NPC Natural Product Communications Flavonoids and their Qualitative Variation in Calystegia soldanella and Related Species (Convolvulaceae) 2015 Vol. 10 No. 3 429 - 432 Yoshinori Muraia,*, Hiroaki Setoguchib, Eiichiro Onoc and Tsukasa Iwashinaa a Department of Botany, National Museum of Nature and Science, Amakubo 4-1-1, Tsukuba, Ibaraki 305-0005, Japan Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606-8501, Japan c Research Institute, Suntory Global Innovation Center Ltd., Shimamoto, Mishima, Osaka 618-8503, Japan b murai@kahaku.go.jp Received: August 11th, 2014; Accepted: September 29th, 2014 Coastal species are exposed to severe environmental stresses, e.g. salt and UV-B. The plants adapt themselves to such harsh environment by controlling morphological features and chemical defense systems. Flavonoids are known as efficient anti-stress polyphenols produced by plants. Most flavonoids show antioxidant activity, and their properties are important for plants to survive under high-stress conditions such as those in a coastal area. Among the compounds, ortho-dihydroxylated flavonoids act as strong antioxidants. In this survey, we elucidated the flavonoid composition of a seashore species Calystegia soldanella, which is distributed not only on the seashore, but also by the inland freshwater lake, Lake Biwa. Seven flavonol glycosides, i.e. quercetin 3-Orutinoside, 3-O-glucoside, 3-O-rhamnoside and 3-O-apiosyl-(1→2)-[rhamnosyl-(1→6)-glucoside], and kaempferol 3-O-rutinoside, 3-O-glucoside and 3-Orhamnoside were isolated from the leaves of C. soldanella. In addition, it was shown that the quercetin (Qu) to kaempferol (Km) ratio of coastal populations was higher than that of lakeshore populations. In general, these differences of Qu/Km ratio depend on flavonoid 3’-hydroxylase (F3’H) transcription. RT-PCR analysis suggested that F3’H of C. soldanella is regulated translationally or post-translationally, but not transcriptionally. Furthermore, quantitative and qualitative differences in flavonoid composition occurred among three Calystegia species, C. soldanella, C. japonica and C. hederacea. Keywords: Calystegia soldanella, Coastal population, Flavonol glycosides, Inland population. Calystegia soldanella (L.) Roem. et Schult. (Convolvulaceae) is a perennial herbaceous species that has a wide distribution in the temperate zone. The species is mainly distributed on sandy seashores, but some populations occur rarely by an inland lake, Lake Biwa, in central Japan. This is a freshwater lake which harbors many coastal plants such as Vitex rotundifolia L.fil. (Verbenaceae), Lathyrus japonicus Willd. (Leguminosae), Arabis kawasakiana Makino (Brassicaceae), Dianthus japonicus Thunb. (Caryophyllaceae), Raphanus sativus L. var. raphanistroides Makino (Brassicaceae) and Pinus thunbergii Parl. [1]. Since this lake has been isolated from the coastal region ca. 4 million years ago, the plants growing around it have evolved specially. So this ancient lake is an useful site for studies of biodiversity, biogeography, ecology and evolution. The seashore species are exposed to harsh environmental stresses, e.g. sea water, wind, and intense sunlight, including UV radiation. It is presumed in some plants that the coastal populations acquire physiological and morphological traits which are different from those of inland populations [2, 3]. Such ecotypic divergence may occur as a consequence of adaptation to the severe environment. Flavonoids are one of the most effective anti-stress compounds that show several functions including anti-oxidation and UV protection [4, 5]. These compounds are especially accumulated in plants growing under harsh environments, such as high mountains and the seashore. For example, an alpine plant, the Himalayan Rheum nobile Hook. f. et Thoms. (Polygonaceae) accumulates quercetin glycosides such as quercetin 3-O-[6’’-(3-hydroxy-3-methylglutaroyl)-glucoside], which has an ortho-dihydroxyl group in ring B, which