Phytochemistry 58 (2001) 12291233 www.elsevier.

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A avonoid sulphate and other compounds from the roots of Centaurea bracteata
Guido Flamini*, Maria Pardini, Ivano Morelli
Dipartimento di Chimica Bioorganica e Biofarmacia,Universita di Pisa, Via Bonanno 33, 56126 Pisa, Italy Received 6 April 2001; received in revised form 26 July 2001

Abstract The roots of Centaurea bracteata Scop. (Asteraceae) have been studied for the rst time. Twenty-three compounds were isolated and identied, namely a sterol glucoside, two quinic acid derivatives, one sugar, and 19 avonoids (ve sulphates), one of which, centaradixin, resulted in a new natural product. Structural elucidation was performed mainly by means of FABMS, 1D and 2D NMR spectroscopy. NMR data of some sulphate avonoids are reported for the rst time. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Centaurea bracteata; Asteraceae; Flavonoids; Axillarin 7-sulphate; Centaradixin

1. Introduction Centaurea bracteata Scop. (Asteraceae), known in Italy as ordaliso bratteato is a herbaceous plant placed by Pignatti (1982) in the C. jacea group, together with 10 other species. The plant is not used in Italian folk medicine, although other species of the same genus (i.e. Centaurea cyanus and Centaurea scabiosa) are used against coughs and as ophthalmic drugs (Poletti, 1978) or vulneraries (Mazza, 2000). The studies on this genus deal mainly with sesquiterpene lactones, while avonoids are less investigated; only our previous study about its aerial parts is reported in literature (Flamini et al., 2001); the roots have never been studied at all. This paper deals with the isolation and characterisation of 23 substances belonging to various classes of compounds, in particular two quinic acid derivatives, a sterol glucoside, a monosaccharide, and 19 avonoids, one of which is a new natural products. Moreover, NMR data of some known avonoid sulphates are reported for the rst time.

2. Results and discussion From the n-hexane residue (RH), by repeated ash and p-TLC chromatographies, four compounds were
* Corresponding author. Tel.: +39-050-44074; fax: +39-050-43321. E-mail address: amini@farm.unipi.it (G. Flamini).

isolated, namely jaceidin, jacein, 6-hydroxyluteolin-6,40 dimethylether and centaureidin, which were identied by comparison of their physical and spectroscopic properties with literature reports (Roitman and James, 1985; Agrawal et al., 1989). Similarly, from the chloroform extract (RC), hispidulin, 6-hydroxykaempferol 3,6dimethylether and b-sitosterol 3-O-glucoside were obtained (Agrawal et al., 1989; Reher and Budensisky, 1992). Fractionation of the CHCl3MeOH (9:1) extract (RCM) by size-exclusion chromatography, column chromatography, and preparative TLC, led to the isolation of further eight pure compounds. These were axillarin, nepetin, 6-hydroxyluteolin-6,40 -dimethylether-7-O-glucoside, jacesiolin, centaurein, nepetin 7-glucoside, 5caeoylquinic acid and fructose (Collado et al., 1985; Iwahashi et al., 1985; Barbera et al., 1986; Berghofer and Holzl, 1987; Wada et al., 1987; Agrawal et al., 1989), all identied by 1H and 13C NMR spectroscopy. The RMAc and RMBu residues, after size-exclusion chromatography and repeated Lobar RP-8, RP-18, ash and preparative TLC chromatographies, gave 3-caeoylquinic acid methyl ester, axillarin 7-glucoside, isokaempferide, nepetin 7-sulphate, patuletin 7-sulphate, hispidulin 7-sulphate, quercetin 3,30 -disulphate and the new avonoid sulphate, which we named centaradixin (1) Table 1. Known isolates were identied by comparison with literature data (Saleh et al., 1971; Redaelli et al., 1980; Iwahashi et al., 1985; Kubo et al., 1986; Ahmed and Mabry, 1987; Barron and Ibrahim,

