Bull. Natl. Mus. Nat. Sci., Ser. B, 35(1), pp. 1–9, March 22, 2009

Ultraviolet-absorbing Substances in the Translucent Bracts ofDavidia involucrata (Davidiaceae)
Seiko Takemura1, Junichi Kitajima2 and Tsukasa Iwashina1,3*
1

Graduate School of Agriculture, Ibaraki University, Ami, 300–0393 Japan
2
Laboratory of Pharmacognosy, Showa Pharmaceutical University,
Higashi-tamagawagakuen 3, Machida, Tokyo, 194–8543 Japan3
Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, 305–0005 Japan* Corresponding author: E-mail: iwashina@kahaku.go.jp

Abstract Ultraviolet-absorbing substances in the bracts of Davidia involucrata, which is endemic to China, were surveyed. Two large translucent bracts of the species put on the inflorescencesuch as a parasol and selectively absorbed UV radiation, though visible light almost completelytransmitted. It was shown by high performance liquid chromatography (HPLC) that much amountsof UV-absorbing substances are accumulated. Seven flavonol glycosides were isolated by various
chromatographic techniques such as paper, column and HPLC, and identified as quercetin 3-Osambubioside, quercetin 3-O-galactoside, quercetin 3-O-glucoside, quercetin 3-O-arabinoside,kaempferol 3-O-galactoside, quercetin 3-O-(2-acetylgalactoside) and quercetin 3-O-(acetylglucoside) by UV spectral survey, LC-MS, 1H and 13C NMR, acid hydrolysis, and direct TLC and HPLC
comparisons with authentic samples. Thus, it was proved that the flavonol glycosides, especiallyquercetin 3-O-galactoside and 3-O-glucoside, act as the important UV shields in the translucent
bracts of D. involucrata and protect the inflorescence from noxious UV radiation.
Key words : Davidiaceae, Davidia involucrata, Flavonols, Quercetin 3-O-glycosides, UVshields.

Introduction
Ultraviolet radiation is noxious for all organisms including human. The plants, especiallygreen plants, are directly exposed to not only visible light but also UV radiation. Photosynthetically active radiation penetrates effectively into
the mesophyll and is attenuated by as little as 20
to 30% as it passes through the epidermis (Caldwell et al., 1983). Nuclei are organelles where
primary lesions can result from UV radiation, especially UV-B (Brandle et al., 1977; Howland,1975). Generally, radiation should be selectively
filtered to remove the short wavelength UV component before it reaches the nuclei and chloroplasts. The wavelength selectivity of absorption
in the epidermis of most plant species is much
more pronounced and can often be attributed toflavonoids and related UV-absorbing substances(Robberecht and Caldwell, 1978). Since many of
the UV-absorbing compounds of plant cells arecontained within the vacuole (McClure, 1975),
this is the site for much of the UV absorption in
the epidermis. Thus, flavonoid compounds as UVshields have been reported from various plant
species, e.g., quercetin 3-O-(3,6-p-coumaroylglucoside) and kaempferol 3-O-(3,6-pcoumaroylglucoside) from Pinus sylvestris L.
(Pinaceae) (Jungblut et al., 1995), quercetin 3-Ogalactoside, myricetin 3-O-rutinoside and two pcoumaroyl kaempferol 3-O-glucosides from
Quercus ilex L. (Fagaceae) (Skaltsa et al., 1994),
and quercetin, apigenin and luteolin glycosides
from Olea europaea L. (Oleaceae) (Karabourniotis et al., 1992).
Recently, eight flavonol glycosides were isolat-

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Seiko Takemura et al.

