Biochemical Systematics and Ecology 54 (2014) 15–18

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Biochemical Systematics and Ecology

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Anthocyanins in the flowers of Ipomoea tricolor Cav.(Convolvulaceae)

Kyeung-Il Park a, Atsushi Hoshino b,c,*, Norio Saito d, Fumi Tatsuzawa e,**

aDepartment of Horticultural & Life Science, College of Natural Resources, Yeungnam University, Gyeongbuk 712-749, Republic of KoreabNational Institute for Basic Biology, Okazaki 444-8585, JapancDepartment of Basic Biology, The Graduate University for Advanced Studies (Sokendai), Okazaki 444-8585, JapandMeiji-Gakuin University, Yokohama 244-8538, Japane Laboratory of Olericultural and Floricultural Science, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan

a r t i c l e i n f o

Article history:Received 24 July 2013Accepted 31 December 2013Available online

Keywords:Ipomoea tricolorConvolvulaceaeAnthocyaninMorning glory

1. Subject and source

The genus Ipomoea includes 600–700 species, which is the largest number of species within the Family Convolvulaceae(Austin and Huáman, 1996). As far as anthocyanins are concerned, Ipomoea is the most studied genus in this family, and aresources of complicated anthocyanins (Eich, 2008). Most anthocyanins from Ipomoea flowers are based on 3-sophoroside-5-glcosides of pelargonidin, cyanidin and peonidin, and are acylated with caffeic acid and r-coumaric acids (Kondo et al., 1987;Lu et al., 1991, 1992a, b; Pomilio and Sproviero, 1972a, b; Saito et al., 1998, 1996, 2005, 1995, 1996, 2005; Toki et al., 2001a, b).Among the anthocyanidins of these anthocyanins, peonidin is limited to the flowers of Ipomoea nil (Lu et al., 1992b), Ipomoeatricolor (Kondo et al., 1987), and Ipomoea congesta (Pomilio and Sproviero, 1972b) in the genus Ipomoea. The three morningglories, I. nil, Ipomoea purpurea, and I. tricolor, are popular ornamental climbing plants, and cultivars with a range of flowercolors have been established. The flower anthocyanins of I. nil and I. purpurea have been studied extensively (Iida et al., 1999,2004; Lu et al., 1992a, b; Morita et al., 2005; Saito et al., 1998, 1996, 2005, 1995, 1996, 2005). On the other hand, knowledge ofanthocyanins of I. tricolor is limited, and only two anthocyanins, Heavenly Blue Anthocyanin (Kondo et al., 1987) andWeddingBells Anthocyanin (Hoshino et al., 2003), have been reported. In extending the study, the anthocyanin constituents of I. tricolorwere investigated in detail for its six cultivars.

The morning glory, I. tricolor, is found in Central and South America, and its wild-type plants have blue flowers. Amongits cultivars showing a variety of flower colors, this study compared anthocyanins in the flowers of ‘Summer Skies’ and

* Corresponding author. National Institute for Basic Biology, Okazaki 444-8585, Japan. Tel./fax: þ81 564 55 7534.** Corresponding author. Tel./fax: þ81 19 621 6145.

E-mail addresses: hoshino@nibb.ac.jp (A. Hoshino), fumi@iwate-u.ac.jp (F. Tatsuzawa).

0305-1978/$ – see front matter � 2014 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bse.2013.12.034

K.-I. Park et al. / Biochemical Systematics and Ecology 54 (2014) 15–1816

‘Flying Saucers’ with those of the cultivars previously studied; ‘Heavenly Blue’, ‘Blue Star’, ‘Pearly Gates’, and ‘Wedding Bells’(Choi et al., 2007; Hoshino et al., 2003; Park et al., 2004). ‘Summer Skies’ blooms light blue flowers, and ‘Flying Saucers’shows variegated flowers with blue spots and sectors. The seeds of ‘Summer Skies’ and ‘Flying Saucers’ were purchasedfrom David Dubowski (NC, USA) and Sakata Nursery (Yokohama, Japan), respectively, and the other cultivars were describedbefore (Choi et al., 2007; Hoshino et al., 2003; Park et al., 2004). The plants used here were grown at our experimental field(Okazaki, Japan) in the summer of 2010 and 2011. Voucher specimens were deposited at the Iwate University Museum(IUM).

