Bull. Natl. Mus. Nat. Sci., Ser. B, 34(4), pp. 175–181, December 22, 2008

Foliar Flavonoids from Two Begonia Species in Japan
Tsukasa Iwashina1,2*, Yukiko Saito2, Ching-I. Peng3,
Masatsugu Yokota4 and Goro Kokubugata1,2
1Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba 305–0005, Japan
2
United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology,
Saiwai-cho 3–5–8, Fuchu-shi, Tokyo 183–8509, Japan
3
Research Center of Biodiversity Academia Sinica, Taipei, Nangang, Taipei 115, Taiwan
4
Laboratory of Ecology and Systematics, Faculty of Science, University of the Ryukyus,
Senbaru 1, Nishihara, Nakagami, Okinawa 903–0213, Japan*Corresponding author: E-mail: iwashina@kakaku.go.jp

Abstract Flavonoid compounds in the leaves of two Japanese Begonia species, B. formosana andB. fenicis, were surveyed. The compounds were isolated by various chromatographic techniques
and identified by UV spectra, LC-MS, acid hydrolysis, and direct TLC and HPLC comparisonswith authentic samples. Four C-glycosylflavones, isoorientin, orientin, vicenin-2 and apigenin
6-C-pentoside-8-C-hexoside (or 6-C-hexoside-8-C-pentoside), and two flavonol O-glycosides,
quercetin 3-O-rutinoside and kaempferol 3-O-gentiobioside, were detected in B. formosana. On
the other hand, only one flavonol O-glycoside, quercetin 3-O-rutinoside was found in B. fenicis.
From the results obtained in this survey, the flavonoid diversity of the genus Begonia including ca.
900 species was presumed.
Key words : Begoniaceae, Begonia fenicis, Begonia formosana, C-glycosylflavones, flavonoids,
flavonol O-glycosides.

Introduction
The genus Begonia consists of ca. 900 species
and is distributed to warm temperate from tropical zone, especially North America (Mabberley,1997). They were hybridized among somespecies, and many cultivars were made andgrown as ornamentals. Only two wild species,
Begonia formosana (Hayata) Masam. and B. fenicis Merr. are distributed in Japan (Hatusima andAmano, 1994), and listed as endangered plants
by the Ministry of Environment, Japan (Environment Agency of Japan, 2000).
Flavonoid components have been reported insome Begonia species. In many cases, the anthocyanins in the leaves and flowers have been surveyed. Cyanidin 3-O-glucoside, 3-O-sambubioside, 3-O-sophoroside, 3-O-glucosylrutinoside,
3-O-xylosylrutinoside were detected in Begonia
coccinea Hook., B. metallica W. G. Smith, B.

pearcei Hook. f., B. haageana Regel et Schmidt
var. frostii, B. glaucophylla Hook. f., B. manicata
Brongn., B. argenteo-guttata Hort. ex L.H. Bailey, B. rex Putz., B. richmondensis, B. fuchsioides Hook. var. rosea, B. martiana Link etOtto, B. cv. “Woodruff’s Scarlet”, B. cv.
“Courbeille de Feu” and B. cv. “President
Carnot” (Harborne and Hall, 1964). Of their anthocyanins, cyanidin 3-O-xylosylrutinoside wascompletely identified as cyanidin 3-O-(2-xylosyl-6-rhamnosylglucoside) which were present
as a major pigment in the leaves of 18 Begonia
species, e.g., B. angularis Raddi, B. dietrichiana
Irmsch. and B. sanguinea Raddi (Langhammer
and Grandet, 1974). Cyanidin 3-O-gentiobiosidehas been detected in 51 Begonia species such as
B. bunchii Hort. ex L. H. Bailey, B. manicata
Brongn. and B. metachroa Fotsch. (Bopp, 1957).
Pelargonidin 3-O-glucoside and peonidin 5-Oglucoside were found in the flowers of B. suther-

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Tsukasa Iwashina et al.

