Bull. Natl. Mus. Nat. Sci., Ser. B, 45(4), pp. 169–174, November 22, 2019

Intermolecular Copigmentation and Color Stabilizationin the Violet Blue Flowers of Tall Bearded Iris Cultivar
Takayuki Mizuno* and Tsukasa Iwashina
Department of Botany, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan* E-mail: tmizuno@kahaku.go.jp(Received 8 August 2019; accepted 25 September 2019)

Abstract To reveal the violet blue color expression in the tall bearded iris cultivar ʻVictoria
Fallsʼ, colorimetric value, absorption spectra, quantitative analysis of phenolic compounds, and pH
value were measured using falls and standards of the ﬂowers. Higher lightness and lower chroma
values were shown in standards than those of falls. Absorption spectral curves of the intact falls
and standards are essentially the same, i.e. λmax at 578.9 nm and 580.2 nm, respectively. Copigment index [total C-glycosylﬂavone (TF) contents per total anthocyanin (TA) contents] in eachpart were calculated as 9.4 to 12.8 from the quantitative analysis. Total C-glycosylxanthone (TX)
contents per TA value were also calculated as 3.1 to 4.1, respectively. Based on these results, invitro examination using isolated anthocyanin, violanin, C-glycosylﬂavone, swertisin 2 -O-arabinoside, and C-glycosylxanthone, mangiferin, was performed. Degradation rate of the optical densityof these solutions was also measured. Bathochromic shift of 34.3 and 37.8 nm was observed by theaddition of 0.3 and 0.9 mM swertisin 2 -O-arabinoside to 0.1 mM violanin solution. Bathochromic
shift of λmax 5.2 nm by the addition of 0.3 mM mangiferin to violanin solution was observed. Furthermore, we were reappeared the absorption spectral curve of the intact falls by the addition of
0.9 mM swertisin 2″-O-arabinoside and 0.3 mM mangiferin to 0.1 mM violanin solution, suggesting that the violet blue color expression and stability of the tall bearded iris cultivar is expressedby mixture of these compounds.
Key words : color expression, C-Glycosylﬂavone, in vitro reconstruction, mangiferin, violanin.

Introduction
Flower coloration containing anthocyanin is
inﬂuenced by several factors such as copigmentation, metal complexation and the vacuolar pH
values of epidermal cell (Yoshida et al., 2009;
Iwashina, 2015). Of these factors, intermolecular
copigmentation, a natural process based on noncovalent (supramolecular) complex (Trouillas etal., 2016), is known as bluing effect (bathochromic shift) and color stabilization in some ﬂowers.
It is known that blue to bluish-purple ﬂower colors of Dutch iris (Iris×hollandica Hort. ex© 2019 National Museum of Nature and Science

Todd.) and Japanese garden iris (Iris ensata
Thunb.) occur by intermolecular copigmentation
between anthocyanins and C-glycosylﬂavones(Asen et al., 1970; Yabuya et al., 1997, 2000;
Mizuno et al., 2013). In the case of Dutch iris,
it was demonstrated using C-glycosylﬂavone,swertisin 2″-O-(4‴-acetylrhamnoside) and delphinidin 3-O-[(4‴-p-coumaroylrhamnosyl)-(1→6)glucoside]-5-O-glucoside (violanin). In Japanesegarden iris, it was reported that C-glycosylﬂavone, isovitexin is caused the intermolecular
copigmentaion with anthocynins such as
malvidin 3-O-[(4‴-p-coumaroylrhamnosyl)-(1→6)-glucoside]-5-O-glucoside (ensatin), petunidin 3O-[(4‴-p-coumaroylrhamnosyl)-(1→6)-glucoside]-170