2. Previous work

Among the I. tricolor cultivars, ‘Heavenly Blue’, which is a wild-type cultivar, shows blue flowers accumulating HeavenlyBlue Anthocyanin (HBA), and ‘Wedding Bells’ blooms red flowers with Wedding Bells Anthocyanin (WBA) (Hoshino et al.,2003; Kondo et al., 1987). Both HBA and WBA are acylated complexes of anthocyanidin 3-sophoroside-5-glcosides. HBAand WBA was determined to be peonidin 3-O-(2-O-(6-O-(3-O-glucosyl-caffeoyl)-glucosyl)-6-O-(4-O-(6-O-(3-O-glucosyl-caffeoyl)-glucosyl)-caffeoyl)-glucoside)-5-O-(glucoside) (Kondo et al., 1987), and pelargonidin 3-O-(2-O-(6-O-(3-O-glucosyl-caffeoyl)-glucosyl)-6-O-(4-O-(6-O-(3-O-glucosyl-caffeoyl)-glucosyl)-caffeoyl)-glucoside)-5-O-(glucoside), respectively(Hoshino et al., 2003; Lu et al., 1992a). The pale blue flowers of ‘Blue Stars’ accumulated HBA (Park et al., 2004), whereas thewhite flowers of ‘Pearly Gates’ contained no anthocyanins (Choi et al., 2007). To the best of our knowledge, HBA andWBA arethe only anthocyanins reported in I. tricolor.

3. Present study

3.1. Isolation and identification of anthocyanins

3.1.1. HBA and WBAEach flower pigment of I. tricolor cultivars was extracted from dried corollas (40 mg) with 2 ml of MeOH:HOAc:H2O (4:1:5,

v/v/v) for 1 h at 25 �C. The extracts (20 ml) were analyzed by high performance liquid chromatography (HPLC). The antho-cyanin pigments extracted from 4 varieties, ‘Heavenly Blue’, ‘Blue Star’, ‘Flying Saucers’ and ‘Wedding Bells’, showed almost asingle peak. Judging from the chromatographic and spectroscopic properties (See in Supplementary Information), the an-thocyanins of the former three cultivars were identified to be HBA, and those of the last cultivar were found to be WBA. Noanthocyanin pigments were detected from ‘Pearly Gates’. On the other hand, three major peaks showing different Rt valuesfrom HBA and WBA were detected in ‘Summer Skies’ (See in Supplementary Information), and anthocyanin pigments 1–3were obtained as follows.

3.1.2. Anthocyanins in ‘Summer Skies’For further analysis, the anthocyanin pigments of ‘Summer Skies’ were extracted from 60 g of dried corollas in 5% HOAc–

MeOH (20 L) for 1 h at 4 �C. The extract was isolated and purified by a series of chromatography steps described by Saito et al.(2011) to yield 15 mg of pigment 1, 31 mg of pigment 2 and 122 mg of pigment 3.

The pigments were analyzed by thin layer chromatography (TLC), HPLC, ultraviolet/visible light (UV–VIS) spectroscopy,fast atom bombardment mass spectroscopy (HR-FABMS), and nuclear magnetic spectroscopy (NMR) spectroscopy, asdescribed previously (Saito et al., 2011). For TLC analysis, microcrystalline cellulose sheets (Merck, Germany) were used withmobile phases, BAW (n-BuOH:HOAc:H2O ¼ 4:1:2, v/v/v), BuHCl (n-BuOH:2 N HCl ¼ 1:1, v/v, upper phase), 1% HCl, and AHW(HOAc:HCl:H2O ¼ 15:3:82, v/v/v). Analytical HPLC was carried out using LC-10A (Shimadzu Co., Ltd., Japan), a C18 column(4.2 � 250 mm, Waters, USA), solvent A (1.5% H3PO4 in H2O) with a linear gradient system from 20% to 85% of solvent B (1.5%H3PO4, 20% HOAc, 25% MeCN in H2O). The UV–VIS spectra were recorded on a UV–VIS Multi Purpose Spectrophotometer(MPS-2450, Shimadzu) in 0.1% HCl–MeOH (from 200 to 700 nm). Authentic anthocyanins, peonidin 3-sophoroside-5-glucoside, pelargonidin 3-sophoroside-5-glucoside, HBA and WBA were used for TLC, HPLC, and UV–VIS analysis. More-over, the elemental components of pigments 2 and 3were confirmed byHR-FABMS and the structures of these pigments wereconfirmed by NMR spectroscopy [400 MHz for 1H and 100 MHz for 13C spectra in CF3COOD-DMSO-d6 (1:9)] including 1H–1Hcorrelation spectroscopy (HH COSY), nuclear Overhauser enhancement and exchange spectroscopy (NOESY), 13C–1H corre-lation spectroscopy (CH COSY) and correlation spectroscopy via long-range coupling spectrum (COLOC) spectroscopy (See inSupplementary Information). The chemical shifts are reported relative to DMSO (2.5 ppm), and the coupling constants (J) arereported in Hz. FAB mass and NMR spectra were obtained by JEOL JMS-700 (JEOL Ltd., Japan) and JEOL AL-400 (JEOL Ltd.),respectively.

