The blue anthocyanin pigments from the blue flowers of Heliophila coronopifolia L. (Brassicaceae)

Article abstract of DOI:10.1016/j.phytochem.2011.07.020

Six acylated delphinidin glycosides (pigments 1-6) and one acylated kaempferol glycoside (pigment 9) were isolated from the blue flowers of cape stock (Heliophila coronopifolia) in Brassicaceae along with two known acylated cyanidin glycosides (pigments 7 and 8). Pigments 1-8, based on 3-sambubioside-5-glucosides of delphinidin and cyanidin, were acylated with hydroxycinnamic acids at 3-glycosyl residues of anthocyanidins. Using spectroscopic and chemical methods, the structures of pigments 1, 2, 5, and 6 were determined to be: delphinidin 3-O-[2-O-(β-xylopyranosyl)-6-O-(acyl)- β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside], in which acyl moieties were, respectively, cis-p-coumaric acid for pigment 1, trans-caffeic acid for pigment 2, trans-p-coumaric acid for pigment 5 (a main pigment) and trans-ferulic acid for pigment 6, respectively. Moreover, the structure of pigments 3 and 4 were elucidated, respectively, as a demalonyl pigment 5 and a demalonyl pigment 6. Two known anthocyanins (pigments 7 and 8) were identified to be cyanidin 3-(6-p-coumaroyl-sambubioside)-5-(6-malonyl- glucoside) for pigment 7 and cyanidin 3-(6-feruloyl-sambubioside)-5-(6-malonyl- glucoside) for pigment 8 as minor anthocyanin pigments. A flavonol pigment (pigment 9) was isolated from its flowers and determined to be kaempferol 3-O-[6-O-(trans-feruloyl)-β-glucopyranoside]-7-O-cellobioside-4′-O- glucopyranoside as the main flavonol pigment. On the visible absorption spectral curve of the fresh blue petals of this plant and its petal pressed juice in the pH 5.0 buffer solution, three characteristic absorption maxima were observed at 546, 583 and 635 nm. However, the absorption curve of pigment 5 (a main anthocyanin in its flower) exhibited only one maximum at 569 nm in the pH 5.0 buffer solution, and violet color. The color of pigment 5 was observed to be very unstable in the pH 5.0 solution and soon decayed. In the pH 5.0 solution, the violet color of pigment 5 was restored as pure blue color by addition of pigment 9 (a main flavonol in this flower) like its fresh flower, and its blue solution exhibited the same three maxima at 546, 583 and 635 nm. On the other hand, the violet color of pigment 5 in the pH 5.0 buffer solution was not restored as pure blue color by addition of deacyl pigment 9 or rutin (a typical flower copigment). It is particularly interesting that, a blue anthocyanin-flavonol complex was extracted from the blue flowers of this plant with H₂O or 5% HOAc solution as a dark blue powder. This complex exhibited the same absorption maxima at 546, 583 and 635 nm in the pH 5.0 buffer solution. Analysis of FAB mass measurement established that this blue anthocyanin-flavonol complex was composed of one molecule each of pigment 5 and pigment 9, exhibiting a molecular ion [M+1] ⁺ at 2102 m/z (C ₉₃H₁₀₅O₅₅ calc. 2101.542). However, this blue complex is extremely unstable in acid solution. It really dissociates into pigment 5 and pigment 9.

Full text of DOI:10.1016/j.phytochem.2011.07.020

Phytochemistry 72 (2011) 2219–2229
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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
The blue anthocyanin pigments from the blue flowers of Heliophila coronopifolia L.
(Brassicaceae)
c
d
a
a
Norio Saito a, Fumi Tatsuzawa b, , Kenjiro Toki , Koichi Shinoda , Atsushi Shigihara , Toshio Honda
⇑
a Faculty of Pharmaceutical Sciences, Hoshi University, Shinagawa, Tokyo 142-8501, Japan
b Laboratory of Olericultural and Floricultural Science, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan
c Laboratory of Floriculture, Faculty of Horticulture, Minami-Kyushu University, Takanabe, Miyazaki 884-0003, Japan
d National Agricultural Research Center for Hokkaido Region, Sapporo, Hokkaido 062-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Six acylated delphinidin glycosides (pigments 1–6) and one acylated kaempferol glycoside (pigment 9)
were isolated from the blue flowers of cape stock (Heliophila coronopifolia) in Brassicaceae along with
two known acylated cyanidin glycosides (pigments 7 and 8). Pigments 1–8, based on 3-sambubioside-
5-glucosides of delphinidin and cyanidin, were acylated with hydroxycinnamic acids at 3-glycosyl resi-
dues of anthocyanidins. Using spectroscopic and chemical methods, the structures of pigments 1, 2, 5,
and 6 were determined to be: delphinidin 3-O-[2-O-(b-xylopyranosyl)-6-O-(acyl)-b-glucopyranoside]-
5-O-[6-O-(malonyl)-b-glucopyranoside], in which acyl moieties were, respectively, cis-p-coumaric acid
for pigment 1, trans-caffeic acid for pigment 2, trans-p-coumaric acid for pigment 5 (a main pigment)
and trans-ferulic acid for pigment 6, respectively. Moreover, the structure of pigments 3 and 4 were elu-
cidated, respectively, as a demalonyl pigment 5 and a demalonyl pigment 6. Two known anthocyanins
(pigments 7 and 8) were identified to be cyanidin 3-(6-p-coumaroyl-sambubioside)-5-(6-malonyl-gluco-
side) for pigment 7 and cyanidin 3-(6-feruloyl-sambubioside)-5-(6-malonyl-glucoside) for pigment 8 as
minor anthocyanin pigments. A flavonol pigment (pigment 9) was isolated from its flowers and deter-
mined to be kaempferol 3-O-[6-O-(trans-feruloyl)-b-glucopyranoside]-7-O-cellobioside-40-O-glucopyran-
oside as the main flavonol pigment.
Received 12 January 2011
Received in revised form 1 May 2011
Available online 6 September 2011
Keywords:
Heliophila coronopifolia L.
Brassicaceae
Blue flower color
Acylated delphinidin and cyanidin 3-
sambubioside-5-glucosides
Acylated kaempferol 3-[6-(feruloyl)-
glucoside]-7-cellobioside-40-glucoside
Copigmentation
Anthocyanin–flavonol complex
On the visible absorption spectral curve of the fresh blue petals of this plant and its petal pressed juice
in the pH 5.0 buffer solution, three characteristic absorption maxima were observed at 546, 583 and
635 nm. However, the absorption curve of pigment 5 (a main anthocyanin in its flower) exhibited only
one maximum at 569 nm in the pH 5.0 buffer solution, and violet color. The color of pigment 5 was
observed to be very unstable in the pH 5.0 solution and soon decayed. In the pH 5.0 solution, the violet
color of pigment 5 was restored as pure blue color by addition of pigment 9 (a main flavonol in this
flower) like its fresh flower, and its blue solution exhibited the same three maxima at 546, 583 and
635 nm. On the other hand, the violet color of pigment 5 in the pH 5.0 buffer solution was not restored
as pure blue color by addition of deacyl pigment 9 or rutin (a typical flower copigment). It is particularly
interesting that, a blue anthocyanin–flavonol complex was extracted from the blue flowers of this plant
with H2O or 5% HOAc solution as a dark blue powder. This complex exhibited the same absorption max-
ima at 546, 583 and 635 nm in the pH 5.0 buffer solution. Analysis of FAB mass measurement established
that this blue anthocyanin–flavonol complex was composed of one molecule each of pigment 5 and pig-
+
ment 9, exhibiting a molecular ion [M+1] at 2102 m/z (C93H105O55 calc. 2101.542). However, this blue
complex is extremely unstable in acid solution. It really dissociates into pigment 5 and pigment 9.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
their flower colors includes white, yellow, orange, red, violet and
violet-blue, but a clear blue color is lacking except for that of Helio-
The Brassicaceae is a large family of natural plant species and
includes many important ornamental plants such as stock, wall
flower, honesty, sweet alyssum and so on. Moreover, the range of
phila (cape stock in English). Heliophila coronopifolia, grown as an
ornamental plant with attractive blue flowers, is endemic to cape
areas.
