Bull. Natl. Mus. Nat. Sci., Ser. B, 45(1), pp. 41–46, February 22, 2019

Anthocyanin-ﬂavonol Copigmentation in Bluish Flowers ofGladiolus×grandiﬂora Cultivar
Tomoko Takemura1, Kosaku Takeda2 and Tsukasa Iwashina3,*1

United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology,
Saiwai-cho, Fuchu 183–8509, Japan
2
Tokyo Gakugei University, Koganei, Tokyo 184–8501, Japan3
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 16 October 2018; accepted 26 December 2018)

Abstract The in vitro identiﬁcation of copigmentation was carried out using anthocyanin, malvidin 3,5-di-O-glucoside, and three ﬂavonol glycosides, kaempferol 3-O-sophoroside, kaempferol
3-O-rutinoside and kaempferol 3-O-glucosyl-(1→2)-rhamnoside. They are major pigments ofGladiolus×grandiﬂora cultivar ʻAriakeʼ. Anthocyanin brought about copigmentation, i.e., thebathochromic shift (bluing effect) of visible λmax due to increased concentrations of kaempferol
3-O-rutinoside and kaempferol 3-O-glucosyl-(1→2)-rhamnoside, and 14–30 and 12–29 nm were
estimated as the magnitude (Δλmax), respectively. However, kaempferol 3-O-sophoroside was notoccurred the bathochromic shift of malvidin 3,5-di-O-glucoside. These results showed that bluing
effect on the ﬂower color of the bluish cultivar ʻAriakeʼ caused at least in part by the copigmentation between anthocyanin and two ﬂavonol glycosides, kaempferol 3-O-rutinoside and kaempferol
3-O-glucosyl-(1→2)-rhamnoside.

Introduction
The ﬂower colors, especially purple to blue,
due to anthocyanins are well known to be inﬂuenced by intermolecular copigmentation (e.g.,Yazaki, 1976; Takeda et al., 1993; Yabuya et al.,
1997; Mizuno et al., 2015), intramolecularcopigmentation (e.g., Terahara et al., 1990;
Figueiredo et al., 1999; Sakaguchi et al., 2013),
metal complexation (Takeda et al., 2005; Shionoet al., 2005, 2008; Yoshida et al., 2006), and thevacuolar pH values of epidermal ﬂower cells(Yoshida et al., 1995; Fukuda-Tanaka et al.,
2000; Takeda et al., 2010).
Gladiolus×grandiﬂora cultivars are widelygrown all over the world as popular ﬂowers.
Flower anthocyanins and their distribution in
many Gladiolus cultivars have been reported by
Takemura et al. (2008). Their anthocyanins wereisolated from the ﬂowers of six selected cultivars

and identiﬁed as 3-O-rutinoside-5-O-glucosidesof cyanidin, malvidin, pelargonidin and peonidin,
and 3,5-di-O-glucosides of petunidin, malvidin,
pelargonidin, cyanidin and peonidin, and pelargonidin 3-O-rutinoside and malvidin 3-O-glucoside (Takemura et al., 2008).
On the other hand, some ﬂavonol glycosides
were isolated from the ﬂowers of Gladiolus cultivars and identiﬁed as kaempferol 3-O-sophoroside,
3-O-glucoside,
3-O-rutinosideand3-O-glucosyl-(1→2)-rhamnoside,
quercetin
3-O-rutinoside, and myricetin 3-O-rutinoside,
3-O-glucoside and 3-O-rhamnoside (Takemuraand Iwashina, 2018). As minor ﬂavonols, kaempferol 3-O-pentosylglucoside, 3-O-rhamnosylrhamnosylglucoside and 3-O-glucosylrhamnoside,laricitrin
3-O-diglucoside,
3-Orhamnosylglucoside and 3-O-rhamnosylhexoside, and syringetin 3-O-rhamnosylglucosidewere also found (Takemura and Iwashina, 2018).

Tomoko Takemura et al.

42ʻChristmasʼ were cultivated in the nursery of theTsukuba Botanical Garden, National Museum ofNature and Science, Tsukuba, Japan. Anothercultivar ʻAriakeʼ was cultivated in Ogawa Town,
Ibaraki, Japan.

Fig. 1.

Gladiolus×grandiﬂora cultivar ʻAriakeʼ.