The chromatographic and spectroscopic characteristics are summarized in the Supplementary information. Among thethree anthocyanins of ‘Summer Skies’, No. 3 was the most abundant (47.7%) followed in order by No. 2 (27.4%) and No. 1(10.3%). Pigment 1 showed identical TLC values and HPLC data to those of peonidin 3-sophoroside-5-glucoside used as theauthentic anthocyanin (See in Supplementary Information), and the FAB-MS spectrometry gave a molecular ion at 787 m/z[M]þ, which is the same value as that of peonidin 3-sophoroside-5-glucoside (Lu et al., 1991). Finally, pigment 1 wasconfirmed to be peonidin 3-sophoroside-5-glucoside.

K.-I. Park et al. / Biochemical Systematics and Ecology 54 (2014) 15–18 17

The molecular ions of pigments 2 and 3 were detected at 1111 and 949 m/z [M]þ, respectively, and their Eacyl/Emax valuessuggest that pigment 2 has two moles of caffeic acid compared to pigment 3, possessing 1 mole (See in supplementaryinformation), which were similar to pigments 4 and 3 of I. nil reported by Lu et al. (1992b).

The acid hydrolysis of pigments 2 and 3 gave peonidin as their anthocyanidin (Harborn, 1984). These anthocyaninscontained glucose as their sugar component according to the results of acid hydrolysis. Trans-caffeic acid was detected in thehydrolysate of 1 and 2 by HPLC.

By alkaline hydrolysis, pigments 2 and 3 yielded peonidin 3-sophoroside-5-glucoside as their deacyl anthocyanin. Thedeacyl anthocyanin structure was identified in a direct comparison of co-TLC and co-HPLC analyses with those of pigment 1.Trans-caffeic acid was detected in the hydrolysate of 2 and 3 by HPLC, respectively.

Pigments 2 and 3 were presumed to be caffeoyl peonidin 3-sophoroside-5-glucosides.

3.1.2.1. Pigment 2. The molecular ion [M]þ of pigment 2was observed atm/z 1111 (C52H55O27) suggesting that pigment 2wascomposed of peonidin with three molecules of glucose and two molecules of caffeic acid. The elemental components wereconfirmed by measuring its HR-FABMA spectrum, and the structure was determined based on an analysis of the 1H and 13CNMR spectra (See in Supplementary Information).

The chemical shifts of the 12 aromatic protons of the peonidin and caffeic acidmoieties with their coupling constants wereassigned, as shown in the Supplementary Information. Two sets of one pair of doublet peaks assigned to four olefinic protonsignals of caffeic acids with their large coupling constant (J¼ 15.9 and 15.9 Hz) showed that the acids were present in the transconfiguration. The chemical shifts of the sugar moieties were observed in the region of d 5.59–3.11, where the three anomericprotons exhibited at d 5.59 (d, J ¼ 7.0 Hz, Glc A), d 5.08 (d, J ¼ 8.1 Hz, Glc B), d 4.72 (d, J ¼ 7.6 Hz, Glc C). Based on the observedcoupling constants, these three sugars were assumed to be in the b-pyranose forms. The linkages and/or positions of theattachments of the sugar and acyl groups were determined based on the HH COSY and NOESY experiments.

Through the application of the NOESY experiment, NOEs between H-1 of Glc A and H-4 (d 8.87) of peonidin, H-1 of Glc Band H-6 (d 6.92) of peonidin, and H-2(d 3.90) of Glc A and H-1 of Glc C were observed (Fig. 1), showing that OH-3 and OH-5 ofpeonidin are glycosylated with Glc A and Glc B, respectively, and OH-2 of Glc A is bonded with the H-1 of Glc C formingsophorose.

Four characteristic proton signals that were shifted to a lower magnetic field were also assigned to the methylene protonsof Glc A (d 4.22 and d 4.34, H-6a and b) and Glc C (d 3.96 and d 4.02, H-6a and b). Therefore, two hydroxyl groups of the sugarmoieties, OH-6s of Glc A and C, were assumed to be acylated with two molecules of the acids.