landii Hook. f. (Harborne and Hall, 1964) andthe leaves of B. pavonina Rindl. (Gould et al.,1995), respectively. Partially characterized cyanidin glycosides were reported from the flowers of
B. heracleifolia Cham. et Schlecht., B. senperflorens Link et Otto, B. socotrana Hook. f. and B.
froebelii A. DC. (Forsyth and Simmonds, 1954).
Six acylated anthocyanins were isolated from theflowers of various Begonia cultivars and identified as cyanidin 3-O-(2-xylosyl-6-trans-caffeoylglucoside), 3-O-(2-xylosyl-6-cis-caffeoylglucoside), 3-O-(2-xylosyl-6-trans-p-coumaroylglucoside), 3-O-(2-xylosyl-6-cis-p-coumaroylglucoside), 3-O-(2-glucosyl-6-trans-pcoumaroylglucoside) and 3-O-(2-glucosyl-6cis-p-coumaroylglucoside) (Chirol and Jay,
1995).
Flavan 3-ols and proanthocyanidins, ()-catechin and procyanidin B1, and ()-epicatechin
and procyanidin B2 were isolated from the leavesof B. glabra Aublet and B. fagifolia Fischer(Ensemeyer et al., 1980; Ensemeyer and Langhammer, 1984). Flavones, luteolin and its 7-Oglycoside, and 5,7,4-trihydroxyflavone 6-O-glucoside were reported from B. erythrophylla
Neum. (Vereskovskii et al., 1988a) and B. evansiana Andr. (Zhang et al., 1997), respectively.
However, this flavonoid may be 5,7,4-trihydroxyflavone 6-C-glucoside, i.e., isovitexin, andmisidentified by Zhang et al. (1997), because hydroxyl group is not present at 6-position of
5,7,4-trihydroxyflavone. In B. erythrophylla,four C-glycosylflavones, orientin, isoorientin,
vitexin and isovitexin were accompanied withflavone O-glycoside (Vereskovskii et al., 1988b).
Flavonols and their glycosides were reported
from almost species which have been surveyed
for flavonoids. Quercetin and its 3-O-rutinoside(rutin), 3-O-rhamnoside (quercitrin), 3-O-glucoside (isoquercitrin) and 3-O-xyloside (reynoutrin), kaempferol 3-O-glucoside (astragalin),isokaempferide (kaempferol 3-methyl ether) andits 7-O-glucoside, quercetin 3-methyl ether andits 7-O-glycoside, pachypodol (quercetin 3,7,3trimethyl ether), and ternatin (gossypetin
3,7,8,3-tetramethyl ether) were isolated fromthe leaves and/or flowers of B. lucerna, B.
metallica, B. haageana var. rosea, B. glaucophylla, B. argenteo-guttata, B. rex, B. richmondensis, B. pearcei, B. manicata and B. martiana(Harborne and Hall, 1964), B. erythrophylla(Vereskovskii et al., 1988a), 30 Begonia species(Bopp, 1957), B. fagifolia (Esemeyer and Langhammer, 1984), and B. glabra Ruiz ex Klotzsch.
(Esemeyer and Langhammer, 1982). However,flavonoids in B. formosana and B. fenicis, whichwere used as plant materials in this experiment,
have not been surveyed. In this paper, the isolation and identification of the foliar flavonoids intheir species are described.
Materials and Methods
Plant materials
Two Begonia species, B. formosana (Hayata)
Masam. (Fig. 1) and B. fenicis Merr. (Fig. 2)
were used as plant materials. Begonia formosana
and B. fenicis were collected in south slope of
Mt. Banna, Ishigaki Is., Okinawa, Japan(GK305), and Utsun River, Iriomote Is., Okinawa, Japan (GK1335), respectively. Voucher
specimens have been depositerd in the National
Museum of Nature and Science, Japan (TNS).
Extraction and isolation of flavonoids
Fresh leaves of B. formosana (6.4 g) and B.
fenicis (95.3 g) were extracted with MeOH, respectively. The concentrated extracts were applied to preparative paper chromatography using
solvent systems, BAW (n-BuOH/HOAc/H2O
4 : 1 : 5, upper phase), 15% HOAc and then BEW(n-BuOH/EtOH/H2O4 : 1 : 2.2). The isolated
flavonoids were purified by Sephadex LH-20(Pharmacia) column chromatography using solvent system, 70% MeOH.
High performance liquid chromatography(HPLC)
Qualitative HPLC was performed with Shimadzu HPLC systems using a PEGASIL ODS
column (I.D. 6.0150 mm: Senshu Scientific Co.
Ltd.), at a flow-rate of 1.0 ml min1. Detection

