Flavonoids from the Leaves of Hydrastis (Hydrastidaceae): the Phytochemical Comparison with Glaucidium
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Bull. Natn. Sci. Mus., Tokyo, Ser. B, 32(1), pp. 29–33, March 22, 2006 Flavonoids from the Leaves of Hydrastis (Hydrastidaceae): the Phytochemical Comparison with Glaucidium Tsukasa Iwashina1 and Hiroshi Tobe2 1 Tsukuba Botanical Garden, National Science Museum, Amakubo 4–1–1, Tsukuba, 305–0005 Japan E-mail: [email protected] 2 Department of Botany, Graduate School of Science, Kyoto University, Kyoto, 606–8502 Japan E-mail: [email protected] Abstract Three flavonoids were isolated from the leaves of Hydrastis canadensis, together with another phenolic compound, chlorogenic acid. They were identified as quercetin 3-O-gentiobio- side, quercetin 3-O-galactoside and quercetin 3-O-glucoside by LC-MS, acid hydrolysis, and direct TLC and HPLC comparisons with authentic specimens. The flavonoids in the leaves of Glaucidi- um palmatum have previously been identified as three rare 3-O-allosides of quercetin, kaempferol and rhamnocitrin by Iwashina and Ootani (1990). It was proved that the flavonoids of both H. canadensis and G. palmatum were flavonol 3-O-hexosides, suggesting the phytochemical affinity between both genera. However, the glycosidic sugars of H. canadensis were the common glucose and galactose, and that of G. palmatum was the rare allose, showing intergeneric differences or ge- ographic isolation between the two genera. Key words: Hydrastis canadensis, Hydrastidaceae, Ranunculaceae sensu lato, flavonols, quercetin glycosides. (Gleye et al., 1974; Messana et al., 1980; Weber Introduction et al., 2003). D-Galactose and a ribitol-like sub- The genus Hydrastis (Hydrastidaceae, Ranu- stance have also been isolated from the same culaceae sensu lato) consists of only one species, organs (Iriki and Minamisawa, 1983). Recently, H. canadensis L., and is endemic to central and two rare flavonoids, 6,8-di-C-methylluteolin 7- northeastern America. The genus Hydrastis is methyl ether and 6-C-methylluteolin 7-methyl commonly treated as Ranunculaceae sensu lato ether, were isolated from commercially available (e.g., Cronquist, 1981). However, the genus was samples of the roots, along with b-sitosterol 3-O- recently classified into Hydrastidaceae with an- b-D-glucoside and some alkaloids described other genus Glaucidium, which is endemic to above (Hwang et al., 2003). However, the chemi- Japan and consists of only one species, G. palma- cal components in the leaves have not been re- tum Sieb.& Zucc. (Tobe, 2003). Their close affin- ported. ity was supported by strong molecular evidence In this paper, the isolation and identification of using 18S rDNA, rbcL and atpB gene sequences flavonoids in the leaves of H. canadensis were (Soltis et al., 2000). described, and the phytochemical relationship As chemical substances contained in H. with the genus Glaucidium, which have already canadensis, nine isoquinoline alkaloids, i.e., been reported its flavonoid composition by us berberine, b-hydrastine, canadine, canadaline, (Iwashina and Ootani, 1990), were chemotaxo- hydrastidine, isohydrastidine, (S)-corypalmine, nomically discussed. (S)-isocorypalmine and (S)-tetrahydropalmatine, have been isolated from the rhizomes and roots 30 Tsukasa Iwashina and Hiroshi Tobe Materials and Methods