Acylated Cyanidin 3,7-Diglucosides in the Red-purple Flowers of Sophronitis wittigiana (Orchidaceae)

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1 doi: jjshs1.CH-084 Acylated Cyanidin 3,7-Diglucosides in the Red-purple Flowers of Sophronitis wittigiana (Orchidaceae) Fumi Tatsuzawa 1 *, Norio Saito 2, Tomohisa Yukawa 3, Toshio Honda 4, Koichi Shinoda 5, Kazuhisa Kato 1 and Kazumitsu Miyoshi 6 1 Faculty of Agriculture, Iwate University, Morioka , Japan 2 Meiji-Gakuin University, Minato-ku, Tokyo , Japan 3 Tsukuba Botanical Garden, National Science Museum, Tsukuba , Japan 4 Faculty of Pharmaceutical Sciences, Hoshi University, Shinagawa-ku, Tokyo , Japan 5 NARO Hokkaido Agricultural Research Center, Sapporo , Japan 6 Faculty of Horticulture, Chiba University, Matsudo , Japan Three new acylated cyanidin 3,7-diglucosides (1 3) were isolated from the red-purple flowers of Sophronitis wittigiana as its main floral anthocyanins. These three pigments were based on cyanidin 3,7-diglucoside as the deacylanthocyanin, and their structures were determined to be cyanidin 3-O-[6-O-(malonyl)-b-glucopyranoside)]- 7-O-[6-O-(trans-caffeoyl)-b-glucopyranoside] as pigment 1, its demalonyl anthocyanin as pigment 2, and cyanidin 3-O-[6-O-(malonyl)-glucoside]-7-O-glucoside as pigment 3 by chemical and spectroscopic methods. On the other hand, five known acylated cyanidin 3,7,3ʹ-triglucosides (4 8) were isolated from orange-red or red flowers of S. acuensis, S. brevipedunculata, S. cernua, S. coccinea var. xanthoglossa, and S. grandiflora as well as those of S. coccinea, and identified to be pigment 4 as Sophronitis coccinea anthocyanin 1 (SCA 1), pigment 5 as SCA 2, pigment 6 as SCA 3, pigment 7 as SCA 4, and pigment 8 as SCA 5 in comparison of the TLC, HPLC, and UV- Vis properties with standard samples of SCAs 1 5. These results showed that the three 3,7,3ʹ-O-hydroxy groups of anthocyanins were all substituted with acylglucose andor glucose residues in the orange-red or red flowers of six taxa of Sophronitis, whereas the 3ʹ-O-hydroxy group was free from glucose in the red-purple flowers of S. wittigiana. Thus, the inactivation of 3ʹ-O-glycosylation in cyanidin units might be involved in causing the blue color direction shift from orange-red or red to red-purple flower color of S. wittigiana. Key Words: acylated cyanidin 3,7-diglucoside, Orchidaceae, red-purple flower color, Sophronitis wittigiana. Introduction Sophronitis species with yellow, orange-red, red, and pink colors are native to Brazil. They were introduced into other areas as ornamentals, which have been hybridized not only between species but also between genera such as Cattleya, Laelia, and Brassabola. Although the genus name Sophronitis was given by Lindley in 1828 (Withner, 1990), recently, the genus Sophronitis was combineded with the genus Laelia and transferred into the genus Cattleya (van den Berg, 2008). However, we herein use the independent generic status of Sophronitis Received; July 14, Accepted; September 9, First Published Online in J-STAGE on December 3, This work was supported in part by a Grant-in-Aid for Scientific Research (C) [No , to F.T.] from the Japan Society for the Promotion of Science (JSPS). * Corresponding author ( fumi@iwate-u.ac.jp). in accordance with Withner (1990). Nine species were proposed in this genus (Withner, 1990). In these species, except for S. wittigiana, most of the flowers are colored red or orange-red (Withner, 1990), and the flowers of S. wittigiana are red-purple. From pigment analysis of the orange-red flowers of S. coccinea, Matsui and Nakamura (1988) presumed that the red-purple flower color of S. wittigiana was only formed with anthocyanin pigments and in the absence of carotenoid pigments. Then, five acylated cyanidin 3,7,3ʹ-triglucosides were isolated from the orange-red flowers of S. coccinea (Tatsuzawa et al., 1998). In the present study, we found eight floral anthocyanin pigments, among which five known acylated cyanidin 3,7,3ʹ-triglucosides and three novel acylated cyanidin 3,7-diglucosides were observed in the red-purple, red, and orange-red flowers of seven taxa of Sophronitis species. Furthermore, three novel acylated cyanidin 3,7-diglucosides was confirmed to be 2014 The Japanese Society for Horticultural Science (JSHS), All right reserved

