This article is an Advance Online Publication of the authors’ corrected proof. Note that minor changes may be made before final version publication.

J. Japan. Soc. Hort. Sci. Preview doi: 10.2503/jjshs1.CH-100

Floral Pigments from the Blue Flowers of Nemophila menziesii ‘Insignis Blue’ and the Purple Flower of its Variants

Fumi Tatsuzawa1, Kenjiro Toki2, Yuko Ohtani3, Kazuhisa Kato1*, Norio Saito4, Toshio Honda5 and Masahiro Mii3

1Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan 2Faculty of Horticulture, Minami-Kyushu University, Takanabe, Miyazaki 884-0003, Japan 3Graduate School of Horticulture, Chiba University, Matsudo 271-8510, Japan 4Meiji-Gakuin University, Minato-ku, Tokyo 108-8636, Japan 5Faculty of Pharmaceutical Sciences, Hoshi University, Shinagawa-ku, Tokyo 142-8501, Japan

Two anthocyanins (pigments 1 and 2) were detected from the blue flowers of Nemophila menziesii ‘Insignis blue’ and the purple flowers of its variants as the main floral anthocyanins. These two anthocyanins were isolated from the blue flowers and elucidated to be petunidin 3-O-[6-O-(cis-p-coumaroyl)-b-glucopyranoside]- 5-O-[6-O-(malonyl)-b-glucopyranoside] (1) and petunidin 3-O-[6-O-(trans-p-coumaroyl)-b-glucopyranoside]-5- O-[6-O-(malonyl)-b-glucopyranoside] (2), respectively, by chemical and spectroscopic means, and pigment 1 was confirmed as a new anthocyanin in plants. Two flavonol glycosides (pigments 3 and 5) and two flavone glycosides (pigments 4 and 6) were also isolated from the blue flowers, and were identified to be kaempferol 3-(6- rhamnosyl)-glucoside-7-glucoside (3), apigenin 7,4′-di-glucoside (4), kaempferol 3-(2,6-di-rhamnosyl)-glucoside (5), and apigenin 7-glucoside-4′-(6-malonyl)-glucoside (6) as major flavonoids. Among these four flavonoids, however, pigments 4 and 6 (flavones) were not detected in the purple flowers. These results might be attributed to color production in blue and purple flowers.

Key Words: acylated petunidin glycoside, Boraginaceae, Nemophila menziesii, purple and blue flower color.

tures of acylated anthocyanin and flavones (Yoshida Introduction et al., 2009). They reported that the relatively stable Nemophila menziesii Hook. & Arn. (Boraginaceae), blue flower color was produced by the co-pigmentation which is native to northern America, is a popular orna- between anthocyanin and flavone with the assistance of mental plant. It is often cultivated in flower gardens and metal ions in an in vitro study. However, there has been no pots in Japan. The flowers have five petals, blue, violet, report on the chemical structures of other major anthocy- black, or white in color, and it is called Baby Blue Eyes anins and flavonols until now. Moreover, no comparative in English. N. menziesii ‘Insignis blue’ is one of the main study has been conducted between flower color and the cultivars with blue flowers, and its variants with purple in vivo distribution of pigments. In this paper, we report flowers are bred in Japan. However, the mechanism of the isolation and structure elucidation of flavonoids from color production in the purple flowers is not clear. the flowers ofN. menziesii ‘Insignis blue’, and the differ- In our series of investigations on flavonoid-based ence in pigment components between purple flowers of flower color variations in ornamental plants, we were its variants and standard blue flowers. interested in the chemical investigation of the blue flo- Materials and Methods ral pigments of N. menziesii ‘Insignis blue’, although another group has already reported the chemical struc- General procedures Thin layer chromatography (TLC) was carried out Received; November 16, 2013. Accepted; February 6, 2014. on plastic sheets or glass plates coated with cellulose First Published Online in J-STAGE on April 26, 2014. (Merck) using nine mobile phases: BAW (n-BuOH- This work was supported in part by a Grant-in-Aid for Scientific HOAc-H O, 4 : 1 : 2), BuHCl (n-BuOH-2N HCl, 1 : 1), Research (C) (No. 25450037 to FT) from the Japan Society for the 2 Promotion of Science (JSPS). AHW (HOAc-HCl-H2O, 15 : 3 : 82), 1% HCl for fla- * Corresponding author (E-mail: [email protected]). vonoids, and BAW, EAA (EtOH-HOAc-H2O, 3 : 1 : 1),

© 2014 The Japanese Society for Horticultural Science (JSHS), All right reserved 2 F. Tatsuzawa, K. Toki, Y. Ohtani, K. Kato, N. Saito, T. Honda and M. Mii

ETN (EtOH-NH4OH-H2O, 16 : 1 : 3), and EFW (EtOAc- ited absorption maxima at 621sh, 568sh, and 537 nm in HCO2H-H2O, 5 : 2 : 1) for sugars with aniline hydro- the visible region of 360–750 nm. gen phthalate spray reagent, BAW and 15% HOAc for hydroxycinnamic acids with UV light, and Forestal Isolation of flavones, flavonols, and anthocyanin

