Bull. Natl. Mus. Nat. Sci., Ser. B, 34(1), pp. 43–51, March 21, 2008

Flower Color and Flavonoid Variation in Clematis patens(Ranunculaceae)
Masanori Hashimoto1, Tsukasa Iwashina1,2* and Sadamu Matsumoto2
1

Graduate School of Agriculture, Ibaraki University, Ami 300–0393, Japan2
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 Anthocyanins and other flavonoids in Clematis patens were surveyed in relation totheir flower colors. Flower colors in the species were divided into three colors, white, pink and purple. Of their flower colors, the absorption maxima of the pink and purple were similar to eachother, suggesting that the colors are due to intra-molecular copigment. Their anthocyanins werecharacterized as delphinidin and cyanidin glycosides in both flower colors. However, delphinidin isa major pigment in the purple flowers, together with minor cyanidin. On the other hand, small
amounts of delphinidin and cyanidin were detected from the pink flowers. Thus, it was shown that
their color differences are due to quantitative ones of anthocyanidins, especially delphinidin. Eightflavonols were detected from the pink flowers and characterized as kaempferol 3-O-rutinoside(F4), kaempferol 3-O-glucoside (F5), kaempferol 3-O-(caffeoylglucoside) (F1), quercetin 3-O-glycoside (F8), kaempferol 3-O-gentiobioside (F2), kaempferol 3-O-alloside (F6), and two kaempferol glycosides (F3 and F7). The white flowers also contained these flavonoids and were divided
into two chemotypes by their flavonoid composition. However, the flavonoids could not be detectedfrom the purple flowers. Thus, it was presumed that the purple flower color of C. patens is due toan intra-molecular copigment.
Key words : anthocyanins, Clematis patens, flavonols, flower colors.

Introduction
Clematis patens Morr. & Decne. (Ranunculaceae) is distributed in Honshu, Shikoku,
Kyushu, Korea and China (Tamura and Shimizu,
1982). The species was listed in “Threatend
Wildlife of Japan, Vascular Plants” (Environment
Agency of Japan, 2000), since many plants havemainly been collected for ornamental in Japan.
The flower colors are various from white, pink topurple.
The anthocyanins of some Clematis cultivars
as flower pigments have been reported by Hosoki
et al. (2003) and tentatively characterized as
cyanidin and delphinidin glycosides. Cyanidin
hexoside was detected from the flowers of C.
japonica Thunb. (Ueno et al., 1969). Cyanidin 3O-glucoside was isolated from the fruits of C.

terniflora DC. (Ishikura, 1975). Other flavonoids
have been reported from some Clematis species,
i.e., quercetin itself from the pollen of C. apiifolia DC., C. japonica and C. stans Sieb. & Zucc.,
and three quercetin glycosides, 3-O-glucoside,
3-O-glucuronide and 3-O-rutinoside from theleaves of C. stans (Kizu et al., 1995). An
acylated flavone glycoside, apigenin 7-O-(pcoumaroylglucoside) have been isolated from C.
terniflora var. robusta Tamura (C. ternifloravar. terniflora) (Aritomi, 1963). The eight taxa of
Clematis subsect. Viornae in USA, C. addisonii
Britton, C. texensis Buckley, C. glaucophylla
Small, C. versicolor Small ex Rydberg, C. viorna
L., C. reticulata Walter, C. pitcheri Torrey &Gray var. pitcheri and C. pitcheri var. dictyota(Greene) Dennis, have chemotaxonomically beensurveyed for foliar flavonoids, and seven flavones,

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Masanori Hashimoto et al.