Fig. 1. The bracts of Davidia involucrata, and UV-A and UV-B contents (w/m2) in sunshine before and after
passing the translucent bract.

ed as UV-absorbing substances from the translucent bracts of Rheum nobile Hook. f. et Thomson(Polygonaceae), which grows in the alpine zoneof the eastern Himalayas (Iwashina et al., 2004).
Their flavonoids were identified as quercetin 3O-[6-(3-hydroxy-3-methylglutaroyl)-glucoside],quercetin 3-O-glucoside, quercetin 3-O-galactoside, quercetin 3-O-rutinoside, quercetin 3-Oarabinoside, quercetin itself, quercetin 7-O-glycoside and kaempferol glycoside. Of their compounds, the former five glycosides were major
ones and have been shown to be the important
UV shields in this species (Iwashina et al.,2004). Quercetin 3-O-[6-(3-hydroxy-3-methylglutaroyl)-glucoside] was a new acylated flavonol
glycoside.
Saussurea involucrata Karel. et Kir. (Asteraceae) also put the large translucent cream-colored bracts concealing the huge inflorescenceand grows in the alpine zone of central Asia suchas Kyrgyz, Kazakhstan, Mongolia and China.
From their bracts and also leaves, much amountof quercetin 3-O-rutinoside, which may be acts
as UV shields, has been isolated together withfour minor flavone glycosides, apigenin, luteolin,
hispidulin and nepetin 7-O-glucosides (Kusano etal., 2007).
Davidia involucrata Baill. is endemic to southwestern China (Mabberley, 1997) and is sometimes cultivated in the gardens and parks as ornamentals. We noticed that the two translucent
bracts, which bear in spring, put on the inflorescence such as a parasol, and presumed that UVabsorbing substances, e.g., flavonoids, are accumulated in the bracts such as Rheum nobile andSaussurea involucrata.
The flavonoid compounds have been isolated
from the leaves of D. involucrata and identified
as quercetin 3-O-glucoside, quercetin 3-O-galactoside, quercetin 3-O-arabinoside and kaempfer-

UV-absorbing substances in the bracts of Davidia

ol 3-O-galactoside (Iwashina and Hatta, 1998).
Ouyang and Zhou (2003) have been surveyed theleaves for flavonoids and isolated kaempferol,kaempferol 3-O-glucoside and quercetin in addition to the above flavonoids. Of their compounds,
quercetin 3-O-galactoside has also been found in
the bracts as a major flavonoid, together withminor quercetin 3-O-rhamnoside (Rast, 1968).
In this paper, we describe the identification of
the flavonoid glycosides in the bracts and function of their compounds as UV filter.
Materials and Methods
Plant materials
Davidia involucrata Baill. is cultivated in the
Tsukuba Botanical Garden, National Museum of
Nature and Science, Tsukuba, Japan (Acc. no.
TBG 43668). The fresh bracts were collected inMay, 2004.
Measurement of UV radiation in sunshine, anddirect UV-visible spectral survey of bracts
UV-A (355 nm) and B (313 nm) radiation insunshine before and after passing the translucent
bract was measured by UV monitor MS-211-I(EKO Co. Ltd.). UV-visible spectra of the fresh
bracts were surveyed on a Shimadzu MPS-2000
multi purpose recording spectrophotometer between 300 and 700 nm.
Qualitative and quantitative HPLC of crude extract and isolated UV absorbing substances
Qualitative HPLC survey of the crude extracts
from the bracts and the isolated UV-absorbing
substances were performed with Shimadzu
HPLC systems using a Shim-pack CLC-ODS(I.D. 6.0150 mm; Shimadzu) at flow-rate:
1.0 ml min1, detection: 190–700 nm and eluent:
MeCN/H2O/H3PO4 (22 : 78 : 0.2). Fresh bracts(0.1 g) were extracted with MeOH (2 ml) for
quantitative HPLC survey. After keeping for 1
day in room temperature, the extracts were filtered and analysed by HPLC.
Preparative HPLC was performed with Tosoh
HPLC systems using a Senshu-pak PEGASIL

