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Flavonoids from the leaves of six Corylopsis species (Hamamelidaceae)
Biochemical Systematics and Ecology 44 (2012) 361–363
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Flavonoids from the leaves of six Corylopsis species (Hamamelidaceae) Tsukasa Iwashina a, *, Junichi Kitajima b, Tomoko Takemura c a b c
Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba, Ibaraki 305-0005, Japan Laboratory of Pharmacognosy, Showa Pharmaceutical University, Higashi-tamagawagakuen, 3 Machida, Tokyo 194-8543, Japan Invasive Species Research Project, National Institute for Agro-Environmental Sciences, 3-1-3 Kan-non-dai, Tsukuba, Ibaraki 305-8604, Japan
a r t i c l e i n f o Article history: Received 23 January 2012 Accepted 3 June 2012 Available online 6 July 2012 Keywords: Corylopsis Hamamelidaceae Dihydrochalcones Flavonols Flavonoids Chemotaxonomy
1. Subject and source The genus Corylopsis (Hamamelidaceae) is distributed to East Asia and the Himalayas, and consists of 26 species (Yamazaki, 1989). All the species bear yellow flowers in early spring. Six Corylopsis species, Corylopsis pauciflora Sieb. & Zucc., Corylopsis gotoana Makino, Corylopsis glabrescens Franch. & Savat., Corylopsis spicata Sieb. & Zucc., Corylopsis sinensis Hemsl. and Corylopsis coreana Uyeki, were used as plant materials in this survey. Of their species, C. gotoana and C. spicata are endemic to Japan, C. pauciflora and C. glabrescens are native to Japan and Korea, and C. sinensis is native to China (Morley and Chao,1977). C. pauciflora and C. spicata are widely cultivated in gardens and parks as ornamentals and cultivated in Tsukuba Botanical Garden, National Museum of Nature and Science, Tsukuba, Japan. C. glabrescens, C. sinensis and C. coreana are growing in Botanical Garden, Graduate School of Science, The University of Tokyo, Japan and C. gotoana in the Kochi Prefectural Makino Botanical Garden, Kochi, Japan. The voucher specimens were deposited in the herbarium of National Museum of Nature and Science (TNS), Japan. 2. Previous work Flavonoids in the leaves of some Corylopsis species have previously been surveyed. Quercetin 3-O-rhamnoside has been found in the leaves of C. glabrescens, C. pauciflora, Corylopsis platypetala Rehd. & Wils., C. sinensis, C. spicata and Corylopsis wilmottae Rehd. & Wils., and kaempferol 3-O-rhamnoside from former two species (Reznik and Egger, 1960; Egger and Reznik, 1961; Hegnauer, 1966). Myricetin has been reported from C. spicata, together with leucodelphinidin, leucocyanidin and ellagic acid (Hegnauer, 1966). Another phenolics, bergenin has been isolated from the leaves of C. glabrescens, C. pauciflora, C.
* Corresponding author. E-mail address: [email protected] (T. Iwashina). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.06.017
362
T. Iwashina et al. / Biochemical Systematics and Ecology 44 (2012) 361–363
Fig. 1. Chemical structures of the flavonols isolated from the leaves of Corylopsis species.