apigenin, apigenin 7-O-glucuronide, luteolin,
luteolin 7-O-glucoside, luteolin 7-O-glucuronide,luteolin 7-O-rhamnosylglucoside and chrysoeriol 7-O-glycoside; three flavone C-glycosides,
isoorientin, isovitexin and vicenin-2; and fiveflavonols, quercetin 3-O-gluoside, quercetin 3O-arabinoside, quercetin 3-O-rhamnosylglucoside, quercetin 3-O-rhamnosylgalactoside andkaempferol 3-methyl ether, were isolated together with two unknown flavonoids (Dennis andBierner, 1980). Quercetin and kaempferol glycosides were detected from the leaves of nine
Clematis species in Europe, i.e., C. cirrosa L., C.
integrifolia L., C. flammula L., C. rehderiana
Craib., C. serratifolia Rehd., C. viorna L., C.
vitalba L., C. montana Buch. and C. alpina Mill.
(Lebreton, 1986). A triperpene, oleanolic acid,has been isolated from the roots of C. patens(Fujita et al., 1974). However, the flower
pigments and flavonoids are not reported fromC. patens.
In this paper, we describe anthocyanins andother flavonoids from the flowers of Clematis
patens in relation to its various flower colors.
Materials and Methods
Plant materials
Eighteen individuals of Clematis patens fromeighteen populations in Japan were used as plantmaterials. Their flower colors, collection sites
and acc. No. were as follows.
White flowers: Miyagi Pref. (TBG142575),
Nikota, Uchihara, Ibaraki-gun, Ibaraki Pref.
(TBG132792), Koibuchi, Uchihara, Ibaraki-gun,Ibaraki Pref. (TBG132786), Muramatsu, Tokai,
Naka-gun, Ibaraki Pref. (TBG143503), Kasumigawa, Ohme-shi, Tokyo (TBG142581), Funabashi-shi, Chiba Pref. (TBG142577), Ashigakubo, Yokose, Chichibu-gun, Saitama Pref.
(TBG123485), Nagano Pref. (TBG141573).
Pink flower: Nikko-shi, Tochigi Pref. (TBG
134741).
Purple flowers: Oshibe, Iwama, Nishi-ibarakigun, Ibaraki Pref. (TBG133548), Nikko-shi,
Tochigi Pref. (TBG134739), Mamada, Oyama-shi, Tochigi Pref. (TBG134738), Ohme-shi,
Tokyo (TBG142576), Inba-mura, Susui, Inbagun, Chiba Pref. (TBG142580), Kawarayu,
Naganohara, Azuma-gun, Gunma Pref. (TBG
137173), Osaka Pref. (TBG134742), Sanda-shi,
Hyogo Pref. (TBG142574), Hiroshima Pref.
(TBG123436).
Plant materials are growing in Tsukuba Botanical Garden, National Museum of Nature andScience, Tsukuba, Japan.
Extraction and isolation of anthocyanin andother flavonoids
Fresh sepals of each individual were extractedwith HCOOH/MeOH (8 : 92). In cases of anthocyanin isolation, the extracts were concentrated
in vacuo, applied to Sephadex LH-20 column
chromatography (solvent system: 5% HOAc),
and applied to preparative paper chromatography(prep. PC) using solvent systems: BAW (nBuOH/HOAc/H2O  4 : 1 : 5, upper phase) andthen 15% HOAc. Isolated anthocyanin was furthermore purified by preparative HPLC (seebelow).
In cases of other flavonoids, the concentrated
extracts were applied to prep. PC using solvent
systems: BAW, 15% HOAc and then BEW(n-BuOH/EtOH/H2O  4 : 1 : 2.2). The obtained
flavonoids were purified by Sephadex LH-20
column chromatography using solvent system:
70% MeOH.
High performance liquid chromatography (HPLC)
Qualitative and quantitative HPLC was performed with Shimadzu HPLC systems using
Shim-Pak CLC-ODS column (I.D. 6.0150 mm:
Shimadzu), at a flow-rate of 1.0 ml min1; detection was 190 – 700 nm and eluents were H3PO4/
HOAc/MeCN/H2O (3 : 8 : 6 : 83) for anthocyanins,and H3PO4/MeCN/H2O (0.2 : 22 : 78) for otherflavonoids. Fresh sepals (0.2 g) were extractedwith FAH (HCOOH/MeCN : H2O  8 : 20 : 72)
for quantitative HPLC survey. Preparative HPLC
was performed with Tosoh HPLC systems using
Senshu Pak PEGASIL ODS column (I.D.
10250 mm: Senshu Scientific Co. Ltd.), at a

Flower color and flavonoids in Clematis patens

flow-rate of 1.5 ml min1; detection was 530 nm
and eluent was HCOOH/MeCN/H2O (5 : 13 : 82).
Direct visible absorption spectra of the sepals
Direct visible absorption spectra of the sepals
from C. patens were measured between 400 and
700 nm using Shimadzu multipurpose recording
spectrophotometer MPS-2000.
Characterization of anthocyanins
After concentration, crude extracts weredirectly hydrolyzed with 12% HCl, 100°C for
3 min. After cooling in water, the solution was
shaken with 3-methyl-1-butanol (isoamyl alcohol). Anthocyandins were identified by direct
TLC and HPLC comparisons with authentic
pelargonidin, cyanidin, peonidin, delphinidin,
petunidin and malvidin.
Identification of other flavonoids
The isolated flavonoids were identified by UV
spectral survey according to Mabry et al. (1970),
and direct TLC and HPLC comparisons of original glycosides and their hydrolysates withauthentic samples. TLC, HPLC and UV spectral
properties of the isolated flavonoids are as follows.
Kaempferol 3-O-(caffeoylglucoside) (F1). TLC:
Rf 0.63 (BAW), 0.66 (BEW), 0.67 (15%HOAc);
UV – dark purple, UV/NH3 – yellow fluorescence.
UV: l max (nm) MeOH 267, 333; NaOMe 274,325sh, 386 (inc.); AlCl3 268, 305, 357;
AlCl3/HCl 276, 303, 336, 390sh; NaOAc
274, 312, 373; NaOAc/H3BO3 267, 305sh, 353.
Kaempferol 3-O-rutinoside (nicotiflorin, F4).
TLC: Rf 0.51 (BAW), 0.57 (BEW), 0.49 (15%
HOAc); UV – dark purple, UV/NH3 – dark yellow.
HPLC: tR 5.88 min. UV: l max (nm) MeOH 266,
352; NaOMe 275, 326, 400 (inc.); AlCl3
274, 304, 354, 396; AlCl3/HCl 275, 303, 350,
392; NaOAc 274, 306, 375; NaOAc/H3BO3
267, 351.
Kaempferol 3-O-glucoside (astragalin, F5).
TLC: Rf 0.68 (BAW), 0.73 (BEW), 0.32 (15%
HOAc); UV – dark purple, UV/NH3 – dark yellow.
HPLC: tR 6.57 min. UV: l max (nm) MeOH 266,45