3

ODS column (I.D. 10.0250 mm, Senshu Scientific Co. Ltd.) at flow-rate: 1.5 ml min1, detection: 350 nm and eluent: HCOOH/MeCN/H2O(1 : 20 : 79).
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 flowe-rate of 0.2 ml min1, eluent:
MeCN/H2O/HCOOH (22 : 78 : 1), and ESI
4.5 kV, ESI 3.5 kV, 250°C.
Extraction and isolation of UV absorbing substances
Fresh bracts (ca. 39 g) were extracted with
MeOH. After evaporation, the extracts wereapplied to preparative paper chromatography(PPC) using solvent systems: BAW (nBuOH/HOAc/H2O4 : 1 : 5, upper phase), 15%
HOAc and then BEW (n-BuOH/EtOH/H2O
4 : 1 : 2.2). The isolated compounds were finallypurified by Sephadex LH-20 column chromatography using solvent system: 70% MeOH. Of
seven compounds (1–7) detected by HPLC survey, 1 and 3 were obtained as pure solutions, and
2 and 3, and 5 and 6 as the mixtures, respectively.
Compound 4 could not be isolated for the minuteamount. For further isolation of minor compounds 5 and 6, fresh bracts (136 g) were collected in May, 2005 and extracted with MeOH. Afterapplication to PPC, they were isolated by preparative HPLC and obtained as pale yellow powder(5) and pure solution (6).
Identification of UV absorbing substances
UV absorbing substances were identified by
UV spectral survey according to Mabry et al.
(1970), LC-MS, acid hydrolysis (in 12% aq.HCl,
100°C, 30 min) and characterization of its products, 1H and 13C NMR (in pyridine-d5, 500 MHz
for 1H NMR and 125 MHz for 13C NMR), direct
TLC and HPLC comparisons with authentic samples. TLC, HPLC, LC-MS, and 1H and 13C NMR
data of UV absorbing substances are as follows.
Quercetin 3-O-sambubioside (1). TLC: Rf
0.42 (BAW), 0.42 (BEW), 0.47 (15% HOAc);

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Seiko Takemura et al.

Fig. 2. UV-visible spectral curve of Davidia
involucrata bract.

UV – dark purple, UV/NH3 – yellow. HPLC: Rt(min) 4.38. UV: l max (nm) MeOH 256, 267 sh,
357; NaOMe 272, 328, 406 (inc.); AlCl3
274, 432; AlCl3 /HCl 269, 298, 361, 397;NaOAc 273, 326, 388; NaOAc/H3BO3 262,
296, 378.
Mixture of quercetin 3-O-galactoside (hyperin,2) and quercetin 3-O-glucoside (isoquercitrin, 3).
TLC: Rf 0.50 (BAW), 0.51 (BEW), 0.22 (15%
HOAc); UV – dark purple, UV/NH3 – yellow.
HPLC: Rt (min) 5.25 (2) and 5.52 (3). UV: l max(nm) MeOH 256, 267 sh, 358; NaOMe 272,
329, 409 (inc.); AlCl3 275, 435; AlCl3/HCl
269, 299, 363, 400; NaOAc 274, 325, 389;NaOAc/H3BO3 262, 297, 378.
Quercetin 3-O-arabinoside (4). TLC: Rf 0.63
(BAW), 0.57 (BEW), 0.12 (15% HOAc);
UV – dark purple, UV/NH3 – yellow. HPLC: Rt(min) 5.96. UV: l max (nm) MeOH 256, 270 sh,
358; NaOMe 273, 329, 407 (inc.); AlCl3
274, 421; AlCl3 /HCl 268, 299, 362, 398;NaOAc 273, 324, 390; NaOAc/H3BO3 261,
296, 377.
Quercetin 3-O-(2-acetylgalactoside) (6).
TLC: Rf 0.71 (BAW), 0.69 (BEW), 0.43 (15%
HOAc); UV – dark purple, UV/NH3 – yellow.
HPLC: Rt (min) 8.86. UV: l max (nm) MeOH
256, 267 sh, 356; NaOMe 273, 329, 407 (inc.);
AlCl3 274, 431; AlCl3 /HCl 270, 298, 359,
397; NaOAc 273, 325, 394; NaOAc/H3BO3

