Bull. Natl. Mus. Nat. Sci., Ser. B, 40(1), pp. 55–59, February 20, 2014

Flavonoids from the Flowers and Aerial Parts ofTorenia concolor var. formosana
Tsukasa Iwashina* and Goro Kokubugata
Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan* E-mail: iwashina@kahaku.go.jp(Received 30 October 2013; accepted 18 December 2013)

Abstract Two ﬂavones and three anthocyanins were isolated from the violet-blue ﬂowers ofTorenia concolor var. formosana. They were characterized as apigenin and luteolin 7-O-glucuronides, and malvidin and peonidin glycosides and malvidin 3,5-di-O-glucoside, respectively, byUV, acid hydrolysis, LC-MS and TLC and HPLC comparisons with authentic samples. On theother hand, C-glycosylﬂavone was isolated from the aerial parts, together with minor two
unknown ﬂavonoids, and identiﬁed as apigenin 6,8-di-C-arabinoside by UV, LC-MS, and TLC andHPLC comparison with authentic sample. Flavonoids were reported from this species for the ﬁrsttime. Flavonoid pattern was the same among the some populations of Japan and Taiwan.
Key words : anthocyanins, apigenin 6,8-di-C-arabinoside, C-glycosylﬂavone, ﬂavones, ﬂavonoids, Torenia concolor var. formosana.

Introduction
The genus Torenia consists of ca. 30–40 species
and is distributed in mainly tropical and subtropicalzones of East Asia (Mabberley, 1987; Yamazaki,1981). In Japan, two taxa, Torenia glabra Osbeck
and T. concolor Lindl. var. formosana Yamazaki,
are growing, and some cultivars of T. fournieri
Linden ex Fourn. are cultivated as ornamentals(Yamazaki, 1981). Of their taxa, T. concolor var.
formosana is native to Amami-Ohshima Is. andMiyako Is., Japan, and Taiwan (Hatusima andAmano, 1994), and is designated to endangered
plant in Japan.
The ﬂavonoids in Torenia species have hardly
been reported. The anthocyanins, 3,5-di-O-glucosides of delphinidin, cyanidin, petunidin, peonidin and malvidin were isolated from the ﬂowersof T. fournieri cultivars and wild type (Aida etal., 2000). Flavones, apigenin 7-O-glucuronide,luteolin 7-O-glucoside and luteolin 7-O-glucuronide, were also found as copigment substances inthe ﬂowers of the same species (Aida et al.,2000). The anthocyanin, malvidin 3-O-glucoside5-O-(6″-p-coumaroylglucoside) was isolated
from the ﬂowers of Torenia hybrida ʻSummerwave Blueʼ together with minor peonidin
3-O-glucoside-5-O-(6″-p-coumaroylglucoside)(Suzuki et al., 2000; Fukusaki et al., 2004).
Though the presence of the anthocyanins wasshowed in the ﬂowers of Torenia baillonii Godefroy ex André, their identiﬁcation was not performed (Lang and Potrykus, 1971).
As a series of ﬂavonoid survey of endangered
plants in Ryukyus, we have studied the ﬂavonoids in Cassytha spp. (Lauraceae) (Murai et al.,2008), Begonia spp. (Begoniaceae) (Iwashina etal., 2008), Pothos chinensis (Raf.) Merr. (Araceae) (Iwashina et al., 2010), Myoporum bontioides (Sieb. et Zucc.) A. Gray (Myoporaceae)(Iwashina and Kokubugata, 2010) and Triumfetta
procumbens G. Forst. (Malvaceae) (Iwashina andKukubugata, 2012).

56

Tsukasa Iwashina and Goro Kokubugata

H2O=4 : 1 : 2.2). The isolated compounds werepuriﬁed by Sephadex LH-20 column chromatography using solvent systems, 70% MeOH (ﬂavones) and MeOH/H2O/HCOOH (65 : 30 : 5)(anthocyanins).

Fig. 1. Torenia concolor Lindl. var. formosana
Yamazaki, in Bitou Cape, Taiwan.

