Chalcone Glycoside in the Flowers of Six Corylopsis Species As Yellow Pigment

Chalcone Glycoside in the Flowers of Six Corylopsis Species as Yellow Pigment

Tsukasa Iwashina1,2*, Tomoko Takemura2 and Tamaki Mishio2

1 Department of Botany, National Museum of Nature and Science, Amakubo, Tsukuba 305-0005, Japan 2 United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Saiwai-cho, Fuchu 183-8509, Japan

A chalcone glycoside was isolated from the flowers of six Corylopsis species, C. pauciflora, C. spicata, C. glabrescens, C. sinensis, C. gotoana, and C. coreana, and identified as chalcononaringenin 2'-O-glucoside by UV spectral survey, liquid chromatograph-mass spectrometry (LC-MS), acid hydrolysis, and characterization of its products, and direct thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) comparison with an authentic sample. Five flavonol glycosides, which were accompanied with chalcone glycoside, were also isolated and identified as quercetin 3-O-rhamnoside, quercetin 3-O-glucoside, myricetin 3-O-rhamnoside, myricetin 3-O- glucoside, and kaempferol 3-O-rhamnoside in the same manner. Chalcone glycosides have been reported from various plant species, Dianthus, Coreopsis, Cosmos, Dahlia, and Bidens as yellow flower pigments. In this survey, it was shown for the first time that the yellow flower color of Corylopsis species is due to a chalcone glycoside, chalcononaringenin 2'-O-glucoside, but other flavonol glycosides hardly act as yellow pigments.

Key Words: chalcone, chalcononaringenin 2'-O-glucoside, Corylopsis, Hamamelidaceae, yellow flower color.

Hydroxyflavonols such as patuletin and quercetagetin Introduction also contribute to the yellow color of the flowers of The genus Corylopsis (Hamamelidaceae) is distrib- Centaurea ruthenica Lam. (Asteraceae), Lotus uted throughout East Asia and the Himalayas, and corniculatus L. (Leguminosae), and Mimulus luteus L. consists of 26 species (Yamazaki, 1989). All species (Scrophulariaceae) (Harborne, 1965; Mishio et al., bear yellow flowers in early spring. Of their species, 2006). More recently, it was clarified that the pale yellow C. pauciflora Sieb. & Zucc. and C. spicata Sieb. & Zucc. flower color of Clematis cultivars (Ranunculaceae) is are widely cultivated in gardens and parks as due to the high amount of common flavonol glycosides, ornamentals; however, wild plants are comparatively quercetin 3-O-glucoside, 3-O-galactoside, and 3-O- rare in nature and three taxa, i.e., C. glabrescens Franch. rutinoside (Hashimoto et al., 2008); however, it was & Savat., C. gotoana Makino var. pubescens (Nakai) shown that kaempferol glycosides do not act as yellow Yamazaki, and C. spicata, have been nominated as pigments, even if they are abundant. Furthermore, it was endangered plants by Ministry of Environment, Japan reported that three common flavonol glycosides, (Environment Agency of Japan, 2000). quercetin 3-O-rutinoside, 7-O-glucoside, and 3-O- The yellow flowers of wild and cultivated plants are glucoside, act as yellow pigments by the addition of mainly due to carotenoid pigments (Harborne, 1993). aluminum ions, in deep yellow flowers of Camellia On the other hand, Cosmos, Dianthus, Dahlia, chrysantha (H.H. Hu) Tuyama (Tanikawa et al., 2008). Coreopsis, Bidens, and Antirrhinum flowers arise from In Corylopsis species, a few flavonoids, quercetin water-soluble yellow pigments, chalcones and aurones and kaempferol 3-O-rhamnosides, myricetin, leuco- (Harborne, 1966; Shimokoriyama, 1957a; Yoshida et al., delphinidin, and leucocyanidin, have been found in the 2004). Yellow Mirabilis jalapa L. (Nyctaginaceae) and leaves of C. pauciflora, C. platypetala Rehd. & Wils. Glottiphyllum longum (Haw.) N.E. Br. (Aizoaceae) and C. spicata (Egger and Reznik, 1961; Hegnauer, belonging to Caryophyllales are due to betaxanthin 1966; Reznik and Egger, 1960); however, flower pigments such as miraxanthin and dopaxanthin pigments have not been surveyed. (Impelizzeri et al., 1973; Piattelli et al., 1965). 6- In this paper, we report for the first time that the yellow flower color of Corylopsis species is expressed Received; February 3, 2009. Accepted; April 21, 2009. by a chalcone glycoside, chalcononaringenin 2'-O- * Corresponding author (E-mail: [email protected]). glucoside, i.e., isosalipurposide.

