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Phenolic compounds from Iris rossii, and their chemotaxonomic and systematic significance
Biochemical Systematics and Ecology 44 (2012) 157–160
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Phenolic compounds from Iris rossii, and their chemotaxonomic and systematic significance Takayuki Mizuno a, *, Yudai Okuyama b, Tsukasa Iwashina a, b a b
United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan Department of Botany, National Museum of Nature and Science, Tsukuba, Ibaraki 305-0005, Japan
a r t i c l e i n f o Article history: Received 29 December 2011 Accepted 28 April 2012 Available online xxx Keywords: Iris rossii Iridaceae Anthocyanin C-glycosylflavones Xanthones Phylogeny
1. Subject and source Iris rossii Baker (Iridaceae) is distributed from south–west Japan and Korea to north–central China. The plants are dwarf and the flowers are ca. 3.5–4.0 cm without crest-like appendages on the outer perianth segments (Satake, 1982). In Japan, this Iris is a relict and threatened plant that has recently declined due to plant hunting, change of land use and also ecological succession following grassland abandonment (Hayashi et al., 1980; Naito and Nakagoshi, 1995). Although the morphological and ecological features of I. rossii have been reported, chemotaxonomic and phylogenetic data are lacking. In this study, we isolated flavonoids including an anthocyanin and C-glycosylflavones, and xanthones from the flowers and leaves of I. rossii. Furthermore, we obtained the matK gene and trnK intron sequence data and determined the phylogenetic tree (Wilson, 2009), which contains 44 species, in which I. rossii is in section Limniris of the genus Iris. The material for chemotaxonomic and phylogenetic analysis was collected from live specimens, cultivated in the Tsukuba Botanical Garden, National Museum of Nature and Science, Japan. A voucher specimen of I. rossii (TM01) was deposited in the herbarium of the National Museum of Nature and Science, Japan (TNS). 2. Previous work Eight Iris species and three varieties are native or have been introduced to Japan (Satake, 1982). Flavonoids have been analyzed from the flowers and leaves of the Iris species Iris japonica Thunb. (Arisawa et al., 1973), Iris pseudacorus Fisch.
* Corresponding author. Tel.: þ81 29 851 5159; fax: þ81 29 853 8998. E-mail address: [email protected] (T. Mizuno). 0305-1978/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.bse.2012.04.022
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T. Mizuno et al. / Biochemical Systematics and Ecology 44 (2012) 157–160
(Williams et al., 1986), Iris gracilipes A. Gray (Hayashi et al., 1984), Iris setosa Pallas and its three varieties (Hayashi et al., 1989; Iwashina and Ootani, 1995), Iris laevigata Fisch. (Iwashina and Ootani, 1996) and Iris ensata Thunb. (Iwashina et al., 1996). Flavonoids of I. rossii have also been isolated, including the anthocyanin, delphinidin glycoside, the C-glycosylflavone, swertisin and the xanthone, mangiferin (Hayashi et al., 1980). Other phenolics have been detected in this species using paper chromatography, but these have not been isolated (Hayashi et al., 1980). The distribution of flavonoids in the genus Iris has been reviewed (Iwashina and Ootani, 1998; Wang et al., 2010).