In this paper, we describe the isolation andcharacterization of the ﬂavonoids including theanthocyanins of the ﬂowers and aerial parts ofTorenia concolor var. formosana in Japan andTaiwan.
Materials and Methods
Plant materials
Torenia concolor Lindl. var. formosana
Yamazaki was used as plant material. Collection
sites and dates of the plants are as follows:
Honohoshi, Amami-Ohshima Is., KagoshimaPref., Japan, 12 Nov. 2006 (GK4512); Tatsugo,Amami-Ohshima Is., Kagoshima Pref., Japan, 6Feb. 2009; Fan Papshan, Taiwan, 12 Nov. 2006(GK8820); Lienhua-yu, Taiwan, 12 Nov. 2006
(GK8821); Tungyen-shan, Taiwan, 6, Nov. 2006;
Bitou Cape, Taiwan, 31 Oct. 2011. Voucher specimens are deposited in the Herbarium of National
Museum of Nature and Science, Japan (TNS).
Extraction and separation
Fresh aerial parts (168.4 g) and violet-blue
ﬂowers (0.9 g) of T. concolor var. formosana
were extracted with MeOH and HCOOH/MeOH(8 : 92), respectively. The concentrated extracts
were applied to preparative paper chromatographyusing solvent systems, BAW (n-BuOH/HOAc/
H2O=4 : 1 : 5, upper phase) and 15% HOAc. Flavone mixtures were, moreover, chromatographedusing solvent system, BEW (n-BuOH/EtOH/

High performance liquid chromatography (HPLC)
HPLC was performed with Shimadzu HPLC
systems using Senshu pak PEGASIL ODS column (I.D. 6.0×150 mm, Senshu Scientiﬁc Co.
Ltd., Japan) at a ﬂow-rate of 1.0 ml min−1.
Detection was 350 nm (ﬂavones) and 530 nm(anthocyanins). Eluents were MeCN/H2O/H3PO4(15 : 85 : 0.2) (ﬂavones) and MeCN/HOAc/H2O/
H3PO4 (6 : 8 : 83 : 3) (anthocyanins).
Liquid chromatograph-mass spectra (LC-MS)
LC-MS was performed with Shimadzu
LC-MS systems using Senshu pak PEGASILODS column (I.D. 2.0×150 mm, Senshu Scientiﬁc Co. Ltd.) at a ﬂow-rate of 0.2 ml min−1,ESI＋ 4.5 kV and ESI− 3.5 kV, 250°C. Eluentswere MeCN/H2O/HCOOH (10 : 85 : 5) (ﬂavones)and (8 : 5 : 82 or 20 : 5 : 75) (anthocyanins).
Acid hydrolysis
Acid hydrolysis was performed in 12% aq.
HCl, 100°C, 30 min. After shaking with diethylether (ﬂavones) and isoamyl alcohol (anthocyanins), aglycones migrated to the organic layer,and sugars and C-glycosylﬂavone were left inaqueous layer.
Thin layer chromatography (TLC)
TLC was performed with Cellulose F plastic
plate (Merck, Germany) using solvent systems,BAW, BEW and 15% HOAc.
Identiﬁcation of ﬂavones and anthocyanins
Flavones were identiﬁed by UV spectral survey according to Mabry et al. (1970), LC-MS,characterization of acid hydrolysates, and TLC
and HPLC comparisons with authentic samples.
Anthocyanins were characterized by UV andLC-MS, and HPLC comparison with authenticsample. Following authentic samples were used;Flavonoids from Torenia concolor var. formosana

Fig. 2.

