acidic hydrolysis of 6a and by TLC comparison of the sugar with an authentic sample. Thus, compound 6, named barbatoside B, is wahlenbergioside-30 -O-(2000 -(p-methoxycinnamoyl))-glucoside. The UV spectrum of 7 was almost the same as that of 6. Moreover, this substance also had a grey-blue colour when sprayed on TLC with Godin's reagent. This suggested a structural relationship between 6 and compound 7. A molecular mass of 810 amu was deduced from the ES±MS spectrum which exhibited a quasimolecular ion [M+Na]+ at m/z 833. Moreover, the peak at m/z 493 corresponded to the loss of the trans-propenyl alcohol moiety linked to a 3,4,5-trisubstituted phenyl residue [(M+Na)-340]+. The 1H and 13C NMR spectra of 7 were very similar to those of 6, with the exception of the signal for the methoxyl group OCH3±C-400 , which was missing in 7. After enzymatic hydrolysis of 7 with b-glucosidase followed by acid hydrolysis, it was possible to conclude the presence of two glucosyl moieties, as in 6. However, the p-coumaroyl group was linked at position C-4 of the C-30 sugar because of the down®eld shift observed for this proton in the 1H NMR spectrum. Thus, compound 7, named barbatoside C, is wahlenbergioside-30 -O- (4000 (p-coumaroyl))- glucoside (trans con®guration). Compound 8 was separated from 7 by semipreparative HPLC. Compound 7 and 8 showed the same UV and MS spectra. However, in 8, the p-coumaroyl moiety was found to possess a cis con®guration, from the 1H NMR signals of H-b and H-g which appeared as two d with a coupling constant of 12.5 Hz. Thus, compound 8, named barbatoside D, is wahlenbergioside-30 -O-(4000 -(p-coumaroyl))-glucoside (cis con®guration). Phenylpropanoid derivatives with structures similar to compounds 5±8 have already been found in the genera Codonopsis, Lobelia and Wahlenbergia in the Campanulaceae family (Ishimaru et al., 1991; Lee and Ko, 1992; Ma et al., 1997). On the other hand, none possessed the p-coumaroyl moiety attached to the glucose at position C-30 , as in compounds 6±8. The APCI±MS spectrum of 9 showed a molecular mass of 524 amu. A fragment observed at m/z 481 [(M H)-42]
633
corresponded to the loss of an acetyl moiety. The loss of a hexosyl group led to the peak at m/z 361 [(M±H)162] . The elimination of both the sugar and the acetyl moieties explained the detected fragment at m/z 319 [(M H)-204] . From the 1H and 13C NMR data, the molecular formula was established to be C24H28O13. The 1H NMR spectrum showed three signals in the low®eld region at 6.75 (1H, d, J=8.0 Hz), 6.80 (1H, dd, J=8.0, 1.5 Hz) and 7.06 (1H, d, J=1.5 Hz) indicative of a 3,4-dihydroxyphenyl moiety attached at C-2. Other signals at 6.16 (1H, s), 5.38 (1H, br. s), 5.01 (1H, s), 2.96 (1H, dd, J=17.5, 4.5 Hz), 2.84 (1H, dd, J=17.5, 2.0 Hz) corresponded to a chromane substituted in positions 3, 5, 7 and 8 (Adinol® et al., 1989; Sakar et al., 1993). The remaining signals were attributed to a methoxyl group at 3.79 (3H, s), to a methyl at 1.87 (3H, s), and to a glucosyl moiety. The 13C NMR spectrum allowed con®rmation of the catechin skeleton (Bennini et al., 1993). A signal at 172.01 for the carbonyl group of the acetyl moiety, together