acts as an UV shield [6]. B-Ring ortho-dihydroxylated flavonoids are well produced in some plants at higher altitudes, which offer severe conditions (e.g. cold and intense UV-B) [7, 8]. They are stronger antioxidants than mono-hydroxylated flavonoids. Vitex rotundifolia which is a coastal species, but also grows by Lake Biwa, as well as C. soldanella, contain large amounts of luteolin glycosides, which are ortho-dihydroxylated flavonoids [9]. In Campanula punctata Lam. (Campanulaceae), a significantly high accumulation of flavonoids such as quercetin 3-O-glucoside was observed in coastal individuals [10]. In this paper, we describe flavonoid isolation and identification from the leaves of coastal and inland individuals of Calystegia soldanella. Furthermore, we quantitatively compared the flavonoid composition of them and their related species C. japonica Choisy (syn. C. pubescens Lindl.) and C. hederacea Wall. by HPLC, and surveyed the expression of related gene flavonoid 3’-hydroxylase (F3’H) between coastal and inland (lake) populations of C. soldanella and C. japonica. Flavonoid composition of Calystegia soldanella Seven flavonoids were obtained from the leaves of C. soldanella and identified as kaempferol 3-O-rutinoside, 3-O-glucoside and 3O-rhamnoside, and quercetin 3-O-rutinoside, 3-O-glucoside, 3-Orhamnoside and 3-O-apiosyl-(1→2)-[rhamnosyl-(1→6)-glucoside] (Table 1). This is the first record of these flavonol glycosides from this species. Quercetin 3-O-apiosyl-(1→2)-[rhamnosyl-(1→6)glucoside] is a rare compound previously isolated from the leaves of Solanum glaucophyllum Desf. (Solanaceae) [11] and the seeds of Chenopodium pallidicaule Aellen (Amaranthaceae) [12]. Of the isolated compounds, quercetin 3-O-rutinoside and 3-O-glucoside were major components in the coastal individuals. The lakeshore individuals mainly contained kaempferol 3-O-rutinoside and 3-Oglucoside together with the two quercetin glycosides mentioned above. Their aglycones were quercetin and kaempferol (Figure 1). In addition, all flavonoids isolated from the leaves of C. soldanella in this survey were flavonol 3-O-glycosides. However, Ahn et al. 430 Natural Product Communications Vol. 10 (3) 2015 Figure 1: Chemical structures of mono- and dihydroxylated flavonoids and their related enzyme F3´H. Figure 2: The ratio of quercetin (Qu) and kaempferol (Km) between coastal and inland populations of Calystegia soldanella. [13] have surveyed the flavonoid composition of C. soldanella in a coastal population in Korea and reported three flavonols, i.e. kaempferol 3-O-galactoside-7-O-glucoside, 3-O-digalactoside-7-Oglucoside and 3,7-di-O-galactoside from the leaves (Table 1). These flavonoids were qualitatively different from those of Japanese populations. It is of particular interest that intraspecific variation in flavonoid composition occurs in the plants from Japan and Korea. Low genetic differentiation, which was affected by long-distance seed dispersal, has been generally estimated in C. soldanella [14, 15]. However, it is likely that genetic structure, especially affecting glycosylation at the 7-position of flavonols, is varied between the Japan and Korea populations. Further studies, i.e. gene phylogeny and chemical profiling of the populations of Japan, Korea and other regions, including Europe, are needed to explain this issue. Quercetin to kaempferol ratio The flavonoid composition surveyed was qualitatively the same for the Japanese populations from the Pacific Ocean coast, the Sea of Japan coast, and the inland Lake Biwa. However, quantitative flavonoid variation occurred between the three sites. Though the total quercetin/total kaempferol ratio of the two seashore sites was similar (4.5:1～10.5:1), that of the inland Lake Biwa populations was 0.8:1 ～ 1.8:1. Thus coastal populations contained larger amounts of quercetin glycosides (Figure 2). In general, the antistress activity of B ring ortho-dihydroxyl flavonoids, such as quercetin, is