Apigenin 7-O-glucuronide (1).

malvidin 3,5-di-O-glucoside from the ﬂowers of
Gladiolus cultivars (Takemura et al., 2008),
apigenin 7-O-glucuronide from the ﬂowers of
Aeginetia indica L. (Orobanchaceae) (Iwashina,2010), luteolin 7-O-glucuronide from the leavesof Uncarina grandidieri (Baill.) Stapf. (Pedaliaceae) (Yamazaki et al., 2007) and apigenin
6,8-di-C-arabinoside from the leaves of Ajuga
decumbens Thunb. (Lamiaceae) (Inomata et al.,2013). TLC, HPLC, UV and LC-MS data of theisolated compounds are as follows.
Apigenin 7-O-glucuronide (1). TLC: Rf 0.59(BAW), 0.45 (BEW), 0.18 (15%HOAc); color
UV (365 nm)–dark purple, UV/NH3–dark greenish yellow. HPLC: tR (min) 11.38. UV: λmax(nm) MeOH 268, 333; ＋NaOMe 273, 379(inc.); ＋AlCl3 274, 299, 348, 375sh; ＋AlCl3/
HCl 275, 298, 340, 375sh; ＋NaOAc 257, 266,390; ＋NaOAc/H3BO3 268, 340. LC-MS: m/z
447 [M＋H]＋, 445 [M−H]− (molecular ionpeaks, apigenin＋1 mol glucuronic acid), m/z 269[M−176−H]− (fragment ion peak, apigenin).
Luteolin 7-O-glucuronide (2). TLC: Rf 0.36(BAW), 0.27 (BEW), 0.11 (15%HOAc); color
UV (365 nm)–dark purple, UV/NH3–dark yellow.
HPLC: tR (min) 7.86. UV: λmax (nm) MeOH
255, 266sh, 348; ＋NaOMe 268, 392 (inc.);＋AlCl3 273, 425; ＋AlCl3/HCl 264, 273sh, 296,359, 384sh; ＋NaOAc 259, 358sh, 401;
＋NaOAc/H3BO3 259, 373. LC-MS: m/z 463[M＋H]＋, 461 [M−H]− (molecular ion peaks,luteolin＋1 mol glucuronic acid), m/z 287[M−176＋H]＋ (fragment ion peak, luteolin).
Apigenin 6,8-di-C-arabinoside (3). TLC: Rf
0.31 (BAW), 0.28 (BEW), 0.35 (15%HOAc);
color UV (365 nm)–dark purple, UV/NH3–darkgreenish yellow. HPLC: tR (min) 7.21. UV:λmax (nm) MeOH 273, 332; ＋NaOMe 283,Fig. 3.

Fig. 4.

57

Luteolin 7-O-glucuronide (2).

Apigenin 6,8-di-C-arabinoside (3).

333, 401 (inc.); ＋AlCl3 280, 305, 350, 385sh;＋AlCl3/HCl 280, 303, 346, 384sh; ＋NaOAc
282, 398; ＋NaOAc/H3BO3 285, 319, 361, 417.
LC-MS: m/z 535 [M＋H]＋, 533 [M−H]−(molecular ion peaks, apigenin＋2 mol pentose).
Malvidin glycoside (A1). HPLC: tR (min)7.69. LC-MS: m/z 801 [M]＋ (molecular ionpeak, malvidin＋1 mol rhamnose or p-coumaricacid, and 2 mol glucose), m/z 655 [M−146]＋(fragment ion peak, malvidin＋2 mol glucose).
Peonidin glycoside (A2). HPLC: tR (min)6.34. LC-MS: m/z 771 [M]＋ (molecular ion
peak, peonidin＋1 mol rhamnose or p-coumaricacid, and 2 mol glucose).
Malvidin 3,5-di-O-glucoside (A3). HPLC: tR(min) 8.84. LC-MS: m/z 655 [M]＋ (molecularion peak, malvidin＋2 mol glucose).
Results and Discussion
Flavones and anthocyanins in the ﬂowers
Two ﬂavones 1 and 2 were isolated from theﬂowers. Flavonoid 1 liberated apigenin and glucuronic acid, which were characterized by HPLC(aglycone) and PC (sugar) comparisons withauthentic samples, by acid hydrolysis. The