(HOAc-HCl-H2O, 30 : 3 : 10) for aglycone (Harborne, The flavonoids in the dried blue flowers (200 g) of 1984). N. menziesii ‘Insignis blue’ were extracted with 5% Analytical high performance liquid chromatography HOAc (30 L). Flavonoids in the extract were absorbed (HPLC) was performed on an LC 10A system (Shimadzu, on a Diaion HP-20 column and, after re-extracting with Kyoto, Japan), using a Spherisorb C18 column (4.6 5% HOAc-MeOH, the eluant was concentrated into a ϕ × 250 mm; Waters, Milford, CT, USA) at 40°C with a small volume. The concentrated extract was fractionated flow rate of 1 mL·min-1 and monitoring at 530 nm. The with Sephadex LH-20 Column chromatography (CC) eluant was applied as a linear gradient elution for 40 min using MeOH-HOAc-H2O (6 : 1 : 12). The fractions were from 20 to 85% solvent B (1.5% H3PO4, 20% HOAc, further purified with paper chromatography (PC) (BAW 25% MeCN in H2O) in solvent A (1.5% H3PO4 in H2O) and 15% HOAc) and preparative HPLC. Prep. HPLC with 5 min of re-equilibration at 20% solvent B, for was performed on a LC 10A system (Shimadzu) using flavonoids and hydroxycinnamic acid (method 1). The a mBondasphere C18 column (19 ϕ × 150 mm, Waters) other eluant for malonic acid was applied as an isocratic at 40°C with a flow rate of 4 mL·min-1. Six pigments elution of solvent A for 10 min and monitored at 210 nm were obtained: 1 (10 mg) and 2 (30 mg) as dark violet (method 2) (Tatsuzawa et al., 2013). powders (anthocyanins), 3 (380 mg) and 5 (150 mg) as UV-Vis spectra were recorded on a UV-Vis spectro- pale yellow powders (flavonols), and 4 (30 mg) and 6 photometer MPS-2450 (Shimadzu) in 0.1% HCl-MeOH (45 mg) as pale pink powders (flavones). for anthocyanins and in MeOH for flavonols and fla- vones. High-resolution fast atom bombardment mass Pigment 1 (Petunidin 3-O-[6-O-(cis-p-coumaroyl)- (HR-FABMS) spectra were determined on a JMS-700 b-glucopyranoside]-5-O-[6-O-(malonyl)-b- mass spectrometer (JEOL, Tokyo, Japan) operating in glucopyranoside]) the positive ion mode using a 1 : 1 mixture of dithioth- Dark violet powder; UV-Vis (in 0.1% HCl-MeOH): reitol and 3-nitrobenzyl alcohol as a matrix. lmax 543, 305, 278 nm, Eacyl/Emax(%) = 59, E440/Emax(%) Nuclear magnetic resonance (NMR) spectra (AL- = 14, AlCl3 shift +; TLC (Rf values × 100); BAW 1 400; JEOL) were acquired at 400 MHz for H spectra 12, BuHCl 11, 1% HCl 20, AHW 50; HPLC: Rt(min) 13 and 100 MHz for C spectra in dimethyl sulfoxide 27.3; HR-FABMS calc. C40H41O22: 873.2089. Found: (DMSO)-d6 for flavonols and flavones and DMSO- 873.2057. d6-deuterium chloride (DCl) (9 : 1) for anthocyanins. Chemical shifts are reported relative to a tetramethyl­ Pigment 2 (Petunidin 3-O-[6-O-(trans-p-coumaroyl)- silane (TMS) internal standard (d), and coupling con- b-glucopyranoside]-5-O-[6-O-(malonyl)-b- stants are in Hz. glucopyranoside]) Dark violet powder; UV-Vis (in 0.1% HCl-MeOH):

Plant materials lmax 541, 306, 282 nm, Eacyl/Emax(%) = 76, E440/Emax(%) Seeds of N. menziesii ‘Insignis blue’ were purchased = 13, AlCl3 shift +; TLC (Rf values × 100); BAW from Sakata Seed (Yokohama, Japan). The plants were 23, BuHCl 13, 1% HCl 5, AHW 34; HPLC: Rt(min) grown in the greenhouses of Minami-Kyushu University 33.4; HR-FABMS calc. C40H41O22: 873.2089. Found: 1 and Iwate University. The variant plants with purple 873.2072. H NMR [400 MHz, DMSO-d6-DCl (9 : 1), flowers were bred and grown in the greenhouse of Chiba an internal standard of TMS]; d Petunidin: 8.88 (s, H-4), University. The fresh flowers were collected in spring, 7.05 (brs, H-6), 7.19 (brs, H-8), 7.91 (d, J = 2.2 Hz, dried at 45°C, and stored in desiccators until use. The H-2′), 7.86 (d, J = 2.2 Hz, H-5′), 3.91 (s, -OCH3), Glc flower colors of these plants were recorded by compar- A: 5.62 (d, J = 7.8 Hz, H-1), 3.60 (t, J = 8.5 Hz, H-2), ing them directly with the Royal Horticultural Society 3.51 (t, J = 8.6 Hz, H-3), 3.39 (t, J = 8.2 Hz, H-4), 3.99 (R.H.S.) Colour Chart (Blue 100B for ‘Insignis blue’ (m, H-5), 4.29 (m, H-6a), 4.33 (brd, J = 12.0 Hz, H-6b), and Purple-Violet N80C for the variant by R.H.S. Colour Glc B: 5.18 (d, J = 7.8 Hz, H-1), 3.53 (t, J = 8.9 Hz, Chart). The chromaticity value of these fresh flowers, H-2), 3.41 (m, H-3), 3.25 (t, J = 9.1 Hz, H-4), 3.79 (m, b*/a* = -31.43/-0.60 = 52.38 for ‘Insignis blue’ and H-5), 4.30 (m, H-6a), 4.38 (brd, J = 11.7 Hz, H-6b), b*/a* = -23.16/18.63 = -1.24 for the variant was mea- trans-p-Coumaric acid: 7.35 (d, J = 8.8, H-2,6), 6.37 sured by CM-700d spectrophotometer (Konica-Minolta, (d, J = 8.8, H-3,5), 6.27 (d, J = 15.8, H-a), 7.38 (d, 13 Tokyo, Japan). The absorption spectrum of the fresh J = 15.8, H-b), Malonic acid: 3.33 (s, -CH2-). C NMR petals of N. menziesii ‘Insignis blue’ exhibited charac- [100 MHz, DMSO-d6-DCl (9 : 1), an internal standard teristic absorption maxima at 715, 630, 590, and 555sh of TMS]; d Petunidin: 162.0 (C-2), 145.1 (C-3), 133.0 nm in the visible region of 360–750 nm. In contrast, the (C-4), 154.8 (C-5), 105.6 (C-6), 168.1 (C-7), 97.7 (C-8), absorption spectrum of the fresh petals of variants exhib- 155.2 (C-9), 111.6 (C-10), 112.9 (C-1′), 108.5 (C-2′), J. Japan. Soc. Hort. Sci. Preview 3