485 486 T. Iwashina, T. Takemura and T. Mishio

using a PEGASIL ODS column (I.D. 6.0 × 150 mm: Materials and Methods Senshu Scientific Co. Ltd., Tokyo, Japan) at a flow-rate Plant materials of 1.0 mL min−1, detection wavelength of 350 and/or Of the six Corylopsis species used in this experiment, 410 nm, and the eluent was acetonitrile/water/phosphoric C. pauciflora Sieb. & Zucc. (Fig. 1B) and C. spicata acid (22 : 78 : 0.2). Sieb. & Zucc. (Fig. 1D) were cultivated in Tsukuba Botanical Garden, National Museum of Nature and Liquid chromatograph-mass spectra (LC-MS) Science, Tsukuba, Japan, C. glabrescens Franch. & LC-MS (Shimadzu) was measured using a PEGASIL Savat. (Fig. 1A), C. sinensis Hemsl. (Fig. 1C), and ODS column (I.D. 2.0 × 150 mm: Senshu Scientific Co. C. coreana Uyeki in the Botanical Gardens, Graduate Ltd.), at a flow-rate of 0.2 mL ⋅ min−1, detection School of Science, The University of Tokyo, Tokyo, wavelength of 350 and 250 nm, and the eluent was formic Japan, and C. gotoana Makino in the Kochi Prefectural acid/acetonitrile/water (0.2 : 15 : 85), ESI+ 4.5 kV, ESI− Makino Botanical Garden, Kochi, Japan. 3.5 kV, 250°C.

UV spectral survey Acid hydrolysis UV spectra of crude methanol extracts (300–700 nm) Acid hydrolysis of the isolated flavonoids was and the isolated flavonoids (220–500 nm) were measured performed in 12% aqueous hydrochloric acid for 30 min on a Shimadzu MPS-2000 multipurpose recording at 100°C. After cooling in water, the solution was shaken spectrophotometer (Shimadzu, Kyoto, Japan). with diethyl ether. The aglycones (ether layer) and glycosidic sugars (aqueous layer) were identified by Extraction and isolation HPLC (aglycones) and paper chromatography (sugars) Fresh flowers of six Corylopsis species, C. pauciflora using solvent systems: BBPW (n-butanol/benzene/ (78 g), C. spicata (95 g), C. glabrescens (3 g), C. sinensis pyridine/water = 5 : 1 : 3 : 3) and BTPW (n-butanol/ (3 g), C. coreana (0.5 g), and C. gotoana (5 g), were toluene/pyridine/water = 5 : 1 : 3 : 3). extracted with methanol, respectively. The concentrated extracts were applied to preparative paper chromatogra- Identification of pigments phy using solvent systems: BAW (n-butanol/acetic acid/ All isolated pigments were flavonoid glycosides and water = 4 : 1 : 5, upper phase), 15% acetic acid and then were identified by UV spectral survey according to BEW (n-butanol/ethanol/water = 4 : 1 : 2.2). The isolated Mabry et al. (1970), LC-MS, characterization of pigments were purified by Sephadex LH-20 (Amersham hydrolysates, and direct thin layer chromatography Bioscience, Uppsala, Sweden) column chromatography (TLC) using solvent systems: BAW, BEW, and 15% (solvent system: 70% methanol). acetic acid, and/or HPLC comparisons with authentic samples. TLC, HPLC, LC-MS, and acid hydrolysis data Qualitative HPLC of the isolated flavonoids are shown in Table 1, and UV Crude extracts and the isolated pigments were spectral data in Table 2. analysed with a Shimadzu HPLC system (Shimadzu)

Fig. 1. Flowers of Corylopsis species used as plant materials. A = C. glabrescens, B = C. pauciflora, C = C. sinensis, and D = C. spicata. J. Japan. Soc. Hort. Sci. 78 (4): 485–490. 2009. 487

Table 1. HPLC, LC-MS, and acid hydrolysis data of flavonoids isolated from the flowers of Corylopsis species.