58

Tsukasa Iwashina and Goro Kokubugata

attachment of 1 mol glucuronic acid to 7-positionof apigenin was proved by UV spectral data and
LC-MS survey. Finally, 1 was identiﬁed as apigenin 7-O-glucuronide by TLC and HPLC comparison with authentic specimen. Apigenin
7-O-glucuronide has been reported from theﬂowers of Torenia fournieri cultivars and wild
type (Aida et al., 2000). It was shown by UV
spectral survey according to Mabry et al. (1970)
that 2 is 5,3′,4′-trihydroxy-7-substituted ﬂavone.
Luteolin and glucuronic acid were produced byacid hydrolysis. The attachment of 1 mol glucuronic acid to luteolin was determined by
LC-MS. Thus, 2 was identiﬁed as luteolin
7-O-glucuronide by direct TLC and HPLC comparison with authentic sample. Luteolin
7-O-glucuronide was also reported from theﬂowers of garden torenia (Aida et al., 2000).
Three anthocyanins A1–A3 were isolated fromthe ﬂowers. Molecular weights, 801, 771 and 655
were assigned to A1, A2 and A3 by LC-MS,
showing that they are malvidin monorhamnosyldihexoside or p-coumaroyl-dihexoside, peonidinmonorhamnosyl-dihexoside or p-coumaroyldihexoside, and malvidin dihexoside, respectively. Malvidin 3-O-glucoside-5-O-(6″-pcoumaroylglucoside) and peonidin 3-O-glucoside5-O-(6″-p-coumaroylglucoside), which are corresponding to their molecular weight, have beenreported from the ﬂowers of Torenia hybrida
ʻSummerwave Blueʼ (Suzuki et al., 2000; Fukusakiet al., 2004). However, their retention times ofHPLC in literature (Suzuki et al., 2000; Fukusakiet al., 2004) are faster compared with those of
A1 and A2, presuming that they are not acylated.
Though A3 was identiﬁed as malvidin 3,5-di-Oglucoside by HPLC comparison with authentic
sample, A1 and A2 could not be identiﬁed forminute amount of the plant materials. Malvidin
3,5-di-O-glucoside has been found in the ﬂowersof T. fournieri (Aida et al., 2000).
C-Glycosylﬂavone from the aerial parts
Only one ﬂavonoid 3 was isolated from theaerial parts of T. concolor var. formosana. UV
spectral properties of 3 showed the presence offree 5-, 7- and 4′-hydroxyl groups in ﬂavone
nucleus. Since 3 was unhydrolyzable by hot acidtreatment, it was proved that this is C-glycosylﬂavone. LC-MS survey showed the attachmentof 2 mol pentose to apigenin. Finally, 3 was identiﬁed as apigenin 6,8-di-C-arabinoside by direct
TLC and HPLC comparison with authentic sample. C-Glycosylﬂavone was reported from Torenia
species for the ﬁrst time. Two very minor ﬂavonoids were found in the aerial parts by HPLC
survey, together with 3. However, they could not
be isolated for minute amount. Though the ﬂavonoids in the ﬂowers has been reported from
Torenia species (Aida et al., 2000; Suzuki et al.,
2000; Fukusaki et al., 2004), those of the aerial
parts were found by this experiment for the ﬁrsttime.
In this survey, both the ﬂowers and aerial parts
were used as plant materials. However, their ﬂavonoid composition was completely different
with each other, i.e. ﬂavone O-glycosides andanthocyanins in ﬂowers and C-glycosylﬂavone inaerial parts. It may be due to the difference of
their function of ﬂavonoids in each plant parts.
Though the plants of two Japanese and three Taiwanese populations were surveyed for ﬂavonoids, their patterns were essentially the samewith each other.
References
Aida, R., Yoshida, K., Kondo, T., Kishimoto, S. and Shibata,
M. 2000. Copigmentation gives bluer ﬂowers on transgenic torenia plants with the antisense dihydroﬂavonol4-reductase gene. Plant Science 160: 49–56.
Fukusaki, E., Kawasaki, K., Kajiyama, S., An C.-I,Suzuki, K., Tanaka, Y. and Kobayashi, A. 2004. Flower
color modulations of Torenia hybrida by downregulation of chalcone synthase gene with RNA interference.
Journal of Biotechnology 111: 229–240.
Hatusima, S. and Amano, T. 1994. Flora of the Ryukyus,
South of Amami Island. The Biological Society ofOkinawa, Nisihara.
Inomata, Y., Terahara, N., Kitajima, J., Kokubugata, G.
and Iwashina, T. 2013. Flavones and anthocyaninsfrom the leaves and ﬂowers of Japanese Ajuga species(Lamiaceae). Biochemical Systematics and Ecology,
51: 123–129.
Iwashina, T., Saito, Y., Peng, C.-I, Yokota, M. andFlavonoids from Torenia concolor var. formosana
Kokubugata, G. 2008. Foliar ﬂavonoids from two
Begonia species in Japan. Bulletin of the National
Museum of Nature and Science, Series B 34: 175–181.
Iwashina, T. 2010. Flavonoids from two parasitic andachlorophyllous plants, Aeginetia indica and Orobanche
minor (Orobanchaceae). Bulletin of the National
Museum of Nature and Science, Series B 36: 127–132.
Iwashina, T. and Kokubugata, G. 2010. Flavonoids in theleaves and ﬂowers of Myoporum bontioides native tonorthernmost region in the Myoporaceae. Bulletin ofthe National Museum of Nature and Science, Series B36: 117–125.
Iwashina, T., Peng, C.-I and Kokubugata, G. 2010. Flavone
O- and C-glycosides from Pothos chinensis (Araceae).
Bulletin of the National Museum of Nature and Science, Series B 36: 27–32.
Iwashina, T. and Kokubugata, G. 2012. Flavone and ﬂavonol glycosides from the leaves of Triumfetta procumbens in Ryukyu Islands. Bulletin of the National
Museum of Nature and Science, Series B 38: 63–67.
Lang, W. and Potrykus, I. 1971. Lichtmikroskopische undPigmentanalytische Untersuchungen zum Ablauf derPetalenenwicklung von Torenia baillonii (Scrophulariac.).
Zeitschrift für Pﬂanzenphysilogie 65: 1–12.
Mabberley, D. J. 1987. The Plant-Book. pp. 718, Cambridge University Press, Cambridge.
Mabry, T. J., Markham, K. R. and Thomas, M. B. 1970.