150.0 (C-3′), 144.6 (C-4′), 144.7 (C-5′), 113.6 (C-6′), an internal standard of TMS); d Apigenin: 164.3 (C-2),

56.4 (-OCH3), Glc A: 101.0 (C-1), 73.2 (C-2), 76.5 104.1 (C-3), 181.4 (C-4), 160.5 (C-5), 99.5 (C-6), 163.5 (C-3), 70.2 (C-4), 75.0 (C-5), 65.1 (C-6), Glc B: 102.3 (C-7), 94.9 (C-8), 157.0 (C-9), 105.5 (C-10), 123.8 (C-1), 73.7 (C-2), 76.2 (C-3), 69.8 (C-4), 74.8 (C-5), (C-1′), 128.3 (C-2′,6′), 116.6 (C-3′,5′), 158.9 (C-4′), Glc 64.1 (C-6), trans-p-Coumaric acid: 125.1 (C-1), 130.6 E: 99.8 (C-1), 73.0 (C-2), 76.5 (C-3), 69.5 (C-4), 77.2 (C-2,6), 115.9 (C-3,5), 160.9 (C-4), 113.9 (C-a), 145.3 (C-5), 60.6 (C-6), Glc F: 99.8 (C-1), 73.1 (C-2), 76.5 (C-b), 167.4 (COOH), Malonic acid: 168.0 (C-1), 41.3 (C-3), 69.5 (C-4), 77.2 (C-5), 60.6 (C-6). (C-2), 168.1 (C-3). Pigment 5 (Kaempferol 3-O-(2,6-di-O-rhamnosyl)- Pigment 3 (Kaempferol 3-O-(6-O-rhamnosyl)-glucoside- glucoside)

7-O-glucoside) Pale yellow powder; TLC (Rf values × 100); BAW Pale yellow powder; TLC (Rf values × 100); BAW 45, BuHCl 60, 1% HCl 72, AHW 80, UV Dark brown,

16, BuHCl 21, 1% HCl 60, AHW 81, UV Dark brown, UV/NH3 yellow, UV (lmax nm); MeOH 346, 322sh, UV/NH3 yellow, UV (lmax nm); MeOH: 348, 324sh, 299sh, 265, +NaOMe 363, 299, 272, +AlCl3 395, 352, 266, +NaOMe: 396, 359, 265, +AlCl3: 396, 354, 300, 304, 273, +AlCl3/HCl 393, 349, 302, 274, +NaOAc 348, 273, +AlCl3/HCl: 394, 349, 299, 274, +NaOAc: 348, 314sh, 266, +NaOAc/H3BO3 347, 319sh, 266, HPLC + 325sh, 266, +NaOAc/H3BO3: 348, 326sh, 266, HPLC (Rt min) 22.1, HR-FABMS calc. for C33H41O19 [M + H] + 1 (Rt min) 16.5, HR-FABMS calc. for C33H41O20 [M + H] 741.2242, Found 741.2213. H NMR (400 MHz, 1 757.2191, Found 757.2180. H NMR (400 MHz, DMSO-d6, an internal standard of TMS); d Kaempferol: DMSO-d6, an internal standard of TMS); d Kaempferol: 6.19 (d, J = 1.9 Hz, H-6), 6.40 (d, J = 1.9 Hz, H-8), 7.95 6.46 (s, H-6), 6.89 (s, H-8), 8.02 (d, J = 8.0 Hz, H-2′,6′), (d, J = 8.8 Hz, H-2′,6′), 6.88 (d, J = 8.8 Hz, H-3′,5′), Glc 6.90 (d, J = 8.0 Hz, H-3′,5′), Glc C: 5.36 (d, J = 7.3 Hz, C: 5.49 (d, J = 7.1 Hz, H-1), 3.41 (t, J = 9.5 Hz, H-2), H-1), 3.19 (m, H-2), 3.17 (m, H-3), 3.05 (m, H-4), 3.19 3.20 (m, H-3), 3.06 (t, J = 9.3 Hz, H-4), 3.36 (m, H-5), (m, H-5), 3.29 (m, H-6a), 3.70 (brd, J = 11.0 Hz, H-6b), 3.21 (m, H-6a), 3.67 (brd, J = 10.0 Hz, H-6b), Rha A: Glc D: 5.08 (d, J = 7.3 Hz, H-1), 3.27 (t, J = 8.2 Hz, 4.32 (s, H-1), 3.36 (m, H-2), 3.25 (m, H-3), 3.06 (t, H-2), 3.19 (m, H-3), 3.15 (m, H-4), 3.43 (m, H-5), 3.48 J = 9.3 Hz, H-4), 3.25 (m, H-5), 0.96 (d, J = 6.4 Hz,

