Triacylated cyanidin 3-(3X-glucosylsambubioside)-5-glucosides from the flowers of Malcolmia maritima

Article abstract of DOI:10.1016/j.phytochem.2007.08.031

Three acylated cyanidin 3-(3^X-glucosylsambubioside)-5-glucosides (1-3) and one non-acylated cyanidin 3-(3^X-glucosylsambubioside)-5-glucoside (4) were isolated from the purple-violet or violet flowers and purple stems of Malcolmia maritima (L.) R. Br (the Cruciferae), and their structures were determined by chemical and spectroscopic methods. In the flowers of this plant, pigment 1 was determined to be cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-3-O-(β-d-glucopyranosyl)-β-d-xy lopyranosyl)-6-O-(trans-p-coumaroyl)-β-d-glucopyranoside]-5-O-[6-O- (malonyl)-(β-d-glucopyranoside) as a major pigment, and a minor pigment 2 was determined to be the cis-p-coumaroyl isomer of pigment 1. In the stems, pigment 3 was determined to be cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-3-O-(β-d-glucopyranosyl)-β-d-xy lopyranosyl)-6-O-(trans-p-coumaroyl)-β-d-glucopyranoside]-5-O- (β-d-glucopyranoside) as a major anthocyanin, and also a non-acylated anthocyanin, cyanidin 3-O-[2-O-(3-O-(β-d-glucopyranosyl)-β-d-xylopyranosyl)-β-d-glucopyranoside]-5-O-(β-d-glucopyranoside) was determined to be a minor pigment (pigment 4). In this study, it was established that the acylation-enzymes of malonic acid has important roles for the acylation of 5-glucose residues of these anthocyanins in the flower-tissues of M. maritima; however, the similar enzymatic reactions seemed to be inhibited or lacking in the stem-tissues.

Full text of DOI:10.1016/j.phytochem.2007.08.031

Available online at www.sciencedirect.com
PHYTOCHEMISTRY
Phytochemistry 69 (2008) 1029–1036
www.elsevier.com/locate/phytochem
Triacylated cyanidin 3-(3X-glucosylsambubioside)-5-glucosides
from the flowers of Malcolmia maritima
a,
b
a
c
Fumi Tatsuzawa *, Norio Saito , Kenjiro Toki , Koichi Shinoda ,
b
b
Atsushi Shigihara , Toshio Honda
a
Faculty of Horticulture, Minami-Kyushu University, Takanabe, Miyazaki 884-0003, Japan
Faculty of Pharmaceutical Sciences, Hoshi University, Shinagawa, Tokyo 142-8501, Japan
National Agricultural Research Center for Hokkaido Region, Sapporo, Hokkaido 062-8555, Japan
b
c
Received 4 July 2007; received in revised form 30 July 2007
Available online 24 October 2007
Abstract
Three acylated cyanidin 3-(3X-glucosylsambubioside)-5-glucosides (1–3) and one non-acylated cyanidin 3-(3X-glucosylsambubioside)-
5-glucoside (4) were isolated from the purple-violet or violet flowers and purple stems of Malcolmia maritima (L.) R. Br (the Cruciferae),
and their structures were determined by chemical and spectroscopic methods. In the flowers of this plant, pigment 1 was determined to be
cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-3-O-(b-D-glucopyranosyl)-b-D-xylopyranosyl)-6-O-(trans-p-coumaroyl)-b-D-glucopyranoside]-
5-O-[6-O-(malonyl)-(b-D-glucopyranoside) as a major pigment, and a minor pigment 2 was determined to be the cis-p-coumaroyl isomer
of pigment 1. In the stems, pigment 3 was determined to be cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-3-O-(b-D-glucopyranosyl)-b-D-xylo-
pyranosyl)-6-O-(trans-p-coumaroyl)-b-D-glucopyranoside]-5-O-(b-D-glucopyranoside) as a major anthocyanin, and also a non-acylated
anthocyanin, cyanidin 3-O-[2-O-(3-O-(b-D-glucopyranosyl)-b-D-xylopyranosyl)-b-D-glucopyranoside]-5-O-(b-D-glucopyranoside) was
determined to be a minor pigment (pigment 4). In this study, it was established that the acylation-enzymes of malonic acid has important
roles for the acylation of 5-glucose residues of these anthocyanins in the flower-tissues of M. maritima; however, the similar enzymatic
reactions seemed to be inhibited or lacking in the stem-tissues.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Malcolmia maritima (L.) R. Br; Cruciferae; Acylated anthocyanins; Cyanidin 3-(3X-glucosylsambubioside)-5-glucoside; p-Coumaric acid;
Sinapic acid; Malonic acid; Flower color
1. Introduction
Lobularia maritima, and Lunaria annua (Tatsuzawa et al.,
2006, 2007). As part of our continuing work, we are inter-
ested in the structures of the floral anthocyanins of M. mari-
tima, since anthocyanins of this plant have not been
thoroughly studied.