with a signal for the methyl group at 20.77 was observed. The linkage position of the acetyl moiety was at C-3 because of the down®eld shift of this carbon compared with an unsubstituted C-3 skeleton (Nonaka et al., 1983). A GHMBC experiment placed the methoxyl group in position C-5 and showed the glucosyl moiety to be linked to the hydroxyl at C-8. Shifts obtained in the 1H and 13C NMR spectra for positions C-2 and C-3 indicated that the stereochemistry of 9 was that of epicatechin (Shen et al., 1993; Harborne, 1994). Thus, compound 9, named barbato¯avan, is 3-acetyl-5-methoxy-7,30 ,40 -trihydroxy-8-O-glucoside-¯avan-3-ol. Compounds 5±9 were tested in a spectrophotometric assay for free radical scavenging activity, using DPPH. Quercetin and BHT [2,6-di(tert-butyl)-4-methylphenol] were used as reference compounds. Wahlenbergioside derivatives 5±8 were inactive. On the other hand, 9 had an activity similar to that of quercetin (Table 3). Antioxidant properties of catechins have been the subject of several studies due to their contribution to the antioxidant properties of black and green teas (Sera®ni et al., 1994). Green tea beverages have demonstrated protective eects against cardio-vasucular and liver diseases (Imai and Nakachi, 1995). Catechins were also found useful as food preservatives. These compounds prevent the formation of toxic oxygen species and peroxides in food (Nakayama et al., 1994). 3. Experimental 3.1. General TLC: Silica gel 60 F254 sheets (Merck); CHCl3± MeOH±H2O (65:35:5). Open column chromatography (CC): Sephadex LH-20 (Pharmacia) and silica gel
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Table 1 1 H NMR spectral data of compounds 5, 6, 7 and 8 (measured in CD3OD). Attributions are supported by GHSQC and GHMBC dataa Assignment
5
H-2 H-6 OCH3 H2-a H-b H-g Ha-20 Hb-20 Ha-40 Hb-40 H-60 H-200 H-300 H-500 H-600 OCH3 H-b0 H-g0 H-1000 H-2000 H-3000 H-4000 H-5000 Ha-6000 Hb-6000 H-10000 H-20000 H-30000 H-40000 H-50000 Ha-60000 Hb-60000
6.61 6.61 3.84 4.71 6.23 6.55 2.99 2.85 2.91 2.85 1.51
a
4.60 3.17 3.28 3.28 3.39 3.82 3.63 4.68 3.49 3.45 3.43 3.28 3.82 3.73
6 (1H, (1H, (3H, (2H, (1H, (1H, (1H, (1H, (1H, (1H, (3H,
(1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, J=3.0) d, J=3.0) s) d, J=6.5) dt, J=16.0, 6.5) d, J=16.0) d, J=15.5) d, J=6.5) d, J=15.5) d, J=6.5) s) ± ± ± ± ± ± ± d, J=8.0) dd, J=9.0, 8.0) m) m) m) dd, J=12.0, 2.5) dd, J=12.0, 4.5) d, J=8.0) m) m) m) m) d, J=12.0) dd, J=12.0, 4.5)
6.56 6.54 3.83 4.60 6.14 6.42 2.86 2.86 2.86 2.86 1.52 7.50 6.90 6.90 7.50 3.80 6.39 7.65 4.87 4.79 3.59
7 (1H, (1H, (3H, (2H, (1H, (1H, (1H, (1H, (1H, (1H, (3H, (1H, (1H, (1H, (1H, (3H, (1H, (1H, (1H, (1H, (1H,
d, J=2.0) d, J=2.0) s) d, J=6.0) dt, J=16.0, 6.0) d, J=16.0) m) m) m) m) s) d, J=8.5) d, J=8.5) d, J=8.5) d, J=8.5) s) d, J=16.0) d, J=16.0) d, J=7.5) t, J=7.5) t, J=9.0)
3.85 (1H, dd, J=12.0, 2.0) 3.67 (1H, dd, J=12.0, 4.5) 4.68 (1H, d, J=7.5)
3.80 (1H, dd, J=12.0, 2.5) 3.72 (1H, dd, J=12.0, 4.5)
6.63 6.61 3.85 4.73 6.26 6.57 2.89 2.89 2.98 2.98 1.50 7.47 6.81 6.81 7.47 ± 6.36 7.66 4.68
8 (1H, (1H, (3H, (2H, (1H, (1H, (1H, (1H, (1H, (1H, (3H, (1H, (1H, (1H, (1H,