2 65 only distributed in the red-purple flowers of S. wittigiana as its main pigments. In this paper we report the occurrence of these three novel acylated cyanidin 3,7-diglucosides in the red-purple flowers of S. wittigiana, and discuss the blue color direction effect on the flower colors of Sophronitis plants. Materials and Methods General procedures Thin-layer chromatography (TLC) was conducted on cellulose-coated plastic sheets (Merck, Darmstadt, Germany) using seven mobile phases: BAW (n-buoh HOAcH 2 O. 4 : 1 : 2, vvv), BuHCl (n-buoh2n HCl, 1 : 1, vv, upper layer), AHW (HOAcHClH 2 O, 15 : 3 : 82, vvv), 1% HCl for anthocyanins, BAW and ETN (EtOHNH 4 OHH 2 O, 16 : 1 : 3, vvv) for organic acid, Forestal (HOAcHClH 2 O, 30 : 3 : 10, vvv) for anthocyanidin, and BAW and EAA (EtOAcHOAc H 2 O, 1 : 1 : 1, vvv) for sugars with detection using UV light andor the aniline hydrogen phthalate spray reagent (Harborne, 1984). Analytical HPLC was performed on an LC 10A system (Shimadzu, Kyoto, Japan), using a C18 ( mm) column (Waters, Milford, MA, USA) at 40 C with a flow rate of 1 ml min -1 and monitoring at 530 nm. The eluant was applied as a linear gradient elution for 40 min from 20 to 85% solvent B (1.5% H 3 PO 4, 20% HOAc, 25% MeCN in H 2 O) in solvent A (1.5% H 3 PO 4 in H 2 O) with 5 min of re-equilibration at 20% solvent B, for anthocyanins, anthocyanidins and hydroxycinnamic acids (method 1). The other eluant for malonic acid was applied as an isocratic elution of solvent A for 10 min and monitoring at 210 nm (Tatsuzawa et al., 2009) (method 2). UV-Vis spectra for purified anthocyanins were recorded on an MPS-2450 (Shimadzu) in 0.1% HCl-MeOH (from 200 to 700 nm). Spectral absorption of the fresh flower was directly measured on the intact petals using a recording spectrophotometer operated as double-beam instrument (MPS-2450) (Saito, 1967; Yokoi and Saito, 1973). Crude carotenoids were extracted from dried perianths (ca. 4 mg) of each plant by soaking in MeOH : acetone (1 : 1; vv) for 30 min at room temperature. UV-Vis spectra of the extracts were recorded on a MPS-2450 spectrophotometer ( nm). Fast atom bombardment mass spectra (FABMS) were obtained in positive ion mode using the magic bullet (5 : 1 mixture of dithiothreitol and dithioerythritol) as a matrix with JMS SX-102 (JEOL Ltd., Tokyo, Japan). NMR spectra were recorded on JMN GX-500 (JEOL Ltd.) at 500 MHz for 1 H spectra in CD 3 OD-CF 3 COOD (9 : 1). Chemical shifts are reported relative to a tetramethylsilane (TMS) internal standard (d), and coupling constants (J) are in Hz. Plant materials The flowers of Sophronitis brevipedunculata, S. coccinea, S. coccinea var. xanthoglossa, S. grandiflora, and S. wittigiana were gifts from the cultivator, Mr. Tamon Suzuki (Amiele Project, Gifu, Japan), and S. acuensis and S. cernua were grown in Tsukuba Botanical Garden, National Museum of Nature and Science, Tsukuba, Japan. The flower colors of the fresh perianths from flowers of each species were recorded by comparing them directly with the RHS Colour Chart (The Royal Horticultural Society, London, UK), and their CIE L* a* b* chromaticity values were recorded on a CM-2002 spectro color meter (Minolta Co., Ltd., Tokyo, Japan). Perianths were picked up by hand, air dried for 2 3 days at 40 C, and kept at -20 C until used. Analysis of anthocyanin distributions in the flowers of seven taxa of Sophronitis Dried perianths (each ca. 20 mg) of seven taxa were extracted with 1 ml MAW (MeOH-HOAc-H 2 O, 4 : 1 : 5, vvv) as the anthocyanin extracts. Quantitative analysis of these anthocyanin extracts was performed by HPLC (method 1). Isolation, purification, and structure determination