Of their Gladiolus cultivars, ʻAriakeʼ may be oneof the most bluish ﬂower cultivars (Fig. 1). As
major anthocyanin, malvidin 3,5-di-O-glucosidehas been isolated from ʻAriakeʼ, together withtwo minor malvidin glycosides (Takemura et al.,2005). Three major ﬂavonols, kaempferol 3-Osophoroside, kaempferol 3-O-rutinoside andkaempferol
3-O-glucosyl-(1→2)-rhamnosidewere also found in this cultivar, together withother kaempferol, quercetin, myricetin, laricitrin
and syringetin 3-O-glycosides (Takemura et al.,2005; Takemura and Iwashina, 2018).
In this survey, the in vitro identiﬁcation ofcopigmentation was carried out using anthocyanin, malvidin 3,5-di-O-glucoside, and three ﬂavonol glycosides, kaempferol 3-O-sophoroside,kaempferol 3-O-rutinoside and kaempferol
3-O-glucosyl-(1→2)-rhamnoside which aremajor pigments in ʻAriakeʼ ﬂowers.
Materials and Methods
Plant materials
Gladiolus×grandiﬂora cultivars ʻViolettaʼ andExtraction, isolation and identiﬁcation of anthocyanin and ﬂavonols
Malvidin 3,5-di-O-glucoside was extracted
and isolated from the ﬂowers of Gladiolus cultivar ʻViollettaʼ according to Takemura et al.
(2008). Three ﬂavonols, kaempferol 3-Oglucosyl-(1→2)-rhamnoside, and kaempferol
3-O-sophoroside and 3-O-rutinoside, wereextracted and isolated from the ﬂowers of Gladiolus cultivars ʻChristmasʼ and ʻAriakeʼ according
to Takemura and Iwashina (2018). They wereidentiﬁed by UV-vis spectra, LC-MS, acid andalkaline hydrolyses, 1H and 13C NMR, and/or
TLC and HPLC comparisons with authentic samples.
Quantitative analysis of anthocyanin and ﬂavonols
Anthocyanin of the perianth was dissolved
with 0.1%HCl–MeOH (1 ml) for one day. Thissolution was diluted to 100, 200 and 400 folds byaddition of 0.1%HCl–MeOH. Absorption spectra(450–600 nm) of their solution were measured byHPLC [Pegasil ODS column (I.D. 6.0×150 mm)(Senshu Scientiﬁc Co. Ltd., Tokyo), eluent,
MeCN/H2O/HOAc/H3PO4 (6 : 83 : 8 : 3)], andabsolute values (4.16×10−3 mol/l) were calculated by calibration curves. On the other hand,
ﬂavonols of the perianth were dissolved with
MeOH (1 ml) and diluted to 100, 200 and 400
folds, and absorption spectra (220–500 nm) weremeasured by HPLC [Pegasil ODS column(I.D. 6.0×150 mm), eluent, MeCN/H2O/H3PO4(22 : 78 : 0.2)]. Absolute values (kaempferol3-O-sophoroside, 3.78×10−2 mol/l, kaempferol
3-O-rutinoside, 1.46×10−2 mol/l, and kaempferol
3-O-glucosyl-(1→2)-rhamnoside,
6.40×10−2 mol/l) of their solution were calculated by calibration curves.

Anthocyanin-ﬂavonol Copigmentation in Gladiolus Cultivar

43

Table 1. The effect of concentration and copigmentation of malvidin 3,5-di-O-glucoside with three ﬂavonol
glycosides
Flavonols

Malvidin 3,5-di-O-glucoside (nm)−4after 1 day

λmax

Δλmax

intensity

λmax

Δλmax

intensity

Kaempferol 3-O-sophoroside02
35

532
532
532
535

—
00
3

0.24
0.29
0.32
0.37

532
532
532
532

—
00
0

0.23
0.27
0.31
0.37

Kaempferol 3-O-rutinoside

02
3
5532
546
556
562

—
142430

0.24
0.37
0.42
0.49

532
542
542
542

—
1010
10

0.23
0.33
0.39
0.46

Kaempferol 3-O-glucosyl(1→2)-rhamnoside

02
3
5

532
544
558
561

—
1226
29

0.24
0.31
0.40
0.43

532
541
543
557

—
9
1125

0.23
0.30
0.33
0.42

Mixture of three ﬂavonols

02.3 : 1.3 : 1.4

532541—90.240.42532540—
80.23
0.43

2×10(M)

3.5
2.650
1.8
700.0

600.0

500.0

400.0
1.8

The intact absorption spectral curve of the bluish perianth of cultivar ʻAriakeʼ was shown inFig. 2. Although absorption maxima of malvidin
3,5-di-O-glucoside is λmax 532 nm, that of theperianth appeared in 562 nm, showing the occurrence of the bathochromic shift of 30 nm. Moreover, since it has only one absorption maxima at
562 nm, and the absence of absorption maxima at

2.650

Results and Discussion

3.5Absorption spectra of fresh perianths
Fully
expandedbluishperianthof
Gladiolus×grandiﬂora cultivar ʻAriakeʼ wasmeasured for visible absorption spectra by Shimadzu multipurpose spectrophotometer MPS2000. To examine the absorption spectra ofanthocyanin-ﬂavonol copigmentation, malvidin
3,5-di-O-glucoside (2×10−4 M) and kaempferol
3-O-sophoroside, kaempferol 3-O-rutinoside andkaempferol
3-O-glucosyl-(1→2)-rhamnoside(4×10−4 M, 6×10−4 M and 10×10−4 M) weredissolved in 0.2M sodium acetate buffer solution(pH 4.8, 1.5 ml) and then mixed. After 5 minfrom mixing the anthocyanin and ﬂavonol solution, the copigmentation absorption spectra weremeasured by the visible absorption with a Shimadzu multipurpose spectrophotometer MPS2000.