platypetala, C. sinensis, C. spicata and C. wilmottae (Fujise, 1959; Plouvier, 1961, 1963). Recently, a chalcone glycoside, chalcononaringenin 20 -O-glucoside was isolated from the flowers of C. pauciflora, C. gotoana, C. glabrescens, C. spicata, C. sinensis and C. coreana as a yellow pigment (Iwashina et al., 2009). However, foliar flavonoids have not been investigated previously by modern techniques such as HPLC. 3. Present study Fresh leaves of C. coreana (59 g), C. glabrescens (61 g), C. gotoana (48 g), C. pauciflora (322 g), C. sinensis (71 g) and C. spicata (208 g) were extracted with MeOH, respectively. After investigation of their flavonoid composition by HPLC, the concentrated extracts were applied to preparative paper chromatography using the solvent systems: BAW (n-BuOH/HOAc/H2O ¼ 4:1:5, upper phase), 15% HOAc and then BEW (n-BuOH/EtOH/H2O ¼ 4:1:2.2). Roughly isolated flavonoids were applied to preparative HPLC using the eluent: 20% MeCN, and purified by Sephadex LH-20 column chromatography using the solvent system: 70% MeOH. Nine flavonoids (1–9) were isolated from the leaves of Corylopsis species. Of their flavonoids, 1, 2, 4 and 5 were found in all six species. Flavonoids 2, 4 and 5 were identified as quercetin 3-O-rhamnoside (quercitrin), myricetin 3-Orhamnoside (myricitrin) and myricetin 3-O-glucoside by UV, LC–MS, characterization of acid hydrolyzates, and direct TLC and HPLC comparisons with authentic quercitrin (Extrasynthèse, Genay, France), myricitrin from the bark of Myrica rubra Sieb. & Zucc. (Myricaceae) (Hattori and Hayashi, 1931) and myricetin 3-O-glucoside from the fronds of Cyrtomium microindusium Sa. Kurata (Dryopteridaceae) (Iwashina et al., 2006). Flavonoid 1 was identified as kaempferol 3-O-rhamnoside by UV, LC–MS and acid hydrolysis. Flavonoid 3 was detected from all species except for C. glabrescens and identified as quercetin 3-O-glucoside (isoquercitrin) by UV, LC–MS, acid hydrolysis and direct TLC and HPLC comparison with authentic isoquercitrin from the aerial
Fig. 2. Chemical structures of the dihydrochalcones isolated from the leaves of Corylopsis species.
T. Iwashina et al. / Biochemical Systematics and Ecology 44 (2012) 361–363
363
Table 1 Distribution of the flavonoids from the leaves of six Corylopsis species. Species Series Pauciflorae C. pauciflora Series Corylopsis C. spicata C. gotoana C. glabrescens C. coreana C. sinensis
1
2
3
4
5
6
þþ
þþ
þ
þ
þ
t
þ þ þ þ þ
þþ þ þ þþ þþ
þ þ
þ þþ þþ þ t
þ þ t t t
þ þ
7
8
9
þþ
t
þ þþ
1, kaempferol 3-O-rhamnoside; 2, quercetin 3-O-rhamnoside; 3, quercetin 3-O-glucoside; 4, myricetin 3-O-rhamnoside; 5, myricetin 3-O-glucoside; 6, quercetin 30 -O-glucoside; 7, quercetin 3-O-rutinoside; 8, phloretin 40 -O-glucoside; 9, phloretin 40 -O-galloylglucoside; þþ, major compounds; þ, presence; t, trace.
parts of Osyris alba L. (Santalaceae) (Iwashina et al., 2008a). Flavonoid 6 was isolated from C. pauciflora as a minor compound and identified as quercetin 30 -O-glucoside by UV, LC–MS and acid hydrolysis. Flavonoid 7 was found in C. sinensis and C. coreana as a major compound and identified as quercetin 3-O-rutinoside (rutin) by UV, LC–MS, acid hydrolysis and direct TLC and HPLC comparison with authentic rutin from the leaves of Begonia fenicis Merr. (Begoniaceae) (Iwashina et al., 2008b). Major flavonoid 8 was isolated from C. pauciflora, together with minor compound 9. It was presumed by UV spectral properties that they are flavanones, dihydroflavonols or dihydrochalcones (Markham, 1982). Finally, flavonoid 8 was identified as a dihydrochalcone, phloretin 40 -O-b-glucopyranoside by UV, LC–MS, acid hydrolysis and 1H and 13C NMR. UV spectral properties of 9 were essentially the same with those of 8. It liberated phloretin 40 -O-glucoside