d, J=1.5) d, J=1.5) s) d, J=6.5) dt, J=16.0, 6.5) d, J=16.0) m) m) m) m) s) d, J=9.0) d, J=9.0) d, J=9.0) d, J=9.0)
(1H, d, J=16.0) (1H, d, J=16.0) (1H, d, J=8.0)
6.63 6.61 3.85 4.73 6.26 6.57 2.89 2.89 2.98 2.98 1.50 7.67 6.75 6.75 7.67 ± 5.79 6.90 4.68
(1H, (1H, (3H, (2H, (1H, (1H, (1H, (1H, (1H, (1H, (3H, (1H, (1H, (1H, (1H,
d, J=1.5) d, J=1.5) s) d, J=6.5) dt, J=16.0, 6.5) d, J=16.0) m) m) m) m) s) d, J=9.0) d, J=9.0) d, J=9.0) d, J=9.0)
(1H, d, J=12.5) (1H, d, J=12.5) (1H, d, J=8.0)
3.65 (1H, t, J=9.5) 4.81 (1H, m)
3.65 (1H, t, J=9.5) 4.81 (1H, m)
3.58 (1H, d, J=12.0)
3.58 (1H, d, J=12.0)
4.68 (1H, d, J=8.0)
4.68 (1H, d, J=8.0)
3.42 (1H, t, J=9.0)
3.42 (1H, t, J=9.0)
3.79 (1H, dd, J=12.0, 2.5) 3.72 (1H, dd, J=12.0, 4.5)
3.79 (1H, dd, J=12.0, 2.5) 3.72 (1H, dd, J=12.0, 4.5)
Proton coupling constants (J in Hz), signal multiplicities and integrations are in parentheses.
(63±200 mm, Merck). Medium-pressure liquid chromatography (MPLC): home-packed LiChroprep-RP-18 column (15±25 mm, 46036 mm i.d., Merck). Anal. HPLC: Hewlett Packard 1050 instrument equipped with a photodiode array detector; Nucleosil RP-18 column (7 mm; 2504 mm i.d., Macherey±Nagel); MeCN±H2O (5:95!50:50 in 30 min, 0.05% CF3COOH, 1 ml/min). UV: Shimadzu UV-160A and Perkin-Elmer Lambda-3 spectrophotometers. Mp: Mettler FP-80/82 hot-stage apparatus; uncorrected. [a]20 D : Perkin-Elmer 241 polarimeter. 1H and 13C NMR: Varian Inova 500 spectrometer; in ppm rel. to TMS; J in Hz; carbon multiplicities from DEPT experiments. FAB±MS: Finnigan-MAT TSQ-700 triple-stage quadrupole instrument. ES±MS, APCI±MS: MAT-LCQ ion trap instrument. 3.2. Plant material The whole plant of Campanula barbata L. was collected in La Creusaz above Les Marecottes, Valais, Switzerland, in July 1996. A voucher specimen (No. 96169) has been deposited at the Institute of Pharmacognosy and Phytochemistry, University of Lausanne, Switzerland.
3.3. Extraction and isolation The dried whole plant (430 g) was ground and extracted at room temp. successively with CH2Cl2 (31500 ml) and MeOH (31500 ml) to yield 14 and 72 g of extract, respectively. A portion (50 g) of the MeOH extract was subjected to CC on silica gel eluted with CHCl3±MeOH (9:1±1:1) to provide 15 fractions (1±15). Fraction 4 contained pure kaempferol-3-O-glucoside (1; 45 mg). Fraction 6 was puri®ed by gel ®ltration on Sephadex LH-20 MeOH±H2O (1:1) to yield 9 (68 mg). Fraction 7 was also separated by ®ltration on Sephadex LH-20 MeOH to give kaempferol-3-O-rutinoside (2; 140 mg) and quercetin-3-O-glucoside (3; 65 mg). Two successive ®ltrations on Sephadex LH-20 (MeOH and MeOH±H2O 1:1) were made on fraction 8 to obtain fractions 8c1 7 and 8d1 6. 8c6 and 8d4 were then mixed and separated by semi-preparative HPLC (RP-18, MeCN±H2O 27:73, ¯ow rate 4 ml/min) aording 7 (3.5 mg) and 8 (1.5 mg). 6 (15 mg) was obtained after separation of fraction 10 by MPLC (RP-18, MeCN± H2O 15:85). Fraction 12 was separated by MPLC (RP18, MeOH±H2O 20:80) to yield 13 fractions. Fractions
M. Cuendet et al. / Phytochemistry 56 (2001) 631±636
635
12e and 12i contained pure compounds 5 (120 mg) and 4 (quercetin-3-O-rutinoside; 180 mg), respectively.
terminal glucose was obtained in the butanolic fraction.