stronger than that of mono-hydroxyl flavonoids, such as kaempferol. This anti-stress activity may be due to their antioxidant activity, because several environmental stresses, especially salt and UV-B, cause oxidative stress to plant cells [16]. Therefore, seashore C. soldanella populations chemically adapted to the environmental stress from sea water and/or UV irradiation by accumulation of quercetin glycosides. Murai et al. Flavonoid composition of related species, Calystegia japonica and C. hederacea We also surveyed the flavonoid composition of the inland species, C. japonica and C. hederacea. The former contained two major flavonoids, kaempferol 3-O-glucoside and 3-O-rutinoside, and five minor flavonoids, quercetin 3-O-glucoside and 3-O-rutinoside, and kaempferol 3-O-galactoside, 3-O-rhamnoside and 3-O-rutinoside (Table 1). The foliar flavonoids of C. japonica have previously been investigated by Ahn et al. [13], as well as those of C. soldanella. They reported two flavonol glycosides, quercetin 3-O-galactoside7-O-glucuronide and kaempferol 3-O-galactoside-7-O-glucoside. A Korean population of this species also contained flavonol 3,7-Oglycoside, as in the case of C. soldanella. However, the flavonoids of another Japanese population have been reported and four kaempferol glycosides, i.e. 3-O-glucoside, 3-O-galactoside, 3-Orhamnoside and 3-O-rutinoside were isolated from the aerial parts [17]. These results indicated that the glycosylation pattern of the two investigated species (C. soldanella and C. japonica) was clearly different between Japan and Korea. On the other hand, two major flavonol glycosides, i.e., quercetin 3O-galatoside and kaempferol 3-O-galactoside, and three minor ones, quercetin 3-O-glucoside, 3-O-arabinoside and kaempferol 3O-glucoside were isolated from the leaves of C. hederacea (Table 1). This is the first report of the presence of flavonol glycosides from this species. Aglycones of major flavonoids from Japanese populations of C. hederacea were glycosylated with galactose. This character differs from those of the other two species. These results indicated that C. soldanella and C. japonica are closely related from the chemotaxonomic viewpoint of flavonoid composition. In addition, it is noteworthy that the flavonoid content of C. japonica and C. hederacea was considerably lower than that of C. soldanella. Low accumulation of efficient anti-stress flavonoids in C. japonica and C. hederacea might reflect that these inland plants are in a mild stress environment compared with the coastal ones. In other cases, a deciduous tree Vitex rotundifolia, which is also distributed in coastal region and by the inland lake, contained orthodihydroxylated luteolin glycosides with stronger anti-stress activity in their leaves [9]. However, there is no quantitative and qualitative difference between the seashore and lakeshore populations. These results indicate that the quantitative variation of flavonoids between coastal and inland lake populations varied in different plant species, and C. soldanella chemically adapted to each environmental condition. Salt treatment Chemical experiments by individuals of natural populations revealed that coastal populations of C. soldanella contained higher levels of quercetin glycosides compared with lakeshore populations. To test whether these quantitative differences occurred by (1) response to each environment or (2) were genetically fixed during the evolution, we surveyed the variation of flavonoids under salt stress conditions using the plants sampled from inland and coastal populations. We collected the plants from seashore and lakeshore populations and cultivated them with freshwater (control) and 200 mM NaCl in freshwater (salt treatment). There was no qualitative and quantitative difference in flavonoid composition between control and salt-treatment plants. As mentioned above, genetic differentiation of C. soldanella is relatively low due to frequent gene