In continuing the work on flower color variations caused by acyl-
ated anthocyanins in the Brassicaceae, the distribution of structur-
ally complicated pelargonidin and cyanidin glycosides in the
⇑
Corresponding author. Tel./fax: +81 19 621 6145.
E-mail address: fumi@iwate-u.ac.jp (F. Tatsuzawa).
0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.07.020

2220
N. Saito et al. / Phytochemistry 72 (2011) 2219–2229
flowers of Matthiola incana (Saito et al., 1995, 1996), Orychophrag-
onus violaceus (Honda et al., 2005), Cheiranthus cheiri, Lobularia mari-
tima and Lunaria annua (Tatsuzawa et al., 2006, 2007, 2010),
Malcolmia maritima (Tatsuzawa et al., 2008a), and Iberis umbellata
(Saito et al., 2008) were previously reported. To the best of our
knowledge, no report in the literature has described an anthocyanin
study of the blue flowers of H. coronopifolia. Therefore, a study was
carried out to elucidate the structures of anthocyanin pigments of
the blue flowers of this plant. The anthocyanins in this flower were
anticipated to be heavily acylated with molecules of hydroxycin-
namic acids and malonic acid. In this paper, the structural elucida-
tion of six new acylated delphinidin 3-sambubioside-5-glucoside
and one acylated kaempferol glycoside isolated from the blue flow-
ers of H. coronopifolia are reported, as well as a blue anthocyanin–fla-
vonol complex comprising one molecule each of delphinidin 3-p-
coumaroylsambubioside-5-malonylglucoside and kaempferol 3-
feruloylglucoside-7-cellobioside-40-glucoside.
acid–water; 4:1:5, v/v/v) extract or 5% HOAc (acetic acid–water;
5:95, v/v) extract from the blue flowers of H. coronopifolia as major
peaks by the HPLC analysis with monitoring at 530 nm for antho-
cyanins and at 350 nm for flavonols (Figs. 1 and 2). The proportions
of anthocyanin peaks were 6.1% (pigment 1), 6.4% (pigment 2),
0.5% (pigment 3), 1.1% (pigment 4), 43.1% (pigment 5), 29.2% (pig-
ment 6), 5.3% (pigment 7) and 3.3% (pigment 8), based on a per-
centage of the total absorbance of anthocyanin peaks. From the
extract with 5% HOAc, these pigments were isolated and purified
using the process described in a previous report (see Tatsuzawa
et al., 2007, 2008a and Section 4.3.). The chromatographic and
spectroscopic properties of these anthocyanins are summarized
in Table 1, and also those of the flavonol glycoside were described
in Section 4.4.2.
Acid hydrolysis of six anthocyanins (pigments 1–6) resulted in
delphinidin, glucose, xylose, and hydroxycinnamic acids in which
p-coumaric acid was detected, respectively, in the hydrolysates
of pigments 1, 3 and 5, caffeic acid in that of pigment 2, and ferulic
acid in those of pigments 4 and 6. In addition, malonic acid was de-
tected in the hydrolysates of pigments 1, 2, 5 and 6. Similarly, acid
hydrolysis of pigments 7 and 8 resulted in cyanidin, glucose, xy-
lose, malonic acid and hydroxycinnamic acids (p-coumaric acid
for pigment 7 and ferulic acid for pigment 8).
2. Results and discussion
Eight anthocyanin peaks (pigments 1–8) and one flavonol peak
(pigment 9) were observed in the MAW (methanol–acetic
5
6
2
1
7
8
4
3
20
30
40
0
10
Retention time (min)
Fig. 1. HPLC profile for anthocyanins (530 nm) in the blue flower extract of Heliophila coronopifolia L. Pigments 1–8 are same as in Table 1.
9
20
30
40
0
10
Retention time (min)
Fig. 2. HPLC profile (350 nm) for flavones and flavonols in the blue flower extract of H. coronopifolia L.

N. Saito et al. / Phytochemistry 72 (2011) 2219–2229
2221
Alkaline hydrolysis of pigments 1–6 resulted in the same
deacylated delphinidin glycoside and hydroxycinnamic acids. Sim-
ilar hydrolysis of pigments 7 and 8 gave the same deacylated
cyanidin glycoside with p-coumaric acid in the hydrolysate of
pigment 7 and ferulic acid in that of pigment 8. The deacylated
cyanidin glycoside was identified to be cyanidin 3-sambubioside-
5-glucoside by the analysis of HPLC in comparison with the
authentic specimen obtained from anthocyanins of L. annua
(Tatsuzawa et al., 2006). The structures of pigments 7 and 8 were
identified to be cyanidin 3-[2-(xylosyl)-6-(p-coumaroyl)-gluco-
side]-5-[6-(malonyl)-glucoside] for pigment 7, and to be cyanidin
3-[2-(xylosyl)-6-(feruloyl)-glucoside]-5-[6-(malonyl)-glucoside] for
pigment 8 by analyses using chromatographic and spectroscopic
methods in comparison with both authentic pigments isolated
from L. annua (Tatsuzawa et al., 2006) (Table 1).
Structure elucidations of these new flavonoid pigments were
performed based on analyses of their 1H NMR spectra (500 MHz)
and 13C NMR (125.78 MHz) in CD3OD-DCl (9:1) for anthocyanins
and DMSO-d6 for flavonol, including 2D COSY, NOESY, 1H–13
C
HMQC and 1H–13C HMBC spectra, as described herein.
2.1. Structural determination of new anthocyanins
2.1.1. Deacylanthocyanin of pigments 1–6
The FAB mass spectrum of the deacylanthocyanin of pigments
1–6 gave a molecular ions [M]+ at 759 m/z (calc. C32H39O21
,
759.198) assuming that the deacylanthocyanin is composed of del-
phinidin with two molecules of glucose and one molecule of
xylose. The 1H NMR spectrum of this deacylanthocyanin exhibited
five aromatic proton signals assigned for those of a delphinidin
moiety (Saito et al., 2007) (Table 2). The anomeric protons of the
sugar moieties were assigned at d5.57 (d, J = 7.3 Hz, H-1 of Glc
A), 5.19 (d, J = 7.9 Hz, H-1 of Glc B) and 4.70 (d, J = 7.6 Hz, H-1 of
xylose). Those sugars were assumed to adopt b-pyranose forms
based on their observed J-values (Table 2). By application of its
NOESY experiment, the long range NOEs between H-1 of Glc A
and H-4 (d9.00) of delphinidin, H-1 of Glc B and H-6 (d7.05) of del-
phinidin, and H-2 (d4.03) of Glc A and H-1 of xylose were observed
(Fig. 3), indicating that the OH-3 and OH-5 of delphinidin are gly-
cosylated with Glc A and Glc B, respectively, and also OH-2 of Glc A
is bonded with xylose at the anomeric position forming sambubi-
ose. Therefore, this deacylanthocyanin was determined to be
delphinidin 3-O-[2-O-(b-xylopyranosyl)-b-glucopyranoside]-5-O-
b-glucopyranoside.
2.1.2. Pigments 2, 5 and 6
The molecular ions [M]+ of pigments 2, 5 and 6 as three major
pigments in this plant were observed at 1007 m/z (C44H47O27 calc.
1007.230) for pigment 2, at 991 m/z (C44H47O26 calc. 991.236) for
pigment 5, and at 1021 m/z (C45H49O27 calc. 1021.246) for pigment
6, indicating that these three pigments are composed of delphini-
din with two molecules of glucose and one molecule each of xy-
lose, malonic acid and hydroxycinnamic acids (caffeic acid for
pigment 2, p-coumaric acid for pigment 5 and ferulic acid for pig-
ment 6). The elemental components of these pigments were con-
firmed by measuring their high resolution FAB MS (HRMS)
(Table 1).
These pigment structures were further elucidated based on the
analysis of their NMR spectra. The 1H NMR spectra of these pig-
ments were mutually superimposed, except for the signals of
hydroxycinnamic acid moieties, as described above. As shown in
Table 2, the proton chemical shifts of the delphinidin moiety with
coupling constants were assigned in these three pigments. Their
values showed good mutual agreement. Signals of the sugar moie-
ties were observed in the region of d5.61–3.01, where three char-
acteristic anomeric protons were observed at d5.56–5.61 for Glc

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N. Saito et al. / Phytochemistry 72 (2011) 2219–2229
Table 2
1H NMR spectroscopic data (d) of acylated anthocyanins isolated from Heliophila coronopifolia (500 MHz, CD3OD-DCl, TMS as an internal standard).
Deacyl pigments Pigment 1
1–6
Pigment 2
Pigment 3 (demalonyl
Pigment 4 (demalonyl
Pigment 5
Pigment 6
pigment 5)
pigment 6)
1H
1H
1H
1H
1H
1H
1H
Delphinidin
4
8.98 s
8.50 s
8.80 s
8.83 s
8.78 s
8.74 s
8.80 s
6
7.04 d(1.8)
7.09 d(1.8)
7.81 s
6.84 d(1.8)
6.74 d(1.8)
7.74 s
6.95 d(1.8)
6.90 d(1.8)
7.79 s
6.97 d(1.8)
6.87 d(1.8)
7.78 s
6.82 b(1.9)
6.88 b(1.9)
7.72 s
6.91 d(1.8)
6.86 d(1.8)
7.77 s
6.92 d(1.8)
6.88 d(1.8)
7.78 s
8
20
60
7.81 s
Hydroxycinnamic acid I
2
3
5
6
7.11 d(8.9)
6.29 d(8.9)
6.29 d(8.9)
7.11 d(8.9)
6.79 d(1.8)
7.26 d(8.9)
6.74 d(8.9)
6.74 d(8.9)
7.26 d(8.9)
6.82 s
7.16 d(8.9)
6.68 d(8.9)
6.68 d(8.9)
7.16 d(8.9)
6.83 brs
6.68 d(8.6)
6.72 dd(1.8,
8.6)
6.68 d(8.6)
6.82 m
6.71 d(8.3)
6.82 dd(1.9, 8.3)
a
b
5.72 d(12.9)
6.38 d(12.9)
6.18 d(15.9)
7.28 d(15.9)
6.21 d(15.9)
7.35 d(15.9)
6.13 d(15.9)
7.26 d(15.9)
3.73 s
6.20 d(15.9)
7.31 d(15.9)
6.21 d(15.9)
7.34 d(15.9)
3.77 s
–OCH3
Malonic acid
–CH2–
3.25–3.35
3.24–3.36
3.30 s
3.30 s
Glucose A
1
2
5.55 d(7.6)
4.02 dd(7.6, 9.2)
5.53 d(7.7)
4.08 dd(7.7,
8.6)
3.81 m
3.50 m
5.56 d(7.4)
4.06 t⁄(8.6)
5.55 d(7.3)
4.07 dd(7.3, 8.8)
5.54 d(7.3)
3.99 dd(7.3, 8.4)
5.61 d(7.3)
4.09 dd(7.3, 8.2)
5.57 d(7.4)
4.08 dd(7.4, 8.9)
3
4
5
3.79 t⁄(9.2)
3.50 t⁄(9.2)
3.67 m
3.81 m
3.54 m
3.96 m
3.80 t⁄(9.2)
3.54 m
3.92 m
3.75 t⁄(8.9)
3.50 t⁄(9.5)
3.87 m
3.85 t⁄(8.9)
3.83 t⁄(8.9)
3.55 t⁄(8.9)
3.99 ddd(2.5, 8.4,
9.0)
3.55 t⁄(9.2)
3.96 m
3.99 ddd(2.5, 7.9,
11.9)
6a
3.74 m
4.32
brd(11.9)
4.72 m
4.42 dd(8.0,
11.9)
4.53 d(11.9)
4.38 m
4.34 dd(7.3, 11.9)
4.44 dd(3.1, 11.9)
4.43 dd(7.9, 11.9)
4.37 dd(8.3, 12.1)
6b
3.93 d(11.9)
4.49 dd(3.4, 12.5)
4.52 dd(2.8, 11.9)
4.58 dd(2.5, 12.1)
Glucose B
1
2
3
4
5
6a
5.16 d(7.6)
3.67 dd(7.6, 9.2)
3.58 m
5.21 d(7.6)
3.73 m
3.59 m
3.44 m
3.77 m
4.15 m
5.16 d(7.7)
3.75 m
3.57 m
3.45 m
3.79 m
5.17 d(7.7)
3.73 m
3.58 m
4.42 m
3.71 m
3.88 m
5.06 d(7.6)
3.64 dd(7.6, 9.2)
3.47 t⁄(9.2)
3.33 t⁄(9.5)
3.48 m
5.17 d(7.9)
5.15 d(7.9)
3.76 dd(7.9, 9.2)
3.57 t⁄(8.0)
3.41 m
3.80 m
4.10 dd(6.4, 11.3)
3.77 dd(7.9, 9.2)
3.59 t⁄(9.2)
3.46 t⁄(9.5)
3.55 m
3.46 t⁄(9.5)
3.82 m
4.23 dd(5.8, 12.1)
3.74 m
4.25 dd(6.1,
12.2)
3.70–3.77
6b
3.93 d(11.9)
4.59
4.56 d(12.2)
3.96 dd(2.5, 12.2)
3.85 d(11.9)
4.51 d(12.1)
4.52 dd(1.9, 11.3)
brd(12.5)
Xylose
1
2
4.71 d(7.7)
3.13 dd(7.7, 9.2)
4.67 d(8.0)
3.16 m
4.70 d(7.7)
3.20 dd(7.7,
8.9)
3.24–3.34
3.35 m
4.69 d(7.6)
3.16 dd(7.6, 8.9)
4.62 d(7.6)
3.11 dd(7.6, 8.9)
4.73 d(7.3)
3.25 dd(7.9, 9.2)
4.71 d(7.4)
3.22 dd(7.4, 8.9)
3
4
5a
3.27 t⁄(9.2)
3.34 m
2.95 t(11.6)
3.27 m
3.20 dd(8.5, 8.9)
3.33 m
3.10 dd(10.1, 11.6)
3.20 m
3.33 t⁄(8.8)
3.39 t⁄(8.5)
3.05 t⁄(11.5)
3.33 m
3.25–3.35
3.23 m
3.37 t⁄(8.7)
3.03 t⁄(11.6)
2.95 t⁄(11.3)
3.01 dd(10.4,
11.3)
2.93 t⁄(11.3)
5b
3.58 m
3.54 m
3.62 dd(5.2,
11.3)
3.63 dd(5.2, 11.6)
3.55 dd(5.2, 11.6)
3.68 dd(5.2, 11.5)
3.65 dd(5.2, 11.6)
Coupling constants (J in Hz) in parentheses.
s = singlet, brs = broad singlet, t⁄ = distorted triplet, d = doublet, dd = double doublet, ddd = double double doublet, m = multiplet.
A, at d5.15–5.17 for Glc B and at d4.70–4.73 for xylose for these
three pigments. Based on the observed coupling constants (Ta-
ble 2), the nine sugars were assigned to have b-pyranose forms.
Chemical shifts of hydroxycinnamic acids were also assigned as
shown in Table 2, and identified to be caffeic acid for pigment 2,
p-coumaric acid for pigment 5 and ferulic acid for pigment 6. The
signals of six olefinic protons of three hydroxycinnamic acids
exhibited large coupling constants (J = 15.9 and 15.9 Hz for caffeic
acid, J = 15.9 and 15.9 Hz for p-coumaric acid, and J = 15.9 and
15.9 Hz for ferulic acid) indicating to be trans configurations (Ta-
ble 2). The linkages and/or positions of the attachments of the su-
gar and acyl groups in these pigments were determined by analysis
of their COSY and NOESY spectra (Fig. 3). Long range NOEs were
observed in the spectrum of each pigment 2, 5 and 6 between H-
1 of Glc A and H-4 of delphinidin, H-1 of Glc B and H-6 of delphin-
idin, and H-1 of xylose and H-2 of Glc A, supporting that OH-3 and -
5 of delphinidin were glycosylated with Glc A and Glc B, respec-
tively. Furthermore, xylose was bonded with Glc A at OH-2 of Glc
A forming sambubiose in these pigments (Fig. 3). Based on results
of analyses of their COSY spectra, four characteristic methylene
proton signals (H-6a and b) being shifted to lower magnetic fields
were assigned to those of Glc A (d4.42 and 4.53 for pigment 2,
d4.43 and 4.52 for pigment 5, and d4.37 and 4.58 for pigment 6)
and Glc B (d4.25 and 4.56 for pigment 2, d4.23 and 4.51 for pigment
5, and d4.10 and 4.52 for pigment 6) in these pigments 2, 5 and 6.
These results indicated that both OH-6 groups of Glc A and B were
acylated with malonic acid and hydroxycinnamic acid in these pig-
ments. However, we were unable to determine the acid moiety of

N. Saito et al. / Phytochemistry 72 (2011) 2219–2229
2223
Fig. 3. Acylated anthocyanins from H. coronopifolia L. Observed main NOEs are indicated by arrows.
OH-6 of Glc A and Glc B, where malonic acid and hydroxycinnamic
acid were bonded alternatively, based on analyses of their NOESY
spectra and also their HMBC spectra. To determine the position
of both acids, demalonyl pigments 5 and 6 were prepared using
the procedures described in a previous report (Saito et al., 2007).
The structures of demalonyl pigments 5 and 6 were determined
(see in the following Section 2.1.3.). The obtained results estab-
lished that demalonyl pigments 5 and 6 were free from malonic
acid at OH-6 of Glc B, and acylated with hydroxycinnamic acid at
OH-6 of Glc A. Therefore, the structures of pigments 5 and 6 were
determined unambiguously to be delphinidin 3-O-[2-O-(b-xylo-
pyranosyl)-6-O-(trans-p-coumaroyl)-b-glucopyranoside]-5-O-[6-
O-(malonyl)-b-glucopyranoside] for pigment 5, and to be
The FAB mass spectra of demalonyl pigment 5 and pigment 3
exhibited a molecular ion [M]+ at 905 m/z in agreement with the
mass C41H45O23 (calc. 905.235). The elemental components were
confirmed by measuring their high-resolution FAB MS (Table 1).
In the 1H NMR spectrum of the demalonyl pigment 5, the chem-
ical shifts were almost identical to those of pigment 5, except for
the proton signals of Glc B and malonic acid moieties (Table 2).
Particularly, the up-field shifts of methylene protons (d3.88 and
3.96, H-6a and b of Glc B) were observed in this spectrum in com-
parison with those of pigment 5 (d4.23 and 4.51, H-6a and b of
Glc B), supporting that the OH-6 of Glc B in demalonyl pigment
5 is free from malonic acid (Fig. 3). Therefore, the structures of
demalonyl pigment 5 and pigment 3 were determined to be
delphinidin 3-O-[2-O-(b-xylopyranosyl)-6-O-(trans-p-coumaroyl)-
b-glucopyranoside]-5-O-b-glucopyranoside, which is a new anth-
ocyanin in plants.
The FAB mass spectra of demalonyl pigment 6 and pigment 4
exhibited a molecular ion [M]+ at 934 m/z, in agreement with the
mass C42H47O24 (calc. 935.246). The elemental components were
confirmed by measuring their high-resolution FAB MS (Table 1).
The 1H NMR spectrum of the demalonyl pigment 6 was superim-
posed on that of pigment 6, except for the proton signals of Glc B
and malonic acid moieties (Table 2). By analysis of its COSY spec-
trum, the chemical shifts of methylene protons of Glc B were
shifted to a higher field at d3.70–3.77 (H-6a) and 3.85 (H-6b) than
those (d4.10, H-6a and d4.52, H-6b) of pigment 6, indicating that
the OH-6 of Glc B is free from malonic acid in demalonyl pigment
6. Furthermore, the signals of methylene protons of malonic acid
disappeared in the 1H NMR spectrum of the demalonyl pigment
6. Therefore, the structures of demalonyl pigment 6 and pigment
4 were determined to be delphinidin 3-O-[2-O-(b-xylopyranosyl)-
6-O-(trans-feruloyl)-b-glucopyranoside]-5-O-b-glucopyranoside,
which is a new anthocyanin in plants.
delphinidin
3-O-[2-O-(b-xylopyranosyl)-6-O-(trans-feruloyl)-b-
glucopyranoside]-5-O-[6-O-(malonyl)-b-glucopyranoside] for pig-
ment 6 (Fig. 3). Both pigments are new anthocyanins in plants
(Harborne and Baxter, 1999; Honda and Saito, 2002; Andersen
and Jordheim, 2006; Veitch and Grayer, 2008). These structures
were confirmed based on analyses of their 13C NMR spectra
(Table 3). For pigment 2, we were unable to prepare its demalonyl
pigment 2 because of the small amounts available. Therefore, H2O2
degradation of pigment 2 was performed to prepare the acyl sugar
residue at the OH-3 of delphinidin, yielding caffeoyl-sambubiose
from degradation products by analyses of TLC and HPLC (See in
the Section 4.4.6.). Consequently, the structure of pigment 2 was
determined to be delphinidin 3-O-[2-O-(b-xylopyranosyl)-6-O-
(trans-caffeoyl)-b-glucopyranoside]-5-O-[6-O-(malonyl)-b-glucopy-
ranoside] (Fig. 3), which is a new anthocyanin in plants.
2.1.3. Demalonyl pigment 5 (pigment 3) and demalonyl pigment 6
(pigment 4)
Demalonyl pigments 5 and 6 were prepared by hydrolysis of
pigments 5 and 6 with 1 N HCl at room temperature for 2 weeks.
They were purified with preparative HPLC by the process described
in a previous report (Saito et al., 2007). The chromatographic and
spectroscopic data of the demalonyl pigments 5 and 6 are pre-
sented in Table 1. The analytical results obtained for demalonyl
pigment 5 were identical to those of pigment 3. These results of
demalonyl pigment 6 were identical to those of pigment 4.
2.1.4. Pigment 1
By analysis of FAB mass spectrum, the molecular ion [M]+ of
pigment 1 was observed at 991 m/z (C44H47O26 calc.991.236),
which is identical to that of pigment 5, indicating that pigment 1
was composed of delphinidin with two molecules of glucose, and

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N. Saito et al. / Phytochemistry 72 (2011) 2219–2229
Table 3
Table 4
13C NMR spectroscopic data of acylated anthocyanins (pigments 5 and 6) from the
flowers H. coronopifolia in CD3OD-DCl (9:1).
NMR spectroscopic data of a flavonol from H. coronopifolia.
Pigment 9
Deacyl pigment 9
Pigment 5
Pigment 6
13C d (ppm)
1H d (ppm)
13C d (ppm)
1H d (ppm)
Delphinidin
2
3
4
5
6
7
8
Kaempferol
2
3
4
5
6
7
8
9
10
10
20,60
30,50
40
164.4
145.8
133.1
156.3
105.9
169.2
97.2
156.6
112.8
119.8
113.3
147.7
145.9
147.7
113.3
164.6
145.8
133.4
156.4
106.0
169.2
97.3
156.7
113.0
120.5
113.0
147.7
146.2
147.7
113.0
156.0
134.2
177.8
160.8
99.2
156.1
134.1
177.8
160.9
99.2
6.33 s
6.78 s
6.48 d(2.1)
6.84 d(2.1)
162.7
94.6
162.8
94.5
9
156.1
105.7
123.5
130.7
115.9
159.4
156.2
105.8
123.6
130.8
115.9
159.4
10
10
20
30
40
50
60
8.16 d(8.6)
7.15 d(8.6)
8.18 d(8.9)
7.15 d(8.9)
Ferulic acid
1
2
3
4
5
6
a
b
125.4
111.1
147.9
149.3
115.5
123.2
113.9
145.1
166.3
55.7
Hydroxycinnamic acid I
1
2
3
4
5
6
a
7.21 s
126.7
131.4
116.7
161.2
116.7
131.4
147.0
114.7
119.9
112.2
149.1
150.6
116.5
124.0
147.3
115.1
56.5
6.79 d(8.9)
6.98 brd(8.0)
6.26 d(15.9)
7.37 d(15.9)
COOH
–OCH3
b
3.81 s
–OCH3
C@O
Glucose A
169.2
169.1
1
2
3
4
5
6a
6b
101.5
71.1
76.8
68.2
73.1
63.1
5.45 d(7.7)
3.54 m
3.38 m
3.67 m
3.72 m
101.5
71.2
76.8
67.9
73.1
60.2
5.43 d(7.7)
3.54 dd(7.6, 9.5)
3.42 m
3.66 m
3.40 m
Malonic acid
–CH2–
C@O
49.3
173.7
168.9
49.2
170.5
169.1
C@O
Glucose A
4.08 m
4.15 d(10.9)
3.50 m
3.72 m
1
2
3
4
5
6
100.6
82.5
77.9
72.0
75.8
64.6
99.9
82.4
77.8
72.0
75.5
64.6
Glucose B
1
2
3
4
5
6a
6b
99.2
77.1
72.8
79.7
75.1
60.0
5.08 d(7.6)
3.33 m
3.45 m
3.43 m
3.60 m
3.61 m
3.76 m
99.4
77.1
74.8
79.7
75.1
60.0
5.19 d(8.0)
3.31 m
3.48 m
3.44 m
3.65 m
3.61 m
3.76 m
Glucose B
1
2
3
4
5
6
102.6
74.8
77.7
71.0
75.9
65.1
102.8
74.7
77.7
71.0
75.9
65.1
Glucose C
1
2
3
4
5
6a
6b
100.1
73.2
76.5
69.6
74.7
60.6
5.01 d(7.1)
3.28 m
3.26 m
3.49 m
3.47 m
3.51 m
3.71 m
100.1
73.2
75.9
69.8
72.8
60.6
5.02 d(7.3)
3.27 m
3.32 m
3.50 m
3.35 m
3.34 m
3.68 m
Xylose
1
2
3
4
5
106.3
75.4
77.7
70.9
67.1
106.2
75.8
77.7
70.9
67.1
Glucose D
1
2
3
4
5
6a
6b
103.1
73.3
69.6
70.0
76.5
61.1
4.30 d(7.6)
3.04 m
3.18 m
3.07 m
3.20 m
3.39 m
3.68 m
103.1
73.3
69.6
70.0
76.5
61.0
4.30 d(8.0)
3.02 t⁄(8.6)
3.17 t⁄(9.2)
3.07 t⁄(9.2)
3.21 m
one molecule each of xylose, malonic acid and p-coumaric acid.
The elemental components were confirmed by measuring its
HRMS, and chemical and spectroscopic data of pigment 1 are pre-
sented in Table 1. The results of chemical and spectroscopic analy-
3.29 m
3.44 m
1H: 500 MHz DMSO-d6, TMS as an internal standard.
13C: 125.78 MHz DMSO-d6, TMS as an internal standard.
Coupling constants (J in Hz) in parentheses.
ses of pigment
1 were identical to those of an authentic
cis-derivative of pigment 5, which was prepared from pigment 5
using the process described previously (Tatsuzawa et al., 2008b
and see in Section 4.4.5. and Table 1).
s = singlet, brs = broad singlet, d = doublet, brd = broad doublet,
dd = double doublet, t⁄ = distorted triplet, m = multiplet.
The 1H NMR spectrum of pigment 1 was the same as that of pig-
ment 5 except for signals of p-coumaric acid moiety (Table 2). Par-
ticularly, the chemical shifts of the olefinic protons of p-coumaric
uration of p-coumaric acid in pigment 1 to be a cis-form. Conse-
quently, pigment 1 was determined to be delphinidin 3-O-[2-O-
(b-xylopyranosyl)-6-O-(cis-p-coumaroyl)-b-glucopyranoside]-5-O-
[6-O-(malonyl)-b-glucopyranoside] (Fig. 3), which is a new antho-
cyanin in plants.
acid were shifted to
J = 12.9 Hz, H- ) and d6.38 (d, J = 12.9 Hz, H-b) with smaller cou-
pling constants than those (d6.20, d, J = 15.9 Hz, H- and d7.31, d,
J = 15.9 Hz, H-b) of pigment 5. These results supported the config-
a higher magnetic field at d5.72 (d,
a
a

N. Saito et al. / Phytochemistry 72 (2011) 2219–2229
2225
Fig. 4. Acylated kaempferol glycoside (pigment 9) isolated from the flowers of H. coronopifolia L. Observed main NOEs are indicated by arrows. Observed main HMBCs are
indicated by dotted arrows.
2.2. Structure of a flavonol (pigment 9)
constants, the four glucoses were of b-pyranose form. By analysis
of its NOESY spectrum, three long-range NOEs were observed be-
tween H-8 and H-6 of kaempferol and H-1 of Glc B, H-30 and -50
of kaempferol and H-1 of Glc C, and H-4 of Glc B and H-1 of Glc
D, suggesting that kaempferol was glycosylated with Glc B at 7-
OH and with Glc C at OH-40, and that Glc B was bonded with Glc
D at OH-4. The glycosylation at OH-3 of kaempferol with Glc A
was concluded by the results of spectroscopic properties as de-
scribed above and confirmed by observation of HMBC connectively
from H-1 of Glc A to C-3 of kaempferol as depicted in Fig. 4. There-
fore, the structure of deacyl pigment 9 was determined to be
As shown in Fig. 2, several flavonol glycosides were detected in
5% HOAc extract from the blue flowers of H. coronopifolia using
HPLC analysis. From these flavonol glycosides, a dominant pigment
(pigment 9) was isolated and then purified as a pale yellow pow-
der. The chromatographic and spectroscopic properties of pigment
9 are summarized in Section 4.4.2. Acid hydrolysis of pigment 9 re-
sulted in kaempferol, glucose and ferulic acid. Alkaline hydrolysis
of pigment 9 resulted in a deacyl kaempferol glycoside and ferulic
acid. The FAB mass spectrum of pigment 9 gave a molecular ion
[M+1]+ at 1111 m/z (C49H58O29, calc. 1110.306) indicating that pig-
ment 9 was composed of kaempferol with four molecules of glu-
cose and one molecule of ferulic acid. The chromatographic and
spectroscopic properties of deacyl pigment 9 are summarized in
Section 4.4.3. The structure of deacyl pigment 9 was presumed to
be kaempferol 3,7,40-tetra-glucoside by its chromatographic and
spectroscopic properties (in Section 4.4.2) and also by analysis of
the 1H NMR spectrum (Table 4). From the 1H NMR spectrum of
deacyl pigment 9, six aromatic protons of kaempferol and four
anomeric protons of glucose were assigned. From their coupling
kaempferol
3-O-(b-glucopyranoside)-7-O-[4-O-(b-glucopyrano-
syl)-b-glucopyranoside]-40-O-(b-glucopyranoside). This structure
was further confirmed by measurement of its 13C NMR spectrum
(Table 4).
The 1H NMR spectrum of pigment 9 was identical to that of
deacyl pigment 9, except for the signals of ferulic acid and Glc A
moieties. Eight chemical shifts of ferulic acid were assigned as
shown in Table 4. Two olefinic protons of this acid with large cou-
pling constants (J = 15.9 and 15.9 Hz) indicated that the geometry
of the olefin was of trans-configuration. By analysis of its COSY
Fig. 5. Visible spectra of H. coronopifolia L.

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N. Saito et al. / Phytochemistry 72 (2011) 2219–2229
Fig. 6. FABMS spectra of anthocyanin–flavonol (pigments 5 and 9) complex pigment.
spectrum, the downshifts of methylene protons (d4.08, H-6a and
d4.15, H-6b) of Glc A were observed, supporting that Glc A was
acylated with ferulic acid at the OH-6 group. Furthermore, the gly-
cosylation on OH-7 and OH-40 of kaempferol with Glc C and Glc D
was confirmed by measurement of its NOESY spectrum as
described for deacyl-pigment 9. Consequently, the structure of
pigment 9 was determined to be kaempferol 3-O-[6-O-(trans-feru-
loyl)-b-glucopyranoside]-7-O-[4-O-(b-glucopyranosyl)-b-glucopyrano-
side]-40-O-(b-glucopyranoside) (Fig. 4), which is a new kaempferol
glycoside in plants (Harborne and Baxter, 1999; Williams, 2006;
Veitch and Grayer, 2008). This structure was confirmed further
by measuring its 13C NMR spectrum (Table 4). Regarding the gly-
cosylation patterns of flavonol glycosides in the Brassicaceae,
kaempferol 3-sophoroside-7-cellobioside has been found in plants
of Brassica oleracea (Nielsen et al., 1998). However, kaempferol 3-
glucoside-7-cellobioside-40-glucoside is a new pattern.
ally observed for non-acylated anthocyanins. Particularly, the extra
band at 635 nm in the visible region is not observed in pH 5.0 solu-
tion of delphinidin 3-sambubioside-5-diglucoside.
In order to recover the original blue color for the solution of pig-
ment 5, copigments such as rutin (Asen et al., 1972) and deacyl
pigment
9
(kaempferol 3,40-diglucoside-7-cellubioside) were
added to the pH 5.0 solution of pigment 5. However, only the violet
color at an absorption maximum (near 570 nm) was produced as
the usually observed co-pigmentation between delphinidin 3,5-
diglycoside and flavone and/or flavonol (Asen et al., 1972; Chen
and Hrazdina, 1981) (Fig. 5b).
On the other hand, the stable and blue color solution of pigment
5 with a strong extra absorption maximum at 635 nm is restored,
as observed for the fresh flower juice by addition of pigment 9,
which is acylated with ferulic acid, as shown in Fig. 5a. The absorp-
tion pattern of this solution showing maxima at 546, 583 and
635 nm is very similar to those of polyacylated anthocyanins (Hon-
da and Saito, 2002). Consequently, it might be considered that both
the hydroxycinnamic acid residues of pigment 9 (ferulic acid) and
of pigment 5 (p-coumaric acid) mutually collaborate for the forma-
tion of the stable and blue anthocyanin–flavonol complex, which
exhibits three characteristic absorption maxima as those of platy-
conin and cinerarin.
At the same time, a blue anthocyanin–flavonol complex powder
was isolated from the blue flowers of this plant with water or 5%
HOAc solution (see in Sections 4.3. and 4.5.). This blue powder
was confirmed to be constituted of pigments 5 and 9 in a ratio of
1:1 by analysis of TLC and HPLC, and also exhibited the same three
absorption maxima at 546, 583 and 635 nm as those of the fresh
flowers of this plant (Fig. 5). By analysis of the FAB mass spectrum,
the anthocyanin–flavonol complex gave a molecular ion [M+1]+ at
2102.1 m/z (calc. C93H105O55, 2101.542) with fragment ion 991
[M]+ of pigment 5 and 1111 [M+1]+ of pigment 9 (Fig. 6). Therefore,
it was revealed that, in the blue flower of H. coronopifolia, delphin-
2.3. Flower color and an anthocyanin–flavonol complex pigment in
blue flowers of H. coronopifolia
As shown in Fig. 5, the blue petal of H. coronopifolia and its fresh
flower juice in the pH 5.0 buffer solution exhibited three character-
istic absorption maxima at 546, 583 and 635 nm in the visible re-
gion of 400–700 nm. These three absorption maxima are similar to
those of platyconin (533, 570 and 618 nm) and cinerarin (537, 575
and 622 nm) in the same buffer solution (Honda and Saito, 2002).
These pigments are composed from delphinidin glycosides with
two molecules of caffeic acid as platyconin and also with three
molecules of caffeic acid as cinerarin. However, anthocyanins of
H. coronopifolia delphinidin 3-sambubioside-5-malonylglucoside
were acylated, respectively, with only one molecule of hydroxycin-
namic acids. A main anthocyanin of this plant, pigment 5, exhibits
only one absorption maximum (569 nm) in the pH 5.0 solution,
and its color is unstable in the solution and soon decayed as usu-

N. Saito et al. / Phytochemistry 72 (2011) 2219–2229
2227
idin
3-[2-(xylosyl)-6-(p-coumaroyl)-glucoside]-5-[6-(malonyl)-
determined at 500 MHz for 1H spectra and at 125.78 MHz for 13C
spectra in DCl-CD3OD (1:9) for anthocyanins and DMSO-d6 for
flavonols. Chemical shifts are reported relative to a TMS internal
standard (d), and coupling constants are in Hz.
glucoside] (pigment 5) and kaempferol 3-[6-(feruloyl)-glucoside]-
7-cellobioside-40-glucoside (pigment 9) play an important role in
forming the blue copigmentation complex by interaction of the
hydroxycinnamic acid residues of both pigments.
4.2. Plant materials
3. Concluding remarks
Seeds of H. coronopifolia L. (Synonym H. longifolia DC.) were pur-
chased from Takii Seed Co., Ltd. (Kyoto, Japan), and grown in green-
houses at Iwate University (Morioka, Iwate, Japan) and
Minamikyushu University (Takanabe, Miyazaki, Japan). Flowers
were collected during winter to spring seasons. The flowers exhib-
ited a blue color [Blue 99C by Royal Horticultural Society (R.H.S.)
Color Chart, and its chromaticity value b ꢂ (ꢁ49.82)/
a ꢂ (31.64) = ꢁ3.46 and L ꢂ (47.83)] was recorded on a SE-2000
Spectro Color Meter (Nippon Denshoku Industries Co., Ltd.). The
flowers collected were dried at 40 °C overnight and stored at
ꢁ20 °C in the refrigerator until needed.
Six acylated delphinidin glycosides were isolated along with
two known acylated cyanidin glycosides from the blue flowers of
cape stock (H. coronopifolia). From the chemotaxonomical point
of view, this report is the first to describe the distribution of del-
phinidin glycosides in Brassicaceae (Harborne, 1967; Timberlake
and Bridle, 1975; Hrazdina, 1982; Harborne and Grayer, 1988;
Strack and Wray, 1994; Andersen and Jordheim, 2006). These
anthocyanins have rather simple acylation patterns and more com-
mon glycosylation patterns than those of polyacylated anthocya-
nins of M. incana (Saito et al., 1995, 1996), O. violaceus (Honda
et al., 2005), Iberis umbellata (Saito et al., 2008), and others in this
family. Among six new acylated anthocyanins, the structure of pig-
ment 5 was determined to be delphinidin 3-p-coumaroylsambu-
bioside-5-malonylglucoside as a main anthocyanin.
At the same time, a new flavonol, kaempferol 3-feruloylgluco-
side-7-cellobioside-40-glucoside (pigment 9), was isolated from
this blue flower as a dominant flavonol. This flavonol is responsible
for producing the blue flower color in this plant as a copigment.
Moreover, a blue anthocyanin–flavonol complex powder was iso-
lated from this blue flower using water. By FAB mass spectro-
graphic analysis, this blue complex exhibited a molecular ion
[M+1]+ at 2102 m/z (C93H105O55 calc. 2101.542) composed from
pigments 5 and 9 (in the ratio of 1:1). Therefore, we propose that
the blue flower color of H. coronopifolia is formed mainly by strong
intermolecular copigmentation between delphinidin 3-p-coumaro-
ylsambubioside-5-malonylglucoside and kaempferol 3-feruloylg-
lucoside-7-cellobioside-40-glucoside.
4.3. Isolation of flower pigments
Dried blue flowers (ca. 300 g) of H. coronopifolia were immersed
in 5% HOAc (30L) at room temp. for 5 h. The extract was passed
through a Diaion HP-20 (Mitsubishi Chemical’s Ion Exchange Res-
ins) column (90 ꢀ 150 mm), on which acylated pigments were ab-
sorbed. The column was thoroughly washed with 5% HOAc (15L),
and eluted with 5% HOAc–MeOH (1L) to recover the pigments.
After concentration, eluates were separated and purified with pa-
per chromatography using BAW.
4.3.1. Pigments 5 and 9
For a further purification of the major pigments 5 and 9, the
crude blue anthocyanin pigment powder (ca. 80 mg) was subjected
to HPLC on a Waters C18 (4.6 ꢀ 250 mm) at 40 °C with a flow rate
of 1 ml/min and monitoring at 330 nm. The solvent used was as
follows: a linear gradient elution for 15 min from 55% to 70% sol-
vent B in solvent A. By this procedure, major pigments 5 and 9
were completely separated from the blue anthocyanin pigment
powder as the two different fractions. Each fraction was trans-
formed to a Diaion HP-20 column, on which pigments were ad-
sorbed. Pigments 5 and 9 were eluted with 5% HOAc–MeOH
followed by addition of excess of Et2O, and then dried. Purified pig-
ment 5 (ca. 30 mg) and pigment 9 (ca. 30 mg) were obtained.
4. Experimental
4.1. General procedures
TLC was carried out on plastic coated cellulose sheets (Merck)
using ten mobile phases: BAW (n-BuOH–HOAc–H2O, 4:1:2), BuHCl
(n-BuOH–2N HCl, 1:1, upper layer), AHW (HOAc–HCl–H2O,
15:3:82), 1% HCl (HCl–H2O, 3:97), 5% HOAc (HOAc–H2O, 5:95),
15% HOAc (HOAc–H2O, 15:85) and H2O for anthocyanins and flavo-
nols, and BAW, EAA (EtOAc–HOAc–H2O, 3:1:1), ETN (EtOH–
NH4OH–H2O, 16:1:3) and EFW (EtOAc–HCOOH–H2O, 5:2:1) for
sugars and organic acid with UV light and aniline hydrogen phthal-
ate spray reagent (Harborne, 1984).
Analytical HPLC was performed on a LC 10A system (Shimadzu),
using a Waters C18 (4.6 ꢀ 250 mm) column at 40 °C with a flow
rate of 1 mL/min and monitoring at 530 nm for anthocyanins and
at 350 nm for flavonols by photodiode array detector. The eluant
was applied as a linear gradient elution for 40 min from 20% to
85% solvent B (1.5% H3PO4, 20% HOAc, 25% MeCN in H2O) in solvent
A (1.5% H3PO4 in H2O).
4.3.2. Pigments 1, 2, 3, 4, 6, 7 and 8
The residual extract solutions in which pigments 5 and 9 were
removed were collected and passed through a Diaion HP-20 col-
umn in order to obtain minor pigments (1–4, 6–8). The column
was thoroughly washed with 5% HOAc and the pigments were
eluted with 5% HOAc–MeOH from the column. After concentration,
the eluates were separated and purified by prep. HPLC. Prep. HPLC
was performed on a Waters C18 (4.6 ꢀ 250 mm) column at 40 °C
with a flow rate of 1 ml/min and monitoring at 530 nm. The sol-
vent used was as follows; a linear gradient elution for 20 min from
55% to 70% solvent B in solvent A. Each fraction was transformed to
a Diaion HP-20 column, on which pigments were absorbed. These
pigments were eluted with 5% HOAc–MeOH, followed by addition
of excess of Et2O, and then dried. The purified pigments were ob-
UV–Vis spectra were recorded on UV–Vis Multi Purpose Spec-
trophotometer (MPS-2450, Shimadzu) in 0.1% HCl–MeOH (from
200 to 700 nm). The spectral absorption of flowers was directly
measured on the intact tissue using a recording spectrophotometer
operating as a double-beam instrument (Type: MPS-2450) (Yokoi
and Saito, 1973; Saito, 1967).
tained as follows; pigment
1 (ca. 5.0 mg), pigment 2 (ca.
10.0 mg), pigment 3 (ca. 1.0 mg), pigment 4 (ca. 2.0 mg), pigment
6 (ca. 20.0 mg), pigment 7 (ca. 2.0 mg) and pigment 8 (ca. 1.5 mg).
4.4. Analyses of anthocyanins
High resolution FAB mass (HR FABMS) spectra were obtained in
the positive ion mode using the magic bullet (5:1 mixture of dithi-
othreitol and dithioerythritol) as a matrix. NMR spectra were
The identification of anthocyanins was carried out by stand-
ard procedures involving deacylation, demalonylation, partial

2228
N. Saito et al. / Phytochemistry 72 (2011) 2219–2229
acid-hydrolysis with acid, both alkaline and acid hydrolyses, and
H2O2 oxidation (Harborne, 1984; Saito et al., 2008).
was isolated and purified by TLC and HPLC to give the purified iso-
mer of pigment 5 (ca. 1 mg). By analysis of chemical and spectro-
scopic methods, this isomer was identical with pigment 1 as
shown in Table 1. The isomer of pigment 5; dark violet powder;
for TLC, HPLC, UV–Vis, see Table 1.
4.4.1. Anthocyanin pigments 1–8
Characterization of pigments 1–8 are as follows: anthocyanin
pigments 1–6 are dark violet powders, and pigments 7 and 8 are
dark purple-red powders: for UV–Vis, TLC, HPLC and HR-FAB MS,
see Table 1: for 1H and 13C NMR spectroscopic assignments, see Ta-
ble 3 and 4. The structure determination of pigments 7 and 8 was
described in the report (Tatsuzawa et al., 2006).
4.4.6. Caffeoyl sambubiose
Pigment 2 (ca. 1 mg) was dissolved in H2O (1 mL), and 35% H2O2
(1 mL) was added to this solution, and the solution was kept stand-
ing for 60 min at 25 °C. A small amount of manganese dioxide was
added to the solution to decompose excess H2O2 and stood over-
night (Saito et al., 1995). After removal of manganese dioxide by
syringe filter, the solution was saturated with NH3 for 60 min
and added EtOH (8 mL). The solution was evaporated to dryness
to give brown residues. From this residue of pigment 2, caffeoyl-
sambubiose (0.6 mg) was obtained. On the acid hydrolysis of this
caffeoylsambubiose, by 2 N HCl at 80–100 °C for 60 min, caffeic
acid, glucose and xylose were detected in its hydrolysate.
Caffeoylsambubiose; TLC (100ꢀ) BAW 40, 15% HOAc 60, AHW
71; color pale blue (under UV).
4.4.2. Flavonol pigment (pigment 9)
Pale yellow powder: for 1H and 13C NMR spectroscopic assign-
ments, see Table 4. TLC: Rf (100ꢀ), BAW 17, BuHCl 12, 1% HCl
11, AHW 48, UV: kmax(nm) MeOH 326, (299), 267; +NaOMe
365, (299), 267; +AlCl3 360, 324, 237; +AlCl3/HCl 360, 318, 238;
+NaOAc 326, (299), 267; +NaOAc/H3BO3 326, (299), 267. HPLC:
Rt(min) 24.1. HR-FAB MS: calc
C49H59O29 1111.3142. Found
1111.3163; for 1H and 13C NMR spectroscopic assignments, see
Table 4.
4.4.3. Deacyl pigments 1–9
4.5. Isolation of a blue anthocyanin–flavonol complex
A deacyl cyanidin glycoside was obtained from pigments 7 and
8, a deacyl delphinidin glycoside was obtained from pigments 1–6,
and also a deacyl kaempferol glycoside was obtained from pigment
9 by the procedure of alkaline hydrolysis (Tatsuzawa et al., 2006).
Dried blue flowers (ca. 10 g) of H. coronopifolia were immersed
in H2O (500 ml) at room temp. for 3 h. The extract was treated with
EtOH (1.5 L) and concentrated to a dark blue syrup. The blue pig-
ment syrup was dissolved in H2O (50 ml), and was passed through
a Sephadex LH-20 column with H2O. The blue fraction was col-
lected, and concentrated to dryness, which was further purified
with TLC. A blue anthocyanin–flavonol complex band of TLC was
eluted with H2O and dried. A dark blue anthocyanin–flavonol com-
plex powder (3 mg) was obtained.
4.4.3.1. Deacyl cyanidin glycoside. Dark purple-red powder; for TLC,
HPLC, and UV–Vis, see Table 1.
4.4.3.2. Deacyl Delphinidin glycoside. Dark violet powder; for TLC,
HPLC, UV–Vis and FAB MS, see Table 1, for 1H NMR spectroscopic
assignments, see Tables 2 and 4.
A dark blue powder which was composed with pigment 5 and
pigment 9 in a ratio of 1:1; Visible spectra in pH 5.0 buffer solution,
kmax 546, 583 and 635 nm; TLC Rf (100ꢀ), BAW 31 (pigment 5) and
17 (pigment 9), BuHCl 40 (pigment 5) and 12 (pigment 9), 1% HCl
34 (pigment 5) and 11 (pigment 9), AHW 62 (pigment 5) and 48
(pigment 9), and Water 88 (blue spot of anthocyanin–flavonol
complex) {92 (pigment 5) and 36 (pigment 9)}; HPLC Rt(min)
4.4.3.3. Deacyl pigment 9. Yellow powder; for 1H and 13C NMR spec-
troscopic assignments, see Table 4. TLC: Rf (100ꢀ), BAW 13, BuHCl
12, 1% HCl 5, AHW 21, UV: kmax(nm) MeOH 342, 316, 267;
+NaOMe (352), 342, (315), (280), 268; +AlCl3 (380), 333, (300),
274; +AlCl3/HCl (380), 331, (300), 274; +NaOAc 341, 321, 266;
+NaOAc/H3BO3 341, 321, 266. HPLC: Rt(min) 10.2. HR-FAB MS:
calc. C39H51O26 935.2669. Found 935.2629; for 1H and 13C NMR
spectroscopic assignments, see Table 4.
28.9 and 24.1. FAB MS m/z 2102 [M+1]+ (calc. for C93H105O55
,
2101.542).
Acknowledgement
4.4.4. Demalonyl pigments 5 and 6 (pigments 3 and 4)
Pigments 5 and 6 (each ca. 8 mg) were dissolved in 1 N HCl
solution (each 10 mL), respectively, and allowed to stand at room
temperature for 2 weeks as described previously (Saito et al.,
2007). Both pigments 5 and 6 were almost demalonylated in these
solutions within this period. Demalonylated pigments 5 and 6
were then absorbed on the resin columns of Diaion HP-20, respec-
tively, and were eluted with 5% HOAc–MeOH from the columns.
After evaporation in vacuo, both concentrate residues demalonyl
pigments 5 and 6 were dissolved in small volumes of 5% HOAc–
MeOH, respectively, followed by addition of excess Et2O, from
which precipitated solids were then dried in vacuo to give demalo-
nyl pigment 5 (ca. 4 mg) and demalonyl pigment 6 (ca. 4 mg). By
analysis of chemical and spectroscopic methods, demalonyl pig-
ment 5 was identical with pigment 3 and demalonyl pigment 6
was identical with pigment 4 as shown in Table 1. Demalonyl pig-
ments 5 and 6; Dark violet powders; for TLC, HPLC, UV–Vis, and
FAB MS, see Table 1; for 1H NMR spectra, see Table 2.
This work was supported in part by a Grant-in-Aid for Science
Research (C) [No. 22580024, to F.T.] from the Japan Society for
the Promotion of Science (JSPS).
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