Takayuki Mizuno and Tsukasa Iwashina

5-O-glucoside (petanin). In addition, it has been
revealed that their bluing effect depend on theamount and structure of these compounds (Asenet al., 1971, 1972; Yabuya et al., 1997).
Tall bearded iris (Iris×germanica L.) is oneof the ornamental plants in the genus Iris as wellas Dutch iris and Japanese garden iris. The iris
grows to more than 70 cm tall and have glamorous ﬂower. Anthocyanin, violanin, six C-glycosylﬂavones such as swertisin 2″-O-arabinoside,swertiajaponin 2″-O-arabinoside, schaftoside,
isoschaftoside, swertiajaponin and swertisin
2″-O-glucoside, and C-glycosylxanthone, mangiferin, were isolated from the violet blue ﬂower
cultivar ʻVictoria Fallsʼ (Mizuno et al., 2012).
However, it is not clear whether those are correlated to the ﬂower color.
In this study, we performed the measurementof colorimetric value, absorption spectra, quantitative analysis, and pH value of falls and standards of the tall bearded iris cultivar ʻVictoria
Fallsʼ. Furthermore, based on these results, invitro examination using isolated anthocyanin,
C-glycosylﬂavone and C-glycosylxanthone was
performed.
Materials and Methods
Plant materials
The tall bearded iris (Iris×germanica) cultivar ʻVictoria Fallsʼ was cultivated in the Tsukuba
Botanical Garden, National Museum of Natureand Science, Tsukuba, Japan. Flowers were collected at full-bloom stage in May 2019 (Fig. 1).
Their fresh falls and standards were used as plantmaterials. The ﬂower color values were recordedby the Royal Horticultural Society (R.H.S.) color
chart and a color reader (CR-10, Konica Minolta
Sensing Inc., Japan) as CIEL*a*b* (Gonnet,
1995, 1998).
Absorption spectra of fresh ﬂower
The fully expanded falls and standards wereused for the measurement of visible absorption
spectra (400–700 nm) with UV-2600 spectrophotometer (Shimadzu, Kyoto) which attached inte-

Fig. 1. Tall bearded iris cultivar ʻVictoria Fallsʼ.
The downward-curving petals are called as
falls. The upright petals are called as standards.

grated sphere ISR-2600 (Shimadzu).
Authentic samples
Authentic violanin (Fig. 2A) was obtained
from the ﬂowers of I. sanguinea Hornem.
(Mizuno et al., 2018). Authentic swertisin
2″-O-arabinoside (Fig. 2B) and mangiferin (Fig.
2C) were obtained from the ﬂowers of tallbearded iris cultivar ʻVictoria Fallsʼ (Mizuno etal., 2012). These compounds were checked by
HPLC before use as the standards of quantitative
analysis and in vitro examination.
Quantitativeevaluationofanthocyanin,
C-glycosylﬂavone and C-glycosylxanthone
Total anthocyanin (TA), total C-glycosylﬂavone (TF) and total C-glycosylxanthone (TX)
contents were measured and calculated by HPLC
and LC-Solution software (Shimadzu) from simple linear regression using violanin for TA contents at 530 nm, swertisin 2″-O-arabinoside for
TF contents at 350 nm, and mangiferin for TX
contents at 350 nm. Each TA, TF and TX contents were revealed as nmol mg−1 fresh weight.
HPLC analyses were performed with Shimadzu HPLC systems equipped with a SPD–20A
UV–Vis detector using InertSustain C18 (I.D.

Copigmentation of Tall Bearded Iris
Table 1.

171

Chromaticity in the falls and standards of tall bearded iris cultivar 'Victoria Fallsʼ

Flower parts

RHSCC

Falls
Standards

VioletBlue 94C
VioletBlue 94D

Chromaticity
L*

a*

b*

Chroma(C)

Hue angle(h°)

47.1±1.856.3±1.917.9±0.8
12.0±1.0−35.2±0.7−29.0±1.2

39.5
31.4

297.0
292.4

The measurement was performed triplicate (n＝3±SE).
The values of chroma (C) and hue angle (h°) were calculated as fallows. C*＝(a*2＋b*2)1/2 and h＝tan−1 (b*/a*).