of anthocyanins for the red-purple flowers of S. wittigiana Dried perianths (ca. 20 g) of S. wittigiana were immersed in 5% HOAc-H 2 O (5 L) at room temperature for 12 hrs and extracted. The extract was passed through a Diaion HP-20 Ion Exchange Resin (Mitsubishi Chemical, Tokyo, Japan) column ( mm), on which anthocyanins were absorbed. The column was then thoroughly washed with 5% HOAc-H 2 O (20 L) and eluted with 5% HOAc-MeOH (500 ml) to recover the anthocyanins. After concentration, the eluates were separated and purified with paper chromatography (No. 590; ADVANTEC, Tokyo, Japan) using BAW. The separated pigments were further purified with preparative HPLC, which was performed on a Waters C18 (19 f 150 mm; Waters) column at 40 C with a flow rate of 1 ml min -1 and monitoring at 530 nm. The solvent used was as follows: a linear gradient elution for 20 min from 50 to 70% solvent B in solvent A. As major anthocyanin pigments, pigments 1 3 were concentrated, and then these concentrated fractions were each dissolved in a small volume of 5% HOAc-EtOH followed by the addition of excess Et 2 O to give precipitated pigments 1 (ca. 17 mg), 2 (ca. 4 mg), and 3 (ca. 4 mg). 1. Pigment 1: Dark red-purple powder: UV-VIS (in 0.1% HCl-MeOH): lmax 529, 331, (279), 284 nm, E acyl = 72%, E 440 = 21%, AlCl 3 - values) BAW 0.36, BuHCl 0.02, 1% HCl 0.08, AHW 0.23; HPLC (method 1): Rt (min) 23.5.; high-resolution FAB mass spectra (HR-FABMS) calc. for C 39 H 39 2 : found: ; 1 H NMR d cyanidin: 8.63 (s, H-4), 6.75 (d, J = 2.1 Hz, H-6), 7.06 (d, J = 2.1 Hz, H-8), 7.84 (brd, J = 2.4 Hz, H-2ʹ), 6.88 (d, J = 8.8 Hz, H-5ʹ), 8.13 (dd, J = 2.4, 8.8 Hz, H-6ʹ). Caffeic acid: 6.48 (d, J = 2.0 Hz, H-2), 6.43 (d, J = 8.1 Hz, H-5), 6.33 (dd, J = 2.0, 8.1 Hz, H-6), 6.03 (d, J = 15.9 Hz,

3 66 F. Tatsuzawa, N. Saito, T. Yukawa, T. Honda, K. Shinoda, K. Kato and K. Miyoshi H-a), 7.14 (d, J = 15.9 Hz, H-b). Glucose A: 5.19 (d, J = 7.6 Hz, H-1), 3.65 (t, J = 8.6 Hz, H-2), 3.53 (t, J = 9.2 Hz, H-3), 3.58 (t, J = 9.5 Hz, H-4), 3.80 (ddd, J = 2.0, 8.9, 9.0, H-5), 3.29 (dd, J = 8.0, 11.9, H-6a), 4.48 (dd, J = 1.7, 11.9, H-6b). Glucose B: 5.23 (d, J = 7.7 Hz, H-1), 3.51 (t, J = 9.2 Hz, H-2), 3.38 (m, H-3), 3.35 (t, J = 9.5, H-4), 3.88 (ddd, J = 2.8, 8.9, 9.0 Hz, H-5), 4.24 (dd, J = 8.2, 11.9 Hz, H-6a), 4.58 (dd, J = 2.6, 11.9 Hz, H-6b). malonic acid: 3.40 (m, -CH 2 -). 2. Pigment 2: Dark red-purple powder: UV-VIS (in 0.1% HCl-MeOH): lmax 528, 329, (279), 283 nm, E acyl = 80%, E 440 = 23%, AlCl 3 values) BAW 0.31, BuHCl 0.01, 1% HCl 0.07, AHW 0.19; HPLC (method 1): Rt (min) 20.5.; high-resolution FAB mass spectra (HR-FABMS) calc. for C 36 H 37 O 19 : found: Pigment 3: Dark red-purple powder: UV-VIS (in 0.1% HCl-MeOH): lmax 524, 282 nm, E 440 = 22%, AlCl 3 values) BAW 0.23, BuHCl 0.02, 1% HCl 0.17, AHW 0.29; HPLC (method 1): Rt (min) 15.1.; high-resolution FAB mass spectra (HR-FABMS) calc. for C 30 H 33 O 19 : found: Acid hydrolysis Acid hydrolysis of pigments 1 3 (ca. 0.5 mg each) was achieved using 2N HCl (1 ml) at 90 C for 2 hrs and resulted in the presences of cyanidin, glucose, malonic acid, and caffeic acid in the hydrolysate of pigment 1, cyanidin, glucose, and caffeic acid in the hydrolysate of pigment 2, and cyanidin, glucose, and malonic acid in the hydrolysate of pigment 3. Moreover, the demalonylation of pigments 1 and 3 (ca. 0.5 mg each) was achieved using 1N HCl (0.5 ml) at room temperature for 5 days, and resulted in the isolation of demalonyl pigment 1 (= pigment 2) and demalonyl pigment 3 (= cyanidin 3,7-diglucoside). 1. Cyanidin. UV-VIS (in 0.1% HCl-MeOH): lmax 536, 273 nm, E 440 = 44%, AlCl 3 - values) Forestal 0.42; HPLC (method 1): Rt (min) Glucose. TLC: (R f -values) BAW 0.23, EAA Caffeic acid. TLC: (R f -values) BAW 0.61, ETN 0.31; HPLC (method 1): Rt (min) Malonic acid. HPLC (method 2): Rt (min) Demalonyl pigment 1 (= pigment 2). See above. 