(m, H-6a), 3.71 (m, H-6b), Rha A: 4.38 (s, H-1), 3.45 -CH3), Rha B: 5.06 (s, H-1), 3.73 (brs, H-2), 3.48 (brd, (brs, H-2), 3.23 (m, H-3), 3.08 (t, J = 9.5 Hz, H-4), J = 9.5 Hz, H-3), 3.14 (t, J = 9.4 Hz, H-4), 3.76 (dd, 13 13 3.30 (m, H-5), 0.99 (d, J = 5.8 Hz, -CH3). C NMR J = 6.1, 9.5 Hz, H-5), 0.81 (d, J = 6.4 Hz, -CH3). C (100 MHz, DMSO-d6, an internal standard of TMS); NMR (100 MHz, DMSO-d6, an internal standard of d Kaempferol: 157.4 (C-2), 133.5 (C-3), 177.6 (C-4), TMS); d kaempferol: 156.9 (C-2), 132.6 (C-3), 177.2 160.9 (C-5), 99.4 (C-6), 162.9 (C-7), 94.7 (C-8), 156.1 (C-4), 161.2 (C-5), 98.7 (C-6), 164.1 (C-7), 93.8 (C-8), (C-9), 105.7 (C-10), 120.7 (C-1′), 131.0 (C-2′,6′), 115.2 156.5 (C-9), 104.0 (C-10), 121.0 (C-1′), 130.7 (C-2′,6′), (C-3′,5′), 160.1 (C-4′), Glc C: 101.2 (C-1), 74.2 (C-2), 115.1 (C-3′,5′), 159.8 (C-4′), Glc C: 98.6 (C-1), 77.1 76.4 (C-3), 70.0 (C-4), 76.4 (C-5), 66.9 (C-6), Glc D: (C-2), 75.6 (C-3), 70.5 (C-4), 77.2 (C-5), 66.9 (C-6), Rha 99.9 (C-1), 73.1 (C-2), 75.9 (C-3), 69.6 (C-4), 77.2 A: 100.8 (C-1), 70.3 (C-2), 70.5 (C-3), 71.8 (C-4), 68.3

(C-5), 60.6 (C-6), Rha A: 100.8 (C-1), 70.4 (C-2), 70.6 (C-5), 17.7 (-CH3), Rha B: 100.6 (C-1), 70.3 (C-2), 70.3 (C-3), 71.8 (C-4), 68.3 (C-5), 17.7 (-CH3). (C-3), 71.8 (C-4), 68.3 (C-5), 17.3 (-CH3).

Pigment 4 (Apigenin 4′,7-di-O-glucoside) Pigment 6 (Apigenin 4′-O-(6-O-malonyl)-glucoside-7-

Pale pink powder; TLC (Rf values × 100); BAW 14, O-glucoside)

BuHCl 12, 1% HCl 39, AHW 54, UV Dark brown, UV/ Pale pink powder; TLC (Rf values × 100); BAW 13, NH3 Dark yellow, UV (lmax nm); MeOH 335sh, 316, BuHCl 14, 1% HCl 10, AHW 42, UV Dark brown, UV/

269, +NaOMe 335sh, 316, 269, +AlCl3 378, 336, 297, NH3 Dark yellow, UV (lmax nm); MeOH 337sh, 316, 278, +AlCl3/HCl 378, 332, 297, 278, +NaOAc 335sh, 270, +NaOMe 337sh, 284, +AlCl3 375, 335, 297, 278, 316, 269, +NaOAc/H3BO3 335sh, 316, 269, HPLC (Rt +AlCl3/HCl 372, 333, 297, 278, +NaOAc 337sh, 316, + min) 18.7, HR-FABMS calc. for C27H31O15 [M + H] 270, +NaOAc/H3BO3 337sh, 316, 270, HPLC (Rt min) 1 + 595.1633, Found 595.1663. H NMR (400 MHz, 24.4, HR-FABMS calc. for C30H33O18 [M + H] 681.1667, 1 DMSO-d6, an internal standard of TMS); d Apigenin: Found 681.1696. H NMR (400 MHz, DMSO-d6, an 7.00 (s, H-3), 6.46 (d, J = 2.2 Hz, H-6), 6.88 (d, internal standard of TMS); d Apigenin: 6.96 (s, H-3), J = 2.2 Hz, H-8), 8.08 (d, J = 9.0 Hz, H-2′,6′), 7.21 (d, 6.44 (d, J = 1.9 Hz, H-6), 6.86 (d, J = 1.9 Hz, H-8), 8.08 J = 9.0 Hz, H-3′,5′), Glc E: 5.04 (d, J = 7.3 Hz, H-1), (d, J = 9.0 Hz, H-2′,6′), 7.21 (d, J = 9.0 Hz, H-3′,5′), 3.29 (t, J = 8.4 Hz, H-2), 3.26 (m, H-3), 3.16 (m, H-4), Glc E: 5.03 (d, J = 7.3 Hz, H-1), 3.32 (t, J = 8.6 Hz, 3.36 (m, H-5), 3.42 (m, H-6a), 3.46 (m, H-6b), Glc F: H-2), 3.25 (t, J = 8.5 Hz, H-3), 3.20 (t, J = 8.5 Hz, H-4), 5.08 (d, J = 7.3 Hz, H-1), 3.18 (t, J = 8.4 Hz, H-2), 3.68 (ddd, J = 1.9, 7.3, 9.4 Hz, H-5), 4.04 (dd, J = 7.2, 3.36 (m, H-3), 3.20 (m, H-4), 3.41 (m, H-5), 3.49 (m, 12.0 Hz, H-6a), 4.42 (dd, J = 1.4, 12.0 Hz, H-6b), Glc 13 H-6a), 3.71 (m, H-6b). C NMR (100 MHz, DMSO-d6, F: 5.04 (d, J = 7.3 Hz, H-1), 3.23 (m, H-2), 3.29 (t, 4 F. Tatsuzawa, K. Toki, Y. Ohtani, K. Kato, N. Saito, T. Honda and M. Mii