pH measurement
The pH values of pressed juice from the falls
and standards were measured using a twin pH
meter AS-212 (HORIBA, Ltd., Tokyo). The measurements were performed in triplicate.
In vitro examination of intermolecular copigmentation
Puriﬁed violanin, swertisin 2″-O-arabinosideand mangiferin were dissolved in citric phosphate buffer (pH 5.2). The proportion of thesecompounds was decided based on the result of
quantitative analysis of the falls. Visible absorption spectra of these solutions were measured
using UV-2600 spectrophotometer at 0, 10 and
30 min after dissolution.
Result and Discussion

Fig. 2. Structures of violanin (A), swertisin
2″-O-arabinoside (B) and mangiferin (C).

10×250 mm, GL Sciences Inc., Tokyo), at a
ﬂow-rate of 1.0 ml min−1. 5% HCOOH in H2O(solvent A) and 5% HCOOH in 90% MeCN (solvent B) were used as the mobile phase under gradient elution conditions. The gradient solution
program is as follows: a linear gradient from
10% to 50% solvent B for 35 min, 50% solvent B
for 10 min.

The values of RHS color chart and colorimetric values were shown in Table 1. Higher L*
value and lower C* value of the standards wererevealed, meaning that the standards are more
lightness than the falls. RHS color chart and thevalue of hue angle, i.e. 297.0° for the falls and292.4° for the standards, revealed that the ﬂower
color is violet blue.
Visible absorption spectra of the intact falls
and standards of ʻVictoria Fallsʼ were shown bysolid and dashed lines in Fig. 3. The λmax ofeach part was shown at 578.9 and 580.2 nm. Twoshoulders at 544.4 and 636.0 nm, and at 544.4
and 635.4 nm were also observed, respectively.
These absorption spectral curves were revealed
as essentially the same to each other.
TA, TF and TX contents in the falls and standards of ʻVictoria Fallsʼ were shown in Table 2.

172

Takayuki Mizuno and Tsukasa Iwashina

Table 2. Quantitative analysis of anthocyanin and C-glycosylﬂavone and C-glycosylxanthone in the ﬂower of thetall bearded iris cultivar 'Victoria Falls'
Flower parts
Falls
Standards

Amount of three phenolic compounds (nmol mg−1)
TA*

TF**

TX***

TF/TA
ratio

TX/TA
ratio

1.50
1.18

14.09
15.06

4.71
4.83

9.4
12.8

3.1
4.1

*TA＝Total anthocyanin contents.
**TF＝Total C-glycosylﬂavone contents.
***TX＝Total C-glycosylxanthone contents.

Fig. 3. Visible absorption spectra of the intact falls(solid line) and standards (dashed line).

The ratios of TA contents per TF contents and TA
contents per TX contents were calculated as TF/
TA values and TX/TA values (Table 2). The
value of TF/TA was used for index of bluing
effect in some ﬂower color (Wang et al., 2001;
Uddin et al., 2002). TA contents of the falls werehigher than those of the standards. TF and TX
contents were generally similar to each other. TF/
TA and TX/TA values were higher in the standards than those of the falls.
For in vitro examination, the pH value of buffer solution was decided as 5.2 from the result ofpressed juice, which is weakly acidic and thesame value with that of Dutch iris (Mizuno et al.,2013). The results of in vitro examination using
puriﬁed violanin in addition of swertisin
2″-O-arabinoside or mangiferin at 10 min after
dissolution were shown in Figs. 4A and 4B, andTable 3. Absorption maxima of buffer solution

Fig. 4. Effect of swertisin 2″-O-arabinoside (A)and mangiferin (B) on the visible absorption
spectra of violanin. Concentration of swertisin
2″-O-arabinoside: 0 (solid line), 0.3 (bluedashed line) and 0.9 (blue bold line) mM, andmangiferin are 0 (solid line) and 0.3 mM (bluedashed line).