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Table 1 13 C NMR data for compound 1 and patuletin and nepetin 7-sulphates (50 MHz)a Carbon 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 3-OMe 6-OMe
a b c d

1b 156.3 s 137.6 s 178.4 s 150.4 s 133.3 s 152.9d s 97.9 d 152.0d s 106.6 s 120.4 s 116.1 d 145.6 s 149.5 s 115.2 d 120.8 d 59.6 q 60.3 q

1c 159.0 s 139.5 s 180.5 s 152.3 s 135.7 s 153.8d s 100.6 d 153.0d s 109.3 s 122.7 s 116.6 d 146.4 s 150.2 s 116.6 d 122.8 d 60.5 q 61.7 q

Patuletin 7-sulphateb 147.9 s 135.7 s 176.2 s 150.1d s 132.4 s 152.7d s 97.7 d 151.3d s 105.3 s 121.8 s 115.7 d 145.2 s 149.9d s 114.6 d 120.4 d 60.2 q

Nepetin 7-sulphateb 164.4 s 102.4 d 182.3 s 151.4d s 133.2 s 152.8d s 98.3 d 152.6d s 106.2 s 120.5 s 116.3 d 146.4 s 151.3d s 113.1 d 119.3 d 60.2 q

Multiplicities deduced by DEPT experiments. Measured in DMSO-d6. Measured in CD3OD. Signals may be exchanged.

1987a; Nishizawa et al., 1988; Tomas-Barberan et al., 1987; Agrawal et al., 1989). Centaradixin (1) was obtained as a yellowish amorphous powder that appears on TLC as a yellow-orange arrow-shaped spot after treatment with Naturstoereagenz A-PEG. The negative FABMS spectrum gave a quasimolecular peak [MH] at 425 m/z, corresponding to the molecular formula C17H14O11S, supported also by elemental analysis (see Experimental); another signicant ion at m/z 448 ([M+NaH]) showed the presence of sodium in the sample. The 13C NMR spectrum showed 17 resonances, sorted by DEPT experiments into 4 CH, 2 CH3 and 11 quaternary C. In the 1H NMR spectrum obtained in DMSO-d6 a three-proton ABM system was present ( 7.56, d, J=2.0 Hz; 7.50, dd, J=8.3, 2.0 Hz; 6.90, d, J=8.3 Hz), typical of the 30 ,40 disubstituted ring B of a avonoid unit. Ring A showed only a resonance as one-proton singlet at  7.28. Moreover, two three-proton singlets were present at  3.73 and 3.78. The situation was very close to that of axillarin, apart from the signal of ring A that resulted deshielded. This circumstance was more evident in the 1 H NMR spectrum recorded in CD3OD, where the resonance of the ring A proton appeared at very low elds ( 7.36). Analysis of the 13C NMR spectrum conrmed the strange situation of ring A with respect to axillarin. Resonances of ring A carbons showed the typical shifts due to the presence of a sulphate group linked to the C-7 hydroxy group (Barron and Ibrahim, 1987b; Barron et al., 1988): 4.6 ppm upeld for the ipso carbon (C-7), 4.3 and 6.6 ppm downeld for the ortho