Quercetin 3-O-gentiobioside (1). TLC (cellu- lose): Rf 0.29 (BAW), 0.41 (BEW), 0.33 Plant materials (15%HOAc); color UV– dark purple, UV/NH – Plant materials were collected in Shannon 3 yellow. HPLC: retention time (Rt) 4.23 min. LC- County near Mountain View, Missouri, along the MS: m/z 625 [MϪH]Ϫ calcd. for C H O Jack’s Fork River in 1990, and cultivated in Shaw 27 30 17 (quercetinϩ2 mol glucose), 463 [MϪmonoglu- Nature Reserve, Missouri Botanical Garden, cosylϪH]Ϫ (quercetinϩ1 mol glucose) and 301 USA. [MϪdiglucosylϪH]Ϫ (quercetin). UV: lmax (nm) MeOH 257, 266sh, 358; ϩNaOMe 274, High performance liquid chromatography 328, 410 (inc.); ϩAlCl 273, 433; ϩAlCl /HCl (HPLC) 3 3 267, 301, 362, 395sh; ϩNaOAc 273, 326, 396; Flavonoid composition of the leaves was sur- ϩNaOAc/H BO 262, 379. veyed by HPLC using Shim-pack CLC-ODS 3 3 Mixture of quercetin 3-O-galactoside (2a) and (I.D. 6.0ϫ150 mm, Shimadzu), at flow-rate: quercetin 3-O-glucoside (2b). TLC (cellulose): 1.0 ml/min, injection: 10 ml, detection: 190–400 Rf 0.61 (BAW), 0.71 (BEW), 0.19 (15%HOAc); nm, and eluent: MeCN/H2O/H3PO4 (22 : 78 : 0.2). color UV– dark purple, UV/NH3–yellow. HPLC: Rt 5.47 (quercetin 3-O-galactoside) and Liquid chromatography-mass spectra (LC-MS) 5.61 (quercetin 3-O-glucoside). LC-MS was surveyed with Symmetry C col- 18 UV: lmax (nm) MeOH 257, 266sh, 360; umn (I.D. 2.1ϫ150 mm, Waters), at flow-rate: ϩNaOMe 273, 331, 410 (inc.); ϩAlCl 274, 0.18 ml/min, eluent: 15% MeCN→45% MeCN 3 ϩ Ϫ 434; ϩAlCl /HCl 267, 301, 363, 396sh; (30 min), ESI 3.5 kV, cone voltage 30 V, ESI 3 ϩNaOAc 274, 325, 392; ϩNaOAc/H BO 262, 3.5 kV, cone voltage 30 V, 400°C, ion energy 3 3 380. 1.0 V. Chlorogenic acid (3). HPLC: Rt 3.67 min. UV: lmax (nm) MeOH 244sh, 297sh, 328; Extraction and isolation of phenolic compounds ϩNaOMe 263, 310sh, 377 (inc.); ϩAlCl 257sh, Dry leaves (8.0 g) were extracted with MeOH. 3 316sh, 360; ϩAlCl /HCl 238, 297sh, 326; After filtration, the extracts were concentrated to 3 ϩNaOAc 296sh, 338, 376sh; ϩNaOAc/H BO a small volume and applied to prep. PC using 3 3 ϭ 256sh, 304sh, 349. solvent systems: BAW (n-BuOH/HOAc/H2O 4:1:5, upper phase), 15% HOAc and then BEW ϭ Results (n-BuOH/EtOH/H2O 4:1:2.2). Flavonoids were eluted with MeOH and purified by Three flavonoids were detected from the leaves Sephadex LH-20 column chromatography (sol- of Hydrastis canadensis by HPLC survey (Fig. 1) vent system: 70% MeOH). and isolated by various chromatographic man- ners. Flavonoid 1 produced quercetin and glu- Identification of phenolic compounds cose, which were characterized by direct TLC Flavonoids and phenolic acid were identified and HPLC comparisons with authentic speci- by UV spectroscopy using shift reagents (Mabry mens, by acid hydrolysis. Liquid chromatogra- et al., 1970), characterization of acid hy- phy-MS survey of 1 indicated the molecular ion, drolysates (aglycones and glycosidic sugars), m/z 625 [MϪH]Ϫ, showing the presence of LC-MS data, and finally direct TLC and HPLC quercetin and 2 mol glucose. The attachment of comparisons with authentic specimens. Thin- sugar to 3-hydroxyl group of quercetin was layer chromatographic, HPLC, LC-MS and UV shown by UV spectroscopy using shift reagents spectral properties of isolated phenolic com- (see Materials and Methods, Mabry et al., 1970). pounds were as follows. Finally, flavonoid 1 was identified as quercetin 3- Flavonoids from Hydrastis canadensis 31 Fig. 1. High performance liquid chromatogram of MeOH extract from the leaves of Hydrastis canadensis. 1ϭquercetin 3-O-gentiobioside, 2aϭquercetin 3-O-galactoside, 2bϭquercetin 3-O-glucoside, 3ϭchlorogenic acid, 4ϭisoqinoline alkaloids such as berberin, and other peaksϭunknown phenolic acids. O-glucosyl-(1→6)-glucoside, i.e., quercetin 3-O- quercitrin) and 3-O-gentiobioside (ϭglucosyl- gentiobioside (Fig. 2), by direct TLC and HPLC (1→6)-glucoside) were found from the leaves of comparison with authentic sample. Hydrastis canadensis. Those glycosides are com- It was shown by HPLC survey that flavonoid 2 mon flavonoids in the plant kingdom. On the is a mixture of two compounds. Quercetin, glu- other hand, the flavonoids in the leaves of Glau- cose and galactose were liberated by acid hydrol- cidium palmatum have previously been identified ysis. UV spectral properties showed that the as three rare 3-O-allosides of quercetin, compound was mixture of quercetin 3-O-glyco- kaempferol and rhamnocitrin (Iwashina and sides. It was shown by direct HPLC comparisons Ootani, 1990) (Fig. 2). Thus, it was proved that with authentic hyperin and isoquercitrin that the flavonoids of both H. canadensis and G. flavonoid 2 was the mixture of quercetin 3-O- palmatum were flavonol 3-O-hexosides, suggest- galactoside (2a) and quercetin 3-O-glucoside ing the phytochemical affinity between both gen- (2b) (Fig. 2). era. However, the glycosidic sugars of H. A phenolic acid 3 was also isolated from the canadensis were the common glucose and galac- leaves. It was identified as 3-caffeoylquinic acid, tose, and that of G. palmatum was the rare allose, i.e., chlorogenic acid, by UV spectral scopy and showing intergeneric differences or geographic direct HPLC comparison with authentic speci- isolation between the two genera. The presence men. Moreover, the peak of a major substance or absence of other chemical components in the appeared on HPLC chromatogram. It was charac- roots, e.g., isoquinoline alkaloids (presence in H. terized as some yellow isoquinoline alkaloids canadensis) (Gleye et al., 1974; Messana et al., such as berberin by UV spectral properties. 1980; Weber et al., 2003, Their alkaloids were also found from the leaves in this survey), C- Discussion methylflavones (presence in H. canadensis) (Hwang et al., 2003), and a coumarin, glaupalol In this survey, three quercetin glycosides, i.e., (presence in G. palmatum) (Irie et al., 1968; Ya- 3-O-galactoside (hyperin), 3-O-glucoside (iso- mamoto et al., 1971), also suggests strong inter- 32 Tsukasa Iwashina and Hiroshi Tobe Fig. 2. Chemical structures of the flavonoids isolated from the leaves of Hydrastis canadensis (left) and Glau- cidium palmatum (right). generic chemical differences between the Hy- tion of Flowering Plants. Columbia University Press, drastis and Glaucidium. New York, p.124. Gleye, J., A. Ahond & E. Stanislas, 1974. La canadaline: nouvel alcaloïde d’Hydrastis canadensis. Phytochem- Acknowledgements istry, 13 (3): 675–676. Hwang, B. Y., S. K. Roberts, L. R. Chadwick, C. D. Wu & The authors thank Mr. Scott Woodbury, Horti- A. D. Kinghorm, 2003. Antimicrobial constituents culture Supervisor, Shaw Nature Reserve, Mis- from goldenseal (the rhizomes of Hydrastis souri Botanical Garden, USA, for providing the canadensis) against selected oral pathogens.