Fig. 2. UV-vis absorption spectral curve of intact
bluish perianth of Gladiolus×grandiﬂora cultivar ʻAriakeʼ.

660–670 nm, this is apparently not due to metal
complexing and pH (Saitô, 1967).
Copigment effects of three ﬂavonol glycosides
on absorption spectra of malvidin 3,5-di-O-glucoside are shown in Table 1 and Figs. 3, 4 and 5.
As the results, malvidin 3,5-di-O-glucosidebrought about copigmentation by the addition of
ﬂavonol glycosides, i.e., the bathochromic shift(bluing effect) in the visible λmax due toincreased
concentrations
ofkaempferol
3-O-rutinoside and kaempferol 3-O-glucosyl-

Tomoko Takemura et al.

4410.500

0.500

0.500

0.500

0700.0

600.0

500.0

450.0

700.0

600.0

500.0

450.0

0001

10.500

0.500

00

700.0

600.0

500.0

450.0

Fig. 4. UV-vis absorption spectral curve of mixture of malvidin 3,5-di-O-glucoside and kaempferol 3-O-rutinoside in CH3COOK buffer.
1＝malvidin
3,5-di-O-glucoside : kaempferol
3-O-rutinoside (1 : 2), 2＝malvidin 3,5-di-Oglucoside : kaempferol 3-O-rutinoside (1 : 3) and
3＝malvidin
3,5-di-O-glucoside : kaempferol
3-O-rutinoside (1 : 5). pH 4.8.

1

11Fig. 3. UV-vis absorption spectral curve of mixture of malvidin 3,5-di-O-glucoside and kaempferol 3-O-sophoroside in CH3COOK buffer.
1＝malvidin 3,5-di-O-glucoside : kaempferol 3O-sophoroside (1 : 2), 2＝malvidin 3,5-di-Oglucoside : kaempferol 3-O-sophoroside (1 : 3)
and 3＝malvidin 3,5-di-O-glucoside : kaempferol 3-O-sophoroside (1 : 5). pH 4.8.

Fig. 5. UV-vis absorption spectral curve of
mixture of malvidin 3,5-di-O-glucoside andkaempferol
3-O-glucosyl-(1→2)-rhamnoside
in CH3COOK buffer. 1＝malvidin 3,5-di-Oglucoside : kaempferol
3-O-glucosyl-(1→2)rhamnoside (1 : 2), 2＝malvidin 3,5-di-Oglucoside : kaempferol
3-O-glucosyl-(1→2)rhamnoside (1 : 3) and 3＝malvidin 3,5-di-Oglucoside : kaempferol
3-O-glucosyl-(1→2)rhamnoside (1 : 5). pH 4.8.

(1→2)-rhamnoside, and 30 and 29 nm were estimated as the magnitude (Δλmax), respectively(Table 1, Figs. 4 and 5). On the other hand, thebathochromic shift did not occur by the additionof kaempferol 3-O-sophoroside (Table 1 and Fig.
3). In the case of Hydrangea macrophylla(Thunb. ex Murray) Ser. ﬂowers, 3-caffeoylquinic acid and 3-p-coumaroyl-quinic acid similarly act as copigments. However, 5-caffeoylquinic acid (chlorogenic acid) obstructs
copigment activity (Takeda et al., 1990). Moreover, it was shown that although isovitexin (apigenin 6-C-glucoside) strongly acts as copigment,vitexin (apigenin 8-C-glucoside) weakly affectagainst cyanidin 3,5-di-O-glucoside (Asen et al.,1972).
We calculated the ratio of malvidin 3,5-di-Oglucoside : kaempferol 3-O-sophoroside : kaempferol 3-O-rutinoside : kaempferol 3-O-glucosyl(1→2)-rhamnoside in cultivar ʻAriakeʼ perianthAnthocyanin-ﬂavonol Copigmentation in Gladiolus Cultivar45110.5000.500

cose.
As the results, it was presumed that althoughkaempferol
3-O-rhamnosylglucoside
and3-O-glucosylrhamnoside are occurred the copigmentation, kaempferol 3-O-sophoroside, whichattach 2 mol glucose but not each 1 mol of glucose and rhamnose, obstruct the copigmentationin Gladiolus cultivar ﬂowers.
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00700.0

600.0

500.0

450.0

Fig. 6. UV-vis absorption spectral curve of mixture of malvidin 3,5-di-O-glucoside and threekaempferol 3-O-glycosides in CH3COOKbuffer. Malvidin 3,5-di-O-glucoside : kaempferol
3-O-sophoroside : kaempferol
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