262, 297, 373. LC-MS: m/z 477 [MH] andm/z 475 [MH]. 1H NMR (500 MHz, pyridined5): d 13.43 (1H, s, 5-OH), 8.35 (1H, d, J2.1
Hz, H-2), 8.20 (1H, dd, J2.1 and 8.4 Hz, H6), 7.30 (1H, d, J8.6 Hz, H-5), 6.71 (1H, d,J1.8 Hz, H-8), 6.62 (1H, d, J2.1 Hz, H-6),
6.29 (1H, d, J7.6 Hz, galactosyl H-1), 6.27
(1H, d, J6.7 Hz, galactosyl H-2), 4.64 (1H, d,J2.7 Hz, galactosyl H-4), 4.39 (1H, m, galactosyl H-6a), 4.38 (1H, m, galactosyl H-3), 4.27
(1H, dd, J5.8 and 11.0 Hz, galactosyl H-6b),4.18 (1H, t, J12.5 Hz, galactosyl H-5), 2.16
(3H, s, acetyl CH3). 13C NMR (125 MHz, pyridine-d5): d (quercetin) 157.4 (C-2), 134.9 (C-3),
178.5 (C-4), 162.9 (C-5), 99.7 (C-6), 165.7 (C7), 94.4 (C-8), 157.4 (C-9), 105.3 (C-10), 123.1(C-1), 116.3 (C-2), 146.8 (C-3), 150.7 (C-4),117.4 (C-5), 122.3 (C-6); d (galactose) 101.6(C-1), 74.4 (C-2), 73.1 (C-3), 69.8 (C-4), 77.6
(C-5), 61.5 (C-6); d (acetic acid) 170.7 (COO–),
21.4 (CH3).
Quercetin 3-O-(acetylglucoside) (7). TLC: Rf
0.71 (BAW), 0.69 (BEW), 0.43 (15% HOAc);
UV – dark purple, UV/NH3 – yellow. HPLC: Rt(min) 9.17. UV: l max (nm) MeOH 256, 266 sh,
356; NaOMe 273, 329, 407 (inc.); AlCl3
274, 431; AlCl3/HCl 270, 298, 359, 397;NaOAc 273, 325, 394; NaOAc/H3BO3 262,
297, 373. LC-MS: m/z 477 [MH] and m/z 475[MH].
Results and Discussion
Absorption of UV radiation with the translucent
bractsWhen the amount of UV radiation (UV-A and
UV-B) in sunshine were measured before andafter passing the bracts, both the amount of UVA and UV-B clearly decreased in the latter case(Fig. 1). Direct UV-visible spectra of the translucent bracts are shown in Fig. 2. Though weak absorption maxima were present in 675 and 480 sh
nm in visible range (400–700 nm), showing thepresence of a very small amount of chlorophylls,
other absorption maxima could not be detected.
On the other hand, UV range (300–400 nm) wasUV-absorbing substances in the bracts of Davidia

strongly absorbed. Thus, it was shown that thetranslucent bracts of D. involucrata selectively
absorb UV radiation, but visible light is almost
completely transmitted, so that we presumed thatthe epidermis of the bracts contain much amountof UV-absorbing substances which are present in
the cells to screen UV radiation.
Identification of UV-absorbing substances in thebractsHPLC patterns of the translucent bracts weremonitored by 280 nm (UV-B range) (Fig. 3).
Some peaks appeared on the chromatogram andwere presumed to be flavonoid compounds by

UV spectral properties. Major compounds 2 and3 were obtained as the mixture. UV spectral survey according to Mabry et al. (1970) showed thatthey are flavonol glycosides having free 5-, 7-,3- and 4-hydroxyl and a substituted 3-hydroxyl
groups. By acid hydrolysis, they liberated
quercetin, glucose and galactose. From the results described above, they were presumed to be
the mixture of the glycosides which attachedgalactose or glucose to 3-position of quercetin.
Finally, they were identified as the mixture ofquercetin 3-O-galactoside (2, Fig. 5) and
quercetin 3-O-glucoside (3, Fig. 6) by direct TLC
and HPLC comparisons with authentic hyperin
and isoquercitrin from the flowers of Astrophytum ornatum (DC.) Web. (Cactaceae) (Iwashina
and Ootani, 1986) and the fronds of Cyrtomium
falcatum (L. f.) C. Presl (Dryopteridaceae)(Iwashina et al., 2006). Quercetin 3-O-galactoside and 3-O-glucoside have been isolated from

Fig. 3. HPLC pattern of MeOH extracts from thebracts of Davidia involucrata. 1quercetin 3O-sambubioside, 2quercetin 3-O-galactoside, 3quercetin 3-O-glucoside, 4quercetin
3-O-arabinoside, 5kaempferol 3-O-galactoside, 6quercetin 3-O-(2-acetylgalactoside),and 7quercetin 3-O-(acetylglucoside).
Table 1.