by mild acid hydrolysis (in 1.2% HCl:MeOH ¼ 1:1, 100 C, 15 min). The attachment of 1 mol gallic acid to phloretin 40 -O-glucoside was shown by the appearance of a molecular ion peak, m/z 589 [M þ H]þ and fragment ion of m/z 275 (M of phloretin is 274, which is a loss of 314 from the molecular ion, representing galloylglucose) by LC–MS. Thus, flavonoid 9 was characterized as phloretin 40 -Ogalloylglucoside, which was found in nature for the first time. UV, LC–MS and HPLC properties of 9 were as follows. UV: lmax 277, 282sh, 326sh; þNaOMe 242, 288, 353; þAlCl3 302, 365; þAlCl3/HCl 277, 286, 297, 362; þNaOAc 276sh, 286, 327sh; þNaOAc/H3BO3 277sh, 284, 309sh. LC–MS: m/z 589 [M þ H]þ, 275 [M-314 þ H]þ. HPLC: tR 17.36 min (eluent: MeCN/ H2O/H3PO4 ¼ 22:78:0.2). 4. Chemotaxonomic significance Seven flavonol glycosides, kaempferol 3-O-rhamnoside (1), quercetin 3-O-rhamnoside (2), quercetin 3-O-glucoside (3), myricetin 3-O-rhamnoside (4), myricetin 3-O-glucoside (5), quercetin 30 -O-glucoside (6) and quercetin 3-O-rutinoside (7) (Fig. 1), and two dihydrochalcones, phloretin 40 -O-glucoside (8) and phloretin 40 -O-galloylglucoside (9) (Fig. 2), were found in the leaves of six Corylopsis species (Table 1). Of their flavonoids, kaempferol and quercetin 3-O-rhamnosides have been reported from the leaves of some Corylopsis species including C. glabrescens, C. pauciflora, C. sinensis and C. spicata (Reznik and Egger, 1960; Egger and Reznik, 1961; Hegnauer, 1966). Other flavonol glycosides and dihydrochalcones were found in Corylopsis species for the first time. Quercetin 3-O-rutinoside (7) was detected from C. sinensis and C. coreana which are native to China and Korea, but is not distributed in Japan. Dihydrochalcone, phloretin 40 -O-glucoside, was isolated from C. pauciflora, together with two minor flavonoids, phloretin 40 -O-galloylglucoside (9) and quercetin 30 -O-glucoside (6). C. pauciflora is the only species belonging to the series Pauciflorae. On the other hand, other five species are in another series Corylopsis (Yamazaki, 1989). Recently, a chalcone glycoside, chalcononaringenin 20 -O-glucoside (isosalipurposide), has been isolated from the yellow flowers of their Corylopsis species as major pigment. However, it could not be found in their leaves which were analyzed in this survey. Flavonoid characters of the genus Corylopsis have been shown to be flavonols such as kaempferol, quercetin and myricetin together with leucoanthocyanidins (now called proanthocyanidins) in leaves (Reznik and Egger, 1960; Egger and Reznik, 1961; Hegnauer, 1966) and chalcone in flowers (Iwashina et al., 2009). In this survey, a new flavonoid character, dihydrochalcone, was also found in the leaves of Corylopsis species. References Egger, K., Reznik, H., 1961. Planta 57, 239. Fujise, 1959. Bull. Chem. Soc. Jpn. 32, 97. Hattori, S., Hayashi, K., 1931. Acta Phytochim. 5, 213. Hegnauer, R., 1966. Chemotaxonomie der Pflanzen. Band 4. Dicotyledoneae: Daphniphyllaceae – Lythraceae. Birkhäuser, Basel. Iwashina, T., Kitajima, J., Matsumoto, S., 2006. Biochem. Syst. Ecol. 34, 14. Iwashina, T., López-Sáez, J.A., Kitajima, J., 2008a. Biochem. Syst. Ecol. 36, 146. Iwashina, T., Saito, Y., Peng, C.-I., Yokota, M., Kokubugata, G., 2008b. Bull. Natl. Mus. Nat. Sci. Ser. B 34, 175. Iwashina, T., Takemura, T., Mishio, T., 2009. J. Japan. Soc. Hort. Sci. 78, 485. Markham, K.R., 1982. Techniques of Flavonoid Identification. Academic Press, London. Morley, B., Chao, J.-M., 1977. J. Arnold Arbor. 58, 382. Plouvier, V., 1961. Compt. Rend. 252, 599. Plouvier, V., 1963. Compt. Rend. 257, 4061. Reznik, H., Egger, K., 1960. Z. Naturforsch. 15b, 247. Yamazaki, T., 1989. Hamamelidaceae. In: Wild Flowers of Japan. Woody Plants. Heibonsha, Tokyo, pp. 154–215.