350; NaOMe 275, 325, 400 (inc.); AlCl3
274, 304, 352, 395; AlCl3/HCl 275, 302, 346,
395; NaOAc 274, 303, 372; NaOAc/H3BO3
267, 351.
Quercetin 3-O-glycoside (F8). UV: l max (nm)
MeOH 257, 265sh, 359; NaOMe 274, 329, 407(inc.); AlCl3 274, 429; AlCl3/HCl 266, 302,
362, 400sh; NaOAc 272, 310sh, 399; NaOAc/
H3BO3 262, 376.
Results and Discussion
Direct visible absorption spectra of the sepals
Eighteen individuals which were used as thematerials in this experiment, were divided into
three flower colors, i.e., white (8 individuals),
pink (1 individual) and purple (9 individuals)(Figs. 1 – 3). Direct visible spectra of the purple
and pink flower colors are shown in Figs. 4 and5. In the purple flowers, its absorption maxima
are present in 571 nm, together with 534 and 617
nm. Though the absorbance intensity of the pinkflower is lower than that of the purple flowers,
their absorption property is similar to each other.
Saito (1967) classified the absorption spectra into
four groups according to number and position of
maxima in the visible region. According to him,
the purple and pink flowers of C. patens belong
to GROUP C, of which the flower colors are due
to inter-molecular or intra-molecular copigment(Honda and Saito, 2002; Saito, 2002).
Characterization of anthocyanidins
Two anthocyanidins were liberated by acidhydrolysis of the crude exracts from the purple andpink flowers. They were identified as delphinidin
and cyanidin (Fig. 6) by direct TLC comparisonswith authentic samples. Their anthocyanidins
were contained in both flower colors. However,delphinidin is major pigment in the purple flowers, together with minor cyanidin. On the other
hand, small amount of delphinidin and cyanidin
were detected from the pink flower. Thus, it was
shown that the color differences between the purple and pink flowers are due to quantitative onesof the anthocyanidin, especially delphinidin. A46

Masanori Hashimoto et al.

Fig. 1.

White flowers of Clematis patens (TBG143503).

Fig. 2.

Pink flowers of Clematis patens (TBG134741).

Flower color and flavonoids in Clematis patensFig. 3.

47

Purple flower of Clematis patens (TBG142576).

Fig. 4. Absorption spectral curve of Clematis
patens purple flower.

Fig. 5. Absorption spectral curve of Clematis
patens pink flower.

delphinidin glycoside was isolated from the purple flowers (TBG133548), and glucose and caffeic acid were liberated by acid hydrolysis, together
with delphinidin. The complete identification of
the anthocyanins are now in progress.

Identification of other flavonoids
Four flavonoids were isolated from the pinkflowers (TBG134741). Of their compounds,
flavonoids F4 and F5 were identified as
kaempferol 3-O-rutinoside and 3-O-glucoside

48

Masanori Hashimoto et al.

(Fig. 7) by UV and TLC properties, and direct
TLC and HPLC comparisons with authentic
nicotiflorin from the aerial parts of Osyris alba
L. (Santalaceae) (Iwashina et al., 2008) and astragalin from the fronds of Cyrtomium falcatum(L. f.) C. Presl (Dryopteridaceae) (Iwashina etal., 2006).
Major flavonoid F1 liberated kaempferol, glucose and caffeic acid by acid hydrolysis. The
presence of free 5, 7 and 4-hydroxyl and asubstituted 3-hydroxyl groups was shown by
UV spectral mesurement in addition to various
shift reagents, suggesting the attachment ofboth glucose and caffeic acid to 3-position of
kaempferol. Thus, flavonoid F1 was characterized as kaempferol 3-O-(caffeoylglucoside) (Fig.
7). The glycoside has only been reported fromthe aerial parts of the fern, Pteridium aquilinum(L.) Kuhn as kaempferol 3-O-(6-caffeoylgluco-side) (Imperato and Minutiello, 1997).
UV spectral properties showed that minor
flavonoid F8 is quercetin 3-O-glycoside. However, its identification could not perform for small
amount of original glycoside. HPLC propertiesof this compound did not agree with those ofcommon quercetin 3-O-glycosides, 3-O-glucoside and 3-O-sophoroside.