carbons (C-6 and C-8, respectively), and 3.1 ppm downeld for the para carbon (C-10), in DMSO-d6; in CD3OD the results are very similar (4.9, +3.1, +5.6 and +3.0 ppm, respectively). This situation is conrmed also by the large downeld shift (0.80 ppm) experienced by the proton attached to the alpha carbon (H-8). The high resonance value of the two methoxyl groups ( 59.6 and 60.3) suggested that both the ortho positions should be substituted, so they must be placed only at the 3 and 6 position (Agrawal et al., 1989). This situation is corroborated by the characteristic downeld shifts observed for C-2, C-3 and C-4 (ca. 10.5, 4.0 and 2.5 ppm, respectively) because the 3-OCH3, and the shifts experienced by C-5, C-6, C-7 and C-9 (ca. 9, +31, 10, 5 ppm, respectively) due to the 6-OCH3 with respect to quercetin (Agrawal et al., 1989) and by comparison with similar methoxy-substituted avonoids (Barbera et al., 1986; Agrawal et al., 1989) (Fig. 1). Moreover, a cross peak was evidenced in the NOESY spectrum between the 3-OCH3 and the H-20 and H-60 resonances. Therefore, 1 is axillarin 7-sulphate sodium salt, which we named centaradixin. This structure is supported by its negative FABMS spectrum where, besides the quasimolecular peak at m/z 425, [MH], and at m/z 448, [M+NaH], there was a peak due to loss of a sulphate group at m/z 345, [MH80]. Finally, the presence of sodium was conrmed by the yellow colour obtained by the ame assay; acid hydrolysis of 1 liberated axillarin and sulphate, identied by TLC with authentic samples and precipitation with barium chloride, respectively. Most of the known avonoid sulphates have been characterized only by UV and MS measurements, so we report their NMR data for the rst time. Summarising, the roots of C. bracteata yielded 23 compounds, among which 19 were avonoids, two caffeoylquinic derivatives, one sugar and one sterol glucoside. Most of the avonoids, according to the pattern of this genus, were polymethoxylated and three compounds have been obtained in great amounts (centaureidin 7800 mg, nepetin-7-glucoside 455 mg, and 6hydroxyluteolin-6,40 -dimethylether-7-glucoside 335 mg; 0.51, 0.03 and 0.02% with respect to the dry plant material). It is quite interesting to note that the main constituent of the roots was centaureidin (7800 mg),

Fig. 1. Signicant CH long range correlations observed in COLOC of compound 1.

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while in the aerial parts was its isomer jaceidin (3650 mg) (Flamini et al., 2001). Five of the avonoids, were sulphate conjugate; these compounds are relatively rare in nature (Williams and Harborne, 1994) and, among the Asteraceae, they are produced only by four tribes: Inuleae, Eupatorieae, Senecioneae and Heliantheae (Barron et al., 1988). We have reported the identication of another avonoid sulphate from the aerial parts of C. bracteata (Flamini et al., 2001). This study denitively conrms the presence of avonoid sulphate esters in the genus Centaurea and that now there are ve producing tribes among the Asteraceae, comprising also the Centaureinae. Flavonoid sulphates, generally, are obtained as potassium salts, while our isolates are all the less common sodium salts. Till now only the genus Lippia (Verbenaceae) seems to produce sodium salts, among which nepetin and hispidulin 7-sulphates (Tomas-Barberan et al., 1987); our study is the rst report, besides the new compound centaradixin (1), of patuletin 7-sulphate and quercetin 3,30 -disulphate sodium salts. Normally, these compounds have been obtained from the aerial parts; Varin et al. (1986) went into detail about Flaveria bidentis arming the presence of the highest amounts of avonoids sulphates in the buds, followed by the leaves, while in the roots they were completely absent. Therefore, the presence of ve avonoid sulphates in the roots of C. bracteata, besides the further one isolated from the aerial parts (Flamini et al., 2001), seems to be noteworthy. Really, on the contrary with respect to F. bidentis, C. bracteata prefers to synthetize/store these substances in the roots. Our ndings seem to conrm the previous observations on Brickellia spp. and Flaveria chloraefolia (Barron et al., 1988) about the competition between the glucosilation and sulphation reactions for the same aglycones. In fact we have observed the concomitant presence of the 7-glucosides and 7-sulphates, of axillarin and nepetin; moreover the aerial parts (Flamini et al., 2001) contained quercetin 3-glucoside-30 -sulphate and the roots quercetin 3,30 -disulphate. The large diusion of avonoid sulphates in water or hydrophyte plants (Tomas-Barberan et al., 1987; Barron et al., 1988; De Beck et al., 1998; Seabra and Correia Alves, 1988) seems to support the view that this conjugation could represent an ecological adaptation to the aquatic habitat. However our ndings, both on the aerial parts (Flamini et al., 2001) and the roots, seems to contradict this hypotesis, in fact C. bracteata has been collected in a very arid environment, the normal habitat according to Pignatti (1982), that describes this species as a xerophytic one. Flavonoids sulphates showed many biological activities, useful both to human health and plant physiology. They are enzymes inhibitors (Haraguchi et al., 1996), allergens (Sallusto et al., 2000), antioxidants (Yagi et al.,