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Flavonoids from the leaves of six Corylopsis species (Hamamelidaceae) Tsukasa Iwashina a, *, Junichi Kitajima b, Tomoko Takemura c a b c
Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba, Ibaraki 305-0005, Japan Laboratory of Pharmacognosy, Showa Pharmaceutical University, Higashi-tamagawagakuen, 3 Machida, Tokyo 194-8543, Japan Invasive Species Research Project, National Institute for Agro-Environmental Sciences, 3-1-3 Kan-non-dai, Tsukuba, Ibaraki 305-8604, Japan
a r t i c l e i n f o Article history: Received 23 January 2012 Accepted 3 June 2012 Available online 6 July 2012 Keywords: Corylopsis Hamamelidaceae Dihydrochalcones Flavonols Flavonoids Chemotaxonomy
1. Subject and source The genus Corylopsis (Hamamelidaceae) is distributed to East Asia and the Himalayas, and consists of 26 species (Yamazaki, 1989). All the species bear yellow flowers in early spring. Six Corylopsis species, Corylopsis pauciflora Sieb. & Zucc., Corylopsis gotoana Makino, Corylopsis glabrescens Franch. & Savat., Corylopsis spicata Sieb. & Zucc., Corylopsis sinensis Hemsl. and Corylopsis coreana Uyeki, were used as plant materials in this survey. Of their species, C. gotoana and C. spicata are endemic to Japan, C. pauciflora and C. glabrescens are native to Japan and Korea, and C. sinensis is native to China (Morley and Chao,1977). C. pauciflora and C. spicata are widely cultivated in gardens and parks as ornamentals and cultivated in Tsukuba Botanical Garden, National Museum of Nature and Science, Tsukuba, Japan. C. glabrescens, C. sinensis and C. coreana are growing in Botanical Garden, Graduate School of Science, The University of Tokyo, Japan and C. gotoana in the Kochi Prefectural Makino Botanical Garden, Kochi, Japan. The voucher specimens were deposited in the herbarium of National Museum of Nature and Science (TNS), Japan. 2. Previous work Flavonoids in the leaves of some Corylopsis species have previously been surveyed. Quercetin 3-O-rhamnoside has been found in the leaves of C. glabrescens, C. pauciflora, Corylopsis platypetala Rehd. & Wils., C. sinensis, C. spicata and Corylopsis wilmottae Rehd. & Wils., and kaempferol 3-O-rhamnoside from former two species (Reznik and Egger, 1960; Egger and Reznik, 1961; Hegnauer, 1966). Myricetin has been reported from C. spicata, together with leucodelphinidin, leucocyanidin and ellagic acid (Hegnauer, 1966). Another phenolics, bergenin has been isolated from the leaves of C. glabrescens, C. pauciflora, C.
* Corresponding author. E-mail address: [email protected] (T. Iwashina). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.06.017
362
T. Iwashina et al. / Biochemical Systematics and Ecology 44 (2012) 361–363
Fig. 1. Chemical structures of the flavonols isolated from the leaves of Corylopsis species.