Flavonoids from Begonia in Japan

Fig. 1.

177

Begonia formosana (Hayata) Masam.

ESI

was 190–700 nm and eluent was MeCN/H2O/
H3PO4 (20 : 80 : 0.2).

MeCN/H2O/HCOOH (15 : 80 : 5),
4.5 kV, ESI 3.5 kV, 250°C.

Liquid chromatograph—mass spectra (LC-MS)
LC-MS was measured using a PEGASIL ODS
column (I.D. 2.0150 mm, Senshu Scientific Co.
Ltd.), at a flow-rate of 0.2 ml min1, eluent:

Identification of flavonoids
The isolated flavonoids were identified by UV
spectra according to Mabry et al. (1970), LCMS, acid hydrolysis and characterization of hy-

Fig. 2.

Begonia fenicis Merr.

and

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Tsukasa Iwashina et al.

drolysates, and direct TLC and HPLC comparisons with authentic samples. TLC, HPLC, UV,
LC-MS and acid hydrolysis data were as follows.
Quercetin 3-O-rutinoside (rutin, 1). TLC: Rf
0.47 (BAW), 0.53 (BEW), 0.46 (15% HOAc);
UV — dark purple, UV/NH3 — yellow. HPLC:tR (min) 7.35. UV: l max (nm) MeOH 257,
265sh, 358; NaOMe 273, 326, 411 (inc.);
AlCl3 274, 432; AlCl3/HCl 267, 300, 363,
396sh; NaOAc 273, 326, 400; NaOAc/
H3BO3 262, 380. LC-MS: m/z 611 [MH], 609[MH] (quercetin  each 1 mol glucose andrhamnose); m/z 465 [M146H] (quercetin 
1 mol glucose); m/z 303 [M308H]
(quercetin). Acid hydrolysis (in 12% HCl,
100°C, 30 min): quercetin, glucose and rhamnose.
Kaempferol 3-O-gentiobioside (2). TLC: Rf
0.41 (BAW), 0.56 (BEW), 0.48 (15%HOAc);
UV — dark purple, UV/NH3 — dark greenish
yellow. HPLC: tR (min) 7.48. UV: l max (nm)
MeOH 266, 297sh, 351; NaOMe 275, 325, 399(inc.); AlCl3 274, 305, 355, 395; AlCl3/HCl
275, 303, 349, 393; NaOAc 275, 312, 391;
NaOAc/H3BO3 267, 297sh, 355. LC-MS: m/z
611 [MH], 609 [MH] (kaempferol  2 mol
glucose). Acid hydrolysis: kaempferol and glucose.
Luteolin 6-C-glucoside (isoorientin, 3).
TLC: Rf 0.43 (BAW), 0.52 (BEW), 0.24 (15%
HOAc); UV — dark purple, UV/NH3 — yellow.
HPLC: tR (min) 5.34. UV: l max (nm) MeOH
257, 270, 349; NaOMe 270, 327sh, 411 (inc.);
AlCl3 275, 422; AlCl3/HCl 263sh, 277,
296sh, 360, 384sh; NaOAc 275, 327sh, 400;
NaOAc/H3BO3 267, 379, 427sh. LC-MS: m/z
449 [MH], 447 [MH] (luteolin  1 mol
glucose). Acid hydrolysis: unhydrolyzable.
Luteolin 8-C-glucoside (orientin, 4). TLC:
Rf 0.22 (BAW), 0.29 (BEW), 0.09 (15% HOAc);
UV — dark purple, UV/NH3 — yellow. HPLC:tR (min) 6.19. UV: l max (nm) MeOH 257, 268,
293sh, 349; NaOMe 271, 327sh, 407 (inc.);
AlCl3 274, 422; AlCl3/HCl 265, 276, 297,
358, 385sh; NaOAc 276, 327sh, 396;NaOAc/H3BO3 266, 377, 427sh. LC-MS: m/z