6. Demalonyl pigment 3 (= cyanidin 3,7-diglucoside). See above. Alkaline hydrolysis Pigments 1 3 (ca. 0.5 mg each) were dissolved in 2N NaOH (1 ml) using degassed syringes to stir for 15 min. These solutions were then acidified with 2N HCl (1.1 ml). These hydrolysates were used for the analysis of TLC and HPLC with authentic cyanidin 3,7-diglucoside (seranin) obtained from Serapias lingua (Strack et al., 1989) and Bletilla striata Murasaki Shikibu (Tatsuzawa et al., 2010), respectively. In addition, caffeic acid and malonic acid were detected in the hydrolysates of pigments 1, 2, and 3, respectively. 1. Deacylanthocyanin (cyanidin 3,7-diglucoside). UV- VIS (in 0.1% HCl-MeOH): lmax 525, 281 nm, E 440 = 25%, AlCl 3 values) BAW 0.10, BuHCl 0.03, 1% HCl 0.13, AHW 0.34; HPLC (method 1): Rt (min) Caffeic acid. see above. 3. Malonic acid. See above. H 2 degradation Pigments 1 3 (ca. 0.5 mg each) were dissolved in H 2 O (100 ml) and oxidized with H 2 (100 ml). The resulting solutions were chromatographed on cellulose plates using BAW and EAA by TLC. The components obtained from pigments 1 3 by H 2 degradation were confirmed to be malonylglucose and glucose by co-tlc analysis with authentic samples, which were obtained from Phalaenopsis anthocyanin 3 by H 2 degradation (Tatsuzawa et al., 1997) and commercial glucose (Wako Chemicals, Osaka, Japan). 1. Malonylglucose TLC: (R f values) BAW 0.25, EAA Glucose. see above. Known anthocyanins of orange-red or red flowers of Sophronitis acuensis, S. brevipedunculata, S. cernua, S. coccinea var. xanthoglossa, and S. grandiflora Mixed dried orange-red or red perianths (ca. 30 g) of six taxa of Sophronitis plants were immersed in 5% HOAc-H 2 O at room temperature for 12 hrs and extracted. The extract was purified by Diaion HP-20 CC, PC, TLC, and HPLC by the previous procedures of S. coccinea (Tatsuzawa et al., 1998) and Bletilla striata (Tatsuzawa et al., 2010). From this extract, five anthocyanin pigments (4 8) were obtained as follows: pigment 4 (ca. 4 mg), pigment 5 (ca. 2 mg), pigment 6 (ca. 1 mg), pigment 7 (ca. 2 mg), and pigment 8 (ca. 1 mg). 1. Pigment 4 = Sophronitis coccinea anthocyanin 1 (SCA 1: cyanidin 3-malonylglucoside-7-caffeoylglucoside- 3 -glucoside). Dark red powder; UV-VIS (in 0.1% HCl-MeOH): lmax 521, 332, 283 nm, E acyl = 60%, E 440 = 29%, AlCl 3 shift 0; TLC: (R f values) BAW 0.28, BuHCl 0.02, 1% HCl 0.13, AHW 0.29; HPLC (method 1): Rt (min) Pigment 5 = SCA 2 (cyanidin 3,3 - diglucoside-7- caffeoylglucoside). Dark red powder; UV-VIS (in 0.1% HCl-MeOH): lmax 521, 332, 283 nm, E acyl = 70%, E 440 = 33%, AlCl 3 shift 0; TLC: (R f values) BAW 0.23, BuHCl 0.02, 1% HCl 0.15, AHW 0.26; HPLC (method 1): Rt (min) Pigment 6 = SCA 3 (cyanidin 3-malonylglucoside- 7-feruloylglucoside-3ʹ-glucoside). Dark red powder; UV-VIS (in 0.1% HCl-MeOH): lmax 524, 331, 284 nm, E acyl = 79%, E 440 = 28%, AlCl 3 shift 0; TLC: (R f values) BAW 0.37, BuHCl 0.04, 1% HCl

4 , AHW 0.30; HPLC (method 1): Rt (min) Pigment 7 = SCA 4 (p-coumaroyl cyanidin 3- malonylg lucoside-7,3 -diglucoside). Dark red powder; UV-VIS (in 0.1% HCl-MeOH): lmax 520, 319, 284 nm, E acyl = 92%, E 440 = 28%, AlCl 3 shift 0; TLC: (R f values) BAW 0.41, BuHCl 0.04, 1% HCl 0.21, AHW 0.32; HPLC (method 1): Rt (min) Pigment 8 = SCA 5 (caffeoyl cyanidin 3,7,3 - triglucoside). Dark red powder; UV-VIS (in 0.1% HCl- MeOH): lmax 523, 332, 283 nm, E acyl = 68%, E 440 = 27%, AlCl 3 shift 0; TLC: (R f values) BAW 0.28, BuHCl 0.02, 1% HCl 0.12, AHW 0.27; HPLC (method 1): Rt (min) Partial acid hydrolysis of S. coccinea anthocyanin (SCA) 2 Partial acid hydrolysis of cyanidin 3,3ʹ- diglucoside- 7-caffeoylglucoside (ca. 0.5 mg, SCA 2) was achieved using 2N HCl (1 ml) at 90 C for 10 min, and resulted in one of the main peaks [HPLC (method 1): Rt (min): 20.5], which was identified as cyanidin 3-glucoside-7-caffeoylglucoside (= pigment 2) by analysis using TLC and HPLC methods. Results and Discussion 1. Flower anthocyanins of seven taxa of Sophronitis 1) Structure determination of three new anthocyanins 1 3 in the red-purple flowers of S. wittigiana By HPLC analysis (method 1), 10 anthocyanin peaks were observed in the MAW extract from red-purple flowers of Sophronitis wittigiana as major anthocyanin peaks. From these peaks, three major anthocyanin peaks (pigments 1 3; pigment 1: 40.5% of the total anthocyanin contents calculated from the HPLC peak area at 530 nm, pigment 2: 9.2%, pigment 3: 20.4%) were isolated from the dried flowers (20 g) with 5% HOAc solvent and purified according to the procedure described previously (Tatsuzawa et al., 1998). The chromatographic and spectroscopic properties of these pigments are summarized in Materials and Methods. Alkaline hydrolysis of pigments 1 3 (ca. 0.5 mg each) yielded only one deacylated anthocyanin. Its structure was identified to be cyanidin 3,7-diglucoside by comparison of TLC and HPLC with authentic cyanidin 3,7-diglucoside (seranin) obtained from the acylated anthocyanins of Serapias lingua (Strack et al., 1989) and Bletilla striata Murasaki Shikibu (Tatsuzawa et al., 2010) by alkaline hydrolysis. Further detailed structural studies of these anthocyanins 1 3 were carried out as follows. (1) Pigment 1 Acid and alkaline hydrolyses of pigment 1 gave cyanidin, glucose, malonic acid, and caffeic acid, and cyanidin 3,7-diglucoside, malonic acid, and caffeic acid, respectively. By H 2 degradation, pigment 1 gave malonylglucose (see Materials and Methods). Moreover, the molecular ion [M] + of pigment 1 was observed at mz 859 (C 39 H 39 2 ) by FABMS measurement, indicating that pigment 1 is composed of cyanidin with two molecules of glucose and one molecule each of caffeic acid and malonic acid. These elemental components of pigment 1 were confirmed by measuring its HR-FABMS. From these results, the structure of pigment 1 was presumed to be caffeoyl cyanidin 3-malonylglucoside-7-glucoside. The detailed structure of pigment 1 was further elucidated by investigation of its 1 H NMR spectra, including 2D COSY and NOESY spectra. The chemical shifts of nine aromatic protons of cyanidin and caffeic acid moieties with their coupling constants were assigned by analysis of its 2D COSY spectrum, as shown in the section pigment 1 in Materials and Methods. The 1 H NMR spectrum exhibited two olefinic proton signals of caffeic acid with large coupling constants (J = 15.9 Hz and 15.9 Hz). Therefore, its caffeic acid was determined to be in trans configuration. Proton chemical shifts of the sugar moieties were observed in the region of d , where the two anomeric protons resonated at d5.19 (d, J = 7.6 Hz, Glc A-H1) and d5.23 (d, J = 7.7 Hz, Glc B-H1), respectively. Based on the observed coupling constants, these two sugars were assumed to be in the b-pyranose form (see section pigment 1 in Materials and Methods). By analysis of its 2D COSY spectrum, four characteristic proton signals shifted to a lower magnetic field were assigned as methylene protons of Glc A (d4.29 and 4.48, H-6a and -6b) and Glc B (d4.24 and 4.58, H-6a and -6b), respectively. These results indicated that two OH-6 groups of Glc A and Glc B were acylated with malonic acid and caffeic acid. 2D NOESY spectrum of this pigment was analyzed to distinguish the attachment positions of acid and glucose with cyanidin aglycone (Fig. 1). The signal of the anomeric protons of Glc A was correlated to the signals of the proton H-4 (d8.63) and that of Glc B was correlated to H-8 and -6 (d7.06 and 6.75) in its NOESY spectrum. These characteristic features revealed that the OH-3 and OH-7 positions of cyanidin were both glycosylated with Glc A and Glc B, respectively. Furthermore, H-6a and b of Glc B weakly correlated with the signals of H-a (d6.03) and b (7.14) of caffeic acid in its NOESY spectrum. This result supported that the OH-6 of Glc B was esterified with caffeic acid (Fig. 1). Moreover, by H 2 oxidation of pigment 1, malonylglucose was detected as a degradation product. Fig. 1. New anthocyanins from the red-purple flowers of Sophronitis wittigiana. Observed main NOEs from pigment 1 are indicated by arrows.