Rcaffeoylglucosyl: kaempferol 3-O-(caffeoylglucoside) (F1)Rgentiobiosyl: kaempferol 3-O-gentiobioside (F2)Rrutinosyl: kaempferol 3-O-rutinoside (nicotiflorin, F4)
Rglucosyl: kaempferol 3-O-glucoside (astragalin, F5)
Rallosyl: kaempferol 3-O-alloside (asiaticalin, F6)

RH: cyanidin
ROH: delphinidinRglycosyl: quercetin 3-O-glycoside (F8)Fig. 6. Chemical structures of anthocyanidinsfrom the purple and pink flowers of Clematis
patens.

Fig. 7. Chemical structures of flavonol glycosides
from the white and pink flowers of Clematis
patens.

Fig. 8.

HPLC chromatogram of the extracts from the pink flowers of Clematis patens.

Flower color and flavonoids in Clematis patensFig. 9.

HPLC chromatogram of the extracts from the white flowers (B) of Clematis patens.

Fig. 10.

HPLC chromatogram of the extracts from the white flowers (A) of Clematis patens.

Fig. 11.

HPLC chromatogram of the extracts from the purple flowers of Clematis patens.

Four flavonoid peaks (F2, F3, F6 and F7),
which were not isolated, appeared on HPLC
chromatograms of crude extracts from thewhite flowers (Fig. 9). Two of them were identified as kaempferol 3-O-gentiobioside (F2) andkaempfrol 3-O-alloside (F6) (Fig. 7) by direct
HPLC comparisons with authentic samples from49

the flowers of Glycine max (L.) Merr. (Leguminosae) (Iwashina et al., 2007) and the leaves ofGlaucidium palmatum Sieb. & Zucc. (Glaucidiaceae) (Iwashina and Ootani, 1990), respectively.
Remained two glycosides F3 and F7 were partially characterized as kaempferol glycosides.

50

Masanori Hashimoto et al.

Table 1.
Flowercolorspurple1pink2white A3
white B4

Distribution of anthocyanidins and flavonols in the purple, pink and white flowers of Clematis patens.
Anthocyanins

Flavonols

Dp

Cy

D1

D2

D3

D4

D5

D6

D7

D8


t



t
t



















t

t







t

t


t

t






1

TBG133548, TBG134739, TBG134738, TBG142576, TBG142580, TBG137173, TBG134742, TBG142574 and
TBG123436.
2
TBG134741.
3
TBG142575, TBG142581, TBG142577 and TBG123485.
4
TBG132792, TBG132786, TBG143503 and TBG141573.
Dpdelphinidin, Cycyanidin, D1kaempferol 3-O-(caffeoylglucoside), D2kaempferol 3-O-gentiobioside,D3kaempferol glycoside, D4kaempferol 3-O-rutinoside, D5kaempferol 3-O-glucoside, D6kaempferol
3-O-alloside, D7kaempferol glycoside and D8quercetin 3-O-glycoside. presence, ttrace and absence.

Other flavonoid composition of white, pink andpurple flowers
Of eighteen individuals examined in this survey, pink flower one (TBG134741) synthesized
six flavonol glycosides (F1 and F4 – F8) (Table 1
and Fig. 8). Of their flavonoids, an acylated
flavonol, kaempferol 3-O-(caffeoylglucoside) was
only found in the pink flowers. Eight whiteflower individuals could be divided into two
chemotypes by the presence (B) (TBG132792,
132786, 143503 and 141573) or absence (A)(TBG142575, 142581, 142577 and 123485) offlavonoids F2 and F3 (Table 1, and Figs. 9 and
10). In the purple flowers, flavonol glycosides
were apparently not recognized (Fig. 11). However, as described above, the purple flowers of C.
patens are colored by copigment effects. Thoughdelphinidin glycosides of the species are notenough characterized the chemical structures,
they may be polyacylated anthocyanins and pigmented by intra-molecular copigments.
Flower color variations are reported from various anthocyanin-containing plants, and they are
due to the polymorphism (Bohm, 1987). In thiscase of Clematis patens, it was also presumed
that their flower color variations are due to polymorphism.

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