Accordingly, the structure of pigment 2was determined to be peonidin 3-O-(2-O-(6-O-(trans-caffeoyl)-b-glucopyranosyl)-6-O-(trans-caffeoyl)-b-glucopyranoside)-5-O-(b-glucopyranoside) (Fig. 1). This structure was confirmed further by an anal-ysis of the 13C NMR spectra, as shown in the Supplementary Information.

3.1.2.2. Pigment 3. The molecular ion [M]þ of pigment 3 was observed atm/z 949 (C43H49O24) suggesting that pigment 3 wascomposed of peonidin with three molecules of glucose and one molecule of caffeic acid. The elemental components were

Fig. 1. Anthocyanins in the sky blue flowers of I. tricolor ‘Summer Skies’. The main NOEs are indicated by the arrows. 1: pigment 1; 2: pigment 2 and 3: pigment 3.

K.-I. Park et al. / Biochemical Systematics and Ecology 54 (2014) 15–1818

confirmed by measuring the HR-FABMAS, and the structure was determined based on an analysis of the 1H and 13C NMRspectra (See in Supplementary Information).

An analysis of the NMR spectra revealed the proton chemical shifts of pigment 3 to be similar to those of pigment 2 exceptfor the proton signals of Glc C and the absence of a caffeic acid moiety (See in Supplementary Information). In particular,upfield shifts of methylene protons (d 3.17 and 3.31, H-6a and b) of Glc C in pigment 3 were observed compared to those (d3.96 and 4.02, H-6a and b) of pigment 2. Therefore, OH-6 of Glc C in pigment 3 was free of caffeic acid. Therefore, pigment 3was determined to be peonidin 3-O-(2-O-(b-glucopyranosyl)-6-O-(trans-caffeoyl)-b-glucopyranoside)-5-O-(b-glucopyrano-side). From the above results, three anthocyanins, 3-sophoroside-5-glucoside, 3-dicaffeoly sphoroside-5-glucoside, and 3-caffeoyl sophoroside-5-glucoside of peonidin, were found for the first time in the flowers of I. tricolor.

4. Chemotaxonomic significance

Three anthocyanins, pigments 1–3, along with HBA andWBAwere found in the flowers of the I. tricolor cultivars. Based onthe glycosylation pattern, these anthocyanins are grouped into anthocyanidin 3-sophoroside-5-glcosides, which aredistributed amongmost of the Ipomoea species studied (Eich, 2008). Therefore, anthocyanidin 3-sophoroside-5-glcosides canbe used as chemotaxonomic markers, even in the Ipomoea cultivars showing a range of colors and pigmentation patterns ofthe flowers. The three anthocyanins 1–3 isolated from ‘Summer Skies’ were first reported in I. tricolor, whereas these an-thocyanins have already been isolated from the violet-blue flowers of I. nil cultivars (Lu et al., 1992b). For the acylation pattern,there are three acylation patterns of anthocyanins in the wild Ipomoea flowers as their main anthocyanin, such as non-acylated anthocyanins from Ipomoea fistulosa (Gupta et al., 1980), diacylated anthocaynins from Ipomoea cairica (Pomilioand Sproviero, 1972a), I. congesta (Pomilio and Sproviero, 1972b), and triacylated anthocyanins from Ipomoea asarifolia(Pale et al., 2003), I. nil (Lu et al., 1992a, b), I. purpurea (Saito et al., 1995, 1996) and I. tricolor (Kondo et al., 1987; Park et al.,2004). Less acylated (non-, mono- and diacylated) anthocyanins have been reported in the three Ipomoea species of I.asarifolia, I. nil and, I. purpurea producing the triacylated anthocyanins, e.g. dicaffeoly cyanidin 3-sophoroside-5-glcosidesfrom I. asarifolia (Pale et al., 1998) and I. purpurea (Saito et al., 1995), dicaffeoly peonidin 3-sophoroside-5-glcosides from I.nil (Lu et al., 1992b). Therefore, I. tricolor is the fourth species of the occurrence of both triacylated and less acylated an-thocyanins in the genus Ipomoea. These Ipomoea species with triacylated anthocyanins might have the ability to diversifyflowers with less acylated anthocyanins.

Acknowledgments

The authors wish to thank Prof. Akihide Takehara (Iwate University) for the useful comments, and Seiko Watanabe andRyoko Nakamura for their technical assistance. This study was supported in part by a Grant-in-Aid for Science Research (C)from the Japan Society for the Promotion of Science (JSPS) and the 2013 Yeungnam University Research Grant.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bse.2013.12.034.

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