1994), regulators of auxin transport (Faulkner and Rubery, 1992). Moreover, rutine sulfate was demonstrated to be not carcinogenic (Habs et al., 1984). The group of C. jacea to which C. bracteata belongs together with other ten species, was also considered as constituted by polymorphic forms of one (C. jacea) or two (C. jacea and C. nigrescens) species (Pignatti, 1982). Comparing the chemicals isolated from the other studied species of this group [C. jacea, C. nigrescens and C. pannonica (Bowie and Cameron, 1965; Bohlmann and Zdero, 1967; Wagner et al., 1969; Rosler et al., 1970; Monya, 1971; Kaj-a-Kamb et al., 1992)], we can see some convergences but also many dierences. Consequently, the separation of the species as suggested by Pignatti (1982) could be correct hypothesis. Sesquiterpene lactones, typical compounds of this genus, are not produced at all by C. bracteata. The same is true for the other studied species of this group. If this nding will be conrmed for the remaining species of the group, it could be of chemotaxonomical interest.

3. Experimental 3.1. General Melting points (uncorrected) were determined with a Koer apparatus; optical rotations were measured on a Perkin-Elmer 241 polarimeter; FABMS were recorded, in the negative mode, with a VG ZAB instrument; 1H and 13C NMR spectra were obtained with a Bruker AC200 spectrometer in CD3OD, DMSO-d6, and CDCl3, using TMS as internal standard. All 1D and 2D NMR experiments were performed using the standard Bruker library of microprograms. The following adsorbents were used for purication: ash chromatography, Merck Kieselgel 60 (230410 mesh); low-pressure chromatography, Merck Lobar Lichroprep RP-8 (31 2.5 cm); size-exclusion chromatography, Pharmacia Fine Chemicals Sephadex LH-20; analytical TLC, Merck Kieselgel 60 F254 precoated plates; chromatograms were visualised under UV light at 254 and 366 nm and/or sprayed with Komarowsky or Cerium Sulphate or Naturstoereagenz A-PEG reagents. 3.2. Plant material The roots were collected during full bloom at Montelaterone, Amiata Mount, South Tuscany, Italy, at 600 m above the sea level, in July 1999. A voucher specimen is deposited in PI (Herbarium of the University of Pisa). 3.3. Extraction and isolation The dried and ground roots (1540 g) were extracted, successively, in a Soxhlet apparatus with n-hexane,