Pigments HPLC Rt (min) LC-MS (m/z) Acid hydrolysis 1z 10.76 435 [M + H]+ (chalcononaringenin + 1 mol hexose) naringenin, glucose 2 7.80 449 [M + H]+ (quercetin + 1 mol rhamnose) quercetin, rhamnose 303 [M − 146 + H]+ (quercetin) 3 5.90 465 [M + H]+ (quercetin + 1 mol hexose) quercetin, glucose 303 [M − 162 + H]+ (quercetin) 4 5.51 465 [M + H]+ (myricetin + 1 mol rhamnose) myricetin, rhamnose 319 [M − 146 + H]+ (myricetin) 5 4.61 481 [M + H]+ (myricetin + 1 mol hexose) myricetin, glucose 319 [M − 162 + H]+ (myricetin) 6 11.49 433 [M + H]+ (kaempferol + 1 mol rhamnose) kaempferol, rhamnose 287 [M − 146 + H]+ (kaempferol) Authentic samples Isosalipurposidey 10.75 435 [M + H]+ (chalcononaringenin + 1 mol hexose) naringenin, glucose Quercitrin 7.79 449 [M + H]+ (quercetin + 1 mol rhamnose) quercetin, rhamnose 303 [M − 146 + H]+ (quercetin) Isoquercitrin 5.91 465 [M + H]+ (quercetin + 1 mol hexose) quercetin, glucose 303 [M − 162 + H]+ (quercetin) Myricitrin 5.50 465 [M + H]+ (myricetin + 1 mol rhamnose) myricetin, rhamnose 319 [M − 146 + H]+ (myricetin) Myricetin 3-glucoside 4.58 481 [M + H]+ (myricetin + 1 mol hexose) myricetin, glucose 319 [M − 162 + H]+ (myricetin) z TLC data: Rf 0.68 (BAW), 0.75 (BEW), 0.88 (15%HOAc); UV—dark purple, UV/NH3—orange. y TLC data: Rf 0.70 (BAW), 0.72 (BEW), 0.88 (15%HOAc); UV—dark purple, UV/NH3—orange.

Table 2. UV spectral data of flavonoids isolated from the flowers of Corylopsis species.

Pigments MeOH +NaOMe +AlCl3 +AlCl3/HCl +NaOAc +NaOAc/H3BO3 1 243sh, 368 242sh, 432 249sh, 328, 249sh, 325sh, 398 324sh, 434 (inc.z) 415 328sh, 399 2 258, 263sh, 272, 323, 274, 426 272, 299, 273, 323, 262, 365 348 396 (inc.) 355, 390 383 3 257, 264sh, 273, 323, 274, 433 267, 299, 272, 329, 263, 380 357 407 (inc.) 361, 394 387 4 257, 263sh, 267, 320, 273, 422 273, 305, 273, 323, 262, 376 355 400 (inc.) 361, 396sh 390 5 256, 264sh, 267, 324sh, 271, 422 273, 308, 273, 321, 262, 384 362 397 (inc.) 364, 400sh 400 6 265, 343 274, 322, 274, 303, 274, 301, 273, 378 266, 347 389 (inc.) 350, 392 343, 388 Authentic samples Isosalipurposide 248sh, 368 242sh, 432 253sh, 326, 250sh, 327sh, 393 324sh, 436 (inc.) 416 324sh, 403 Quercitrin 258, 263sh, 272, 323, 274, 426 272, 299, 273, 323, 262, 365 348 396 (inc.) 355, 390 383 Isoquercitrin 257, 265sh, 273, 328, 275, 433 269, 299, 273, 326, 261, 379 357 409 (inc.) 361, 399 399 Myricitrin 257, 263sh, 270, 322, 273, 430 271, 300, 272, 322, 261, 376 356 404 (inc.) 362, 399 390 Myricetin 3-glucoside 257, 264sh, 269, 324sh, 271, 422 273, 310, 273, 324, 259, 383 364 393 (inc.) 366sh, 407 397 z inc. = increase in intensity relative to methanol spectrum.

from the yellow flowers of C. spicata and C. pauciflora Results and Discussion are shown in Figure 2. In the visible range (400–700 nm), UV-visible absorption curves and HPLC pattern of crude their extracts had weak absorption maxima in 468 and extracts from the flowers of Corylopsis species 664 nm, showing the presence of a trace of chlorophylls The absorption curves of crude methanolic extracts (Harborne, 1984). However, the presence of carotenoids, 488 T. Iwashina, T. Takemura and T. Mishio

Fig. 3. HPLC patterns of methanolic extracts from the flowers of Corylopsis pauciflora. Eluent: MeCN/H2O/H3PO4 (22 : 78 : 0.2); Flow-rate: 1.0 mL min−1; Detection: 410 nm. 1 = chalcononaringenin 2'-O-glucoside, 2 = quercetin 3-O-rhamnoside, 3 = quercetin 3-O-glucoside, 4 = myricetin 3-O-rhamnoside, 5 = myricetin 3-O-glucoside, and 6 = kaempferol 3-O-rhamnoside.