3.4. Reduction of 2,2-diphenyl-1-picrylhydrazyl (2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl; DPPH) radical
3.6. Acid hydrolysis of 2, 4, 6 and 7
Thirty microlitres of a soln. containing the compound to be tested and 200 ml of MeOH were added to 50 ml of a 0.02% MeOH soln. of DPPH. Absorbance at 517 nm was determined after 30 min, and the percentage of activity was calculated. 3.5. Enzymatic hydrolysis of 3, 6 and 7 One milligramme of compound with 1 mg of b-glucosidase (Sigma G-0395) in 2 ml of buer (NaOAc 0.5 M), adjusted to pH 5 with acetic acid, was left at room temp. for 14 h; the reaction mixt. was partitioned successively with AcOEt and n-BuOH. The compound without the
Two milligrammes of pure compound (2 and 4), or the residue obtained in the butanolic extract after enzymatic hydrolysis (6 and 7), in 10 ml 2 N HCl aqueous solution was heated at 100 C for 3 h. The reaction mixture was diluted with water (5 ml) and successively extracted with AcOEt and BuOH. A TLC with organic fractions was run with reference substances to identify the aglycone. The aqueous phase, containing free sugars, was neutralised with NaHCO3 to pH 6, then dried. The residue was checked for sugars by TLC (comparison with authentic samples) using AcOEt±MeOH±H2O±HOAc (65:15:15:20) and anisidine phthalate as the detection reagent. The presence of glucose (2, 4, 6 and 7) and rhamnose (2 and 4) was con®rmed. 3.7. Wahlenbergioside-30 -O-glucoside (barbatoside A; 5)
Table 2 13 C NMR spectral data of compounds 5, 6, and 7 (measured in CD3OD). Attributions are supported by GHSQC and GHMBC data Carbon no.
5
6
7
1 2 3 4 5 6 OCH3 a b g 20 30 40 50 60 100 200 300 400 500 600 OCH3 a0 b0 g0 1000 2000 3000 4000 5000 6000 10000 20000 30000 40000 50000 60000
135.05 108.86 154.20 135.12 151.84 103.48 56.75 66.06 124.17 134.89 44.33 77.59 44.33 172.46 24.80 ± ± ± ± ± ± ± ± ± ± 98.22 75.04 77.65 71.43 77.89 62.75 106.69 75.28 77.51 70.78 78.24 62.05
135.11 108.92 154.22 135.17 151.88 103.44 56.75 65.89 124.47 134.32 44.59 77.82 44.59 171.86 24.19 128.39 130.98 115.43 163.11 115.43 130.98 55.88 167.93 116.47 146.23 96.57 75.18 76.39 71.62 77.88 62.71 106.79 75.35 77.68 70.83 78.33 62.08
135.12 108.91 154.28 135.21 151.94 103.48 56.75 66.09 124.19 134.99 44.28 77.68 44.28 172.45 24.85 127.15 131.26 116.85 161.44 116.85 131.26 ± 168.58 114.83 147.21 98.36 75.24 75.72 72.45 75.97 62.52 106.77 75.35 77.68 70.85 78.34 62.09