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CHCl3, CHCl3MeOH (9:1) (6.0 l 35 h) and, at room temperature, with MeOH (7.0 l 7 days 3 times). After removal of solvents, in vacuo at up to 40  C, the following residues were obtained: RH (14.1 g), RC (14.7 g), RCM (67.1 g), and RM (18.4 g). RM was suspended in MeOHH2O (7:3) and extracted, in turn, with EtOAc and n-BuOH obtaining, after removal of the solvents, the residues RMAc and RMBu. The RH residue, after repeated SiO2 ash chromatographies and preparative TLCs, gave jaceidin (6 mg; yield 0.0004%), jacein (6 mg; yield 0.0004%), 6hydroxyluteolin-6,40 -dimethylether (3 mg; yield 0.0002%) and centaureidin (7800 mg; yield 0.51%). In a similar fashion, from the RC residue, hispidulin (18 mg; yield 0.001%), 6-hydroxykaempferol 3,6-dimethylether (8 mg; yield 0.0005%) and b-sitosterol-3-O-b-d-glucoside (29 mg; yield 0.02%) were obtained. The RCM residue was submitted to Sephadex LH-20 size-exclusion chromatography with MeOHCHCl3 (9:1) as eluent and the fractions were submitted to SiO2 column chromatographies and p-TLC, obtaining eight pure compounds: axillarin (48 mg; yield 0.02%), nepetin (20 mg; yield 0.0002%), 6-hydroxyluteolin-6,40 -dimethylether-7-O-b-d-glucopyranoside (335 mg; yield 0.03%), jacesiolin (13 mg; yield 0.0008%), centaurein (24 mg; yield 0.0002%), nepetin-7-O-b-d-glucopyranoside (455 mg; yield 0.03%), 5-caeoylquinic acid (11 mg; yield 0.0008%), and fructopyranose (18 mg; yield 0.001%). The RMAc residue was submitted to a Sephadex LH20 column with MeOH as eluent, obtaining 18 main fractions (AR). From these fractions, after repeated Lobar RP-8 and RP-18 chromatography, 3-caeoylquinic acid methyl ester (6 mg; yield 0.0004%), axillarin 7-O-b-d-glucopyranoside (7 mg; yield 0.0005%), isokaempferide (6 mg; yield 0.0004%), nepetin 7-sulphate (15 mg; yield 0.001%) and patuletin 7-sulphate (12 mg; yield 0.0008%) were isolated. RMBu was chromatographed over Sephadex LH-20 column with MeOH as eluent, obtaining 26 main fractions (AZ). Fraction H gave a precipitate constituted by pure quercetin 3,30 -disulphate (12 mg; yield 0.0008%). From fractions LM, by ash chromatography and SiO2 p-TLC, hispidulin 7-sulphate (6 mg; yield 0.0004%) was puried. Fraction K was submitted to p-TLC obtaining pure compound 1 (18 mg; yield 0.001%). 3.4. Centradixin (1) Yellowish amorphous solid; 1H NMR spectral data (DMSO-d6, 200 MHz)  3.73 (3H, s, 6-OMe), 3.78 (3H, s, 3-OMe), 6.90 (1H, d, J=8.3 Hz, H-50 ), 7.28 (1H, s, H8), 7.50 (1H, dd, J=8.3, 2.0 Hz, H-60 ), 7.56 (1H, d, J=2.0 Hz, H-20 ) ; 1H NMR (CD3OD, 200 MHz)  3.82 (3H, s, 3-OMe), 3.92 (3H, s, 6-OMe), 6.92 (1H, d, J=8.3 Hz, H-50 ), 7.36 (1H, s, H-8), 7.59 (1H, br d, J= 8.3 Hz,

H-60 ), 7.66 (1H, br s, H-20 ); 13C NMR data see Table 1; FABMS (negative mode) m/z 448 [M+NaH], 425 [MH], 345 [MH80]; elemental analysis: found: C 44.60%, H 3.44%, requires: C 47.89, H 3.31. 3.5. Patuletin 7-sulphate H NMR spectral data (DMSO-d6, 200 MHz)  3.74 (3H, s, 6-OMe), 6.87 (1H, d, J=8.3 Hz, H-50 ), 7.29 (1H, s, H-8), 7.65 (1H, dd, J=8.3, 2.0 Hz, H-60 ), 7.67 (1H, d, J=2.0 Hz, H-20 ). 3.6. Nepetin 7-sulphate H NMR spectral data (DMSO-d6, 200 MHz)  3.73 (3H, s, 6-OMe), 6.75 (1H, s, H-3), 6.86 (1H, d, J=8.8 Hz, H-50 ), 7.32 (1H, s, H-8), 7.45 (1H, m, H-60 ), 7.46 (1H, br s, H-20 ). 3.7. Hispidulin 7-sulphate H NMR spectral data (DMSO-d6, 200 MHz)  3.78 (3H, s, 6-OMe), 6.82 (1H, s, H-3), 6.91 (2H, d, J=8.8 Hz, H-20 and H-60 ), 7.32 (1H, s, H-8), 7.93 (2H, d, J=8.8 Hz, H-30 and H-50 ).
1 1 1