containing 0.1 mM violanin were revealed as
λmax 539.5 nm. On the other hand, those of buffer solution of mixture of 0.1 mM violanin andCopigmentation of Tall Bearded Iris

173

Table 3. Intermolecular copigmentation and color stability of violanin and swertisin 2″-O-arabinoside or mangiferin under buffer solution*
Anthocyanin(0.1 mM)
Violanin

Copigment compounds
(mM)

λmax
(nm)

Δλmax
(nm)

Degradation rate of
the optical density (%)**

Swertisin 2″-O-arabinoside

0
0.3
0.9

539.5
573.8
577.3

—
34.3
37.8

95.7
80.4
63.2

Mangiferin

0.3

544.7

5.2

84.0

*Absorption spectra were measured at 10 min after dissolution.
**The values were determined by the degradation rate of optical density of 30 min after dissolution when the
optical density of 0 min is 100%.

0.3 or 0.9 mM swertisin 2″-O-arabinoside wereshown as λmax 573.8 and 577.3 nm, respectively.
They were occurred bathochromic shift (Δλmax) at 34.3 and 37.8 nm, respectively. Whenthe optical density of 0 min after dissolution is100%, degradation rate of optical density of 0,0.3 and 0.9 mM were shown to be 95.7%, 80.4%
and 63.2%, respectively (Table 3).
The absorption maxima of the mixture solution of 0.1 mM violanin and 0.3 mM mangiferin
were revealed as λmax 544.7 nm. Degradation
rate of optical density of 30 min after dissolution
was shown to be 84.0% (Table 3). It was
revealed that Δ λmax value of the mixture solution of 0.1 mM violanin and 0.3 mM swertisin
2″-O-arabinoside was larger than that of the mixture solution of 0.1 mM violanin and 0.3 mM
mangiferin. Furthermore, the value of degradation rate of optical density was higher in 0.1 mM
violanin and 0.3 mM mangiferin solution thanthat of 0.1 mM violanin and 0.3 mM swertisin
2″-O-arabinoside.
To reconstruct the color expression of the falls
under in vitro condition, we mixed 0.1 mM violanin, 0.9 mM swertisin 2″-O-arabinoside and
0.3 mM mangiferin under buffer solution (Fig.
4). Absorption maxima were observed at λmax
577.9 nm with shoulders at 539.8 and 629.4 nm.
These absorption curves were essentially thesame with those of the intact falls (Fig. 2). Furthermore, degradation rate of optical density of
30 min after dissolution was revealed as to be
59.3%.
In this study, we conﬁrmed the effect of C-gly-

Fig. 5. Flower color reconstruction under in vitro
condition from the falls of tall bearded iris ʻVictoria Fallsʼ. 0.1 mM violanin, 0.9 mM swertisin
2″-O-arabinoside and 0.3 mM mangiferin weredissolved in buffer solution (pH 5.2).

cosylﬂavons on the ﬂower color and color stability. It was shown that C-glycosylﬂavones arepresent as intermolecular copigementation substance in the tall bearded iris cultivar, as well as
Dutch iris and Japanese garden iris (Asen et al.,1970; Yabuya et al., 1997, 2000; Mizuno et al.,2013). Furthermore, we examined the inﬂuenceof mangiferin on the ﬂower color. Thus, it was
shown that mangiferin is slightly shifted theabsorption maxima of violanin.
Mangiferin is widely distributed in plants(Richardson, 1983). The compound is known as
one of antioxidant substance (Dar et al., 2005).
In the genus Iris section Iris species, includingtall bearded iris, mangiferin and other xanthones

174

Takayuki Mizuno and Tsukasa Iwashina

are present in the leaves and ﬂowers (Bate-Smithand Harborne, 1963; Ashtakala and Forward,
1971; Williams et al., 1997). This study suggested that mangiferin slightly contribute to theﬂower color expression and stability in some Iris
species along with C-glycosylﬂavones.
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