5 68 F. Tatsuzawa, N. Saito, T. Yukawa, T. Honda, K. Shinoda, K. Kato and K. Miyoshi Consequently, the structure of pigment 1 was elucidated to be cyanidin 3-O-[6-O-(malonyl)-b-glucopyranoside]- 7-O-[6-O-(trans-caffeoyl)-b-glucopyranoside] (Fig. 1), which is a new anthocyanin in plants (Andersen and Jordheim, 2006; Harborne and Baxter, 1999; Honda and Saito, 2002; Veitch and Grayer, 2008, 2011). (2) Pigment 2 Acid and alkaline hydrolyses of pigment 2 gave cyanidin, glucose, and caffeic acid, and cyanidin 3,7-diglucoside and caffeic acid, respectively. By H 2 degradation of pigment 2, only glucose was detected, and glycosylcaffeic acid was not observed in its degradation solution. Moreover, the molecular ion [M] + of pigment 2 was observed at mz 773 (C 36 H 37 O 19 ) using FABMS indicating that pigment 2 is composed of cyanidin with two molecules of glucose and one molecule of caffeic acid. These elemental components of pigment 2 were confirmed by measuring its HR-FABMS. From these results, the structure of pigment 2 was presumed to be caffeoyl cyanidin 3,7-diglucoside. Moreover, the analytical data of pigment 2 were identical to those of the demalonylation of pigment 1 and also one intermediate anthocyanin obtained by partial acid hydrolysis from SCA 2 (cyanidin 3,3ʹ-diglucoside-7-caffeoyl-glucoside). The partial acid hydrolysate from SCA 2 was identical to pigment 2 by HPLC analysis [HPLC (method 1): Rt (min): 20.5, see Partial acid hydrolysis in Materials and Methods] and assumed to be cyanidin 3-glucoside-7-caffeoyl-glucoside. Therefore, the structure of pigment 2 was determined to be cyanidin 3-O-(glucoside)-7-O-[6-O-(trans-caffeoyl)- glucoside] (Fig. 1), which is a new anthocyanin in plants (Andersen and Jordheim, 2006; Harborne and Baxter, 1999; Honda and Saito, 2002; Veitch and Grayer, 2008, 2011). (3) Pigment 3 Acid and alkaline hydrolyses of pigment 3 gave cyanidin, glucose, and malonic acid, and cyanidin 3,7- diglucoside and malonic acid, respectively. Moreover, the molecular ion [M] + of pigment 3 was observed at mz 697 (C 30 H 33 O 19 ) using FABMS indicating that pigment 3 is composed of cyanidin with two molecules of glucose and one molecule of malonic acid. These elemental components of pigment 3 were confirmed by measuring its HR-FABMS. From these results, the structure of pigment 3 was presumed to be malonyl cyanidin 3,7-diglucoside. Moreover, by H 2 degradation of pigment 3, 6-(malonyl)-glucose was detected in its degradation products, similar to the result for pigment 1. Therefore, the structure of pigment 3 was determined to be cyanidin 3-[6-(malonyl)-glucoside]-7-glucoside (Fig. 1). The pigment is also a new anthocyanin in plants. 2) Five known anthocyanins (4 8) in six orange-red or red flowers of Sophronitis plants Five anthocyanin pigments (4 8) were isolated from dried mixed perianths (ca. 30 g) of six orange-red or red Sophronitis taxa with 5% HOAc, except that of S. wittigiana. These pigments were purified according to the procedure described previously (Tatsuzawa et al., 1998), and small amounts of five anthocyanins (4 8) were obtained, respectively. These five anthocyanin structures were investigated by a similar process as before, and were identified to be cyanidin 3-malonylglucoside- 7-caffeoylglucoside-3ʹ-glucoside (Sophronitis coccinea anthocyanin 1 = SCA 1) as pigment 4, cyanidin 3,3ʹdiglucoside-7-caffeoylglucoside (SCA 2) as pigment 5, cyanidin 3-malonylglucoside-7-feruloylglucoside-3ʹglucoside (SCA 3) as pigment 6, p-coumaroyl cyanidin 3-malonylglucoside-7,3ʹ-diglucoside (SCA 4) as pigment 7, and feruloyl cyanidin 3,7,3ʹ-triglucoside (SCA 5) as pigment 8, as shown in the section Materials and Table 1. Flower color and distribution of pigments in the Sophronitis species. Species Flower colors z Anthocyanins (as %) y Carotenoids x RHSCC b*a* S. wittigiana Red-Purple 67C S. acuensis Orange-Red 33A S. brevipedunculata Red 52A S. cernua Orange-Red 34A S. coccinea Orange-Red 33A S. coccinea v. xanthoglossa Orange-Red 33A S. grandiflora Orange-Red 34A z RHSCC; Royal Horticultural Society Colour Chart No. b*a*=hue. y 1: pigment 1: cyanidn 3-malonylglucoside-7-caffeoylglucoside, Rt(min) = : pigment 2: cyanidn 3-glucoside-7-caffeoylglucoside, Rt(min) = : pigment 3: malonyl cyanidin 3,7-diglucoside, Rt(min) = : pigment 4: Sophronitis coccinea anthocyanin 1; cyanidin 3-malonylglucoside-7-caffeoylglucoside-3 -glucoside, Rt(min) = : pigment 5: Sophronitis coccinea anthocyanin 2; cyanidin 3,3 -diglucoside-7-caffeoylglucoside, Rt(min) = : pigment 6: Sophronitis coccinea anthocyanin 3; cyanidin 3-malonylglucoside-7-feruloylglucoside-3 -glucoside, Rt(min) = : pigment 7: Sophronitis coccinea anthocyanin 4; trans-p-coumaroyl cyanidin 3-malonylglucoside-7,3 -diglucoside, Rt(min) = : pigment 8: Sophronitis coccinea anthocyanin 5; feruloyl cyanidin 3,7,3 -triglucoside, Rt(min) = x Carotenoids: + = presence, - = no presence.