References
Ahmed, A.A., Mabry, T.J., 1987. Flavonoids from Iphiona scabra. Phytochemistry 26, 15171518. Agrawal, P.K., Takur, R.S., Bansal, M.C., 1989. Flavonoids. In: Agrawal P.K. (Ed.), Carbon-13 NMR of Flavonoids. Elsevier, Amsterdam, p. 159. Barbera, O., Marco, J.A., Sanz, J.F., Sanchez-Parareda, J., 1986. 3-Methoxyavones and coumarins from Artemisia incanescens. Phytochemistry 25, 23572360. Barron, D., Ibrahim, R.K., 1987a. Quercetin and patuletin 3,30 -disulfates from Flaveria chloraefolia. Phytochemistry 26, 11811184. Barron, D., Ibrahim, R.K., 1987b. Synthesis of avonoids sulfates: I. Stepwise sulfation of positions 2, 7, and 40 using N,N0 -dicyclohexylcarbodiimide and tetrabutylammonium hydrogen sulfate. Tetrahedron 43, 5197-5202. Barron, D., Varin, L., Ibrahim, R.K., Harborne, J.B., Williams, C.A., 1988. Sulphated avonoids-an update. Phytochemistry 27, 23752395. Berghofer, B., Holzl, J., 1987. Biavonoids in Hypericum perforatum; part I. Isolation of I3,II8-biapigenin. Planta Med. 53, 216218. Bohlmann, F., Zdero, C., 1967. Uber Flavone aus Centaurea-Arten. Tetrahedron Lett. 33, 32393242. Bowie, J.H., Cameron, D.W., 1965. Colouring matters of the Aphididae. Part XXV. A comparison of aphid constituents with those of their host plants. A glyceride of sorbic acid. J. Chem. Soc 5651 5657. Collado, I.G., Macias, F.A., Massanet, G.M., Luis, F.R., 1985. Flavonoids from Centaurea clementei. J. Nat. Prod 48, 819822. De Beck, P.O., Dijoux, M.-G., Cartier, G., Mariotte, A.-M., 1998. Quercitrin-30 -sulfate from leaves of Leea guinensis. Phytochemistry 47, 11711173. Faulkner, I.J., Rubery, P.H., 1992. Flavonoids and avonoid sulphates as probes of auxin-transport regulation in Cucurbita pepo hypocotyl segments and vesicles. Planta 186, 618625.

