Flavonoids and Their Distribution Patterns in the Flowers of Gladiolus Cultivars

The Gladiolus is the largest genus in the family Iridaceae that contains many bulbous plants from southern Africa. Flower coloration varies from white, yellow, pink, red to purple. Some anthocyanins have been isolated as flower pigments, but other flavonoids are hardly reported. In this survey, the anthocyanins and other flavonoids of the bluish cultivar, G. x grandiflora ‘Ariake’ were isolated and identified. When absorption spectra of the fresh perianth of ‘Ariake’ was compared with that of isolated anthocyanin, the former absorption maxima bathochromically shifted than the latter one, so it was presumed that the flower color was changed to more purple by the presence of copigments. It was shown by HPLC analysis that three anthocyanins and some flavonoids are present in the crude extract. The major anthocyanin was identified as malvidin 3,5-di-O-glucoside (malvin) and the two minor ones were characterized as malvidin glycosides. Of the flavonoids, three were identified as kaempferol 3-O-rutinoside, kaempferol 3-O-sophoroside and quercetin 3-O-rutinoside by LC-MS, UV, Hand C-NMR spectra, and direct PC and HPLC comparisons with authentic specimens. The remaining pigments were characterized as flavonol 3-O-glycosides based on kaempferol, quercetin, myricetin, laricitrin and syringetin. The pigment patterns of representative red, purple, pink, white and yellow cultivars were surveyed by HPLC. The purple flower cultivar contained many flavonols compared with other flower colors, e.g. pink and red suggesting that the flavonol glycosides represent the more purplish color as copigments. INTRODUCTION Twenty-three anthocyanins, i.e., 3-O-rhamnosylglucoside-5-O-glucosides and 3,5-di-O-glucosides and 3-O-rhamnosylglucosides of cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin, and delphinidin triglucoside, malvidin 3-O-glucoside, pelargonidin 3-O-diglucoside-5-O-glucoside, pelargonidin 3-O-glucoside and petunidin 3-O-glucoside have been detected from the flowers of Gladiolus cultivars (Shibata and Nozaka, 1963; Yatomi and Arisumi, 1968; Arisumi and Kobayashi, 1971; Akavia et al., 1981). On the other hand, 3-galactosides of kaempferol, quercetin, myricetin, laricitrin (myricetin 3’-methyl ether) and syringetin (myricetin 3’,5’-dimethyl ether) were only found from the flowers of a wild species, Gladiolus tristis as well as other flavonoids (Williams et al., 1986). We hypothesized that bluing effect of the flower color was induced by copigmentation with other substances which could be quantitatively detected by high performance liquid chromatography (HPLC). Therefore, it was proved that some UV absorbing compounds are more abundantly present in bluish purple flowers than other color ones. In this paper, the isolation and identification of the flower pigments in G. x grandiflora ‘Ariake’ which may be one of the most bluish cultivars, is described. In addition, the pigment patterns of five cultivars which are representative red, purple, pink, white and yellow, were preparatively surveyed by HPLC. Proc. IX Intl. Symp. on Flower Bulbs Eds.: H. Okubo, W.B. Miller and G.A. Chastagner Acta Hort. 673, ISHS 2005 488 MATERIALS AND METHODS Plant Materials The flowers of the representative five garden cultivars of Gladiolus x grandiflora were used for HPLC survey in this experiment (Fig. 4). G. x grandiflora ‘Ariake’ was collected in Ogawa Town, Ibaraki Pref., Japan, and the other gladioli were grown in Tsukuba Botanical Garden, National Science Museum, Tsukuba, Japan. Isolation of Anthocyanins Anthocyanins were extracted with MAW (MeOH/AcOH/H2O = 50:5:45) from the perianth of G. x grandiflora ‘Ariake’. The extract was applied to preparative paper chromatography (PPC) with solvent systems: BAW (n-BuOH/HOAc/H2O = 4:1:5, upper phase) and 15% HOAc. The isolated anthocyanin A1 was purified by preparative HPLC (solvent system: HCOOH/MeCN/H2O = 5:11:84 and 5:13:82) and then Sephadex LH-20 column chromatography (solvent system: HOAc/MeOH/H2O = 5:70:25). Isolation of Other Flavonoids Other flavonoids were extracted with MeOH. After HPLC investigation of the concentrated extracts, the extract was applied to PPC with BAW, 15% HOAc and then BEW (n-BuOH/EtOH/H2O = 4:1:2.2). Three components (F1, F2 and F3) isolated were purified by preparative HPLC (solvent system: MeCN: H2O = 20:80 and 22:78) and Sephadex LH-20 column chromatography (solvent system: 70% MeOH). The major components F1 and F3 were obtained as pale yellow powder from EtOAc/MeOH solution. HPLC Qualitative HPLC analyzes of the isolated compounds were performed with Shimadzu HPLC systems using Shim-pack CLC-ODS [I.D. 6.0 x 150 mm (Shimadzu)], at a flow-rate of 1.0 ml/min, detection wavelength of 190-530 nm and eluent of MeCN/HOAc/H3PO4/H2O (6:8:3:83) for anthocyanins, and 190-360 nm and MeCN/H2O/H3PO4 (22:78:0.2) for other flavonoids according to Iwashina et al. (1996). In quantitative HPLC analyses, each 0.2 g of perianth was extracted with 2 ml of 5% HOAc for anthocyanins, and 2 ml of MeOH for other flavonoids. The extracts were directly analyzed with HPLC systems described above, after filtration with Maishori-disk H-13-5 (Tosoh). Preparative HPLC Preparative HPLC separation of the compounds was performed with Tosoh preparative HPLC using Senshu Pak PEGASIL ODS [I.D. 10 x 250 mm (Senshu)], at flow rate of 1.5 ml/min, detection wavelength of 530 nm and eluent of HCOOH/MeCN/H2O (5:11:84) for anthocyanins, and 350 nm and MeCN/H2O (20:80 and 22:78) for other flavonoids. Acid Hydrolysis The anthocyanin was hydrolyzed in 12% aq. HCl, 100°C, for 3 min., and the flavonol glycosides in 12% aq. HCl, 100°C, for 30 min. Identification of Anthocyanin The anthocyanin was identified by direct TLC (solvent systems: BAW, BuH (n-BuOH/HCl/H2O = 7:2:5) and 1% HCl) and HPLC comparison with authentic specimen, characterization of acid hydrolysates, and UV-visible spectral properties. HPLC and UV-visible spectral data of the isolated anthocyanin are as follows: 1. Malvidin 3,5-di-O-glucoside (Malvin, A1). TLC Rf: 0.06 (BAW), 0.11 (BuH), 0.08 (1% HCl). HPLC retention time (Rt): 10.52 min. UV λmax (nm): 0.1% HCl-MeOH 272, 520; +AlCl3 274, 520.