J = 7.8 Hz, H-3), 3.17 (t, J = 8.1 Hz, H-4), 3.43 (m, H-5), UV) Violet; HPLC (method 1): Rt(min) 17.2 and 16.2 3.48 (m, H-6a), 3.73 (brd, J = 10.4 Hz, H-6b), Malonic (trans and cis). 13 acid: 3.17 (s, -CH2-). C NMR (100 MHz, DMSO-d6, an internal standard of TMS); d Apigenin: 163.7 (C-2), Malonic acid

104.1 (C-3), 182.1 (C-4), 161.1 (C-5), 98.6 (C-6), 163.1 HPLC (method 2): Rt(min) 4.1. (C-7), 94.7 (C-8), 157.0 (C-9), 105.5 (C-10), 123.9 Results and Discussion (C-1′), 128.4 (C-2′,6′), 116.7 (C-3′,5′), 160.3 (C-4′), Glc E: 99.6 (C-1), 73.1 (C-2), 76.3 (C-3), 69.9 (C-4), 74.0 Two major HPLC peaks were detected in each group (C-5), 63.4 (C-6), Glc F: 99.8 (C-1), 73.1 (C-2), 76.5 of anthocyanins, flavones, and flavonols, respectively,

(C-3), 69.7 (C-4), 77.2 (C-5), 60.6 (C-6), Malonic acid: in 5% HOAc-H2O extract from the blue flowers of 168.6 (C-1), 43.4 (C-2), 168.8 (C-3). N. menziesii ‘Insignis blue’ by HPLC analysis with monitoring at 530 nm for anthocyanins and at 350 nm Aglycones, sugars, and acids for both flavonols and flavones, respectively. The pro- Acid hydrolysis of pigments (ca. 0.5 mg each) was portions of these anthocyanin peaks were 9.3% (pigment achieved by 2N HCl (1 mL) at 90°C for 2 h. Moreover, 1) and 78.3% (pigment 2) based on the percentage of the alkaline hydrolysis of pigments (ca. 0.5 mg each) was total absorbance of peaks (530 nm), those of flavonols achieved by 2N NaOH (1 mL) using a degassed syringe peaks were 28.6% (pigment 3) and 8.1% (pigment 5), to stir for 15 min. The solution was then acidified with and those of flavones peaks were 8.1% (pigment 4) and 2N HCl (1.1 mL). These solutions were used for TLC 16.7% (pigment 6), respectively, based on the percent- and HPLC (Tatsuzawa et al., 2012). Products in these age of the total absorbance of peaks (350 nm) (Fig. 1). hydrolysates were identified in comparison with the In contrast, the proportions of flavonoid peaks of purple authentic standards of glucose, rhamnose, p-coumaric flowers were 18.1% (pigment 1) and 59.3% (pigment 2) acid, malonic acid, kaempferol, and apigenin, which based on the percentage of the total absorbance of peaks were of commercial origin. Petunidin and petunidin (530 nm), and those of flavonol peaks were 28.5% (pig- 3,5-di-glucoside were obtained from the flowers of ment 3) and 13.4% (pigment 5) based on the percentage Torenia fournieri (Tatsuzawa and Shinoda, 2005). of the total absorbance of peaks (350 nm), respectively (Fig. 1). Petunidin 3,5-di-glucoside (deacylanthocyanin) The six pigments 1–6 were purified using Diaion

UV-Vis: lmax 537, 274 nm, E440/Emax = 12%, AlCl3 HP-20 resin CC, PC, Sephadex LH-20 CC, and prepar- shift +; TLC (Rf values × 100); BAW 6, BuHCl 1, 1% ative HPLC, as described previously (Toki et al., 2009). HCl 7, AHW 24; HPLC (method 1): Rt(min) 15.0. Chromatographic and spectroscopic properties of these pigments are shown in Table 1 and in the Experimental Petunidin Section.

UV-Vis: lmax 547, 272 nm, E440/Emax = 20%, AlCl3 Acid hydrolysis of anthocyanins (pigments 1 and shift +; TLC (Rf values × 100); Forestal 47; HPLC 2) resulted in petunidin, malonic acid, and glucose. (method 1): Rt(min) 26.9. Moreover, cis-p-coumaric acid and trans-p-coumaric acid were detected in the hydrolysate of pigments 1 and Kaempferol 2, respectively, by analysis of TLC and HPLC. Acid

UV-Vis: lmax 368, 268 nm, TLC (Rf values × 100); hydrolysis of flavonols and flavones (pigments 3–6) Forestal 56; HPLC (method 1): Rt(min) 38.0. resulted in kaempferol for pigments 3 and 5, and api- genin for pigment 4 and 6 as their aglycones. In their Apigenin sugar components, glucose and rhamnose for pigments 3

UV-Vis: lmax 336, 269 nm, TLC (Rf values × 100); and 5 and glucose for pigments 4 and 6 were observed in Forestal 84; HPLC (method 1): Rt(min) 39.2. hydrolysates, but malonic acid was detected only in the hydrolysate of pigment 6. Furthermore, alkaline hydro- Glucose lysis of pigments 1 and 2 resulted in one deacylanthocy-

TLC (Rf values × 100); BAW 24, EAA 18, ETN 62, anin, which was identical to petunidin 3,5-diglucoside EFW 49; Color (aniline hydrogen phthalate (AHP)) on the basis of the comparison of TLC and HPLC pro- Brown. files (Tatsuzawa and Shinoda, 2005). From these results, the structures of pigments Rhamnose 1–6 were presumed to be cis-p-coumaroyl-malonyl-