In this paper, we report the structure elucidation of
cyanidin 3-(3X-glucosylsambubioside)-5-glucoside, a novel
cyanidin glycoside pattern, and also its three acylated
anthocyanin derivatives in M. maritima of the Cruciferae.
Malcolmia maritima (L.) R. Br. (Virginia stock in Eng-
lish) is a plant species native mainly to the Mediterranean
region, and cultivated as a popular annual garden plant
with white, pink, purple-violet, and violet flowers.
In the continuing work on flower color variation due to
acylated anthocyanins of the ornamental plants in the Cru-
ciferae, we have already reported the distribution of struc-
turally complicated acylated anthocyanins in the flowers of
Matthiola incana (Saito et al., 1995, 1996), Orychophrag-
onus violaceus (Honda et al., 2005) and Cheiranthus cheiri,
2. Results and discussion
*
Four anthocyanin pigments (1 –4) (Figs. 1 and 2) were
Corresponding author. Tel./fax: +81 983 22 6835.
found in the methanol–acetic acid–water (MAW: 4:1:5,
E-mail address: fumi@nankyudai.ac.jp (F. Tatsuzawa).
0031-9422/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.08.031

1030
F. Tatsuzawa et al. / Phytochemistry 69 (2008) 1029–1036
v/v/v) extracts from purple-violet to violet flowers, and also
purple stems of M. maritima by HPLC analysis (Fig. 2).
Their frequencies as estimated by HPLC were pigment 1
(64.4%), pigment 2 (8.6%) and pigment 3 (2.3%) in the
MAW extract from their flowers. On the other hand, in
the MAW extract from its stems pigment 1 (1.5%), pigment
3 (42.3%) and pigment 4 (3.8%) were obtained together
with three other small pigment peaks.
These four pigments (1–4) were extracted from the mix-
ture of flowers and stems with 5% HOAc, 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
(Tatsuzawa et al., 2006, 2007). The chromatographical
data and spectral properties of these pigments are shown
in Table 1.
Acid hydrolysis of all four pigments (1–4) resulted in
cyanidin, glucose, and xylose. Moreover, p-coumaric acid
and sinapic acid were detected in the hydrolysates of pig-
ments 1, 2 and 3, and also malonic acid was detected in
the hydrolysates of pigments 1 and 2 by TLC (Harborne,
1984). Alkaline hydrolysis of pigments 1–3 resulted in only
one deacylated anthocyanin, whose structure was identified
to be pigment 4 by the analyses of TLC and HPLC (Table
1). The deacylanthocyanin was hydrolyzed with 2 N HCl
by heating in a water bath of about 80–100 ꢁC for 1, 5,
and 10 min. By the analysis of TLC and HPLC, the four
intermediary glucosides (3-glucoside, 5-glucoside, 3,5-dig-
lucoside, 3-sambubioside-5-glucoside of cyanidin) as well
as deacylanthocyanin were detected in three partial hydrol-
ysates. From these results, the structure of the deacylanth-
ocyanin (pigment 4) was presumed to be a glucosyl
cyanidin 3-sambubioside-5-glucoside. The structures of
the four anthocyanins (pigments 1–4) were further eluci-
dated based on the analyses of their 1H NMR spectra
(500 MHz) and 13C NMR (125.78 MHz) in DMSO-d6–
CFCOOD (9:1), including 2D COSY, NOESY, 1H–13C
HMQC, 1H–13C HMBC and negative difference NOE
(DIFNOE) spectra.
2.1. Pigment 4 and deacyl pigments 1, 2 and 3
The molecular ions [M]+ of pigment 4 and deacyl pig-
ments 1, 2 and 3 were observed at m/z 905 (C38H49O25),
indicating that these pigments are composed of cyanidin
with three molecules of glucose and one molecule of xylose.
The elemental components of pigment 4 were confirmed by
measuring its high-resolution FABMS (HRMS), and the
mass data obtained are summarized in Section 4.4. As men-
tioned before deacyl pigments 1, 2 and 3 are identical with
pigment 4. Therefore, only the structure elucidation of pig-
ment 4 was carried out as follows.