flow by seed dispersal [15, 16]. The Lake Biwa population, however, shows high differentiation from coastal populations in Japan in cpDNA haplotypes and nuclear DNA microsatellite loci [18], leaf blade thickness [3] and photosynthesis in response to salt stress [2]. These results indicated that Lake Biwa populations have Flavonoids from Calystegia soldanella and related species been isolated from coastal populations not only geographically but also genetically and they adapted to milder conditions in which they are not in need of such an amount of the anti-stress flavonoid, quercetin. Therefore, the Qu/Km ratio observed in coastal and inland populations was genetically fixed. These results indicated that Lake Biwa populations have been isolated from coastal populations not only geographically but also genetically and they adapted to milder conditions in which they are not in need of much of the anti-stress flavonoid, quercetin. Therefore, the Qu/Km ratio observed in coastal and inland populations was genetically fixed. Several environmental factors in the coastal region could influence the flavonoid metabolism. In addition, the plants would acquire adaptive traits to inhabit the coastal environment. One of the important morphological features is leaf thickness. Seashore and lakeshore populations of C. soldanella develop thicker and thinner leaves, respectively [3]. Figure 3: Gene expression of flavonoid 3’-hydroxylase (F3’H) in the leaves and petals of Calystegia soldanella and C. japonica. Seashore: Koshien-hama in Hyogo pref., Lakeshore: Maiami-hama, Inland: Shimamoto-cho in Osaka Pref. Flavonoid 3´-hydroxylase Flavonoid 3’-hydoxylase (F3’H) is the key cytochrome P450 enzyme responsible for ortho-dihydroxylated flavonoids since it catalyzes hydroxylation of the 3’-position of the B ring (orthohydroxylation) of flavonoids, leading to biosynthesis of quercetin and cyanidin (Figure 1). Flavonoid analysis shown above indicated that geographical segregation of C. soldanella promotes structural diversity of the B ring of flavonols. The ortho-dihydroxylation of flavonols was observed in petals as well as leaves of the seashore population. Furthermore, the ortho-dihydroxylated anthocyanin, cyanidin, was the dominant floral pigment in both seashore and lakeshore populations (data not shown). Therefore, leaf-specific reduction of the Qu/Km ratio specifically observed in the lakeshore population predicted that F3’H activity is compromised specifically in the leaves of lakeshore populations. To assess the involvement of F3’H in the structural differences of flavonoids observed among Calystegia plants, a reverse transcription-polymerase chain reaction (RT-PCR) was performed on the Calystegia F3’H gene. In all Calystegia plants, the F3’H gene was highly expressed in petals compared with leaves. However, no obvious difference was observed in expression level of Calystegia F3’H genes between seashore and lakeshore populations (Figure 3). Thus, this result suggests a possibility that enzymatic activity of Calystegia F3’H is regulated translationally or post-translationally. Conclusion In this study, we demonstrated that seashore populations of C. soldanella accumulated higher levels of the anti-stress flavonoid, quercetin, compared with the lakeshore populations. C. soldanella populations in Lake Biwa have been isolated from the coastal area, and released from the environmental constraint of the coastal region (seashore), and adapted to the inland environment, which is milder than that of the seashore. Therefore, their chemical profile is different from that of coastal populations. This is a striking case that Natural Product Communications Vol. 10 (3) 2015 431 geographical environment affects phytochemical metabolites, highlighting the plasticity of specialized metabolism of land plants. Experimental General: NMR spectra were measured in pyridine-d5 at 600 MHz (1H NMR) and 150 MHz (13C NMR). LC-ESI-MS were measured using a Senshu