TLC (Rf values × 100); BAW 41, EAA 37, ETN 71, petunidin 3,5-diglucoside (pigment 1), trans-p- EFW 52; Color (AHP) Brown. coumaroyl-malonyl-petunidin 3,5-diglucoside (pigment 2), di-glucosyl-mono-rhamnosyl-kaempferol (pigment p-Coumaric acid 3), di-glucosyl-apigenin (pigment 4), mono-glucosyl-

TLC (Rf values × 100); BAW 91 and 91 (trans and di-rhamnosyl-kaempferol (pigment 5), and malonyl- cis), 15% HOAc 49 and 74 (trans and cis); Color (under di-glucosyl-apigenin (pigment 6), respectively. J. Japan. Soc. Hort. Sci. Preview 5

Fig. 1. HPLC profile of anthocyanins (530 nm) and flavonols and flavones (350 nm) in the purple and blue flowers of Nemophila menziesii. 1: Petunidin 3-O-[6-O-(cis-p-coumaroyl)-b-glucopyranoside]-5-O-[6-O-(malonyl)-b-glucopyranoside]. 2: Petunidin 3-O-[6-O-(trans-p- coumaroyl)-b-glucopyranoside]-5-O-[6-O-(malonyl)-b-glucopyranoside]. 3: Kaempferol 3-O-(6-O-rhamnosyl)-glucoside-7-O-glucoside. 4: Apigenin 4′,7-di-O-glucoside. 5: Kaempferol 3-O-(2,6-di-O-rhamnosyl)-glucoside. 6: Apigenin 4′-O-(6-O-malonyl)-glucoside-7-O-glucoside.

The elemental components of these pigments were two olefinic proton signals of the p-coumaric acid moi- also confirmed by measuring their HR-FABMS spec- ety, indicated a cis configuration for the acid on the basis tra (see Materials and Methods). The structures of these of their coupling constants (J = 13.2 Hz) (Table 1). The pigments were further elucidated as follows based on chemical shifts of the sugar moiety protons were observed the analysis of their 1H and 13C NMR spectra [400 MHz in the region of d 5.70–3.27, with the two anomeric pro- 1 13 for H and 100 MHz for C spectra in DCl-DMSO-d6 ton resonances at 5.70 (d, J = 7.8 Hz, Glc A) and 5.18 (d, (1 : 9) or DMSO-d6, including 2D correlation spectros- J = 7.6 Hz, Glc B). Based on the observed coupling con- copy (COSY), 2D nuclear Overhauser enhancement stants (Table 1), these two sugars were assumed to be in spectroscopy (NOESY), hetero-nuclear multiple quan- their b-pyranose forms. The linkages and/or positions of tum coherence (HMQC), and hetero-nuclear multiple the attachments of the sugar and acyl groups were deter- bond correlation (HMBC) spectra] (see Materials and mined based on 2D COSY and NOESY experiments. By Methods). application of a NOESY experiment, NOEs between H-1 of Glc A and H-4 (d 8.72) of petunidin, H-1 of Glc B 1. Pigment 1 (anthocyanin) and H-6 (d 6.96) of petunidin were observed (Fig. 2), The FABMS of pigment 1 gave a molecular ion [M]+ supporting the presence of the glycosylation of C-3 and at 873 m/z (calc. for C40H41O22), indicating that pigment 1 C-5 petunidin hydroxyl groups with Glc A and Glc B, is composed of petunidin with two molecules of glucose respectively. and one molecule each of p-coumaric acid and malo- Four characteristic downfield shifted proton signals nic acid. The elemental components were confirmed by were assigned to the methylene protons of Glc A (d 4.38 measuring its HR-FABMS (calc. C40H41O22: 873.2089, and 4.27, H-6a and b) and Glc B (d 4.30 and 4.47, H-6a found 873.2057). and b), indicating acylation of C-6 OHs (Glc A and B) The 1H NMR spectrum of pigment 1 exhibited nine with two acid molecules. HMBC spectra were studied aromatic protons identified with petunidin and p- to identify the attachment sites of acid moieties (Fig. 2). coumaric acid moieties, together with their coupling The signals of the methylene protons of Glc A and Glc constants, and assigned as shown in Table 1. Three pro- B was correlated with those of the COOH carbons of tons were assigned to a methoxyl group of petunidin. p-coumaric acid (d 167.0) and malonic acid (d 167.5) in A set of one pair of doublet resonance, assigned to the the HMBC spectrum (Fig. 2). Therefore, the structure of 6 F. Tatsuzawa, K. Toki, Y. Ohtani, K. Kato, N. Saito, T. Honda and M. Mii