From the analysis of the 1H NMR spectra of pigment 4,
the chemical shifts of six aromatic protons of cyanidin were
assigned as shown in Table 2. Also four signals of anomeric
protons were observed at d 5.59 (d, J = 7.6 Hz, glu A), d
5.09 (d, J = 7.6 Hz, glu B), d 4.80 (d, J = 8.0 Hz, xylose)
and d 4.34 (d, J = 7.7 Hz, glu C). Based on the observed
coupling constants (Table 2), the four sugars were assumed
to have a b-pyranose form.
The linkages and/or positions of the attachment of the
sugar groups in this pigment whose structure was presumed
to be glucosyl cyanidin 3-sambubioside-5-glucoside by the
analysis of its partial hydrolysate were mainly determined
by using 2D COSY, NOEDIF and HMBC experiments.
By NOEDIF experiments, strong NOEs were observed
between H-4 of cyanidin and H-1 of glu A, and H-6 of
cyanidin and H-1 of glu B indicating that cyanidin was gly-
cosylated with glu A at OH-3 of cyanidin and also glycos-
ylated with glu B at OH-5 of cyanidin. A proton signal (d
3.94, t, J = 8.2 Hz) shifted to a lower magnetic field was
assigned to H-2 of glu A by the analysis of 2D COSY spec-
trum of pigment 4. This resonance was correlated to the
13C-1 (d 103.9) of xylose and also to the resonance of
H-1 (xylose, d 4.80) was correlated to the 13C-2 (d 80.5)
of glu A in the HMBC spectrum, supporting that xylose
was linked to OH-2 of glu A forming sambubiose. Further-
more, a strong NOE was observed at H-3 of xylose by the
irradiation of NOEDIF experiment at H-1 of glu C
indicating that glu C was linked to the OH-3 of xylose. This
result was confirmed by the analysis of HMBC spectrum
(Fig. 1).
Therefore, pigment 4 and deacyl pigments 1–3 were
determined to be cyanidin 3-O-[2-O-(3-O-(b-D-glucopyr-
anosyl)-b-D-xylopyranosyl)-b-D-glucopyranoside]-5-O-b-D-
glucopyranoside, which is a new cyanidin glycoside in
plants (Harborne and Baxter, 1999; Andersen and Jord-
heim, 2006).
2.2. Pigment 1
The molecular ion [M]+ of pigment 1 was observed at
m/z 1343 (C61H67O34) indicating that pigment 1 is com-
posed of cyanidin with three molecules of glucose, one mol-
ecule each of xylose, as well as sinapic, p-coumaric and
malonic acids. The elemental components were confirmed
by measuring its HRMS, and the mass data obtained were
Fig. 1. Pigment 1, 2 and 3 in the flowers and stems of Malcolmia mantima.
Pigment 1 and 3 R = trans, Pigment 2, R = cis. Observed main NOE’s are
indicated by arrows. Observed HMBCs are indicated by dotted arrows.

F. Tatsuzawa et al. / Phytochemistry 69 (2008) 1029–1036
1031
Fig. 2. Comparative HPLC profiles of anthocyanin extracts of Malcolmia maritima. 1: cyanidin 3-[2-(2-(trans-sinapoyl)-3-(glucosyl)-xylosyl)-6-(trans-p-
coumaroyl)- glucoside]-5-[6-(malonyl)-glucoside] 2: cyanidin 3-[2-(2-(trans-sinapoyl)-3-(glucosyl)-xylosyl)-6-(cis-p-coumaroyl)-glucoside]-5-[6-(malonyl)-
glucoside] 3: demalonyl pigment 1: cyanidin 3-[2-(2-(trans-sinapoyl)-3-(glucosyl)-xylosyl)-6-(trans-p-coumaroyl)-glucoside]-5-glucoside 4: deacylanthocy-
anin: cyanidin 3-[2-(3-(glucosyl)-xylosyl)-glucoside]-5-glucoside.
summarized in Section 4.4. The detailed structure was elu-
cidated based on the analysis of the NMR spectra.
moieties were observed in the region of d 5.71–3.06, where
the four anomeric protons exhibited at d 5.71 (d,
J = 8.0 Hz, glu A), d 5.20 (d, J = 8.2 Hz, xylose), d 5.19
(d, J = 8.0 Hz, glu B), and d 4.34 (d, J = 8.0 Hz, glu C),
respectively. Based on the observed coupling constants
(Table 2), the four sugars were assumed to be in the b-pyra-
nose form. The linkages and/or positions of the attach-
ments of the sugar and acyl groups were determined
based on 2D COSY, NOESY, NOEDIF and HMBC
experiments.