59

The Systematic Identiﬁcation of Flavonoids. Springer,Berlin.
Murai, Y., Kokubugata, G., Yokota, M., Kitajima, J. andIwashina, T. 2008. Flavonoids and anthocyanins fromsix Cassytha taxa (Lauraceae) as taxonomic markers.
Biochemical Systematics and Ecology 36: 745–748.
Suzuki, K., Xue, H., Tanaka, Y., Fukui, Y., Fukuchi-Mizutani, M., Murakami, Y., Katsumoto, Y., Tsuda, S. andKusumi, T. 2000. Flower color modiﬁcations of Torenia
hybrida by cosuppression of anthocyanin biosynthesis
genes. Molecular Breeding 6: 239–246.
Takemura, T., Takatsu, Y., Kasumi, M., Marubashi, W.
and Iwashina, T. 2008. Anthocyanins of Gladiolus cultivars and their contribution to ﬂower colors. Journal ofthe Japanese Society for Horticultural Science 77:
80–87.
Yamazaki, T., 1981. Scrophulariaceae. In: Satake, Y.,
Ohwi, J., Kitamura, S., Watari, S. and Tominari, T.
(eds.), Wild Flowers of Japan. Herbaceous Plants(including Dwarf Subshrubs), pp. 97–121, Heibonsha,Tokyo.
Yamazaki, K., Iwashina, T., Kitajima, J., Gamou, Y.,
Yoshida, A. and Tannowa, T. 2007. External and internal ﬂavonoids from Madagascarian Uncarina species(Pedaliaceae). Biochemical Systematics and Ecology
35: 743–749.