3. Present study Fresh flowers (17.0 g) and fresh leaves (86.6 g) of I. rossii were extracted with HCOOH/MeOH (8:92) and MeOH in room temperature, respectively. After filtration and concentration, the each residue was resolved in HCOOH/MeOH (8:92) or MeOH, and was applied to preparative paper chromatography (PPC) using solvent systems: BAW (n-BuOH/HOAc/H2O ¼ 4:1:5, upper phase) and 15% HOAc. The crude isolated compounds were purified through Sephadex LH-20 column chromatography using solvent system: MeOH/HOAc/H2O (70:5:25) or 70% MeOH, and then applied to preparative HPLC, which was performed with a Tosoh HPLC system using Inertsil ODS-4 (I.D. 10 250 mm; GL Science Inc., Japan) at a flow rate of 3.0 ml/min; injection of 300–350 ml; detection wavelength of 530 or 340 nm; and eluent of FMW1 (HCOOH/MeCN/H2O ¼ 5:15:80) or FMW2 (HCOOH/ MeCN/H2O ¼ 0.2:18:82). Ten compounds (i.e. one anthocyanin, seven flavones and two xanthones) were obtained as pure solutions or powders. Of these isolated compounds, the anthocyanin (1, ca. 2 mg) was identified as delphinidin 3-O-(pcoumaroylrutinoside)-5-O-glucoside (delphanin) by UV–Vis spectrometry, LC-MS, characterization of alkaline and acid hydrolysates and HPLC comparison with an authentic sample from the fruits of Solanum melongena L. (Takeda et al., 1963). Seven C-glycosylflavones (2–8) and two xanthones (9 and 10) were identified as isoorientin (2, ca. 4 mg), swertiajaponin (3, ca. 2 mg), isovitexin (4, ca. 5 mg), swertisin (5, ca. 7 mg), schaftoside (6, ca. 2 mg), isoschaftoside (7, ca. 2 mg), apigenin 6,8-di-
Fig. 1. Chemical structures of anthocyanin (1), flavones (2–8) and xanthones (9 and 10).
T. Mizuno et al. / Biochemical Systematics and Ecology 44 (2012) 157–160
159
Fig. 2. A maximum likelihood (ML) tree based on chloroplast DNA sequences of matK and trnL-F regions (logL ¼ 7922.1, 2971). The tree has a topology almost identical to that of Wilson (2009), except for the inclusion of I. rossii, which forms a sister clade to series Chinenses.
C-arabinoside (8, ca. 3 mg) and mangiferin (9, ca. 7 mg) by UV spectrophotometry according to Mabry et al. (1970), LC-MS, direct TLC (BAW, 15% HOAc and BEW) and HPLC comparisons with authentic samples, i.e. isoorientin, isovitexin and swertisin from the leaves of I. laevigata (Iwashina and Ootani, 1996), swertiajaponin and mangiferin from the leaves of I. setosa var. nasuensis Hara (Hayashi et al., 1989), schaftoside and isoschaftoside from the aerial parts of Osyris alba L. (Santalaceae) (Iwashina et al., 2008) and apigenin 6,8-di-C-arabinoside from the leaves of Ajuga decumbens Thunb. (Lamiaceae) (Inomata et al., unpublished data). UV spectral properties of compound 10 (ca. 2 mg) were the same with those of mangiferin. However, protonated molecule peaks, m/z 465 [M þ H]þ and 463 [M þ H], appeared on LC-MS, showing the attachment of 1 mol acetic acid to mangiferin; thus, 10 was characterized as mangiferin X00-acetate. The chemical structures of the compounds are shown in Fig. 1. Total genomic DNA of I. rossii was directly extracted from the fresh leaves using the CTAB procedure for phylogenetic analysis (Doyle and Doyle, 1987). Nucleotide sequences of chloroplast matK, trnK and trnL-F regions were obtained using BigDye Terminator v3.0 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol on a 3130xl Genetic Analyzer (Applied Biosystems). In addition to the sequenced DNA of this species, 88 (44 þ 44) nucleotide sequences of corresponding chloroplast DNA regions of 44 species belonging to the section Limniris, genus Iris, were obtained from GenBank (Wilson, 2009). Alignment was conducted using ClustalW implemented in Seaview (Galtier et al., 1996) and obvious errors were corrected manually. Maximum likelihood (ML) trees were constructed using PhyML (Guindon and Gascuel, 2003) implemented in Seaview with heuristic search [using subtree-pruning and regrafting (SPR) algorithm for branch swapping]. To obtain branch support for the phylogenetic tree, bootstrap analysis was conducted with 300 replicates using the same tree search algorithm as the original tree search (Fig. 2). 