Pak PEGASIL ODS column (I.D. 2.0×150 mm, Senshu Scientific Co. Ltd., Japan) at a flow-rate of 0.2 mL min-1, eluting with HCOOH/MeCN/H2O (5:15:80), ESI+ 4.5 kV, ESI- 3.5 kV, 250Ԩ. Plant materials: Three populations of Calystegia soldanella were sampled from Pacific Ocean side (Sakajiri-kaigan, Takasu-hama and Misato-hama, Fukui Prefecture), Sea of Japan side (Uzuekaigan, Nishi-no-hama, Koji-ga-hama, Aichi Prefecture) and inland Lake Biwa (Maiami-hama, Sabae-hama, Shingai-hama, Shiga Prefecture). S. japonica and S. hederaceae were collected from the Tsukuba Botanical Garden, Tsukuba, and Ryugasaki City, Ibaraki Pref. Japan, respectively. Plant collections were carried out on sunny days in the summer of 2010. To minimize ontogenetic effects, mature leaves were randomly selected. Voucher specimens were deposited in the Herbarium of the Kyoto University, Japan (KYO), and National Museum of Nature and Science, Japan (TNS) Qualitative analysis: Fresh leaves (566 g) of C. soldanella were extracted with MeOH. The concentrated extracts were separated by preparative PC using the solvent systems, BAW (n-BuOH/HOAc/H2O = 4:1:5, upper phase), 15% HOAc and BEW (n-BuOH/EtOH/H2O = 4:1:2.2). The isolated flavonoids were purified by Sephadex LH-20 column chromatography. Seven flavonoids were isolated and identified by UV, LC-MS, acid hydrolysis, and direct TLC and HPLC comparisons with authentic samples. The sugar-sugar linkage of quercetin 3-O-apiosyl-(1→2)[rhamnosyl-(1→6)-glucoside] was determined by HMBC correlation in 1H and 13C NMR spectra. Quantitative analysis: Fresh leaves (5 g) of 90 Calystegia soldanella individuals were extracted with MeOH (40 mL) for quantitative HPLC analysis. After filtration, the extracts were directly applied to a Shimadzu HPLC system using a Senshu Pak PEGASIL ODS column (I.D. 6.0 × 150 mm), at a flow-rate of 1.0 mL min-1, detection: 190-400 nm and eluents: MeCN/H2O/H3PO4 (18:82:0.2). Flavonoid content in each individual was estimated from the peak area of each compound at a wavelength of 350 nm. Salt treatment: C. soldanella plants were collected from coastal and inland populations. The plants were transplanted and grown treated with 200 mM NaCl (watering per 3 days). Fresh leaves (2.5 g) of treated and untreated control plants (5 individuals in each treatment) were extracted with 20 mL MeOH. The extracts were applied to quantitative HPLC. Reverse transcription-polymerase chain reaction (RT-PCR): cDNAs were synthesized from the total RNA of each tissue with SuperScript III (Invitrogen). The PCR with ExTaq DNA polymerase (TaKaRaBio) was run at 94ºC for 3 min, followed by 32-38 cycles at 94ºC for 1 min, 58ºC for 1 min, and 72ºC for 2 min. RT-PCR was performed using specific primer sets for C. soldanella F3’H (AB563485) and C. japonica F3’H (synonym: C. pubescens (AB571798); Fw 5’-CACATTGCTTATAACTATCAAGA-3’ and Rv 5’-ATCACTTCAAAGTCATTCCCTTT-3’, and for Calystegia 16S rRNA (AB681270); Fw 5’GATTAGATAGTTGGTGAGGTAAC-3’ and Rv 5’ATTACTAGCGATTCCAGCTTCATAG-3’). PCR cycles were 432 Natural Product Communications Vol. 10 (3) 2015 determined to be 36 cycles for F3'H and 32 cycles for rRNA, respectively, since PCR-products were not saturated in our PCR condition in these cycles. Each PCR product was separated on a Murai et al. 0.8% agarose gel and detected by ethidium bromide staining. Amplified PCR-products were quantified by Analyze_Gels in ImageJ software version 1.43r (http://rsb.info.nih.gov/ij/). Table 1: Flavonoid composition of Calystegia soldanella and related species. Species (population) Major flavonoids Minor flavonoids C. soldanella Japan Seashore Lake Biwa Korea (Ahn et al.) [13] Qu 3Rut, Qu 3G Qu 3Rut, Qu 3G, Km 3Rut, Km 3G Km 3Gal7G, Km 3diGal7G, Km 3G7Gal Qu 3Rham, Km 3Rut, Km 3ApiRut, Km 3G, Km 3Rut Qu 3Rham, Km 3ApiRut, Km 3Rut C. japonica Japan (Tsukuba) Japan (Takagi et al.)