Amorphous white powder. Mp 115±118 C. [a]20 24 D (MeOH, c 0.3). UV lmax (MeOH) nm (log "): 221 (4.38), 266 (4.06). ES±MS (positive ion mode) m/z 687.3 [(M+H)+Na]+, 525.1 [(M+Na)-162]+, 363.1 [(M+Na)324]+. 1H and 13C NMR : Tables 1 and 2. 3.8. Wahlenbergioside- 30 -O-(2000 -(p-methoxycinnamoyl))glucoside (barbatoside B; 6) Amorphous white powder. Mp 124±125 C. [a]20 40 D (MeOH, c0.1). UV lmax (MeOH) nm (log "): 222 (4.69), 271 (4.43), 309 (4.33). ES±MS (negative ion mode) m/z 823.1 [M-H] , 483 [(M H)-340] . 1H and 13C NMR: Tables 1 and 2. 3.9. Wahlenbergioside-30 -O-(4000 -p-coumaroyl)-glucoside (trans) (barbatoside C; 7) Amorphous white powder. Mp 121±122 C. UV lmax (MeOH) nm (log "): 222 (5.71), 266 (4.48), 314 (4.32). ES±MS (positive ion mode) m/z 833.0 [(M+H)+Na]+, 493 [(M+Na)-340]+. 1H and 13C NMR: Tables 1 and 2. Table 3 Percentage of DPPH radical reduction in a spectrophotometric assay caused by compound 9 at dierent concentrations. Measurement at 517 nm, determination after 30 min Concentration (mM)
Quercetin
BHT
9
80.0 40.0 20.0 10.0 5.0 2.5
97.1 96.5 94.9 53.7 25.8 15.3
41.3 29.5 14.9 8.1 3.3 3.3
95.9 89.7 49.9 26.5 16.2 8.8
636
M. Cuendet et al. / Phytochemistry 56 (2001) 631±636
3.10. Wahlenbergioside-30 -O-(4000 -p-coumaroyl)glucoside (cis) (barbatoside D; 8) Amorphous white powder. 1H NMR: Table 1. 3.11. 3-Acetyl-5-methoxyl-7,30 ,40 -trihydroxyl-8-O-glucoside-¯avan-3-ol (barbato¯avan; 9) Amorphous cream powder. Mp 143±145 C. [a]20 78 D (MeOH, c 0.2). UV lmax (MeOH) nm (log "): 230 (sh) (4.02), 281 (3.54). APCI±MS (negative ion mode) m/z 523.1 [M H] , 481.3 [(M-H)-42] , 361.4 [(M-H)-162] , 319.5 [(M H)-204] . 1H NMR (CD3OD) : 7.06 (1H, d, J=1.5 Hz, H-20 ); 6.80 (1H, dd, J=8.0, 1.5 Hz, H-60 ); 6.75 (1H, d, J=8.0 Hz, H-50 ); 6.16 (1H, s, H-6); 5.38 (1H, br. s, H-3); 5.01 (1H, s, H-2); 4.68 (1H, d, J=7.5 Hz, H-100 ); 3.79 (3H, s, OCH3); 3.76 (2H, m, H-600 ); 3.39±3.48 (3H, non resolved, H-200 , H-300 , H-400 ); 3.24 (1H, m, H-500 ); 2.96 (1H, dd, J=17.5, 4.5 Hz, Ha-4); 2.84 (1H, dd, J=17.5, 2.0 Hz, Hb-4); 1.87 (3H, s, CH3). 13 C NMR (CD3OD): 172.01 (CO); 153.33 (C-7); 152.55 (C-5); 149.43 (C-9); 146.01 (C-40 ); 145.98 (C-30 ); 130.92 (C-10 ); 128.68 (C-8); 119.04 (C-60 ); 115.98 (C-50 ); 115.01 (C-20 ); 106.66 (C-100 ); 101.16 (C-10); 93.79 (C-6); 78.38 (C-2); 78.00 (C-500 ); 77.66 (C-300 ); 75.75 (C-200 ); 71.13 (C400 ); 69.33 (C-3); 62.51 (C-600 ); 56.83 (OCH3); 26.62 (C-4); 20.77 (CH3). Acknowledgements Financial support was provided by the Swiss National Science Foundation. References Adinol®, M., Aquila, T., Barone, G., Lanzetta, R., Parrilli, M., 1989. Homoiso¯avanones from Bellevalia romana. Phytochemistry 28, 3244±3246. Bennini, B., Chulia, A.J., Kaouadji, M., Delage, C., 1993. (2R,3R)Dihydro¯avonol aglycone and glycosides from Erica cinerea. Phytochemistry 33, 1233±1236.
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