449 [MH], 447 [MH] (luteolin  1 mol
glucose). Acid hydrolysis: unhydrolyzable.
Apigenin 6,8-di-C-glucoside (vicenin-2, 5).
TLC: Rf 0.22 (BAW), 0.32 (BEW), 0.39 (15%
HOAc); UV — dark purple, UV/NH3 — darkgreenish yellow. HPLC: tR (min) 4.12. UV:l max (nm) MeOH 273, 332; NaOMe 283,
334, 400 (inc.); AlCl3 280, 306, 353, 385sh;
AlCl3/HCl 280, 304, 348, 383sh; NaOAc
282, 397; NaOAc/H3BO3 277sh, 285, 321, 355,
411. LC-MS: m/z 593 [MH] (apigenin 
2 mol glucose). Acid hydrolysis: unhydrolyzable.
Apigenin 6-C-pentoside-8-C-hexoside (or 6-Chexoside-8-C-pentoside, 6). TLC: Rf 0.26
(BAW), 0.32 (BEW), 0.32 (15%HOAc); UV —dark purple, UV/NH3 — dark greenish yellow.
HPLC: tR (min) 5.10. UV: l max (nm) MeOH
274, 334; NaOMe 283, 333, 401 (inc.);
AlCl3 227, 306, 356, 383sh; AlCl3/HCl 275,
305, 353, 383sh; NaOAc 283, 396;NaOAc/H3BO3 276, 285, 321, 352, 413. LCMS: m/z 565 [MH], m/z 563 [MH] (apigenin  each 1 mol hexose and pentose). Acidhydrolysis: unhydrolyzable.
Results and Discussion
Six flavonoids were isolated from the leaves of
B. formosana (Table 1). On the other hand, only
one flavonoid (1) was found in B. fenicis.
Flavonoid 1, which is present in both species,
was obtained as pale yellow powder. Quercetin,
glucose and rhamnose were liberated by acid hydrolysis. UV spectral survey in addition to various shift reagents showed the presence of free 5-,7-, 3- and 4-hydroxyl and a substituted 3-hydroxyl groups. The attachment of each 1 mol glucose and rhamnose to quercetin was shown by
LC-MS. Finally, flavonoid 1 was identified as
quercetin 3-O-rutinoside (Fig. 3) by direct TLC
and HPLC comparison with authentic rutin fromthe aerial parts of Osyris alba L. (Santalaceae)(Iwashina et al., 2008a). Quercetin 3-O-rutinoside has been found in Begonia species (Bopp,
1957; Harborne and Hall, 1964; Vereskovskii etal., 1988a).

Flavonoids from Begonia in Japan
Table 1. Distribution of flavonoids in the leaves ofBegonia formosana and B. fenicis
Flavonoids
Species
1

2

3

4

5

6

B. formosana 
B. fenicis












1quercetin 3-O-rutinoside, 2kaempferol 3-Ogentiobioside, 3isoorientin, 4orientin, 5vicenin-2
and 6apigenin 6-C-pentoside-8-C-hexoside or 6-C-hexoside-8-C-pentoside.