Table 1. 1H and 13C NMR spectroscopic data of anthocyanin pigment 1 from Nemophila menziesii. Pigment 1 1H d (ppm) 13C d (ppm) Petunidin 2 161.2 3 144.6 4 8.72 s 131.6 5 154.6 6 6.96 d(1.2) 103.5 7 167.9 Fig. 2. Structure of pigment 1 isolated from the blue flowers of 8 7.06 d(1.2) 95.8 Nemophila menziesii ‘Insignis blue’. Main NOEs observed are 9 154.9 indicated by arrows. Main HMBC correlations observed are 10 111.5 indicated by dotted arrows. 1′ 108.9 2′ 7.89 d(2.2) 113.0 3′ 148.6 plants (Andersen and Jordheim, 2006; Harborne and 4′ 144.2 Baxter, 1999; Veitch and Grayer, 2008, 2011). 5′ 146.2 6′ 7.86 d(2.2) 107.9 2. Pigment 2 (anthocyanin) + -OMe 3.93 s 56.3 The FABMS of pigment 2 gave a molecular ion [M] Glc A at 873 m/z (calc. for C40H41O22), indicating that pigment 1 5.70 d(7.8) 99.6 2 is composed of petunidin with two molecules of glu- 2 3.62 t(8.4) 74.0 cose and one molecule each of p-coumaric acid and 3 3.50 t(8.8) 77.3 malonic acid. 1 4 3.28 t(9.2) 69.6 The H NMR spectrum of pigment 2 was similar 5 4.02 ddd(2.0, 8.2, 10.2) 75.5 to that of pigment 1, except for the signals of the p- 6a 4.38 dd(8.6, 12.0) 64.1 coumaric acid moiety (see Materials and Methods). The 6b 4.27 m olefinic protons of pigment2 were shifted to lower fields d = Glc B of 6.27 and 7.38 (d, J 15.8 Hz each) in comparison 1 5.18 d(7.6) 100.6 with those of pigment 1, establishing pigment 2 as petuni- b 2 3.56 t(8.4) 74.3 din 3-O-[6-O-(trans-p-coumaroyl)- -glucopyranoside]- b 3 3.43 t(8.7) 76.4 5-O-[6-O-(malonyl)- -glucopyranoside], which has 4 3.27 t(9.4) 70.3 been found in Hyacinthus orientalis (Hosokawa et al., 5 3.85 ddd(2.3, 7.3, 9.7) 74.8 1995). Moreover, this pigment was found in N. menziesii 6a 4.30 m 63.1 by K. Yoshida and her co-workers, and named nemoph­ 6b 4.47 dd(1.6, 11.8) ilinin, whose structure was also confirmed by analysis of 13 cis-p-Coumaric acid its C, HMQC, and HMBC NMR spectra. 1 125.1 2,6 7.27 d(8.8) 132.2 3. Pigments 3 and 5 (flavonols) 3,5 6.52 d(8.8) 114.6 Acid hydrolysis of pigments 3 and 5 yielded kaemp- 4 158.6 ferol, rhamnose, and glucose. The FABMS of pigments 3 + + a 5.70 d(13.2) 115.1 and 5 gave their molecular ions [M H] at 757 m/z (calc. b 6.45 d(13.2) 143.1 C33H41O20) and at 741 m/z (calc. C33H41O19), respectively, COOH 167.0 indicating that pigment 3 is composed of kaempferol Malonic acid with one molecule of rhamnose and two molecules of 1 167.5 glucose, and pigment 5 is composed of kaempferol with 2 3.45 s 41.4 two molecules of rhamnose and one molecule of glucose. 3 168.2 Their elemental components were confirmed by measur- ing their HR-FABMS (see Materials and Methods). 1H NMR (400 MHz) and 13C NMR (100 MHz) (DCl-DMSO-d 6 The 1H NMR spectrum of pigment 3 exhibited six aro- (1 : 9)), at 25°C, an internal standard of TMS. Coupling constants (J in Hz) in parentheses. matic proton signals of kaempferol (see Materials and Methods). The anomeric protons of Glc C, Glc D, and Rha A were observed at d 5.36 (d, J = 7.3 Hz), 5.08 (d, pigment 1 was determined to be petunidin 3-O-[6-O-(cis- J = 7.3 Hz), and 4.38(s). The binding patterns of these p-coumaroyl)-b-glucopyranoside]-5-O-[6-O-(malonyl)- compounds were confirmed by NOESY and/or HMBC b-glucopyranoside], which is a new anthocyanin in experiments. NOEs between H-1 of Rha A and H-6a and J. Japan. Soc. Hort. Sci. Preview 7 b of Glc C and H-1 of Glc D and H-6 and 8 of kaempferol which has been found in some Salvia species (Yoshida were observed. Moreover, correlations between H-1 of et al., 2009). Moreover, this pigment has been found in Glc C and C-3 of kaempferol, H-1 of Rha A and C-6 of N. menziesii by Yoshida et al. (2009), and its structure Glc C, and H-1 of Glc D and C-7 of kaempferol were was also confirmed by analysis of its 13C, HMQC, and observed in the HMBC spectrum. These results suggest HMBC NMR spectra. that OH-3 and 7 of kaempferol and OH-6 of Glc C were The 1H NMR spectrum of pigment 6 was identical glycosylated with Glc C, Glc D, and Rha A, respectively. to that of pigment 4 except for the signals of malonic Therefore, pigment 3 was determined to be kaempfe- acid moiety (see Materials and Methods). The methy- rol 3-O-(6-O-rhamnosyl)-glucoside-7-O-glucoside. This lene proton of malonic acid was assigned at 3.17 (s). is the first report of the isolation of this pigment from By analysis of its NOESY spectrum, NOEs between the genus Nemophila, although the pigment has pre- CH2 of malonic acid and CH2 of Glc E were observed viously been reported in the plant Equisetum palustre to support that OH-4′ of apigenin was glycosylated with (Beckmann and Geiger, 1963). malonylglucose. Therefore, pigment 6 was determined The 1H NMR spectrum of pigment 5 exhibited six aro- to be apigenin 4′-O-(6-O-malonyl)-glucoside-7-O- matic proton signals of kaempferol (see Materials and glucoside, which has been found in Centaurea cyanus Methods). The anomeric protons of Glc C, Rha A, and (Yoshida et al., 2009). This structure was also confirmed Rha B were observed at d 5.49 (d, J = 7.1 Hz), 4.32 (s), by analysis of its 13C, HMQC, and HMBC NMR spec- and 5.06 (s), respectively. The binding patterns of these tra. Moreover, this pigment has been reported from compounds were confirmed by NOESY and/or HMBC N. menziesii by Yoshida et al. (2009) as a flavone com- experiments. NOEs between H-1 of Rha A and H-6a ponent in the metal complex pigment (Nemophilin). and b of Glc C were observed. Moreover, correlations An in vitro restoration study of the blue flower color between H-1 of Glc C and C-3 of kaempferol, H-1 of of N. menziesii was recently performed by mixing pig- Rha A and C-6 of Glc C, and H-1 of Rha B and C-2 ments 2 (anthocyanin) and 6 (flavone) with Fe and Mg of Glc C were observed in the HMBC spectrum. These (Yoshida et al., 2009). The flower color of the variants results suggested that OH-3 of kaempferol, OH-6 of Glc used in this study, which did not contain pigment 6, was C, and OH-2 of Glc C were glycosylated with Glc C, Rha purple like that of Commelina communis (Kondo et al., A, and Rha B, respectively. Therefore, pigment 3 was 1991). Pigment 4 (flavone) was not also accumulated in determined to be kaempferol 3-O-(2,6-di-O-rhamnosyl)- the purple flowers of N. menziesii ‘Insignis blue’s vari- glucoside. This is the first report of the isolation of this ants. Therefore, it might be considered that pigments 4 pigment from the genus Nemophila, although the pig- and 6 (flavones) play a role as co-pigments in producing ment has previously been reported in the plant Clitoria the blue flower color ofN. menziesii ‘Insignis blue’ as an ternatea (Kazuma et al., 2003). important component in vivo.