6 69 Fig. 2. Absorption spectral curves of fresh perianthes of Sophronitis wittigiana (solid line) and S. coccinea (dotted line). Fig. 3. HPLC analysis of anthocyanins in red-purple flowers of Sophronitis wittigiana and orange-red flowers of S. coccinea. 1: pigment 1: cyanidn 3-malonylglucoside-7-caffeoylglucoside. 2: pigment 2: cyanidn 3-glucoside-7-caffeoylglucoside. 3: pigment 3: malonyl cyanidin 3,7-diglucoside. 4: pigment 4: Sophronitis coccinea anthocyanin 1; cyanidin 3-malonylglucoside-7-caffeoylglucoside-3 -glucoside. 5: pigment 5: Sophronitis coccinea anthocyanin 2; cyanidin 3,3 -diglucoside-7-caffeoylglucoside. 6: pigment 6: Sophronitis coccinea anthocyanin 3; cyanidin 3-malonylglucoside-7-feruloylglucoside-3 -glucoside. 7: pigment 7: Sophronitis coccinea anthocyanin 4; trans-p-coumaroyl cyanidin 3-malonylglucoside-7,3 -diglucoside.

7 70 F. Tatsuzawa, N. Saito, T. Yukawa, T. Honda, K. Shinoda, K. Kato and K. Miyoshi Methods. These structures were further confirmed by comparison of their TLC, HPLC and spectroscopic properties with authentic anthocyanins SCAs 2 5 obtained from the orange-red flowers of S. coccinea (Tatsuzawa et al., 1998). 2. Flower colors and anthocyanin structures of seven taxa in Sophronitis plants Fresh perianths of S. wittigiana exhibited a red-purple color with a chromaticity value (b*a*) of (Table 1). The visible absorption curve of its intact perianths (Fig. 2) exhibited a lmax at 548 nm, similar to those of common red flowers containing cyanidin 3,5-diglucoside (Yokoi and Saito, 1973), but the perianths of S. coccinea were orange-red (b*a* = 1.13) and exhibited a lmax at 503 nm and, furthermore, four extra shoulder peaks at nm due to mainly carotenoid pigments (Fig. 2). For the anthocyanin distribution of Sophronitis plants, the occurrence of cyanidin was first reported in the orange-red flowers of S. coccinea as its aglycone (Yokoi, 1975). Then, five acylated cyanidin 3,7,3ʹ-triglucosides were isolated from its orange-red flowers as the main anthocyanin pigments, and their structures were elucidated in detail (Tatsuzawa et al., 1998). This study revealed that the occurrence of five acylated cyanidin 3,7,3ʹ-triglucosides was usually observed in the orange-red or red flowers of Sophronitis plants, S. acuensis, S. brevipedunculata, S. cernua, S. coccinea var. xanthoglossa, and S. grandiflora, except for the red-purple flowers of S. wittigiana containing acylated cyanidin 3,7-diglucosides (Table 1). These results indicated that a blue color direction shift took place for the flower colors of Sophronitis plants by B-ring substitution in their anthocyanins. Similar phenomena were observed in the case of cyanidin 3ʹ-glucose substitution giving scarlet coloration in plants of the Bromeliaceae (Saito and Harborne, 1983), and also in the case of Bletilla flower colors, such as the bluish variety Murasaki Shikibu containing acylated cyanidin 3,7-diglucoside, and common red-purple plants of Bletilla striata containing acylated cyanidin 3,7,3ʹ-glycoside (Saito et al., 1995; Tatsuzawa et al., 2010). As a matter of course, carotenoid pigments except for acylated cyanidin 3,7,3ʹ-triglucosides take part in producing orange-red flower colors, as observed in the orange-red flower colors of five Sophronitis plants (Table 1; Figs. 2 and 3), whereas the flower color lacking carotenoid pigments, such as that of S. brevipedunculata, is only red, but more hypsochromic than the red-purple flower colors of S. wittigiana (Table 1). Therefore, in Sophronitis plants, there are two characteristic methods for producing red-purple flower plants from the common orange-red or red flower color plants as follows: (1) the 3ʹ-OH of cyanidin is free from glycosylation, and (2) decreasing the carotenoid pigment concentration. 3. Anthocyanin distributions of 3,7- and 3,7,3ʹ-glycosides in orchids To the best of our knowledge, the occurrence of cyanidin 3,7-glucoside has been reported in plants of Anacamptis, Barlia, Cephalanthera, Dactylorhiza, Epipactis, Gymnadenia, Himantoglossum, Limodorum, Neottianthe, Nigrirella, Ophrys, Orchis, Serapias, and Traunsteinera for the Orchidaceae, in company with cyanidin 3- and 3,5-glucosides (Strack et al., 1986, 1989). These plants were grouped into the European orchids. On the other hand, the distributions of anthocyanin 3,7,3ʹ-glycosides were reported in the plants of Bletilla (Saito et al., 1995), Dendrobium (Saito et al., 1994; Tatsuzawa et al., 2005, 2006; Williams et al., 2002), Laeliocattleya (Tatsuzawa et al., 1994, 1996), Laelia (Tatsuzawa et al., 1996), Cattleya (Tatsuzawa et al., 1996), Phalaenopsis (Tatsuzawa et al., 1997), Sophronitis (Tatsuzawa et al., 