4. Chemotaxonomic and systematic significance Ten compounds were isolated and characterized from the flowers and leaves of I. rossii. Of these, 1–5, 9 and 10 were detected in the flowers and 2, 4 and 6–10 in the leaves. An anthocyanin, delphanin (1) and four C-glycosylflavones, isoorientin (2), swertiajaponin (3), isovitexin (4) and swertisin (5), have been widely reported from the flowers and leaves of the genus Iris (Iwashina and Ootani, 1998; Wang et al., 2010). The C-glycosylxanthone, mangiferin (9), has also been found in many Iris
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T. Mizuno et al. / Biochemical Systematics and Ecology 44 (2012) 157–160
species (Iwashina and Ootani, 1996). However, three C-arabinosylflavones, schaftoside (6), isoschaftoside (7) and apigenin 6,8-di-C-arabinoside (8), were isolated from Iris for the first time (Iwashina and Ootani, 1998; Wang et al., 2010). These compounds may be characteristic C-glycosylflavones of I. rossii. Phylogeny revealed relationships between I. rossii and other Iris species; Iris odaesanensis Y. Lee, Iris koreana Nakai and Iris minutoaurea Makino, which do not grow in Japan, formed a monophyletic clade that has a sister relationship with I. rossii (Fig. 2). These species compose the series Chinenses of the genus Iris (Wilson, 2009) and are identical to the morphological classification (Brearley and Ellis, 1997). However, flavonoids have not been reported from other species in the series Chinenses. Further flavonoid investigations of the species are necessary for chemotaxonomy. Acknowledgments The authors thank to Mr. Haruhiko Kimura and Hisao Kaneko for plant materials and precious information. References Arisawa, M., Morita, N., Kondo, Y., Takemoto, T., 1973. Yakugaku Zasshi. 93, 1655. Brearley, C., Ellis, J.R., 1997. A Guide to Species Irises: Their Identification and Cultivation. The Press Syndicate of The University of Cambridge, p. 122. Doyle, J.J., Doyle, J.L., 1987. Phytochem. Bull. 19, 11. Galtier, N., Gouy, M., Gautier, C., 1996. Compt. Appl. Biosci. 12, 543. Guindon, S., Gascuel, O., 2003. Syst. Biol. 52, 696. Hayashi, K., Ootani, S., Iwashina, T., 1980. Sci, Rep. Res. Inst. Evolut. Biol. 1, 17. Hayashi, K., Iwashina, T., Kawasaki, M., Ootani, S., 1984. Sci, Rep. Res. Inst. Evolut. Biol. 2, 75. Hayashi, K., Ootani, S., Iwashina, T., 1989. Sci, Rep. Res. Inst. Evolut. Biol. 6, 30. Iwashina, T., Ootani, S., 1995. Ann. Tsukuba Bot. Gard. 14, 35. Iwashina, T., Ootani, S., 1996. Sci, Rep. Res. Inst. Evolut. Biol. 8, 41. Iwashina, T., Ootani, S., 1998. Ann. Tsukuba Bot. Gard. 17, 147. Iwashina, T., Kamenosono, K., Yabuya, T., 1996. J. Jap. Bot. 71, 281. Iwashina, T., López-Sáez, J.A., Kitajima, J., 2008. Biochem. Syst. Ecol. 36, 146. Mabry, T.J., Markham, K.R., Thomas, M.B., 1970. The Systematic Identification of Flavonoids. Springer, Berlin. Naito, K., Nakagoshi, N., 1995. J. Plant Res. 108, 477. Satake, Y., 1982. Wild Flowers of Japan, Herbaceous Plants (Including Dwarf Subshrubs), vol. 1. Heibonsha, Tokyo, p. 60. Takeda, K., Abe, Y., Hayashi, K., 1963. Proc. Jap. Acad. 39, 225. Wang, H., Cui, Y., Zhao, C., 2010. Mini Rev. Med. Chem. 10, 643. Williams, C.A., Harborne, J.B., Goldblatt, P., 1986. Phytochemistry 25, 2135. Wilson, C.A., 2009. Syst. Bot. 34, 277.
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Phenolic compounds from Iris rossii, and their chemotaxonomic and systematic significance Takayuki Mizuno a, *, Yudai Okuyama b, Tsukasa Iwashina a, b a b
United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan Department of Botany, National Museum of Nature and Science, Tsukuba, Ibaraki 305-0005, Japan
a r t i c l e i n f o Article history: Received 29 December 2011 Accepted 28 April 2012 Available online xxx Keywords: Iris rossii Iridaceae Anthocyanin C-glycosylflavones Xanthones Phylogeny
1. Subject and source Iris rossii Baker (Iridaceae) is distributed from south–west Japan and Korea to north–central China. The plants are dwarf and the flowers are ca. 3.5–4.0 cm without crest-like appendages on the outer perianth segments (Satake, 1982). In Japan, this Iris is a relict and threatened plant that has recently declined due to plant hunting, change of land use and also ecological succession following grassland abandonment (Hayashi et al., 1980; Naito and Nakagoshi, 1995). Although the morphological and ecological features of I. rossii have been reported, chemotaxonomic and phylogenetic data are lacking. In this study, we isolated flavonoids including an anthocyanin and C-glycosylflavones, and xanthones from the flowers and leaves of I. rossii. Furthermore, we obtained the matK gene and trnK intron sequence data and determined the phylogenetic tree (Wilson, 2009), which contains 44 species, in which I. rossii is in section Limniris of the genus Iris. The material for chemotaxonomic and phylogenetic analysis was collected from live specimens, cultivated in the Tsukuba Botanical Garden, National Museum of Nature and Science, Japan. A voucher specimen of I. rossii (TM01) was deposited in the herbarium of the National Museum of Nature and Science, Japan (TNS). 2. Previous work Eight Iris species and three varieties are native or have been introduced to Japan (Satake, 1982). Flavonoids have been analyzed from the flowers and leaves of the Iris species Iris japonica Thunb. (Arisawa et al., 1973), Iris pseudacorus Fisch.
* Corresponding author. Tel.: þ81 29 851 5159; fax: þ81 29 853 8998. E-mail address: [email protected] (T. Mizuno). 0305-1978/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.bse.2012.04.022
158
T. Mizuno et al. / Biochemical Systematics and Ecology 44 (2012) 157–160
(Williams et al., 1986), Iris gracilipes A. Gray (Hayashi et al., 1984), Iris setosa Pallas and its three varieties (Hayashi et al., 1989; Iwashina and Ootani, 1995), Iris laevigata Fisch. (Iwashina and Ootani, 1996) and Iris ensata Thunb. (Iwashina et al., 1996). Flavonoids of I. rossii have also been isolated, including the anthocyanin, delphinidin glycoside, the C-glycosylflavone, swertisin and the xanthone, mangiferin (Hayashi et al., 1980). Other phenolics have been detected in this species using paper chromatography, but these have not been isolated (Hayashi et al., 1980). The distribution of flavonoids in the genus Iris has been reviewed (Iwashina and Ootani, 1998; Wang et al., 2010).
3. Present study Fresh flowers (17.0 g) and fresh leaves (86.6 g) of I. rossii were extracted with HCOOH/MeOH (8:92) and MeOH in room temperature, respectively. After filtration and concentration, the each residue was resolved in HCOOH/MeOH (8:92) or MeOH, and was applied to preparative paper chromatography (PPC) using solvent systems: BAW (n-BuOH/HOAc/H2O ¼ 4:1:5, upper phase) and 15% HOAc. The crude isolated compounds were purified through Sephadex LH-20 column chromatography using solvent system: MeOH/HOAc/H2O (70:5:25) or 70% MeOH, and then applied to preparative HPLC, which was performed with a Tosoh HPLC system using Inertsil ODS-4 (I.D. 10 250 mm; GL Science Inc., Japan) at a flow rate of 3.0 ml/min; injection of 300–350 ml; detection wavelength of 530 or 340 nm; and eluent of FMW1 (HCOOH/MeCN/H2O ¼ 5:15:80) or FMW2 (HCOOH/ MeCN/H2O ¼ 0.2:18:82). Ten compounds (i.e. one anthocyanin, seven flavones and two xanthones) were obtained as pure solutions or powders. Of these isolated compounds, the anthocyanin (1, ca. 2 mg) was identified as delphinidin 3-O-(pcoumaroylrutinoside)-5-O-glucoside (delphanin) by UV–Vis spectrometry, LC-MS, characterization of alkaline and acid hydrolysates and HPLC comparison with an authentic sample from the fruits of Solanum melongena L. (Takeda et al., 1963). Seven C-glycosylflavones (2–8) and two xanthones (9 and 10) were identified as isoorientin (2, ca. 4 mg), swertiajaponin (3, ca. 2 mg), isovitexin (4, ca. 5 mg), swertisin (5, ca. 7 mg), schaftoside (6, ca. 2 mg), isoschaftoside (7, ca. 2 mg), apigenin 6,8-di-
Fig. 1. Chemical structures of anthocyanin (1), flavones (2–8) and xanthones (9 and 10).
T. Mizuno et al. / Biochemical Systematics and Ecology 44 (2012) 157–160
159
Fig. 2. A maximum likelihood (ML) tree based on chloroplast DNA sequences of matK and trnL-F regions (logL ¼ 7922.1, 2971). The tree has a topology almost identical to that of Wilson (2009), except for the inclusion of I. rossii, which forms a sister clade to series Chinenses.
C-arabinoside (8, ca. 3 mg) and mangiferin (9, ca. 7 mg) by UV spectrophotometry according to Mabry et al. (1970), LC-MS, direct TLC (BAW, 15% HOAc and BEW) and HPLC comparisons with authentic samples, i.e. isoorientin, isovitexin and swertisin from the leaves of I. laevigata (Iwashina and Ootani, 1996), swertiajaponin and mangiferin from the leaves of I. setosa var. nasuensis Hara (Hayashi et al., 1989), schaftoside and isoschaftoside from the aerial parts of Osyris alba L. (Santalaceae) (Iwashina et al., 2008) and apigenin 6,8-di-C-arabinoside from the leaves of Ajuga decumbens Thunb. (Lamiaceae) (Inomata et al., unpublished data). UV spectral properties of compound 10 (ca. 2 mg) were the same with those of mangiferin. However, protonated molecule peaks, m/z 465 [M þ H]þ and 463 [M þ H], appeared on LC-MS, showing the attachment of 1 mol acetic acid to mangiferin; thus, 10 was characterized as mangiferin X00-acetate. The chemical structures of the compounds are shown in Fig. 1. Total genomic DNA of I. rossii was directly extracted from the fresh leaves using the CTAB procedure for phylogenetic analysis (Doyle and Doyle, 1987). Nucleotide sequences of chloroplast matK, trnK and trnL-F regions were obtained using BigDye Terminator v3.0 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol on a 3130xl Genetic Analyzer (Applied Biosystems). In addition to the sequenced DNA of this species, 88 (44 þ 44) nucleotide sequences of corresponding chloroplast DNA regions of 44 species belonging to the section Limniris, genus Iris, were obtained from GenBank (Wilson, 2009). Alignment was conducted using ClustalW implemented in Seaview (Galtier et al., 1996) and obvious errors were corrected manually. Maximum likelihood (ML) trees were constructed using PhyML (Guindon and Gascuel, 2003) implemented in Seaview with heuristic search [using subtree-pruning and regrafting (SPR) algorithm for branch swapping]. To obtain branch support for the phylogenetic tree, bootstrap analysis was conducted with 300 replicates using the same tree search algorithm as the original tree search (Fig. 2). 4. Chemotaxonomic and systematic significance Ten compounds were isolated and characterized from the flowers and leaves of I. rossii. Of these, 1–5, 9 and 10 were detected in the flowers and 2, 4 and 6–10 in the leaves. An anthocyanin, delphanin (1) and four C-glycosylflavones, isoorientin (2), swertiajaponin (3), isovitexin (4) and swertisin (5), have been widely reported from the flowers and leaves of the genus Iris (Iwashina and Ootani, 1998; Wang et al., 2010). The C-glycosylxanthone, mangiferin (9), has also been found in many Iris
160
T. Mizuno et al. / Biochemical Systematics and Ecology 44 (2012) 157–160
species (Iwashina and Ootani, 1996). However, three C-arabinosylflavones, schaftoside (6), isoschaftoside (7) and apigenin 6,8-di-C-arabinoside (8), were isolated from Iris for the first time (Iwashina and Ootani, 1998; Wang et al., 2010). These compounds may be characteristic C-glycosylflavones of I. rossii. Phylogeny revealed relationships between I. rossii and other Iris species; Iris odaesanensis Y. Lee, Iris koreana Nakai and Iris minutoaurea Makino, which do not grow in Japan, formed a monophyletic clade that has a sister relationship with I. rossii (Fig. 2). These species compose the series Chinenses of the genus Iris (Wilson, 2009) and are identical to the morphological classification (Brearley and Ellis, 1997). However, flavonoids have not been reported from other species in the series Chinenses. Further flavonoid investigations of the species are necessary for chemotaxonomy. Acknowledgments The authors thank to Mr. Haruhiko Kimura and Hisao Kaneko for plant materials and precious information. References Arisawa, M., Morita, N., Kondo, Y., Takemoto, T., 1973. Yakugaku Zasshi. 93, 1655. Brearley, C., Ellis, J.R., 1997. A Guide to Species Irises: Their Identification and Cultivation. The Press Syndicate of The University of Cambridge, p. 122. Doyle, J.J., Doyle, J.L., 1987. Phytochem. Bull. 19, 11. Galtier, N., Gouy, M., Gautier, C., 1996. Compt. Appl. Biosci. 12, 543. Guindon, S., Gascuel, O., 2003. Syst. Biol. 52, 696. Hayashi, K., Ootani, S., Iwashina, T., 1980. Sci, Rep. Res. Inst. Evolut. Biol. 1, 17. Hayashi, K., Iwashina, T., Kawasaki, M., Ootani, S., 1984. Sci, Rep. Res. Inst. Evolut. Biol. 2, 75. Hayashi, K., Ootani, S., Iwashina, T., 1989. Sci, Rep. Res. Inst. Evolut. Biol. 6, 30. Iwashina, T., Ootani, S., 1995. Ann. Tsukuba Bot. Gard. 14, 35. Iwashina, T., Ootani, S., 1996. Sci, Rep. Res. Inst. Evolut. Biol. 8, 41. Iwashina, T., Ootani, S., 1998. Ann. Tsukuba Bot. Gard. 17, 147. Iwashina, T., Kamenosono, K., Yabuya, T., 1996. J. Jap. Bot. 71, 281. Iwashina, T., López-Sáez, J.A., Kitajima, J., 2008. Biochem. Syst. Ecol. 36, 146. Mabry, T.J., Markham, K.R., Thomas, M.B., 1970. The Systematic Identification of Flavonoids. Springer, Berlin. Naito, K., Nakagoshi, N., 1995. J. Plant Res. 108, 477. Satake, Y., 1982. Wild Flowers of Japan, Herbaceous Plants (Including Dwarf Subshrubs), vol. 1. Heibonsha, Tokyo, p. 60. Takeda, K., Abe, Y., Hayashi, K., 1963. Proc. Jap. Acad. 39, 225. Wang, H., Cui, Y., Zhao, C., 2010. Mini Rev. Med. Chem. 10, 643. Williams, C.A., Harborne, J.B., Goldblatt, P., 1986. Phytochemistry 25, 2135. Wilson, C.A., 2009. Syst. Bot. 34, 277.