G. Flamini et al. / Phytochemistry 58 (2001) 12291233 Flamini, G., Antognoli, E., Morelli, I., 2001. Two new avonoids and other compounds from the aerial parts of Centaurea bracteata from Italy. Phytochemistry 57, 559564. Habs, M., Habs, H., Berger, M.R., Schmahl, D., 1984. Negative doseresponse study for carcinogenicity of orally administered rutin sulfate in Sprague-Dawley rats. Cancer Lett. 23, 103108. Haraguchi, H., Ohmi, I., Sakai, S., Fukuda, A., Toihara, Y., Fujimoto, T., Okamura, N., Yagi, A., 1996. Eect of Polygonum hydropiper sulfated avonoids on lens aldose reductase and related enzymes. J. Nat. Prod. 59, 443445. Iwahashi, H., Morishita, H., Osaka, N., Kido, R., 1985. 3-O-Feruloyl4-O-caeoylquinic acid from coee beans. Phytochemistry 24, 630 632. Ka j-a-Kamb, M., Amoros, M., Girre, L., 1992. Chimie et activites biologiques du genre Centaurea. Pharm. Acta Helv. 67, 178188. Kubo, M., Sasaki, H., Endo, T., Taguchi, H., Yoshioka, I., 1986. The constituents of Schizonepeta tenuifolia Briq. Structure of a new monoterpene glucoside, schizonepetoside C. Chem. Pharm. Bull. 34, 30973101. Mazza, F., 2000. Itinerari alla scoperta delle erbe ocinali del Monte Amiata. Stampa 2000 Ed., Abbadia S. Salvatore, Siena. Monya, M., 1971. Caeoylquinic derivative contents in plant species of the Centaurea genus. Farmacia 19, 4550. Nishizawa, M., Izuhara, R., Kaneko, K., Koshihara, Y., Fujimoto, Y., 1988. 5-Lipoxygenase inhibitors isolated from Gardeniae fructus. Chem. Pharm. Bull. 36, 8795. Pignatti, S., 1982. Flora d0 Italia. Edagricole, Bologna. Poletti, A., 1978. Fiori e Piante Medicinali. Musumeci Editore, Milano. Redaelli, C., Formentini, L., Santaniello, E., 1980. Apigenin 7-glucoside and its 200 - and 600 -acetates from ligulate owers of Matricaria chamomilla. Phytochemistry 19, 985986. Reher, G., Budensisky, M., 1992. Triterpenoids from plants of the Sanguisorbeae. Phytochemistry 31, 39093914. Roitman, J.N., James, L.F., 1985. Chemistry of toxic range plants.

1233

Highly oxygenated avonol methyl ethers from Gutierrezia microcephala. Phytochemistry 24, 835848. Rosler, H., Star, A.E., Mabry, T.J., 1970. New 6-methoxyavonols from Centaurea jacea. Phytochemistry 10, 450451. Saleh, N.A.M., Bohm, B.A., Orndu, R., 1971. Flavonoids from Lasthenia conjugens and Lasthenia fremonti. Phytochemistry 10, 611614. Sallusto, F., Poupot, R., Clergue, M., DePalma, R., Fournie, J.J., 2000. A avonoid sulfate antigen activates human alphabeta CD8+ Th2 lymphocytes in pollen allergy. Eur. J. Immunol. 30, 964968. Seabra, R.M., Correia Alves, A., 1988. Quercetin 3-glucuronide-30 sulphate from Hypericum elodes. Phytochemistry 27, 30193020. Tomas-Barberan, F.A., Harborne, J.B., Self, R., 1987. Twelve 6-oxygenated avone sulfates from Lippia nodiora and L. canescens. Phytochemistry 26, 22812284. Varin, L., Barron, D., Ibrahim, R.K., 1986. Identication and biosynthesis of glucosylated and sulphated avonols in Flaveria bidentis. Z. Naturforsch. C: Biosci. 41, 813819. Wada, H., Fujita, M., Murakami, T., Saikai, Y., Chen, C.M., 1987. Chemical and chemotaxonomical studies of ferns. LXXIII. New avonoids with modied B-ring from the genus Pseudophegopteris (Thelypteridaceae). Chem. Pharm. Bull 35, 47574762. Wagner, H., Horhammer, L., Hoer, R., Murakami, T., Farkas, L., 1969. Untersuchungen uber die Glykoside von Centaurea jacea L. Isolierung, Struktur und Synthese von 4,5,7-Trihydroxy-30 ,6-dimethoxy-avon-7-mono-b-d-glukopyranosid, einem neuen Flavonolglykosid aus den Wurzeln von Centaurea jacea L. Tetrahedron Lett. 39, 34113414. Williams, C.A., Harborne, J.B., 1994. Flavone and avonol glycosides. In: Harborne J.B. (Ed.), The Flavonoids: Advances in Research since 1986. Chapman and Hall, Cambridge, p. 344. Yagi, A., Uemura, T., Okamura, N., Haraguchi, H., Imoto, T., Hashimoto, K., 1994. Antioxidative sulphated avonoids in leaves of Polygonum hydropiper. Phytochemistry 35, 885887.