Flavonoid 2 was obtained as a minor compound from B. formosana. UV spectral survey
according to Mabry et al. (1970) showed thepresence of free 5-, 7- and 4-hydroxyl and a substituted 3-hydroxyl groups. Since kaempferol andglucose were produced by acid hydrolysis, it isclear that glucose is attached to 3-position of
kaempferol. LC-MS was appeared the molecularion peak, m/z 611 [MH], showing the attachment of 2 mol glucose to kaempferol. Thus,
flavonoid 2 was characterized as kaempferol 3O-diglucoside and finally identified as kaempferol 3-O-glucosyl-(1→6)-glucoside, i.e. kaempferol 3-O-gentiobioside (Fig. 4) by direct TLC andHPLC comparison with authentic sample fromthe fronds of Asplenium incisum Thunb. (Aspleniaceae) (Iwashina et al., 2000). Kaempferol 3O-gentiobioside was isolated from Begonia
species for the first time.
Flavonoids 3 and 4 were obtained as pale yel-Fig. 3.

Quercetin 3-O-rutinoside (1).

179

low powders from B. formosana. UV spectral
properties of them were those of 5,7,3,4tetrahydroxyflavones. Since they were not hydrolyzable by hot acid treatment, it is clear that
their flavonoids are C-glycosylflavones. Molecular ion peak, m/z 449 [MH] appeared by LCMS, showing the attachment of 1 mol hexose to
5,7,3,4-tetrahydroxyflavone. Finally, flavonoids
3 and 4 were identified as luteolin 6-C-glucoside(Fig. 5) and luteolin 8-C-glucoside (Fig. 6) by direct TLC and HPLC comparisons with authentic
isoorientin and orientin from the leaves of Acerpalmatum Thunb. (Aceraceae) (Iwashina andMurai, 2008). Isoorientin and orientin have beenreported from the leaves of B. erythrophylla(Vereskovskii et al., 1988b).

Fig. 4.

Kaempferol 3-O-gentiobioside (2).

Flavonoids 5 and 6 were also C-glycosylflavones which were shown by hot acid treatment. It was shown by LC-MS survey that theformer is apigenin di-C-hexoside. Finally,
flavonoid 5 was identified as apigenin 6,8-di-Cglucoside (vicenin-2, Fig. 7) by direct TLC andHPLC comparison with authentic sample fromthe fronds of Asplenium normale D. Don (Aspleniaceae) (Iwashina et al., 2008b). Vicenin-2 wasisolated from Begonia species for the first time. It
was shown by LC-MS that flavonoid 6 is apigenin attached each 1 mol pentose and hexose.
The attachment of their sugars to both 6- and 8-

Fig. 5.

Isoorientin (3).

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Tsukasa Iwashina et al.

1984; Forsyth and Simmonds, 1954; Gould et al.,
1995; Harborne and Hall, 1964; Langhammer
and grandet, 1974; Vereskovskii et al., 1987a,
1987b; Zhang et al., 1997). From the results obtained in this survey and also references,flavonoid diversity of the Begonia was presumed
and we are now in progress for flavonoid surveyof the genus.
Fig. 6.

Orientin (4).

positions was determined by UV spectral survey
according to Mabry et al. (1970). However, complete identification of the glycoside was not performed, since NMR spectra was not measured
for meager amount of the isolated compound.
Thus, flavonoid 6 was partially characterized asapigenin 6-C-pentoside-8-C-hexoside or 6-Chexoside-8-C-pentoside such as schaftoside andisoschaftoside (Iwashina et al., 2008a), whichhave not been found in Begonia species.

Fig. 7.

Vicenin-2 (5).

Only two species, B. formosana and B. fenicis
are distributed in Japan. However, their flavonoid
composition was remarkably different to eachother, i.e., flavonol only in B. fenicis andflavonols and C-glycosylflavones in B.
formosana. Flavonoids have fragmentarily beensurveyed among some Begonia species in spite of
the big genus including ca. 900 species. However, five flavonoid classes, i.e. anthocyanins,
flavones, C-glycosylflavones, flavonols, and flavans and proanthocyanidins have been reported(Bopp 1957; Chirol and Jay, 1995; Ensemeyer etal., 1980; Ensemeyer and Langhammer, 1982,

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