4. Pigments 4 and 6 (flavones) Literature Cited Acid hydrolysis of pigments 4 and 6 yielded apigenin Andersen, Ø. M. and M. Jordheim. 2006. The anthocyanins. and glucose. Moreover, malonic acid was detected in p. 471–551. In: Ø. M. Andersen and K. R. Markham (eds.). the hydrolysate of pigment 6 by analysis using TLC Flavonoids: chemistry, biochemistry and applications. CRC and HPLC. The FABMS of pigments 4 and 6 gave their Press, Boca Raton. + Beckmann, S. and H. Geiger. 1963. Über zwei kämpferolglyko- molecular ions [M + H] at 595 m/z (calc. C27H31O15) and at 681 m/z (calc. C H O ), respectively, indicating that side des sumpfschachtelhalmes (Equisetum palustre). 30 33 18 Phytochemistry 2: 281–287. pigment 4 is composed of apigenin with two molecules Harborne, J. B. 1984. Phytochemical methods, second ed. of glucose, and pigment 6 is composed of apigenin with Chapman and Hall, London. two molecules of glucose and one molecule of malo- Harborne, J. B. and H. Baxter. 1999. Anthocyanins. p. 1–114. In: nic acid. Their elemental components were confirmed J. B. Harborne and H. Baxter (eds.). The Handbook of Natural by measuring their HR-FABMS (see Materials and Flavonoids, vol. 2. John Wiley & Sons, Chichester. Methods). Hosokawa, K., Y. Fukunaga, E. Fukushi and J. Kawabata. 1995. The 1H NMR spectrum of pigment 4 exhibited seven Seven acylated anthocyanins in the blue flowers ofHyacinthus orientalis. Phytochemistry 38: 1293–1298. aromatic proton signals of apigenin (see Materials and Kazuma, K., N. Noda and M. Suzuki. 2003. Malonylated fla- Methods). The anomeric protons of Glc E and Glc F vonol glycosides from the petals of Clitoria ternatea. were observed at d 5.04 (d, J = 7.3 Hz) and 5.08 (d, Phytochemistry 62: 229–237. J = 7.3 Hz). The binding patterns of these compounds Kondo, T., K. Yoshida, M. Yoshikane and T. Goto. 1991. were confirmed by NOESY experiments. NOEs between Mechanism for color development in purple flower of H-1 of Glc E and H-3′ and 5′ of apigenin and H-1 of Glc Commelina communis. Agric. Biol. Chem. 55: 2919–2921. F and H-6 and 8 of apigenin were observed. These results Tatsuzawa, F. and K. Shinoda. 2005. Comparison between identi- fication of anthocyanin by HPLC analysis with a photodiode suggest that OH-4′ and 7 of apigenin were glycosylated array detector and that using TLC combined with UV-Vis with Glc E and Glc F, respectively. Therefore, pigment spectral analysis. Hort. Res. (Japan) 4: 225–228 (In Japanese 4 was determined to be apigenin 4′,7-di-O-glucoside, with English abstract). 8 F. Tatsuzawa, K. Toki, Y. Ohtani, K. Kato, N. Saito, T. Honda and M. Mii

Tatsuzawa, F., S. Ito, M. Sato, H. Muraoka, K. Kato, Y. flowers of Nigella damascena. Heterocycles 78: 2287–2294. Takahata and S. Ogawa. 2013. A tetra-acylated cyanidin 3- Veitch, N. C. and R. J. Grayer. 2008. Flavonoids and their glyco- sophoroside-5-glucoside from the purple-violet flowers of sides, including anthocyanins. Nat. Prod. Rep. 25: 555–611. Moricandia arvensis (L.) DC. (Brassicaceae). Phytochemistry Veitch, N. C. and R. J. Grayer. 2011. Flavonoids and their gly- Lett. 6: 170–173. cosides, including anthocyanins. Nat. Prod. Rep. 28: 1626– Tatsuzawa, F., N. Saito, K. Toki, K. Shinoda and T. Honda. 2012. 1695. Flower colors and their anthocyanins in Matthiola incana cul- Yoshida, K., M. Mori and T. Kondo. 2009. Blue flower color tivars (Brassicaceae). J. Japan. Soc. Hort. Sci. 81: 91–100. development by anthocyanins: from chemical structure to cell Toki, K., N. Saito, A. Nogami, F. Tatsuzawa, A. Shigihara and T. physiology. Nat. Prod. Rep. 26: 884–915. Honda. 2009. Flavonoid glycosides isolated from the blue