* [17] Korea (Ahn et al.) [13] Km 3Rut, Km 3G Km 3Rut, Km 3G Km 3Gal7GC, Km 3Gal7G Qu 3Rut, Q 3G, Km 3Gal, Km 3Rham Km 3Gal, Km 3Rham C. hederacea Q 3Gal, Km 3Gal Q 3Ara, Q 3G, Km 3G Qu: quercetin, Km: kaempferol, G: glucoside, Gal: galactoside, GC: glucuronide, Rut: rutinoside (= 6-rhamnosylglucoside), ApiRut: apiosylrutinoside, Ara: arabinoside, Rham: rhamnoside. *Isolated from aerial parts. The amounts of flavonoids isolated from each species in this survey were estimated by HPLC survey. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] Kimura S. (1968) Flora of Ohmiensis. Hoikusha, Osaka. Noda A, Nomura N, Setoguchi H. (2009). Chlorophyll fluorescence in response to salt stress in the coastal plant Calystegia soldanella: a comparison between coastal and freshwater populations. Bulletin of the National Museum of Nature and Science, Series B, 35, 123-129. Setoguchi H, Yamada S, Sugimoto Y. (2009) Geographical differentiation in leaf thickness in the coastal plant Calystegia soldanella (Convolvulaceae): a comparison between coastal and freshwater lakeshore populations. Journal of Phytogeography and Taxonomy, 57, 89-93. Ryan KG, Swinny EE, Winefield C, Markham KR. (2001) Flavonoids and UV photoprotection in Arabidopsis mutants. Zeitschrift für Naturforschung, 56c, 745-754. Ryan KG, Swinny EE, Markham KR, Winefield C. (2002) Flavonoid gene expression and UV photoprotection in transgenic and mutant Petunia leaves. Phytochemistry, 59, 23-32. Iwashina T, Omori Y, Kitajima J, Akiyama S, Suzuki T, Ohba H. (2004) Flavonoids in translucent bracts of the Himalayan Rheum nobile (Polygonaceae) as ultraviolet shields. Journal of Plant Research, 117, 101-107. Murai Y, Takemura S, Takeda K, Kitajima J, Iwashina T. (2009) Altitudinal variation of UV-absorbing compounds in Plantago asiatica. Biochemical Systematics and Ecology, 37, 378-384. Murai Y, Iwashina T. (2010) Flavonol glucuronides from Geum calthifolium var. nipponicum and Sieversia pentapetala (Rosaceae). Biochemical Systematics and Ecology, 38, 1081-1082. Iwashina T, Setoguchi H, Kitajima J. (2011) Flavonoids from the leaves of Vitex rotundifolia (Verbenaceae), and their qualitative and quantitative comparison between coastal and inland populations. Bulletin of the National Museum of Nature and Science, Series B, 37, 87-94. Hashiba K, Iwashina T, Matsumoto S. (2006) Variation in the quality and quantity of flavonoids in the leaves of coastal and inland Campanula punctata. Biochemical Systematics and Ecology, 34, 854-861. Rappaportt I, Giacopello D, Seldes AM, Blanco MC, Deulofeu V. (1977) Phenolic glycosides from Solanum glaucophyllum: a new quercetin triglycoside containing D-apiose. Phytochemistry, 16, 1115-1116. Rastrelli L, Saturnino P, Schettino O, Dini A. (1995) Studies on the constituents of Chenopodium pallidicaule (Canihua) seeds. Isolation and characterization of two new flavonol glycosides. Journal of Agricultural and Food Chemistry, 43, 2020-2024. Ahn N-R, Ko J-M, Cha H-C. (2012) Comparison of flavonoid profiles between leaves and stems of Calystegia soldanella and Calystegia japonica. American Journal of Plant Science, 3, 1073-1076. Chung MG, Kim ST, Chung HG, Chung MS. (1995) Allozyme diversity in Korean populations of Calystegia soldanella and C. japonica (Convolvulaceae): Implications for conservation. Journal of Plant Biology, 38, 173-180. Arafeh R, Kadereit JW. (2006) Long-distance seed dispersal, clone longevity and lack of phylogeographical structure in the European distributional range of the coastal Calystegia soldanella (L.) R.Br. (Convolvulaceae). Journal of Biogeography, 33, 1461-1469. Mittler R. (2002) Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science, 7, 405-410. Takagi S, Yamaki M, Masuda K, Kubota M. (1977) Studies on the purgative drugs. IV. On the constituents of Calystegia japonica Choisy. Yakugaku Zasshi, 97, 1369-1371. Noda A, Mitsui Y, Ikeda H, Setoguchi H. (2011) Long-term isolation of the coastal plant Calystegia soldanella (Convolvulaceae) in ancient freshwater Lake Biwa, Japan. Biological Journal of the Linnean Society, 102, 51-66.