The chemical shifts of 12 aromatic protons of cyanidin,
sinapic acid and p-coumaric acid moieties with their cou-
pling constants were assigned as shown in Table 2. Six pro-
tons were assigned to two methyl groups of sinapic acid.
Four olefinic proton signals of sinapic and p-coumaric
acids, with their large coupling constants (J = 15.9 and
15.9 Hz), showed that those acids were present in the trans
configuration (Table 2). The chemical shifts of the sugar

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F. Tatsuzawa et al. / Phytochemistry 69 (2008) 1029–1036
Table 1
Chromatographic and spectral properties of anthocyanins in flowers of Malcolmia maritima
Anthocyaninsa
Rf value (·100)
Spectral data in 0.1% HCl–MeOH
AHW kmax (nm) Eacyl E440
HPLC
Rt(min)
FAB mass
[M]+
BAW BuHCl 1%
/
/
AlCl3
HCl
Emax
Emax
Pigment 1
Pigment 2
Pigment 3
Pigment 4
Demalonyl pigment 1
Deacylanthocyanin
Cy 3-sambubioside-5-
glucoside
18
13
19
3
19
3
21
18
15
4
15
4
41
81
86
78
64
78
64
58
532,320,298,282
535,316,(298),279 121
529,319,299,281
526,278
529,319,299,281
526,278
112
14
18
13
14
13
14
17
+
+
+
+
+
+
+
31.9
25.0
30.4
13.2
30.4
13.2
12.5
1343
1343
1257
905
1257
905
–
57
36
32
36
32
23
128
–
128
–
5
6
528,273
–
Cy 3-sophoroside-5-glucoside
a
4
4
38
66
526,279
–
13
+
10.9
–
Pigment 1: cyanidin 3-[2-(2-(trans-sinapoyl)-3-(glucosyl)-xylosyl)-6-(trans-p-coumaroyl)-glucoside]-5-[6-(malonyl)-glucoside].Pigment 2: cyanidin 3-[2-
(2-(trans-sinapoyl)-3-(glucosyl)-xylosyl)-6-(cis-p-coumaroyl)-glucoside]-5-[6-(malonyl)-glucoside].Pigment 3: demalonyl pigment 1: cyanidin 3-[2-(2-(trans-
sinapoyl)-3-(glucosyl)-xylosyl)-6-(trans-p-coumaroyl)-glucoside]-5-glucoside.Pigment 4: deacylanthocyanin: cyanidin 3-[2-(3-(glucosyl)-xylosyl)-glucoside]-
5-glucoside.
A proton signal (d 4.05 dd, J = 8.0 and 8.9 Hz) detected
at a lower magnetic field, was assigned to H-2 of glu A by
the analysis of its 2D COSY spectrum. This result sup-
ported that the xylose group was linked to OH-2 of glu
A by forming sambubiose group at OH-3 of cyanidin. Five
characteristic proton signals shifted to a lower magnetic
field were also assigned to the methylene protons of glu
A (d 4.26 and 4.37, H-6a and b), glu B (d 4.03 and 4.39,
H-6a and b), and to a methine proton (d 4.83, dd, J = 8.2
and 8.8 Hz, H-2) of xylose. Thus, three hydroxyl groups
of the sugar moieties, OH-6s of glu A and B and OH-2
of xylose, were assumed to be acylated with three molecules
of acids. By irradiation at H-1 of glu A, a strong NOE was
observed at H-4 of cyanidin together with rather weak
NOEs at H-2, ꢀ6 and -a of p-coumaric acid supporting
that glu A was attached to OH-3 of cyanidin through a gly-
cosidic bond. Moreover, glu A was presumed to be acyl-
ated with p-coumaric acid at OH-6.