quercetin 3-O-rhamnoside (2), quercetin 3-O-glucoside Fig. 2. Absorption curves of crude methanolic extracts from the (3), myricetin 3-O-rhamnoside (4), and myricetin 3-O- flowers of Corylopsis spicata and C. pauciflora. glucoside (5) (Fig. 4) by UV, LC-MS, acid hydrolysis, and direct HPLC comparisons with authentic quercitrin which generally appear as three peaks and/or shoulders from the leaves of Cornus spp. (Cornaceae) (Iwashina between ca. 400 and 540 nm (Davies, 1976), were not and Hatta, 1994), isoquercitrin from the fronds of recognized by spectral survey. On the other hand, high Cyrtomium spp. (Dryopteridaceae) (Iwashina et al., absorption was widely observed in the UV range. The 2006), myricitrin from the bark of Myrica rubra Sieb. HPLC pattern of methanolic extract from the flowers of & Zucc. (Myricaceae) (Hattori and Hayashi, 1931), and C. pauciflora is shown in Figure 3. Flavonoid composi- myricetin 3-O-glucoside from the fronds of Cyrtomium tion of six Corylopsis species was qualitatively the same microindusium Sa. Kurata (Iwashina et al., 2006) except for C. sinensis, i.e., appearance of a major peak (Tables 1 and 2). Kaempferol 3-O-rhamnoside (6) was (1) together with minor peaks, which were characterized identified by UV spectral properties showing to be 3- as flavonol glycosides by their isolation and identifica- substituted kaempferol (Table 2) (Mabry et al., 1970), tion. Major peak 1 had absorption in the yellow range acid hydrolysis (liberation of kaempferol and rhamnose) (410 nm) (Fig. 3), showing that this compound acts as a and measurement of molecular weight by LC-MS yellow pigment. Thus, it was clear that the yellow flower (attachment of 1 mol rhamnose to kaempferol, Table 1). color of Corylopsis species is due to a water-soluble Their flavonol glycosides were found in all species pigment 1. except for C. sinensis, which does not synthesize two myricetin glycosides 4 and 5 (Table 3). The percentage Identification of water soluble pigments of chalcononaringenin 2'-O-glucoside was between A yellow water-soluble compound 1 had absorption 92.5% (C. sinensis) and 86.2% (C. coreana) to total maxima in 368 nm in methanol solution (Table 2), flavonoid contents in the flowers of each Corylopsis showing that the compound is an anthochlor pigment species (Table 3). (Mabry et al., 1970). The yellow color of the original Yellow to orange flower colors are usually due to the compound disappears by acid hydrolysis, and a presence of carotenoids (Britton, 1983; Goodwin, 1976). flavanone, naringenin, which was converted to flavanone On the other hand, anthochlor pigments, such as from chalcone by cyclization (Shimokoriyama, 1957b), chalcones and aurones, have been reported from the was liberated together with glucose, showing that the yellow flowers of various plants, e.g., Cosmos, original glycoside is chalcononaringenin glucoside. Anthirrhinum (Harborne, 1966), Dahlia (Giannasi, Attachment of 1 mol glucose to chalcononaringenin was 1975), Coreopsis (Shimokoriyama, 1957a) etc. Of their shown by LC-MS. Finally, a water-soluble pigment 1 anthochlors, chalcononaringenin 2'-O-glucoside occurs was identified as chalcononaringenin 2'-O-glucoside as a yellow pigment in the flowers of Helichrysum (Fig. 4) by direct TLC and HPLC comparison with arenarium (L.) Moench. (Asteraceae), Paeonia authentic isosalipurposide from the yellow flowers of trollioides Stapf. ex Stern (Paeoniaceae), Dianthus carnation (Dianthus caryophyllus) (Table 1) (Yoshida et caryophyllus, Asystasia gangetica (L.) T. Anderson al., 2004). (Acanthaceae), and Aeschynanthus parviflora R. Br. Five flavonol glycosides were also isolated from the (Gesneriaceae) (Harborne, 1966). In this survey, it was flowers of Corylopsis species. They were identified as shown for the first time that the yellow flower color of J. Japan. Soc. Hort. Sci. 78 (4): 485–490. 2009. 489

Fig. 4. Chemical structures of six flavonoid glycosides obtained from the flowers of Corylopsis species.

Table 3. The percentage (%) of yellow pigments in the flowers of each Corylopsis species.

Pigments Species 123456 C. pauciflora 90.2 2.4 2.6 2.2 2.6 t C. spicata 92.9 2.2 2.2 1.0 1.7 t C. glabrescens 88.8 3.9 2.7 1.8 2.8 t C. sinensis 92.5 2.4 5.1 — — t C. coreana 86.2 3.4 4.5 2.5 3.3 0.1 C. gotoana 93.5 1.4 2.2 0.7 1.9 0.3

The percentage of peak area of each pigments to those of total flavonoids at 410 nm. t: <0.1%, —: absence. 1: chalcononaringenin 2'-O-glucoside, 2: quercetin 3-O-rhamnoside, 3: quercetin 3-O-glucoside, 4: myricetin 3- O-rhamnoside, 5: myricetin 3-O-glucoside, and 6: kaempferol 3-O-rhamnoside.

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