1998), and Vanda (Tatsuzawa et al., 2004) as their main anthocyanin glycosides. These plants are the majority of tropical orchids. As pointed out by Harborne (1993), 3ʹ-glucosylation of a cyanidin derivative appears to be an alternative evolutionary pathway towards the scarlet and orange colors than the loss mutation of the B-ring hydroxylation at position 3ʹ (from cyanidin to pelargonidin) preferred by bird and butterfly pollinators of tropical plants. So, opposite to the scarlet and orange colors where cyanidin 3,7-glycosides were produced in bluish-red flowers, such as those of S. wittigiana and a bluish variety of Bletilla, it can presumed that the gene deletion of anthocyanin 3ʹ-glucosyltransferase occurred in these plants as spontaneous mutations. Acknowledgements We thank Mr. Tamon Suzuki (Amiele Project, Japan) for the donation of plant materials. Literature Cited Andersen, Ø. M. and M. Jordheim The anthocyanins. p In: Ø. M. Andersen and K. R. Markham (eds.). Flavonoids: Chemistry, biochemistry and applications. CRC Press, Boca Raton. Harborne, J. B Phytochemical methods, second ed. Chapman and Hall, London. Harborne, J. B Introduction to ecological biochemistry, fourth ed. p Academic Press, London. Harborne, J. B. and H. Baxter The handbook of natural flavonoids, vol. 2. p John Wiley & Sons, Chichester. Honda, T. and N. Saito Recent progress in the chemistry of polyacylated anthocyanins as flower color pigments. 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8 71 and T. Honda Acylated cyanidin glycosides in the purple-red flowers of Bletilla striata. Phytochemistry 40: Saito, N., K. Toki, K. Uesato, A. Shigihara and T. Honda An acylated cyanidin glycoside from the red-purple flowers of Dendrobium. Phytochemistry 37: Strack, D., E. Busch and E. Klein Anthocyanin patterns in European orchids and their taxonomic and phylogenetic relevance. Phytochemistry 28: Strack, D., E. Busch, V. Wray, L. Grotjshn and E. Klein Cyanidin 3-oxalylglucoside in Orchids. Z. Naturforsch. 41c: Tatsuzawa, F., N. Saito, H. Seki, R. Hara, M. Yokoi and T. Honda Acylated cyanidin glycosides in the red-purple flowers of Phalaenopsis. Phytochemistry 45: Tatsuzawa, F., N. Saito, H. Seki, M. Yokoi, T. Yukawa, K. Shinoda and T. Honda Acylated anthocyanins in the flowers of Vanda (Orchidaceae). Biochem. Syst. Ecol. 32: Tatsuzawa, F., N. Saito, Y. Mikanagi, K. Shinoda, K. Toki, A. Shigihara and T. Honda An unusual acylated malvidin 3-glucoside from flowers of Impatiens textori Miq. (Balsaminaceae). Phytochemistry 70: Tatsuzawa, F., N. Saito, A. Shigihara, T. Honda, K. Toki, K. Shinoda, T. Yukawa and K. Miyoshi An acylated cyanidin 3,7-diglucoside in the bluish flowers of Bletilla striata Murasaki Shikibu (Orchidaceae). J. Japan. Soc. Hort. Sci. 79: Tatsuzawa, F., N. Saito, M. Yokoi, A. Shigihara and T. Honda An acylated cyanidin glycoside in the red-purple flowers of xlaeliocattleya cv Mini Purple. Phytochemistry 37: Tatsuzawa, F., N. Saito, M. Yokoi, A. Shigihara and T. Honda Acylated cyanidin 3,7,3 -triglucosides in flowers of Laeliocattleya cv. Mini Purple and its relatives. Phytochemistry 41: Tatsuzawa, F., N. Saito. M. Yokoi. A. Shigihara and T. Honda Acylated cyanidin glycosides in the orange-red flowers of Sophronitis coccinea. Phytochemistry 49: Tatsuzawa, F., N. Saito, T. Yukawa, K. Shinoda, A. Shigihara and T. Honda Acylated cyanidin 3,7,3 -triglucoside with p-hydroxybenzoic acid from the flowers of Dendrobium. Heterocycles 68: Tatsuzawa, F., T. Yukawa, K. Shinoda and N. Saito Acylated anthocyanins in the flowers of genus Dendrobium section Phalaenanthe (Orchidaceae). Biochem. Syst. Ecol. 33: van den Berg, C New combinations in the genus Cattleya Lindl. (Orchidaceae). Neodiversity 3: Veitch, N. C., R. J. Grayer Flavonoids and their glycosides, including anthocyanins. Nat. Prod. Rep. 25: Veitch, N. C., R. J. Grayer Flavonoids and their glycosides, including anthocyanins. Nat. Prod. Rep. 28: Williams, C. A., J. Greenham, J. B. Harborne, J. M. Konk, L. S. Chia, N. K. Goh, N. Saito, K. Toki and F. Tatsuzawa Acylated anthocyanins and flavonols from purple flowers of Dendrobium cv. Pompadour. Biochem. Syst. Ecol. 30: Withner, C. L The Cattleyas and their Relatives, Volume Ш. Schomburgkia, Sophronitis, and Other South American Genera. p Timber Press, Inc., Portland Yokoi, M Colour and pigment distribution in the cultivars of selected ornamental plants, with special reference to their contribution to the ornamental value of plants. The Transaction of Faculty of Horticulture, Chiba University 14: Yokoi, M. and N. Saito Light absorption patterns of intact Rosa flowers in relation to the flower colour. Phytochemistry 12: