Tetra-acylated cyanidin 3-sophoroside-5-glucosides from the flowers of Iberis umbellata L. (Cruciferae)

Article abstract of DOI:10.1016/j.phytochem.2008.04.010

The structures of 11 acylated cyanidin 3-sophoroside-5-glucosides (pigments 1-11), isolated from the flowers of Iberis umbellata cultivars (Cruciferae), were elucidated by chemical and spectroscopic methods. Pigments 1-11 were acylated with malonic acid,

Full text of DOI:10.1016/j.phytochem.2008.04.010

Phytochemistry 69 (2008) 3139–3150
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Phytochemistry
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Tetra-acylated cyanidin 3-sophoroside-5-glucosides from the flowers
of Iberis umbellata L. (Cruciferae)
b
c
d
Norio Saito a, Fumi Tatsuzawa b, , Eri Suenaga , Kenjiro Toki , Koichi Shinoda ,
*
Atsushi Shigihara a, Toshio Honda a
a Faculty of Pharmaceutical Sciences, Hoshi University, Shinagawa, Tokyo 142-8501, Japan
b Experimental Farm, Faculty of Horticulture, Minami-Kyushu University, Takanabe, Miyazaki 884-0003, 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:
Received 7 February 2008
Received in revised form 8 April 2008
The structures of 11 acylated cyanidin 3-sophoroside-5-glucosides (pigments 1–11), isolated from the
flowers of Iberis umbellata cultivars (Cruciferae), were elucidated by chemical and spectroscopic methods.
Pigments 1–11 were acylated with malonic acid, p-coumaric acid, ferulic acid, sinapic acid and/or glucos-
ylhydroxycinnamic acids.
Pigments 1–11 were classified into four groups by the substitution patterns of the linear acylated res-
idues at the 3-position of the cyanidin. In the first group, pigments 1–3 were determined to be cyanidin
3-O-[2-O-(2-O-(acyl)-b-glucopyranosyl)-6-O-(trans-p-coumaroyl)-b-glucopyranoside]-5-O-[6-O-(malonyl)-
b-glucopyranoside], in which the acyl moiety varied with none for pigment 1, ferulic acid for pigment 2 and
sinapic acid for pigment 3. In the second one, pigments 4–6 were cyanidin 3-O-[2-O-(2-O-(acyl)-b-gluco-
pyranosyl)-6-O-(4-O-(b-glucopyranosyl)-trans-p-coumaroyl)-b-glucopyranoside]-5-O-[6-O-(malonyl)-b-
glucopyranoside], in which the acyl moiety varied with none for pigment 4, ferulic acid for pigment 5 and
sinapic acid for pigment 6. In the third one, pigments 7–9 were cyanidin 3-O-[2-O-(2-O-(acyl)-b-gluco-
pyranosyl)-6-O-(4-O-(6-O-(trans-feruloyl)-b-glucopyranosyl)-trans-p-coumaroyl)-b-glucopyranoside]-5-
O-[6-O-(malonyl)-b-glucopyranoside], in which the acyl moiety varied with none for pigment 7, ferulic
acid for pigment 8, and sinapic acid for pigment 9. In the last one, pigments 10 and 11 were cyanidin
3-O-[2-O-(2-O-(acyl)-b-glucopyranosyl)-6-O-(4-O-(6-O-(4-O-(b-glucopyranosyl)-trans-feruloyl)-b-gluco-
pyranosyl)-trans-p-coumaroyl)-b-glucopyranoside]-5-O-[6-O-(malonyl)-b-glucopyranoside], in which
acyl moieties were none for pigment 10 and ferulic acid for pigment 11.
Keywords:
Iberis umbellata L.
Cruciferae
Flower color
Acylated cyanidin 3-sophoroside-5-
glucosides
Malonic acid
p-Coumaric acid
Ferulic acid
Sinapic acid
The distribution of these pigments was examined in the flowers of four cultivars of I. umbellata by HPLC
analysis. Pigment 1 acylated with one molecule of p-coumaric acid was dominantly observed in purple-
violet cultivars. On the other hand, pigments (9 and 11) acylated with three molecules of hydroxycinnamic
acids were observed in lilac (purple-violet) cultivars as major anthocyanins. The bluing effect and stability
on these anthocyanin colors were discussed in relation to the molecular number of hydroxycinnamic acids
in these anthocyanin molecules.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
and Saito, 2002). As described previously, there are two different
glycosyl patterns at the 3-position of anthocyanidins isolated from
Many complicated anthocyanins acylated with organic acids
have been isolated from the flowers of Matthiola incana (Saito
et al., 1995, 1996), Orychophragonus violaceus (Honda et al.,
2005), Lobularia maritima, Cheiranthus cheiri and Lunaria annua
(Tatsuzawa et al., 2006, 2007), and Malcolmia maritima (Tatsuzawa
et al., 2008). These anthocyanins were acylated with one or more
hydroxycinnamic acids, and their color stability and blueness de-
pends on the numbers of hydroxycinnamic acids present (Honda
the plants of the Cruciferae, such as 3-sophoroside-5-glucosides
and 3-sambubioside-5-glucosides (Tatsuzawa et al., 2006). Re-
cently, a 3-glucosylsambubioside pattern was newly identified in
the flowers of Virginia stock (Tatsuzawa et al., 2008).
Distribution of 3-sophoroside-5-glucosides of anthocyanidins is
highly restricted to the plants of only two genus Brassica and Raph-
anus in this family (Harborne, 1967; Andersen and Jordheim,
2006). Furthermore, as the characteristic features of these anthocy-
anins, their 3-sophoroside residue are acylated with hydroxycin-
namic acids or glucosylhydroxycinnamic acids at the 6-position
of the inner glucose. The other hydroxycinnamic acids are linked
* Corresponding author. Tel./fax: +81 983 22 6835.
E-mail address: fumi@nankyudai.ac.jp (F. Tatsuzawa).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.04.010

3140
N. Saito et al. / Phytochemistry 69 (2008) 3139–3150
to the either 2- and/or 6-positions of the outer glucose, and, rarely,
acylated at the 3-position of the outer glucose in these anthocya-
nins (Andersen and Jordheim, 2006; Idaka et al., 1987b).
2005; Tatsuzawa et al., 2006). The chromatographic and spectro-
scopic properties of these pigments are summarized in Table 2.
Acid hydrolysis of pigments 1–11 resulted in cyanidin, glucose,
and p-coumaric acid. Moreover, ferulic acid was detected in the
hydrolysates of pigments 2, 5, 7–11, and also sinapic acid was de-
tected in the hydrolysates of pigments 3, 6 and 9, respectively. The
acylations with malonic acid were presumed to occur in 1–11 by
their measurements of FAB mass spectra (Table 2). Alkaline hydro-
lysis of pigments 1–11 resulted in only one deacylanthocyanin,
whose structure was identified to be cyanidin 3-sophoroside-5-
glucoside by direct comparison of TLC and HPLC with authentic
sample obtained from Ipomoea purpurea anthocyanins by alkaline
hydrolysis (Saito et al., 1995b). Its structure was confirmed by
the analysis of its NMR spectra (see in Section 2.2 and Tables 5
and 6). In the alkaline hydrolysates, 4-glucosyl-p-coumaric acid
was detected in those of pigments 4–11 and also 4-glucosyl-ferulic
acid was detected in those of pigments 10 and 11 except for free
hydroxycinnamic acids. Both 4-glucosyl-p-coumaric acid and 4-
glucosyl-ferulic acid were identified by direct comparison with
the authentic samples, 4-glucosyl-p-coumaric acid, obtained from
anthocyanins of Lobularia maritima and 4-glucosyl-ferulic acid, ob-
tained from anthocyanins of Orychophragonus violaceus by alkaline
hydrolyses (Tatsuzawa et al., 2006; Honda et al., 2005).
FAB mass measurement of pigments 1–11 gave their molecular
ions [M]+ (m/z) at 1005 (pigment 1), 1181 (pigment 2), 1211 (pig-
ment 3), 1167 (pigment 4), 1343 (pigment 5), 1373 (pigment 6),
1343 (pigment 7), 1519 (pigment 8), 1549 (pigment 9), 1505 (pig-
ment 10) and 1681 (pigment 11), respectively. The elemental com-
ponents of pigments 1–11 were confirmed by the measurements of
their high-resolution FAB mass spectra, and their chemical compo-
sitions based on HR-FAB MS were determined as shown in Table 3.
By partial hydrolysis of pigments 1–11, four intermediary pig-
ment products, cyanidin 3-sophoroside-5-glucoside, cyanidin 3-
In our previous studies, we isolated 33 acylated anthocyanidin
3-sambubioside-5-glucosides from the flowers in the Cruciferae
(Honda et al., 2005; Saito et al., 1995, 1996; Tatsuzawa et al.,
2006, 2007, 2008). As a separate work from the previous papers,
we wish to report the isolation and structure elucidation of 11
new acylated cyanidin 3-sophoroside-5-glucosides from the flow-
ers of Iberis umbellata cultivars, and discussed the color stability
and bluing effect of these anthocyanins.
2. Results and discussion
2.1. Anthocyanins of Iberis umbellata
Eleven major anthocyanin peaks were observed in the acetic
acid extracts from the flowers of four I. umbellata cultivars on high
performance liquid chromatography (HPLC) (Table 1 and Fig. 1).
The relative frequencies of their occurrence in four I. umbellata cul-
tivars were 2.5–26.6% (pigment 1), 1.7–9.4% (pigment 2), 7.6–
18.1% (pigment 3), 1.1–2.7% (pigment 4), 4.6–10.4% (pigment 5),
3.5–20.1% (pigment 6), 1.9–4.6% (pigment 7), 4.9–14.7% (pigment
8), 1.6–17.7% (pigment 9), 1.7–4.7% (pigment 10) and 1.7–8.3%
(pigment 11). These 11 anthocyanin pigments (1–11) were isolated
from the flowers of its four cultivars with 5% HOAc as solvent, and
purified using Diaion HP-20 (Mitsubishi Chemical’s Ion Exchange
Resins) column chromatography (CC), preparative HPLC and TLC
according to the procedures described previously (Honda et al.,
[2-(glucosyl)-6-(p-coumaroyl)-glucoside]-5-glucoside,
cyanidin
3-[2-(2-(feruloyl)-glucosyl)-glucoside]-5-glucoside and cyanidin
3-[2-(2-(sinapoyl)-glucosyl)-glucoside]-5-glucoside were detected
in their hydrolysates as the main products by HPLC analysis as well
as cyanidin 3-sophoroside, cyanidin 3,5-diglucoside and cyanidin
3-glucoside (Table 4). As other major intermediary products, pig-
ment 1 was detected in the hydrolysate of pigment 4. Similarly,
pigment 2 from the hydrolysate of pigment 5, pigment 3 from pig-
ment 6, pigments 1 and 4 from pigment 7, pigments 2 and 5 from
pigment 8, pigment 3 from pigment 9, pigments 1, 4 and 7 from
pigment 10, and pigments 2 and 8 from pigment 11, were also de-
tected, respectively (Table 4).
From the above results, the structures of pigments 1–11 were
presumed to be based on cyanidin 3-[2-(2-(acyl-I)-glucosyl)-6-
(acyl-II)-glucoside]-5-[(malonyl)-glucoside], in which the acyl-I
moiety was none, ferulic acid and/or sinapic acid and acyl-II moiety
was p-coumaric acid, glucosyl-p-coumaric acid, feruloyl-glucosyl-
Fig. 1. HPLC profile (530 nm) of anthocyanins present in the extract from flowers of
Iberis umbellata cv. Purple Flash. (1) pigment 1, (2) pigment 2, (3) pigment 3, (4)
pigment 4, (5) pigment 5, (6) pigment 6, (7) pigment 7, (8) pigment 8, (9) pigment 9,
(10) pigment 10, (11) pigment 11. Structures of pigments are shown in Fig. 2 and
Table 2.
Table 1
Distribution of anthocyanins in the flower extracts of I. umbellata cultivars
Florala color
Hueb b /a
Anthocyanin (as %)c
*
*
Cultivar
1
2
3
4
5
6
7
8
9
10
11
Lilacina
Candycane lilac
Purple flash
PV82D
PV81D
PV81A
PV81A
ꢀ0.79
ꢀ0.78
ꢀ0.57
ꢀ0.57
2.5
3.7
26.6
20.4
1.7
2.4
8.8
9.4
7.6
1.7
1.1
1.8
2.7
5.5
4.6
9.2
13.6
20.1
3.5
1.9
2.8
3.3
4.6
4.9
6.9
14.7
8.0
11.3
17.7
1.6
4.7
3.0
1.8
1.7
8.3
7.6
2.7
1.7
10.5
13.5
18.1
Candycane purple
10.4
11.1
4.5
Percentage of total absorbance of all detected anthocyanins at 530 nm in HPLC analysis.
Anthocyanin pigment number and HPLC condition are same as in Table 2.
Rts of these peaks are shown in Table 2.
a
Royal Horticultural Society Colour Chart.
Obtained from C.I.E. diagram Hunter values.
The extract solution: dried flowers (ca. 3 mg) of each cultivar were immersed in the MAW solution (1 mL) at room temperature for 2 h and extracted.
b
c

N. Saito et al. / Phytochemistry 69 (2008) 3139–3150
3141
Table 2
Chromatographic and spectroscopic data for anthocyanins of I. umbellata cultivarsa
Anthocyaninb
Rf values (ꢁ100)
Spectral data in 0.1% HCl–MeOH
HPLC Rt (min)
FAB-MS [M]+
BAW
BuHCl
1% HCl
AHW
kmax (nm)
Eacyl=Emax (%)
E440/Emax (%)
AlCl3
1
2
3
4
5
6
7
8
44
55
52
29
42
40
30
45
43
19
34
24
17
41
36
3
13
10
3
16
14
2
39
30
29
53
39
41
21
19
26
43
39
46
70
69
70
81
76
79
51
51
59
76
79
70
529, 316, 297, 281
537, 321, 294, 284
534, 320, 297, 283
531, (307), (294), 282
535, (313), 296, (285)
537, (309), 296, (285)
532, (307), (291), 283
536, (313), 296, 284
536, (313), 297, (286)
537, (308), (295), 282
539, (312), (295), 284
525, 279
91
163
141
96
111
109
107
131
126
112
138
–
12
19
19
14
13
14
14
13
13
14
14
13
+
+
+
+
+
+
+
+
+
+
+
+
29.9
32.9
32.2
23.1
26.2
25.7
33.5
36.1
35.9
24.6
27.7
11.0
1005
1181
1211
1167
1343
1373
1343
1519
1549
1505
1681
773
9
10
11
4
3
Deacylanthocyanin
a
The composition of solvents is given in Section 4.1.
Pigment 1: cyanidin 3-[2-(glucosyl)-6-(p-coumaroyl)-glucoside]-5-[6-(malonyl)-glucoside]. Pigment 2: cyanidin 3-[2-(2-(feruloyl)-glucosyl)-6-(p-coumaroyl)-gluco-
b
side]-5-[6-(malonyl)-glucoside]. Pigment 3: cyanidin 3-[2-(2-(sinapoyl)-glucosyl)-6-(p-coumaroyl)-glucoside]-5-[6-(malonyl)-glucoside]. Pigment 4: cyanidin 3-[2-(gluco-
syl)-6-(4-(glucosyl)-p -coumaroyl)-glucoside]-5-[6-(malonyl)-glucoside]. Pigment 5: cyanidin 3-[2-(2-(feruloyl)-glucosyl)-6-(4-(glucosyl)-p -coumaroyl)-glucoside]-5-
[6-(malonyl)-glucoside]. Pigment 6: cyanidin 3-[2-(2-(sinapoyl)-glucosyl)-6-(4-(glucosyl)-p -coumaroyl)-glucoside]-5-[6-(malonyl)-glucoside]. Pigment 7: cyanidin
3-[2-(glucosyl)-6-(4-(6-(feruloyl)-glucosyl)-p -coumaroyl)-glucoside]-5-[6-(malonyl)-glucoside]. Pigment 8: cyanidin 3-[2-(2-(feruloyl)-glucosyl)-6-(4-(6-(feruloyl)-gluco-
syl)-p -coumaroyl)-glucoside]-5-[6-(malonyl)-glucoside]. Pigment 9: cyanidin 3-[2-(2-(sinapoyl)-glucosyl)-6-(4-(6-(feruloyl)-glucosyl)-p -coumaroyl)-glucoside]-5-[6-
(malonyl)-glucoside]. Pigment 10: cyanidin 3-[(2-(glucosyl)-6-(4-(6-(4-glucosyl)-feruloyl)-glucosyl)-p -coumaroyl)-glucoside]-5-[6-(malonyl)-glucoside]. Pigment 11:
cyanidin 3-[2-(2-(feruloyl)-glucosyl)-6-(4-(6-(4-(glucosyl)-feruloyl)-glucosyl)-p-coumaroyl)-glucoside]-5-[6-(malonyl)-glucoside].
Table 3
The estimated molecular formulae of acylated anthocyanins from I. umbellata cvs and their chemical composition based on HR-FAB MS
Anthocyanin
HR-FAB MS
Mf
Chemical compositiona
Calc.
[M]+ found
Cy
Glu
Mal
pC
Fer
1
Sin
1
2
3
4
5
6
7
8
C45H49O26
C55H57O29
C56H59O30
C51H59O31
1005.2512
1181.2986
1211.3091
1167.3040
1343.3514
1373.3619
1343.3514
1519.3987
1549.4093
1505.4042
1681.4516
1005.2515
1181.2988
1211.3113
1167.3059
1343.3489
1373.3586
1343.3523
1519.3978
1549.4097
1505.4023
1681.4557
1
1
1
1
1
1
1
1
1
1
1
3
3
3
4
4
4
4
4
4
5
5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
C61H67O34
1
C62H69O35
C61H67O34
C71H75O37
C772H77O38
C67H77O39
C77H85O42
1
2
1
1
2
9
10
11
[M]+ and Mf are molecular ion mass values observed, and estimated molecular formulae as flavylium forms of anthocyanins isolated from I. umbellata cvs based on HR-FAB
mass data.
Anthocyanin numbers are same to those in Table 2.
On the alkaline hydrolysis of these pigments, glucosyl-p-coumaric acid was detected in the hydrolysates of pigments 4–11, and also glucosyl-ferulic acid was detected in
those of pigments 10 and 11.
a
Cy:Glu:Mal:pC:Fer:Sin = molecular numbers of their components: Cy = cyanidin; Glu = glucose; Mal = malonic acid; pC = p-coumaric acid; Fer = ferulic acid; Sin = sinapic
acid.
Table 4
Main intermediary anthocyanin products of Iberis anthocyanin pigments 1–11 by the partial acid hydrolysis
Main intermediary productsa
Cy3S5G
Cy3PS5G
Cy3FS5G
Cy3SS5G
Pigment 1
Pigment 2
Pigment 3
Pigment 4
Pigment 5
Pigment 6
Pigment 7
Pigment 8
Pigment 1
Pigment 2
Pigment 3
Pigment 4
Pigment 5
Pigment 6
Pigment 7
Pigment 8
Pigment 9
Pigment 10
Pigment 11
ꢂ
+
+
ꢂ
+
+
ꢂ
+
+
ꢂ
+
ꢂ
+
+
ꢂ
+
+
ꢂ
+
+
ꢂ
+
ꢃ
+
+
ꢂ
+
+
ꢂ
+
+
ꢂ
+
ꢂ
ꢃ
ꢂ
ꢂ
ꢂ
ꢃ
ꢂ
+
ꢃ
+
+
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢃ
+
+
ꢃ
ꢃ
ꢂ
ꢃ
ꢂ
+
Pigments 1–11 are the same as in Table 2.
a
For key to abbreviation: Methods see Section 4.4.4; the relative frequency of intermediary products by the partial acid hydrolysis: value < 10% = ‘‘ꢂ”, value < 2% = ‘‘+”,
ꢃ = no reaction residue. Percent of total absorbance of all detected anthocyanins at 530 nm by HPLC analysis. Cy3S5G: cyanidin 3-sophoroside-5-glucoside; Cy3PS5G: cyanidin
3-(2-glucosyl-6-p-coumaroyl-glucoside)-5-glucoside; Cy3FS5G: cyanidin 3-[2-(2-feruloyl-glucosyl)-glucoside]-5-glucoside; Cy3SS5g: cyanidin 3-[2-(2-sinapoyl-glucosyl)-
glucoside]-5-glucoside.

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N. Saito et al. / Phytochemistry 69 (2008) 3139–3150
p-coumaric acid, and/or glucosyl-feruloyl-glucosyl-p-coumaric
acid (Fig. 2). The structures of these pigments were further eluci-
dated on the basis of analyses on their 1H and 13C NMR spectra
[500 MHz for 1H and 125.78 MHz for 13C spectra in CF3CO2D–
DMSO-d6 (1:9) or CF3CO2D-CD3OD (1:9)], including 2D COSY, 2D
NOESY, HMQC and HMBC spectra as follows.
J = 15.9 Hz and 7.40, d, J = 15.9 Hz) with the trans configuration
(Table 5 and Fig. 2). Therefore, the structure of demalonyl pig-
ment 1 was determined to be cyanidin 3-O-[2-O-(b-glucopyrano-
syl)-6-O-(trans-p-coumaroyl)-b-glucopyranoside]-5-(b-glucopyran-
oside), which has been reported in the plant of Brassica oleracea
(Idaka et al., 1987).
2.2. Deacylanthocyanin and demalonyl pigment 1
2.3. Pigments 1–3
The FAB mass spectrum of cyanidin 3-sophoroside-5-glucoside
(deacylanthocyanin of Iberis anthocyanins) gave a molecular ion
[M]+at 773 m/z (calc. for C33H41O21, 773.2140), indicating that
deacylanthocyanin is composed of cyanidin with three molecules
of glucose. The 1H NMR spectrum (500 MHz in CF3CO2D–DMSO-
d6, 1:9) of deacylanthocyanin exhibited six aromatic protons being
identified as those of a cyanidin moiety (Saito et al., 1995b) (Table
5). The anomeric protons of sugar moieties were assigned at d 5.60
(d, J = 7.4 Hz, H-1 of Glc A), d 5.15 (d, J = 7.7 Hz, H-1 of Glc B), and d
4.68 (d, J = 7.7 Hz, H-1 of Glc C), and the glucose moieties of Glc A, B
and C were determined to be in the b-glucopyranose form based on
their observed J values. By application of the NOESY experiment,
the long-range NOEs between H-1 of Glc A and H-4 (d 8.85) of
cyanidin, H-1 of Glc B and H-6 (d 7.13) of cyanidin, and H-2 (d
4.06) of Glc A and H-1 of Glc C were observed (Fig. 2) suggesting
that OH-3 and OH-5 of cyanidin are glycosylated with Glc A and
Glc B, respectively, and also OH-2 of Glc A is bonded with Glc C
forming sophorose. Therefore, deacylanthocyanin was determined
to be cyanidin 3-O-[2-O-(b-glucopyranosyl)-b-glucopyranoside]-5-
(b-glucopyranoside). This structure was also confirmed by analysis
of its 13C NMR spectra (Table 6).
The demalonyl pigment 1 (cyanidin 3-[2-(glucosyl)-6-(p-cou-
maroyl)-glucoside]-5-glucoside) was obtained by the process pre-
viously described (Tatsuzawa et al., 2008 and see Section 4.4.3.).
The FAB mass spectrum of the demalonyl pigment 1 gave a
molecular ion [M]+ at 919 m/z (calc. for C42H47O23, 919.2508)
indicating, that the demalonyl pigment 1 was composed of cyani-
din with three molecules of glucose and one molecule of p-cou-
maric acid. The 1H NMR spectrum of this pigment was identical
with that of deacylanthocyanin except for the signals of Glc A
and p-coumaric acid moieties (Table 5). In the 1H NMR spectrum
of Glc A of demalonyl pigment 1, H-6a (d 4.36) and H-6b (d 4.44)
were shifted into the lower magnetic field than those (d 3.59 and
3.77) of deacylanthocyanin, due to an acylation of Glc A with p-
coumaric acid at its OH-6 group. The olefinic proton signals of
p-coumaric acid (I) exhibited large coupling constants (d 6.29, d,
The FAB mass spectra of pigments 1–3 gave their molecular ions
at 1005, 1181 and 1211 m/z, respectively, in agreement with the
masses calculated for C45H49O26 (calc., 1005.2512), C55H57O29
(calc., 1181.2986) and C56H59O30 (calc., 1211.3091). These values
indicated that this pigments 1–3 were composed of cyanidin with
three molecules of glucose and one molecule each of p-coumaric
acid and malonic acid. Moreover, one molecule of ferulic acid
was contained in pigment 2, and also one molecule of sinapic acid
was contained in pigment 3 (Table 3). By partial hydrolysis exper-
iments as shown in Table 4, cyanidin 3-sophoroside-5-glucoside
and cyanidin 3-[2-(glucosyl)-6-(p-coumaroyl)-glucoside]-5-gluco-
side (demalonyl pigment 1) were detected in the hydrolysate of
pigment 1 as the major intermediary pigment products. Thus, the
structure of pigment 1 was presumed to be malonyl cyanidin 3-
[2-(glucosyl)-6-(p-coumaroyl)-glucoside]-5-glucoside. The struc-
ture of pigment 1 was further elucidated on the basis of the anal-
ysis of its 1H and 13C NMR spectra as described in Section 2.2. The
1H NMR spectrum of pigment 1 was superimposed on that of dem-
alonyl pigment 1 except for the signals of 5-glucose (Glc B) and
malonic acid moieties (Table 5-1). The proton signals d 4.07 (H-
6a) and 4.41 (H-6b) of the methylene of Glc B were shifted to the
lower magnetic field than those (d 3.25–3.85) of demalonyl pig-
ment 1 (see in Section 4.4.3). The proton signals of –CH2– of malonic
acid were additionally observed at d 3.37. Therefore, the structure
of pigment 1 was determined to be cyanidin 3-O-[2-O-(b-glucopyr-
anosyl)-6-O-(trans-p-coumaroyl)-b-glucopyranoside]-5-O-[6-O-
(malonyl)-b-glucopyranoside] (Fig. 2), which is a new anthocyanin
in plants.
As shown in Table 4, pigment 1 was detected in the partial
hydrolysates of pigments 2 and 3 as the intermediary pigment
product as well as the deacylanthocyanin and demalonyl pigment
1. Therefore, the structures of pigments 2 and 3 were presumed to
be feruloyl pigment 1 for pigment 2 and sinapoyl pigment 1 for
pigment 3, which were further supported from the results of their
FAB mass values and acid hydrolysis (Tables 3 and 4).
The 1H NMR spectra of pigments 2 and 3 were identical with
those of pigment 1 except for the signals of Glc C and ferulic acid
(III) moieties in pigment 2 and the signals of Glc C and sinapic acid
(III) moieties in pigment 3 (Fig. 2 and Table 5-1). The proton signals
of H-2 of Glc C in pigment 2 (d 4.68) and pigment 3 (d 4.71) were
clearly shifted to a lower field than that of pigment 1 (d 3.00), indi-
cating that the OH-2 of Glc C was acylated with ferulic acid (III) in
pigment 2, and also the OH-2 of Glc C was acylated with sinapic
acid (III) in pigment 3. The olefinic proton signals of both hydroxy-
cinnamic acids exhibited large coupling constants (ferulic acid (III)
d 6.45, J = 15.9 Hz and d 7.52, J = 15.9 Hz; sinapic acid (III) d 6.51,
J = 15.9 Hz and d 7.53, J = 15.9 Hz), supporting the presence of trans
configuration. Consequently, the structure of pigment 2 was deter-
mined to be cyanidin 3-O-[2-O-(2-O-(trans-feruloyl)-b-glucopyr-
anosyl)-6-O-(trans-p-coumaroyl)-b-glucopyranoside]-5-O-[6-O-
(malonyl)-b-glucopyranoside] (Fig. 2), which is a new anthocyanin
in plants, and the structure of pigment 3 was determined to
be cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-b-glucopyranosyl)-6-
O-(trans-p-coumaroyl)-b-glucopyranoside]-5-O-[6-O-(malonyl)-b-
glucopyranoside] (Fig. 2), which is also a new anthocyanin in
plants. The structure of pigment 2 was confirmed by the analysis
of its 13C NMR spectrum (Table 6-1).
Fig. 2. Iberis acylated anthocyanins from the purple-violet flowers of I. umbellata.
Observed main NOEs are indicated by arrows. Pigments: Group I – 1 = pigment 1,
2 = pigment 2 R = H, 3 = pigment 3 R = OCH3; Group II – 4 = pigment 4, 5 = pigment
5 R = H, 6 = pigment 6 R = OCH3; Group III – 7 = pigment 7, 8 = pigment 8 R = H,
9 = pigment 9 R = OCH3; Group IV – 10 = pigment 10, 11 = pigment 11 R = H.

N. Saito et al. / Phytochemistry 69 (2008) 3139–3150
3143
Table 5-1
1H NMR spectroscopic data of acylated anthocyanins (Deacyl pigment and pigments 1–5) from the flowers of I. umbellata
1H NMR (500 MHz), an internal standard of TMS. Coupling constants (J in Hz) in parentheses.
*
s = singlet; br s = broad singlet; d = doublet; br d = broad doublet; dd = double doublet; t = distoted triplet; m = multiplet.
Anthocyanin numbers is same in Table 2 and Fig. 2.
2.4. Pigments 4–6
masses calculated for C51H59O31 (MW = 1167.3040), C61H67O34
(MW = 1343.3514) and C62H69O35 (MW = 1373.3619). These values
indicated that pigments 4–6 were composed of cyanidin with four
molecules of glucose and one molecule each of p-coumaric acid
The FAB mass spectra of pigments 4–6 gave their molecular ions
at 1167, 1343 and 1373 m/z, respectively, in agreement with the

3144
N. Saito et al. / Phytochemistry 69 (2008) 3139–3150
Table 5-2
1H NMR spectroscopic data of acylated anthocyanins (Pigments 6–11) from the flowers of I. umbellata
1H NMR (500 MHz), an internal standard of TMS. Coupling constants (J in Hz) in parentheses.
*
s = singlet; br s = broad singlet; d = doublet; br d = broad doublet; dd = double doublet; t = distoted triplet; m = multiplet.

N. Saito et al. / Phytochemistry 69 (2008) 3139–3150
3145
Table 6-1
13C NMR spectroscopic data of acylated anthocyanins (Deacyl pigment and pigments 1,3,4,5,7) from the flowers of I. umbellata in DMSO-d6/CF3CO2D (9:1)
Deacyl pigment
Pigment 1
Pigment 3
Pigment 4
Pigment 5a
Pigment 7
Cyanidin
2
3
4
5
6
7
8
162.6
144.8
132.1
155.1
104.1
167.4
96.1
162.5
144.5
131.1
154.9
106.2
167.3
96.3
161.9
144.7
131.7
154.9
104.3
167.1
96.1
162.6
144.6
131.4
154.9
104.7
167.2
96.3
164.6
146.8
133.8
156.4
105.9
168.8
97.4
162.8
146.5
131.6
155.1
105.1
167.6
96.7
9
155.2
111.5
119.7
117.7
146.3
155.1
117.1
127.7
155.5
111.5
119.6
117.7
146.4
155.3
117.1
127.7
155.2
113.9
120.0
116.7
146.7
155.0
116.9
127.7
155.0
111.5
119.6
117.0
146.4
155.3
116.4
127.7
156.5
115.2
121.1
118.4
149.9
157.0
117.5
129.7
155.1
111.2
119.8
116.9
146.5
155.5
117.3
128.8
10
10
20
30
40
50
60
p-Coumaric acid (I)
1
2
3
4
5
6
a
b
125.0
130.4
115.7
159.9
115.7
130.4
113.7
145.2
166.7
125.1
130.6
115.9
160.1
115.9
130.6
113.8
145.9
166.2
127.7
130.1
116.0
159.4
116.0
130.1
115.5
144.6
166.5
121.1
131.0
117.8
160.8
117.8
131.0
115.8
145.8
167.4
125.9
130.2
115.8
159.3
115.8
130.2
114.7
144.5
166.8
COOH
Ferulic acid (II)
1
2
3
4
5
6
a
b
127.2
111.4
149.2
150.9
116.5
124.1
115.3
146.9
168.2
56.5
123.3
111.7
148.6
149.6
115.8
123.7
114.7
145.3
166.6
55.9
COOH
OCH3
Ferulic acid or sinapic acid (III)
1
2
3
4
5
6
a
b
125.0
106.5
138.5
148.4
138.5
106.5
116.1
145.3
166.2
56.4
COOH
OCH3
OCH3
56.4
Malonic acid
CH2
COOH
41.3
166.9
168.1
41.0
167.0
168.3
41.3
167.0
168.1
41.0
168.6
169.1
41.0
167.1
168.2
COOH
Glucose (A)
1
2
3
4
5
6
99.6
81.0
76.5
69.1
77.4
60.5
98.9
80.9
76.3
69.9
73.7
63.2
98.6
–
–
–
–
64.1
99.0
80.8
75.9
69.7
73.7
63.4
99.8
80.0
76.2
–
74.9
64.4
99.3
81.1
74.2
70.3
73.4
63.6
Glucose (B)
1
2
3
4
5
6
101.4
77.5
76.0
69.6
73.1
60.6
101.8
73.2
76.1
69.7
74.2
64.1
100.2
69.8
–
–
–
101.7
73.3
77.2
76.2
74.2
64.2
102.0
74.7
76.9
78.0
77.7
63.5
102.1
73.4
–
69.9
74.8
64.3
64.2
Glucose (C)
1
2
3
103.7
74.5
76.2
104.8
75.9
76.9
102.2
–
–
103.8
74.6
76.1
100.2
75.2
–
104.0
74.8
76.1
(continued on next page)

3146
N. Saito et al. / Phytochemistry 69 (2008) 3139–3150
Table 6-1 (continued)
Deacyl pigment
69.6
76.8
60.6
Pigment 1
Pigment 3
Pigment 4
Pigment 5a
Pigment 7
4
5
6
69.7
74.6
60.6
–
73.4
64.2
69.5
76.7
60.6
–
–
62.6
74.0
77.1
60.9
Glucose (D)
1
2
3
4
5
6
100.1
76.9
–
69.5
73.3
60.8
101.6
74.7
–
–
–
101.0
76.4
74.2
70.2
74.7
63.7
62.6
a
In CD3OD/CF3CO2D (9:1).
and malonic acid, and one additional molecule of ferulic acid in
pigment 5, and also one additional molecule of sinapic acid in pig-
ment 6 (Table 3). By partial acid hydrolysis, pigments 1–3 were de-
tected in the hydrolysates of pigments 4–6, respectively, as the
main intermediary pigment products (Table 4).
one molecule each of p-coumaric acid, malonic acid and ferulic
acid, respectively. Pigment 8 also contained an additional molecule
of ferulic acid and pigment 9 contained an additional molecule of
sinapic acid (Table 3). On the partial acid hydrolysis experiment,
pigments 4 and 5 were detected in the hydrolysates of pigments
7 and 8, respectively, and pigments 3 and 6 were detected in that
of pigment 9 as the main intermediary pigment products, respec-
tively (Table 4). Therefore, the structures of pigments 7–9 were
presumed to be a feruloyl pigment 4 for pigment 7, a feruloyl pig-
ment 5 for pigment 8, and a feruloyl pigment 6 for pigment 9. The
structures of these pigments were further elucidated on the basis
of analysis on their NMR spectra as follows.
The 1H NMR spectrum of pigment 4 was identical with that of
pigment 1 except for the signals of Glc D moiety (Table 5-1). The
resonance of the anomeric proton of Glc D appeared at d 4.95
and its coupling constant is J = 7.7 Hz. Thus, Glc D must be of b-glu-
copyranose form. By analysis of its NOESY spectrum, strong long-
range NOEs between H-1 of Glc D and H-3 and -5 of p-coumaric
acid (I) were observed (Fig. 2), indicating that OH-4 of
p-coumaric acid (I) was glycosylated with Glc D. Therefore, the
structure of pigment 4 was determined to be cyanidin 3-O-[2-O-
(b-glucopyranosyl)-6-O-(4-O-(b-glucopyranosyl)-trans-p-couma-
royl)-b- glucopyranoside]-5-O-[6-O-(malonyl)-b-glucopyranoside],
which is a new anthocyanin in plants. This structure was further
confirmed by the measurement of its 13C NMR spectra (Table 6-1).
The 1H NMR spectrum of pigment 5 was identical with that of
pigment 4 except for the signals of Glc C and ferulic acid (III) moie-
ties. The chemical shift (d 4.77) of H-2 of Glc C was shifted to a lower
magnetic field than that (d 3.00) of pigment 4, supporting that OH-2
of Glc C was acylated with ferulic acid (III) in pigment 5 (Fig. 2). The
olefinic proton signals of ferulic acid (III) exhibited large coupling
constants (J = 15.9 and 15.9 Hz) suggesting that ferulic acid (III)
was in a trans-form. Based on these results, the structure of pigment
5 was determined to be cyanidin 3-O-[2-O-(2-O-(trans-feruloyl)-
b-glucopyranosyl)-6-O-(4-O-(b-glucopyranosyl)-trans-p-couma-
royl)-b-glucopyranoside]-5-O-[6-O-(malonyl)-b-glucopyranoside],
which is a new anthocyanin in plants. This structure was also sup-
ported by its 13C NMR spectra (Table 6-1).
The 1H NMR spectrum of pigment 7 was identical with that of
pigment 4 except for the signals of ferulic acid (II) and Glc D moi-
eties. The chemical shifts (d 4.25 H-6a and 4.48 H-6b) of methylene
protons of Glc D were shifted to a lower magnetic field than those
(d 3.51 H-6a and 3.73 H-6b) of pigment 4 (Tables 5-1 and 5-2),
indicating that OH-6 of Glc D was acylated with ferulic acid (II)
in pigment 7. The olefinic protons of ferulic acid (II) exhibited large
coupling constants (J = 15.9 and 15.9 Hz) indicator of a trans-form
(Table 5-2). Therefore, the structure of pigment 7 was determined
to be cyanidin 3-O-[2-O-(b-glucopyranosyl)-6-O-(4-O-(6-O-(trans-
feruloyl)-b-glucopyranosyl)-trans-p-coumaroyl)-b-glucopyrano-
side]-5-O-[6-O-(malonyl)-b-glucopyranoside] (Fig. 2), which is a
new anthocyanin in plants. This structure was confirmed by the
measurement of its 13C NMR spectra (Table 6-1).
The 1H NMR spectra of pigments 8 and 9 were quite similar to
that of pigment 7 except for the signals of Glc C and ferulic acid (III)
moieties for pigment 8, and also except for the signals of Glc C and
sinapic acid (III) moieties for pigment 9 (Table 5-2). The chemical
shifts (d 4.70 in pigment 8 and d 4.70 pigment 9) of H-2 of Glc C
in both pigments 8 and 9 were shifted to a lower magnetic field
than that of pigment 7 (d 3.03), indicating that pigments 8 and 9
were acylated with ferulic acid (III) and sinapic acid (III) at OH-2
of Glc C, respectively (Fig. 2). Both hydroxycinnamic acids exhib-
ited large coupling constants (J = 15.9 and 15.9 Hz at ferulic acid,
and J = 15.9 and 15.9 Hz at sinapic acid) of both olefinic proton sig-
nals, supporting that both hydroxycinnamic acids have the trans
configurations. Therefore, the structure of pigment 8 was deter-
mined to be cyanidin 3-O-[2-O-(2-O-(trans-feruloyl)-b-glucopyr-
anosyl)-6-O-(4-O-(6-O-(trans-feruloyl)-b-glucopyranosyl)-trans-p-
coumaroyl)-b-glucopyranoside]-5-O-[6-O-(malonyl)-b-glucopy-
ranoside] (Fig. 2), which is a new anthocyanin in plants. The struc-
ture was further confirmed by the measurement of its 13C NMR
spectra (Table 6-2). The structure of pigment 9 was also deter-
mined to be cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-b-glucopyr-
anosyl)-6-O-(4-O-(6-O-(trans-feruloyl)-b-glucopyranosyl)-trans-p-
coumaroyl)-b-glucopyranoside]-5-O-[6-O-(malonyl)-b-glucopyran-
oside] (Fig. 2), which is a new anthocyanin in plants. The structure
was also confirmed by the measurement of its 13C NMR spectra
(Table 6-2).
The 1H NMR spectrum of pigment 6 was similar to that of pig-
ment 5 changing the signals of ferulic acid (III) to sinapic acid (III)
(Tables 5-1 and 5-2). The two aromatic proton signals of sinapic acid
(III) were observed at d 6.96 (H-2 and H-6) with large coupling con-
stants (J = 15.9 and 15.9 Hz) of olefinic proton signals (d 6.49 H-a and d
7.53 H-b) indicating a trans-sinapic acid. From these results, the
structure of pigment 6 was determined to be cyanidin 3-O-[2-O-
(2-O -(trans-sinapoyl)-b-glucopyranosyl)-6-O-(4-O-(b-glucopyran-
osyl)-trans-p-coumaroyl)-b-glucopyranoside]-5-O-[6-O-(malonyl)-
b-glucopyranoside] (Fig. 2), which is a new anthocyanin in plants.
2.5. Pigments 7–9
The FAB mass spectra of pigments 7–9 gave their molecular ions
at 1343, 1519 and 1549 m/z, respectively, in agreement with the
mass calculated for C61H67O34 (pigment 7; MW = 1343.3514),
C71H75O37 (pigment 8; MW = 1519.3987) and C72H77O38 (pigment
9; MW = 1549.4093). These values indicated that pigments 7–9
were composed of cyanidin with four molecules of glucose and

N. Saito et al. / Phytochemistry 69 (2008) 3139–3150
3147
Table 6-2 (continued)
Table 6-2
13C NMR spectroscopic data of acylated anthocyanins (Pigments 8, 9, 11) from the
Pigment 8
Pigment 9
Pigment 11
flowers of I. umbellata in DMSO-d6/CF3CO2D (9:1)
4
5
6
70.7
74.2
61.5
–
–
61.5
–
–
60.8
Pigment 8
Pigment 9
Pigment 11
Cyanidin
2
3
4
5
6
7
8
9
10
10
20
30
40
50
60
162.6
146.7
131.8
155.1
106.5
167.5
96.4
155.1
110.0
119.9
116.9
146.7
155.6
117.3
128.8
162.6
146.7
131.9
155.0
106.5
167.5
96.4
155.1
111.2
119.9
117.2
148.4
155.6
117.3
128.8
162.5
146.8
131.6
155.0
106.5
167.6
96.5
155.0
111.0
119.8
115.3
148.8
155.8
117.1
129.0
Glucose (D)
1
2
3
4
5
6
100.1
76.7
73.9
70.2
74.5
63.6
100.1
76.8
73.9
70.2
74.3
63.7
100.1
–
–
–
–
64.1
Glucose (E)
1
2
3
4
5
6
99.7
–
–
–
–
61.5
p-Coumaric acid (I)
1
2
3
4
5
6
a
b
127.9
130.2
116.5
159.0
116.5
130.2
115.4
144.5
166.9
127.9
130.2
116.6
159.1
116.6
130.2
116.1
144.5
166.9
127.9
130.2
116.1
159.1
116.1
130.2
114.3
144.6
166.7
2.6. Pigments 10 and 11
The FAB mass spectra of pigments 10 and 11 gave their molecu-
lar ions [M]+ at 1505, and 1681 m/z, respectively, in agreement with
their masses calculated for C67H77O39 (pigment 10; MW =
1505.4040) and C77H85O42 (pigment 11; MW = 1681.4515). These
values indicated that pigments 10 and 11 were composed of cyani-
din with five molecules of glucose and one molecule each of p-cou-
maric acid, malonic acid and ferulic acid, and pigment 11 contained
additional one molecule of ferulic acid (Table 3). By partial acid
hydrolysis, pigment 7 was detected in the hydrolysate of pigment
10, and pigment 8 was detected in that of pigment 11 as the main
intermediary pigment products, respectively (Table 4). Thus, the
structures of pigments 10 and 11 were presumed to be a glucosyl
pigment 7 for pigment 10, and to be a glucosyl pigment 8 for
pigment 11. The detailed structures of pigments 10 and 11 were
elucidated on the basis of analysis on their NMR spectra as follows.
The 1H NMR spectrum of pigment 10 was identical with that of
pigment 7 except for the signals of Glc E moiety of pigment 10
(Table 5-2). Since the chemical shifts of anomeric proton of Glc E
exhibited at d 5.06 (d, J = 7.0 Hz), Glc E of pigment 10 must be of
b-glucopyranose form. In the NOESY spectrum of pigment 10,
strong long-range NOE between H-1 of Glc E and H-5 of ferulic acid
(II) was observed (Fig. 2), supporting that OH-4 of ferulic acid (II)
was glycosylated with Glc E. Therefore, the structure of pigment
10 was determined to be cyanidin 3-O-[2-O-(b-glucopyranosyl)-
6-O-(4-O-(6-O-(4-O-(b-glucopyranosyl)-trans-feruloyl)-b-glucopyr-
anosyl)-trans-p-coumaroyl)-b-glucopyranoside]-5-O-[6-O-(malonyl)-
COOH
Ferulic acid (II)
1
2
3
4
5
6
a
125.9
111.1
148.2
148.4
115.8
123.6
114.8
144.7
166.2
55.8
125.0
111.6
148.3
148.4
115.8
123.3
113.8
144.7
166.7
55.9
128.2
111.4
148.6
149.6
115.8
123.2
113.9
144.8
166.2
56.0
b
COOH
OCH3
Ferulic acid or sinapic acid (III)
1
2
3
4
5
6
a
126.1
111.5
148.3
149.6
115.9
123.2
114.8
145.4
166.7
56.0
125.9
115.9
138.6
149.6
138.6
115.9
114.8
145.4
166.2
56.4
126.0
111.4
148.3
149.5
115.9
123.2
113.9
144.8
166.7
56.4
b
COOH
OCH3
OCH3
56.4
Malonic acid
CH2
COOH
41.0
167.5
168.3
41.5
167.2
168.3
41.5
167.1
168.3
b-glucopyranoside] (Fig. 2), which is
plants.
a new anthocyanin in
COOH
The 1H NMR spectrum of pigments 11 was identical with that of
pigment 8 except for the signals of Glc E moiety (Table 5-2). The
anomeric proton of Glc E was assigned at d 5.04 (d, J = 7.0 Hz),
and its coupling constant suggested Glc E to be b-glucopyranose.
By analysis of its NOESY spectrum, a strong long-range NOE
between H-1 of Glc E and H-5 of ferulic acid (II) was observed
to support that OH-4 of ferulic acid (II) was glycosylated with
Glc E. Therefore structure of pigment 11 was determined to be
Glucose (A)
1
2
3
4
5
6
98.6
80.0
76.5
70.8
74.2
63.7
98.6
77.7
–
70.9
74.5
63.7
98.8
–
–
–
–
63.8
Glucose (B)
1
2
3
4
5
6
102.1
73.4
74.9
69.8
74.2
64.3
102.1
73.4
74.9
69.8
74.3
64.7
102.0
cyanidin
3-O-[2-O-(2-O-(trans-feruloyl)-b-glucopyranosyl)-6-O-
–
–
–
–
(4-O-(6-O-(4-O- (b-glucopyranosyl)-trans-feruloyl)-b-glucopyran-
osyl)-trans-p-coumaroyl)-b-glucopyranoside]-5-O-[6-O-(malonyl)-
b-glucopyranoside] (Fig. 2), which is a new anthocyanin in plants.
63.8
Glucose (C)
2.7. The color stability and bluing effect of Iberis anthocyanins
1
2
3
99.9
77.7
76.0
99.9
77.6
–
104.7
–
–
As shown in Fig. 3, the color stability of Iberis anthocyanins was
compared with each other. Each pigment was dissolved in buffer

3148
N. Saito et al. / Phytochemistry 69 (2008) 3139–3150
onus violet-blue anthocyanins whose long-linear side chains were
acylated with at the 2-position of xylose moiety in their 3-sambu-
biose residues (Honda et al., 2005). Orychophragonus violet-blue
anthocyanins were isolated from the flowers of Orychophragonus
violaceus in the Cruciferae. Similar acylated anthocyanins with glu-
cosyl-hydroxycinnamoyl-glucosyl-hydroxycinnamic
acids
or
hydroxycinnamoyl-glucosyl-hydroxycinnamic acids at the 3-gly-
coside residues were recognized to be present in the plants of Ipo-
moea (Pharbitis) and Evolvulus (Convolvulaceae), Petunia
(Solanaceae), Phacelia (Hydrophyllaceae) and Triteleia (Liliaceae)
(Andersen and Jordheim, 2006; Honda and Saito, 2002; Mori
et al., 2006). These complicating structures of acyl groups are con-
sidered to be an advanced character in the Cruciferae (Harborne,
1967; Honda and Saito, 2002).
4. Experimental
4.1. General procedures
TLC was carried out on plastic coated cellulose sheets (Merck)
using seven mobile phases: BAW (n-BuOH–HOAc–H2O, 4:1:2),
BuHCl (n-BuOH–2 N HCl, 1:1, upper layer), AHW (HOAc–HCl–H2O,
15:3:82), 1% HCl for anthocyanins, 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 phthalate spray reagent (Harborne, 1984).
Analytical HPLC was performed on 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. The eluant was ap-
plied as a linear gradient elution for 40 min from 20% to 85% sol-
vent B (1.5% H3PO4, 20% HOAc, 25% MeCN in H2O) in solvent A
(1.5% H3PO4 in H2O).
Fig. 3. Stability of Iberis acylated anthocyanins in buffer solution (pH 6.86, path
length 10 mm, ca. 25 °C). Anthocyanin number is same in Table 2 and Fig. 2. ( ):
ꢂ
Pigment 11 (3 molecules of hydroxycinnamic acid, kmax 593 nm); ( ) pigment 8 (3
ꢃ
molecules of hydroxycinnamic acid, kmax 591 nm); ( ) pigment 5 (2 molecules of
M
hydroxycinnamic acid, kmax 594 nm); ( ) pigment 7 (2 molecules of hydroxycin-
namic acid, kmax 585 nm); (h) pigment 4 (1 molecules of hydroxycinnamic acid,
kmax 565 nm); (j) pigment 1 (1 molecules of hydroxycinnamic acid, kmax 566 nm);
N
( ) deacyl pigment (0 molecules of hydroxycinnamic acid, kmax 572 nm).
*
solution (phosphate–citrate buffer pH 6.9), kept at room tempera-
ture (ca. 25 °C), and measured at intervals of 20 min. The stabilities
of these pigments appeared to increase with increasing content of
hydroxycinnamic acids. Thus, pigments 8 and 11 (three molecules
of hydroxycinnamic acids) were more stable than pigments 5 and 7
(two molecules of hydroxycinnamic acids). Pigments 4 and 1 were
very unstable as well as that of cyanidin 3-sophoroside-5-gluco-
side (Fig. 3).
The bluing color effect of pigments 1–11 was also increased
with increasing content of hydroxycinnamic acids as shown in Ta-
ble 2. The kmax values of VIS region in 0.1% HCl–MeOH solution
were 529 and 531 nm at pigments 1 and 4 (one molecule of
hydroxycinnamic acid), 532–537 nm at pigments 2, 3, 5, 6, 7 and
10 (two molecules of hydroxycinnamic acids) and 536–539 nm at
pigments 8, 9 and 11 (three molecules of hydroxycinnamic acids)
(Table 2). These results indicated that the intramolecular co-pig-
mentation between cyanidin and hydroxycinnamic acids, which
might be formed in these polyacylanthocyanins, of I. umbellata is
similar to that from the flowers of Ipomoea (Pharbitis), Petunia
and so on (Honda and Saito, 2002).
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).
High resolution FAB mass (HR-FAB MS) spectra were obtained
in the positive ion mode using the magic bullet (5:1 mixture of
dithiothreitol and dithioerythritol) as a matrix.
NMR spectra were determined at 500 MHz for 1H spectra and at
125.78 MHz for 13C spectra in CF3COOD–DMSO-d6 (1:9). Chemical
shifts are reported relative to a TMS internal standard (d), and cou-
pling constants are in Hz.
4.2. Plant materials and flower colors
Seeds of I. umbellate cultivars ‘Candycane Lilac’ and ‘Candycane
Purple’ were purchased from Murakami Seed Co. Ltd. (Ibaraki, Ja-
pan) and those of ‘Lilacina’ and ‘Purple Flash’ were purchased from
Fukukaen Nursery & Bulb Co. Ltd. (Nagoya, Japan), and Sakata Co.
Ltd. (Yokohama, Japan), respectively. Seeds were planted in green-
houses at the experimental farm of Minami-Kyushu University.
The flower colors of these plants were recorded by comparing di-
rectly with the Royal Horticultural Society (R.H.S.) color chart
and their chromaticities were recorded on a SE-2000 Spectro Color
Meter (Nippon Denshoku Industries Co. Ltd.). These values are
shown in Table 1. The fresh flowers were collected from winter
to spring seasons in Japan, and dried overnight at 40 °C, and kept
in a refrigerator at about 4 °C.
3. Concluding remarks
In this study, anthocyanins of I. umbellata were proved to be
composed from polyacylated anthocyanins as its main anthocya-
nins, and those of I. umbellata are classified into the group of acyl-
ated anthocyanidin 3-sophoroside-5-glucoside pattern as new
members. To the best of our knowledge, the plants containing
anthocyanins of this group are Brassica, Iberis and Raphanus in
the Cruciferae.
4.3. Isolation of anthocyanins
On the acylation pattern of these anthocyanins, pigments 10
and 11 are acylated at the 6-position of inner glucose in the 3-sop-
horoside with a long-linear side chain of glucosyl-feruloyl-gluco-
syl-p-coumaric acid. This long-linear side chain is comparable to
that of glucosyl-caffeoyl-glucosyl-caffeic acid in the Orychophrag-
Dried mixed flowers (ca. 300 g) of four cultivars were immersed
in 5% HOAc–H2O (5 L) at room temperature overnight, and ex-
tracted. The extract was passed through a Diaion HP-20 (Mitsubishi
Chemical’s Ion Exchange Resins) column (90£ ꢁ 150 mm), on

N. Saito et al. / Phytochemistry 69 (2008) 3139–3150
3149
which acylated anthocyanins were adsorbed. Then, the column was
thoroughly washed with 5% HOAc–H2O (10 L), and eluted with 5%
HOAc–MeOH (500 mL) to recover the anthocyanins. After concen-
tration, the crude anthocyanins were roughly separated into four
bands with paper chromatography (PC; ADVANTEC 590,
60 ꢁ 60 cm) using BAW. The pigments of the above four bands were
further purified by TLC (15% HOAc) and prep. HPLC. Prep. HPLC was
performed on a Waters C18 (19£ ꢁ 150 mm) column at 40 °C with
a flow rate of 1 mL/min and monitoring at 530 nm. The solvent used
was as follows: a linear gradient elution for 60 min from 40% to 85%
solvent B in solvent A. Each fraction was transformed to a Diaion
HP-20 column, on which pigments were adsorbed. Pigments were
eluted with 5% HOAc–MeOH followed by addition of an excess of
Et2O and then dried. The anthocyanin pigments finally obtained
were as follows; pigment 1 (ca. 50 mg), pigment 2 (ca. 5 mg), pig-
ment 3 (ca. 5 mg), pigment 4 (ca. 5 mg), pigment 5 (ca. 15 mg), pig-
ment 6 (ca. 25 mg), pigment 7 (ca. 15 mg), pigment 8 (ca. 15 mg),
pigment 9 (ca. 10 mg), pigment 10 (ca. 7 mg), pigment 11 (ca.
35 mg) and deacylanthocyanin (ca. 5 mg).
44; color green-blue (UV), violet (UV + NH3 gas); HPLC (Rt (min))
9.6.
4.4.3. Demalonyl pigment 1
Pigment 1 (ca. 10 mg) was dissolved in 1 N HCl solution (2 mL)
and allowed to stand at room temperature for 2 weeks as described
previously (Tatsuzawa et al., 2008). Pigment 1 was almost demal-
onylated in this solution within this period. Demalonylated pig-
ment 1 was then absorbed on the resin column of Diaion HP-20,
and was eluted with 5% HOAc–MeOH from the column. After evap-
oration in vacuo, the concentrate residue was dissolved in a small
volume of 5% HOAc–MeOH followed by addition of excess Et2O,
from which solids were then dried in vacuo to give demalonyl pig-
ment 1 powder (ca. 4 mg). Demalonyl pigment 1 was already re-
ported to be present in B. oleracea (Idaka et al., 1987a).
UV–Vis (in 0.1% HCl–MeOH), kmax 527, 315, 297, 282 nm, Eacyl
/
Emax (%) 143, E440/Emax (%) 13, +AlCl3 + shift; TLC (Rf ꢁ100) BAW
50, BuHCl 22, 1% HCl 32, AHW 65; HPLC (Rt (min)) 27.6; FAB-MS
m/z 919 [M]+ (calc. for C42H47O23); HR-FAB MS calc. for 919.2508.
Found 919.2568; 1H NMR (500 MHz, CF3CO2D–DMSO-d6 = 1:9, an
internal standard of TMS); d cyanidin: 8.78 (s, H-4), 7.04 (br s, H-
6), 7.01 (br s, H-8), 8.06 (d, J = 2.5 Hz, H-20), 7.11 (d, J = 8.6 Hz, H-
50), 8.25 (dd, J = 2.5 and 8.6 Hz, H-60). p-Coumaric acid: 7.41 (2H,
d, J = 8.6 Hz, H-2 and -6), 6.80 (2H, d, J = 8.6 Hz, H-3 and -5), 6.29
(d, J = 15.9 Hz, H-a), 7.40 (d, J = 15.9 Hz, H-b). Glucose A: 5.70 (d,
J = 7.4 Hz, H-1), 4.13 (m, H-2), 3.78 (m, H-3), 3.52 (m, H-4), 4.04
(m, H-5), 4.36 (m, H-6a), 4.44 (br d, J = 11.0 Hz, H-6b). Glucose B:
5.10 (d, J = 8.0 Hz, H-1), 3.58 (m, H-2), 3.42 (m, H-3), 3.25–3.85 (rest
of the sugar protons). Glucose C: 4.71 (d, J = 8.0 Hz, H-1), 3.03 (m,
H-2), 3.14 (t, J = 8.9 Hz, H-3), 3.07 (m, H-4), 2.76 (m, H-5), 3.20
(m, H-6a), 3.30 (m, H-6b).
4.4. Analyses of anthocyanin
Dried petals (ca. 10 mg) of each cultivar were extracted with
0.5 mL MAW (MeOH–HOAc–H2O, 4:1:5, v/v/v). The anthocyanin
quantitative analysis of these extracts was performed by HPLC 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 for anthocyanin pigments.
Solvent system used were linear gradient elution for 40 min from
20% to 85% solvent B in solvent A. The identification of anthocya-
nins was carried out by standard procedures involving deacylation
with acid, and hydrolyses with alkaline and acid (Harborne, 1984).
The data of TLC (Rf value), HPLC (Rt (min)), UV–Vis (kmax), and FAB-
MS spectra are shown in Tables 2 and 3, and 1H and 13C NMR spec-
troscopic data are shown in Tables 5 and 6 (see in Sections
4.4.1,4.4.2,4.4.3,4.4.4).
4.4.4. Partial acid hydrolysis of anthocyanin pigments
Pigments 1–11 (each ca. 1 mg) were dissolved in each 0.5 mL of
2 N HCl, and hydrolyzed by heating on a water bath (ca. 90 °C) for
10 min. The partial hydrolysates were quickly analyzed by HPLC
with authentic cyanidin glycosides. As shown in Table 4, cyani-
din 3-sophoroside-5-glucoside (deacylanthocyanin), cyanidin 3-
[2-(glucosyl)-6-(p-coumaroyl)-glucoside]-5-glucoside (demalonyl
pigment 1), cyanidin 3-[2-(2-sinapoyl-glucosyl)-glucoside]-5-glu-
coside and cyanidin 3-[2-(2-feruloyl-glucosyl)-glucoside]-5-gluco-
side were detected in the hydrolysates as the main intermediary
pigment products, respectively. As authentic samples, cyanidin 3-
sophoroside-5-glucoside (deacylanthocyanin) and demalonyl pig-
ment 1 were obtained by the processes described above. Cyanidin
3-[2-(2-sinapoyl-glucosyl)-glucoside]-5-glucoside was isolated
from red cabbage (Idaka et al., 1987b). Cyanidin 3-[2-(2-feruloyl-
glucosyl)-glucoside]-5-glucoside was obtained from the partial
hydrolysates of pigments 2, 5, 8 and 11.
4.4.1. Anthocyanin pigments 1–11
Characterization of pigments 1–11 are as follows: all anthocya-
nins are dark purple-red powders; for UV–Vis, TLC and HPLC, see
Table 2; for HR-FAB MS, see Table 3; for 1H and 13C NMR spectro-
scopic assignments, see Tables 5 and 6.
4.4.2. Deacylanthocyanin and 4-O-glucosylhydroxycinnamic acids
Mixed pigments (ca. 20 mg) were individually dissolved in 2 N
NaOH (2 mL) using a degassed syringe with each allowed to stand
for 15 min. Each solution was then acidified with 2 N HCl and
evaporated in vacuo to dryness, with the resulting residue dis-
solved in 1% HCl–MeOH and applied on TLC (BAW) to yield a
deacylanthocyanin (ca. 5 mg), 4-O-glucosyl-p-coumaric acid (ca.
1 mg), and also small amounts of 4-O-glucosyl-ferulic acid as de-
scribed previously (Honda et al., 2005; Tatsuzawa et al., 2006).
4.4.4.1.
Cyanidin
3-[2-(2-sinapoyl-glucosyl)-glucoside]-5-gluco-
side. UV–Vis (in 0.1% HCl–MeOH), kmax 528, 321, 295, 281 nm,
Eacyl/Emax (%) 74, E440/Emax (%) 14, +AlCl3 + shift; TLC (Rf ꢁ100)
BAW 33, BuHCl 7, 1% HCl 38, AHW 70; HPLC (Rt (min)) 15.3;
FAB-MS m/z 979 [M]+ (calc. for C44H51O25); HR-FAB MS calc.
979.2719. Found 979.2705; 1H NMR (500 MHz, CF3CO2D–DMSO-
d6 = 1:9, an internal standard of TMS); d cyanidin: 8.89 (s, H-4),
6.69 (br s, H-6), 7.04 (br s, H-8), 7.98 (br s, H-20), 7.16 (d,
J = 8.9 Hz, H-50), 8.40 (br d, J = 8.9 Hz, H-60). Sinapic acid: 6.93
(2H, s, H-2 and -6), 6.45 (d, J = 15.9 Hz, H-a), 7.48 (d, J = 15.9 Hz,
H-b), 3.85 (s, 2 ꢁ OCH3). Glucose A: 5.58 (d, J = 7.6 Hz, H-1), 4.09
(dd, J = 7.6 and 9.2 Hz, H-2), 3.59 (m, H-3), 3.31 (t, J = 9.2 Hz, H-
4), 3.55–3.85 (H-5, H-6a and -6b). Glucose B: 5.14 (d, J = 7.7 Hz,
H-1), 3.51 (m, H-2), 3.42 (m, H-3), 3.50–3.60 (H-4), 3.31 (t,
J = 9.2 Hz, H-5), 3.55–3.85 (H-6a and -6b). Glucose C: 5.20 (d,
4.4.2.1.
Deacylanthocyanin
(cyanidin
3-sophoroside-5-gluco-
side). Dark purple-red powders: for UV–Vis and TLC see Table 1;
For 1H and 13C NMR spectra, see Tables 5 and 6, HR-FAB MS calc.
for 773.2140 (C33H41O21). Found 773.2126.
4.4.2.2. 4-O-Glucosyl-p-coumaric acid. UV–Vis in MeOH (kmax) 314,
296, (240), (217) nm; TLC (Rf ꢁ100) BAW 86, BuHCl 75, 1% HCl
19, AHW 51; color dark-blue (UV), dark-violet (UV + NH3 gas);
HPLC (Rt (min)) 7.1.
4.4.2.3. 4-O-Glucosyl-ferulic acid. UV–Vis in MeOH (kmax) 321, (298),
233, (214) nm; TLC (Rf ꢁ100) BAW 83, BuHCl 72, 1% HCl 13, AHW

3150
N. Saito et al. / Phytochemistry 69 (2008) 3139–3150
Harborne, J.B., 1984. Phytochemical Methods, second ed. Chapman and Hall,
London.
J = 7.9 Hz, H-1), 4.67 (dd, J = 7.9 and 9.5 Hz, H-2), 3.37 (m, H-3), 3.19
(m, H-4), 3.08 (m, H-5), 3.55–3.85 (H-6a and -6b).
Honda, T., Saito, N., 2002. Recent progress in the chemistry of polyacylated
anthocyanins as flower color pigments. Heterocycles 56, 633–692.
Honda, T., Tatsuzawa, F., Kobayashi, N., Kasai, H., Nagumo, S., Shigihara, A., Saito, N.,
2005. Acylated anthocyanins from the violet-blue flowers of Orychophragonus
violaceus. Phytochemistry 66, 1844–1851.
Idaka, E., Suzuki, K., Yamakita, H., Ogawa, T., Kondo, T., Goto, T., 1987a. Structure of
monoacylated anthocyanins isolated from red cabbage, Brassica oleracea.
Chemistry Letters, 145–148.
Idaka, E., Yamakita, H., Ogawa, T., Kondo, T., Yamamoto, M., Goto, T., 1987b.
Structure of three diacylated anthocyanins isolated from red cabbage, Brassica
oleracea. Chemistry Letters, 1213–1216.
Mori, M., Kondo, T., Toki, K., Yoshida, K., 2006. Structure of anthocyanin from the
blue petals of Phacelia campanularia and its blue flower color development.
Phytochemistry 67, 622–629.
Saito, N., Tatsuzawa, F., Nishiyama, A., Yokoi, M., Shigihara, A., Honda, T., 1995a.
Acylated cyanidin 3-sambubioside-5-glucoside in Matthiola incana.
Phytochemistry 38, 1027–1032.
Saito, N., Tatsuzawa, F., Yoda, K., Yokoi, M., Kasahara, K., Iida, S., Shigihara, A., Honda,
T., 1995b. Acylated cyanidin glycosides in the violet-blue flowers of Ipomoea
purpurea. Phytochemistry 40, 1283–1289.
Saito, N., Tatsuzawa, F., Hongo, A., Win, K.W., Yokoi, M., Shigihara, A., Honda, T.,
1996. Acylated pelargonidin 3-sambubioside-5-glucoside in Matthiola incana.
Phytochemistry 41, 1613–1630.
4.4.4.2.
Cyanidin
3-[2-(2-feruloyl-glucosyl)-glucoside]-5-gluco-
side. UV–Vis (in 0.1% HCl–MeOH), kmax 529, (322), 296, 282 nm,
Eacyl/Emax (%) 110, E440/Emax (%) 15, +AlCl3 + shift; TLC (Rf ꢁ100)
BAW 39, BuHCl 8, 1% HCl 36, AHW 69; HPLC (Rt (min)) 15.5;
FAB-MS m/z 949 [M]+ (calc. for C43H49O24, 949.2613); 1H NMR
(500 MHz, CF3CO2D–DMSO-d6 = 1:9, an internal standard of
TMS); d cyanidin: 8.89 (s, H-4), 6.99 (br s, H-6), 7.05 (br s, H-8),
8.00 (br s, H-20), 7.17 (d, J = 8.9 Hz, H-50), 8.41 (br d, J = 8.9 Hz, H-
60). Ferulic acid: 7.24 (br s, H-2), 6.82 (d, J = 8.3 Hz, H-5), 7.07 (br
d, J = 8.3 Hz, H-6), 6.42 (d, J = 15.9 Hz, H-a), 7.50 (d, J = 15.9 Hz, H-
b), 3.84 (s, OCH3). Glucose A: 5.58 (d, J = 8.0 Hz, H-1), 4.10 (m, H-
2), 3.57 (m, H-3), 3.39 (m, H-4), 3.55–3.90 (H-5, H-6a and -6b). Glu-
cose B: 5.14 (d, J = 7.3 Hz, H-1), 3.51 (m, H-2), 3.39 (m, H-3), 3.30
(m, H-4), 3.54 (m, H-5), 3.55–3.90 (H-6a and -6b). Glucose C:
5.19 (d, J = 8.0 Hz, H-1), 4.66 (m, H-2), 3.41 (m, H-3), 3.18 (m, H-
4), 3.09 (m, H-5), 3.55–3.90 (H-6a and -6b).
Tatsuzawa, F., Saito, N., Shinoda, K., Shigihara, A., Honda, T., 2006. Acylated cyanidin
3-sambubioside-5-glucosides in three garden plants of the Cruciferae.
Phytochemistry 67, 1287–1295.
References
Tatsuzawa, F., Toki, K., Saito, N., Shinoda, K., Shigihara, A., Honda, T., 2007. Four
acylated cyanidin 3-sambubioside-5-glucosides from the purple-violet flowers
of Lobularia maritima. Heterocycles 71, 1117–1135.
Tatsuzawa, F., Saito, N., Toki, K., Shinoda, K., Shigihara, A., Honda, T., 2008.
Triacylated cyanidin 3-(3X-glucosylsambubioside)-5-glucosides from the
flowers of Malcolmia maritima. Phytochemistry 69, 1033–1040.
Andersen, Ø.M., Jordheim, M., 2006. The anthocyanins. In: Andersen, Ø.M.,
Markham, K.R. (Eds.), Flavonoids: Chemistry, Biochemistry and Applications.
CRC Press, Boca Raton, pp. 471–551.
Harborne, J.B., 1967. Comparative Biochemistry of Flavonoids. Academic Press,
London and New York.