5

Fig. 4.

Quercetin 3-O-samububioside (1).

Quantitative HPLC analysis of UV-absorbing substances in the bracts of Davidia involucrata

Values12 and 3

4

5

6

7

Area
Fresh weight*(mg/g)
Ratio

225,972

9,623,359

1,701,153

174,864

1,846,709

498,730

0.09

3.65

0.65

0.07

0.70

0.19

1.6%

68.5%

12.1%

1.2%

13.1%

3.5%

Fresh bract (0.1 g) was extracted with MeOH (2 ml). Measurement was 280 nm.
*Calculated from the absorption coefficient of authentic quercetin 3-O-galactoside.
1quercetin 3-O-sambubioside, 2quercetin 3-O-galactoside, 3quercetin 3-O-glucoside, 4quercetin 3-O-arabinoside, 5kaempferol 3-O-galactoside, 6quercetin 3-O-(2-acetylgalactoside) and 7quercetin 3-O-(acetylglucoside).

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Seiko Takemura et al.

Fig. 5.

Quercetin 3-O-galactoside (hyperin, 2).

Fig. 6. Quercetin 3-O-glucoside (isoquercitrin,3).

the leaves of this species (Iwashina and Hatta,
1998).
It was shown by UV spectral survey that compound 1 is also 3-O-substituted 5,7,3,4-tetrahydroxyflavone. Quercetin as an aglycone, and xylose and glucose as glycosidic sugars were produced by acid hydrolysis. Thus, compound 1 wascharacterized as quercetin 3-O-xylosylglucoside.
Finally, the compound was completely identified
as quercetin 3-O-xylosyl-(1→2)-glucoside, i.e.,
quercetin 3-O-sambubioside (Fig. 4) by direct
TLC and HPLC comparison with authentic sample from the leaves of Aucuba japonica Thunb.
(Cornaceae) (Iwashina et al., 1997). Quercetin 3O-sambubioside has been reported from D. involucrata for the first time.
Acid hydrolysis of compound 4 liberated
quercetin and arabinose. Since UV spectral survey of the original glycoside showed the presenceof free 5-, 7-, 3- and 4-hydroxyl groups, it wasdetermined that arabinose is attached to 3-posi-

Fig. 7.

Quercetin 3-O-arabinoside (4).

tion of quercetin. By direct TLC and HPLC comparison with authentic sample, which has been
isolated from the bracts of Rheum nobile(Iwashina et al., 2004), compound 4 was shown
to be quercetin 3-O-arabinoside (Fig. 7). The
compound has already been detected from theleaves of this species (Iwashina and Hatta, 1998;
Ouyang and Zhou, 2003).
Though minor compound 5 could not be isolated for small amount, it was presumed to be
kaempferol derivative by UV spectral properties(appearance of l max 266 and 345 nm) on HPLC.
It was identified as kaempfeol 3-O-galactoside(Fig. 8) by HPLC comparison with authentic
trifolin from the leaves of Cornus canadensis
L. (Cornaceae) (Iwashina and Hatta, 1993).
Kaempferol 3-O-galactoside has also been reported from D. involucrata (Iwashina and Hatta,
1998; Ouyang and Zhou, 2003).
Compound 6 was obtained by pale yellow
powder. It was shown to be 3-O-substituted
quercetin by UV spectral survey according to
Mabry et al. (1970). Quercetin and galactose wasliberated by acid hydrolysis. However, its retention time does not agreed with that of authentic
hyperin. The molecular ion peaks, m/z 477[MH] and m/z 475 [MH], showing the attachment of each 1 mol galactose and acetic acidto quercetin, appeared on the chromatogram by
LC-MS survey. In 1H NMR spectrum, a singlet(d 2.16) corresponding to acetyl CH3 appearedwith five aromatic protons (H-6, H-8, H-2, H-5and H-6) and a galactosyl anomeric proton (d6.29, J7.6 Hz). The attachment of acetyl group

UV-absorbing substances in the bracts of Davidia

Fig. 8.