platypetala, C. sinensis, C. spicata and C. wilmottae (Fujise, 1959; Plouvier, 1961, 1963). Recently, a chalcone glycoside, chalcononaringenin 20 -O-glucoside was isolated from the flowers of C. pauciflora, C. gotoana, C. glabrescens, C. spicata, C. sinensis and C. coreana as a yellow pigment (Iwashina et al., 2009). However, foliar flavonoids have not been investigated previously by modern techniques such as HPLC. 3. Present study Fresh leaves of C. coreana (59 g), C. glabrescens (61 g), C. gotoana (48 g), C. pauciflora (322 g), C. sinensis (71 g) and C. spicata (208 g) were extracted with MeOH, respectively. After investigation of their flavonoid composition by HPLC, the concentrated extracts were applied to preparative paper chromatography using the solvent systems: BAW (n-BuOH/HOAc/H2O ¼ 4:1:5, upper phase), 15% HOAc and then BEW (n-BuOH/EtOH/H2O ¼ 4:1:2.2). Roughly isolated flavonoids were applied to preparative HPLC using the eluent: 20% MeCN, and purified by Sephadex LH-20 column chromatography using the solvent system: 70% MeOH. Nine flavonoids (1–9) were isolated from the leaves of Corylopsis species. Of their flavonoids, 1, 2, 4 and 5 were found in all six species. Flavonoids 2, 4 and 5 were identified as quercetin 3-O-rhamnoside (quercitrin), myricetin 3-Orhamnoside (myricitrin) and myricetin 3-O-glucoside by UV, LC–MS, characterization of acid hydrolyzates, and direct TLC and HPLC comparisons with authentic quercitrin (Extrasynthèse, Genay, France), myricitrin from the bark of Myrica rubra Sieb. & Zucc. (Myricaceae) (Hattori and Hayashi, 1931) and myricetin 3-O-glucoside from the fronds of Cyrtomium microindusium Sa. Kurata (Dryopteridaceae) (Iwashina et al., 2006). Flavonoid 1 was identified as kaempferol 3-O-rhamnoside by UV, LC–MS and acid hydrolysis. Flavonoid 3 was detected from all species except for C. glabrescens and identified as quercetin 3-O-glucoside (isoquercitrin) by UV, LC–MS, acid hydrolysis and direct TLC and HPLC comparison with authentic isoquercitrin from the aerial
Fig. 2. Chemical structures of the dihydrochalcones isolated from the leaves of Corylopsis species.
T. Iwashina et al. / Biochemical Systematics and Ecology 44 (2012) 361–363
363
Table 1 Distribution of the flavonoids from the leaves of six Corylopsis species. Species Series Pauciflorae C. pauciflora Series Corylopsis C. spicata C. gotoana C. glabrescens C. coreana C. sinensis
1
2
3
4
5
6
þþ
þþ
þ
þ
þ
t
þ þ þ þ þ
þþ þ þ þþ þþ
þ þ
þ þþ þþ þ t
þ þ t t t
þ þ
7
8
9
þþ
t
þ þþ
1, kaempferol 3-O-rhamnoside; 2, quercetin 3-O-rhamnoside; 3, quercetin 3-O-glucoside; 4, myricetin 3-O-rhamnoside; 5, myricetin 3-O-glucoside; 6, quercetin 30 -O-glucoside; 7, quercetin 3-O-rutinoside; 8, phloretin 40 -O-glucoside; 9, phloretin 40 -O-galloylglucoside; þþ, major compounds; þ, presence; t, trace.
parts of Osyris alba L. (Santalaceae) (Iwashina et al., 2008a). Flavonoid 6 was isolated from C. pauciflora as a minor compound and identified as quercetin 30 -O-glucoside by UV, LC–MS and acid hydrolysis. Flavonoid 7 was found in C. sinensis and C. coreana as a major compound and identified as quercetin 3-O-rutinoside (rutin) by UV, LC–MS, acid hydrolysis and direct TLC and HPLC comparison with authentic rutin from the leaves of Begonia fenicis Merr. (Begoniaceae) (Iwashina et al., 2008b). Major flavonoid 8 was isolated from C. pauciflora, together with minor compound 9. It was presumed by UV spectral properties that they are flavanones, dihydroflavonols or dihydrochalcones (Markham, 1982). Finally, flavonoid 8 was identified as a dihydrochalcone, phloretin 40 -O-b-glucopyranoside by UV, LC–MS, acid hydrolysis and 1H and 13C NMR. UV spectral properties of 9 were essentially the same with those of 8. It liberated phloretin 40 -O-glucoside