Kaempferol 3-O-galactoside (trifolin, 5).

Fig. 9.

Quercetin 3-O-(2-acetylgalactoside) (6).

to 2-position of galactose was proved by 13C
NMR spectrum, i.e., the diagnostic shift to lower
field of carbon signal (d 74.4) corresponding togalactosyl C-2 (Agrawal, 1989). Moreover, it was
confirmed by 1H-1H COSY, FG-HSQC and FGHMBC (Fossen and Andersen, 2006). Thus,
compound 6 was completely identified as
quercetin 3-O-b -D-(2-acetylgalactopyranoside)(Fig. 9). The rare acylated flavonoid has only
been reported from the whole plants of Hypericum perforatum L. (Guttiferae) (Jürgenliemk
and Nahrstedt, 2002) and similar acylated
quercetin glycosides, quercetin 3-O-(3-acetylgalactoside) and quercetin 3-O-(6-acetylgalactoside), have been found in Ledum palstre L. (Ericaceae) (Harborne and Baxter, 1999).
LC-MS survey of compound 7 indicated m/z
477 [MH] and m/z 475 [MH], showing theattachment of each 1 mol hexose and acetic acid7

to quercetin. TLC, LC-MS and UV spectral data
showed that 7 is quercetin 3-O-(acetylhexoside),so that we presumed this compound as to bequercetin 3-O-(acetylglucoside). Complete identification of the compound is now in progress.
Quantitative HPLC survey of the bract isshown in Table 1. Of their flavonoids isolated
from the bracts, major ones were quercetin 3-Ogalactoside (2) and 3-O-glucoside (3). Their relative amount was 68.5% (3.65 mg/g fresh bract).
On the other hand, other flavonoids (1, 4–7) were
between 13.1–1.2% (0.70–0.07 mg/g).
UV-absorbing substances in the bracts of D.
involucrata were all flavonol glycosides, especially quercetin 3-O-glycosides. Quercetin glycosides have been reported as the predominantflavonoid in response to UV-B radiation in someplants species, e.g., Anethum graveolens L. (Umbelliferae) (Möhle et al., 1985), Brassica napus
L. (Brassicaceae) (Olsson et al., 1998; Wilson
and Greenberg, 1993), Petunia hybrida(Solanaceae) (Ryan et al., 1998, 2002), Trifolium
repens L. (Leguminosae) (Hofmann et al., 2000)
and Betula pendula Roth (Betulaceae) (Lavola etal., 1997). Petunia and wild-type of Arabidopsis
thaliana L. leaves exposed to low UV-B conditions contained predominantly kaempferol glycosides, with low levels of quercetin glycosides(Ryan et al., 1998, 2001). However, when theleaves were treated with UV-B, an increase ofquercetin was observed. These results suggestthat quercetin glycosides protect the plants fromUV-B damage. Recently, it was suggested thatquercetin glycoside function as UV protecting
substances in the translucent bracts of Rheum nobile, which grows in the alpine zone of the Himalayas (Iwashina et al., 2004). In also the caseof Davidia involucrata, quercetin glycosides predominantly occurred in the bracts. Thus, it was
shown that quercetin glycosides, especiallyquercetin 3-O-galactoside and quercetin 3-O-glucoside, mainly act as UV shields in the translucent bracts of D. involucrata, and protect the inflorescence from noxious UV radiation. More recently, it has been reported that the bracts of D.
involucrata contain much amounts of flavonoids,

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Seiko Takemura et al.

which were not qualitatively characterized, andact as pollination attractants to insects, especially
Hymenoptera (Hu et al., 2007), so that the bracts
may be functional for both pollination attractantsand UV shields.
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