by mild acid hydrolysis (in 1.2% HCl:MeOH ¼ 1:1, 100 C, 15 min). The attachment of 1 mol gallic acid to phloretin 40 -O-glucoside was shown by the appearance of a molecular ion peak, m/z 589 [M þ H]þ and fragment ion of m/z 275 (M of phloretin is 274, which is a loss of 314 from the molecular ion, representing galloylglucose) by LC–MS. Thus, flavonoid 9 was characterized as phloretin 40 -Ogalloylglucoside, which was found in nature for the first time. UV, LC–MS and HPLC properties of 9 were as follows. UV: lmax 277, 282sh, 326sh; þNaOMe 242, 288, 353; þAlCl3 302, 365; þAlCl3/HCl 277, 286, 297, 362; þNaOAc 276sh, 286, 327sh; þNaOAc/H3BO3 277sh, 284, 309sh. LC–MS: m/z 589 [M þ H]þ, 275 [M-314 þ H]þ. HPLC: tR 17.36 min (eluent: MeCN/ H2O/H3PO4 ¼ 22:78:0.2). 4. Chemotaxonomic significance Seven flavonol glycosides, kaempferol 3-O-rhamnoside (1), quercetin 3-O-rhamnoside (2), quercetin 3-O-glucoside (3), myricetin 3-O-rhamnoside (4), myricetin 3-O-glucoside (5), quercetin 30 -O-glucoside (6) and quercetin 3-O-rutinoside (7) (Fig. 1), and two dihydrochalcones, phloretin 40 -O-glucoside (8) and phloretin 40 -O-galloylglucoside (9) (Fig. 2), were found in the leaves of six Corylopsis species (Table 1). Of their flavonoids, kaempferol and quercetin 3-O-rhamnosides have been reported from the leaves of some Corylopsis species including C. glabrescens, C. pauciflora, C. sinensis and C. spicata (Reznik and Egger, 1960; Egger and Reznik, 1961; Hegnauer, 1966). Other flavonol glycosides and dihydrochalcones were found in Corylopsis species for the first time. Quercetin 3-O-rutinoside (7) was detected from C. sinensis and C. coreana which are native to China and Korea, but is not distributed in Japan. Dihydrochalcone, phloretin 40 -O-glucoside, was isolated from C. pauciflora, together with two minor flavonoids, phloretin 40 -O-galloylglucoside (9) and quercetin 30 -O-glucoside (6). C. pauciflora is the only species belonging to the series Pauciflorae. On the other hand, other five species are in another series Corylopsis (Yamazaki, 1989). Recently, a chalcone glycoside, chalcononaringenin 20 -O-glucoside (isosalipurposide), has been isolated from the yellow flowers of their Corylopsis species as major pigment. However, it could not be found in their leaves which were analyzed in this survey. Flavonoid characters of the genus Corylopsis have been shown to be flavonols such as kaempferol, quercetin and myricetin together with leucoanthocyanidins (now called proanthocyanidins) in leaves (Reznik and Egger, 1960; Egger and Reznik, 1961; Hegnauer, 1966) and chalcone in flowers (Iwashina et al., 2009). In this survey, a new flavonoid character, dihydrochalcone, was also found in the leaves of Corylopsis species. References Egger, K., Reznik, H., 1961. Planta 57, 239. Fujise, 1959. Bull. Chem. Soc. Jpn. 32, 97. Hattori, S., Hayashi, K., 1931. Acta Phytochim. 5, 213. Hegnauer, R., 1966. Chemotaxonomie der Pflanzen. Band 4. Dicotyledoneae: Daphniphyllaceae – Lythraceae. Birkhäuser, Basel. Iwashina, T., Kitajima, J., Matsumoto, S., 2006. Biochem. Syst. Ecol. 34, 14. Iwashina, T., López-Sáez, J.A., Kitajima, J., 2008a. Biochem. Syst. Ecol. 36, 146. Iwashina, T., Saito, Y., Peng, C.-I., Yokota, M., Kokubugata, G., 2008b. Bull. Natl. Mus. Nat. Sci. Ser. B 34, 175. Iwashina, T., Takemura, T., Mishio, T., 2009. J. Japan. Soc. Hort. Sci. 78, 485. Markham, K.R., 1982. Techniques of Flavonoid Identification. Academic Press, London. Morley, B., Chao, J.-M., 1977. J. Arnold Arbor. 58, 382. Plouvier, V., 1961. Compt. Rend. 252, 599. Plouvier, V., 1963. Compt. Rend. 257, 4061. Reznik, H., Egger, K., 1960. Z. Naturforsch. 15b, 247. Yamazaki, T., 1989. Hamamelidaceae. In: Wild Flowers of Japan. Woody Plants. Heibonsha, Tokyo, pp. 154–215.