In NOESY spectrum, the correlations between H-6 of
cyanidin and H-1 of glu B, H-2 of glu A and H-1 of xylose,
and H-3 of xylose and H-1 of glu C were observed, respec-
tively, revealing the glycosylation patterns; such as OH-5 of
cyanidin was glycosylated with glu B, OH-2 of glu A with
xylose, and OH-3 of xylose with glu C. These results were
confirmed by NOEDIF experiments. Irradiation of H-2 of
xylose gave NOEs at H-a, -b, -2 and -6 of sinapic acid as
well as H-1 and H-3 of xylose. Therefore, OH-2 of xylose
was acylated with sinapic acid. These results were also con-
firmed by the analysis of HMBC spectrum (Fig. 1). Unfor-
tunately, the linkage between glu B and malonic acid could
not be confirmed by NOEDIF, NOESY and HMBC exper-
iments. To confirm the acyl position of malonic acid, dem-
alonyl pigment 1 was prepared according to the procedure
described previously (Saito et al., 2007). The structure
determination of demalonyl pigment 1 is performed as
described in the following Section 2.3 by the same proce-
dure as for pigment 3, and the structure of demalonyl pig-
ment 1 was confirmed to be identical with that of pigment 3
by the analysis of TLC, HPLC and spectroscopic data
(Table 1). Since demalonyl pigment 1 was confirmed to
be free from malonic acid at OH-6 of glu B, the structure
of pigment 1 was determined to be cyanidin 3-O-[2-O-(2-
O-(trans-sinapoyl)-3-O-b-D-glucopyranosyl)-b-D-xylopyr-
anosyl)-6-O- (trans-p-coumaroyl)-b-D-glucopyranoside]-5-
O-[6-O-(malonyl)-b-D-glucopyranoside], which is a new
anthocyanin in plants (Andersen and Jordheim, 2006;
Harborne and Baxter, 1999; Honda and Saito, 2002).
2.3. Pigment 3
The FAB mass spectrum of pigment 3 exhibited a
molecular ion at 1257 m/z in agreement with the mass
C58H65O31 (1257.350). The elemental components were
also confirmed by measuring its high-resolution FABMS:
calc. for C58H65O31: 1257.3501. Found: 1257.3523. As
shown in Table 1, pigment 3 was identical with demalonyl
pigment 1, therefore, structure determination of pigment 3
was carried out as follows.
1
The H NMR spectrum of pigment 3 showed the pres-
ence of one molecule of cyanidin, three molecules of glucose,
and one molecule each of xylose, p-coumaric acid and sina-
pic acid. By the analysis of its NMR spectra, it was revealed
that proton chemical shifts of pigment 3 were almost in
agreement with those of pigment 1 except for the proton sig-
nals of glu B and malonic acid moieties (Tables 2 and 3). In
particular, upfield shifts of methylene protons (d 3.78–3.45,
H-6a and b) of glu B in pigment 3 were observed in compar-
ison to those (d 4.03 and 4.39, H-6a and b) of pigment 1.
Therefore, OH-6 of glu B in pigment 3 was free from malon-
ic acid. Other proton signals of pigment 3 were assigned by
the same process as described for pigment 1 (Tables 2 and 3).
Thus, pigment 3 was determined to be cyanidin 3-O-[2-O-(2-
O-(trans-sinapoyl)-3-O-(b-D-glucopyranosyl)-b-D-xylopyr-
anosyl)-6-O- (trans-p-coumaroyl)-b-D-glucopyranoside]-5-
O-(b-D-glucopyranoside), which is a new anthocyanin
in plants (Andersen and Jordheim, 2006; Harborne and

F. Tatsuzawa et al. / Phytochemistry 69 (2008) 1029–1036
1033
Table 2
1H NMR spectroscopic data (d) of anthocyanins isolated from Malcolmia maritima (500 MHz, DMSO-d6–CF3COOD, TMS as an internal standard)
Pigment 1
Pigment 2
Pigment 3
Pigment 4
Cyanidin
4
8.71 s
8.56 s
8.67 s
8.79 s
6
8
20
50
60
6.99 brs
7.02 brs
8.01 brs
7.07 d(8.6)
8.47 brd(8.6)
6.86 d(2.2)
7.18 d(2.2)
7.96 d(2.2)
7.06 d(8.3)
8.41 dd(2.2, 8.3)
6.93 brs
6.96 brs
7.96 d(2.2)
7.02 d(8.9)
8.41 dd(2.2, 8.9)
6.95 d(1.8)
7.08 d(1.8)
8.03 d(2.2)
7.02 d(8.9)
8.34 dd(2.2, 8.9)
p-coumaric acid
2,6
3,5
a
7.30 d(8.0)
6.71 d(8.0)
6.25 d(15.9)
7.35 d(15.9)
7.29 d(8.3)
6.50 d(8.3)
5.69 d(12.8)
6.46 d(12.8)
7.33 d(8.6)
6.73 d(8.6)
6.21 d(15.9)
7.30 d(15.9)
b
Sinapic acid
2,6
a
b
7.02 s
7.01 s
6.96 s
6.54 d(15.9)
7.57 d(15.9)
3.80 s
6.51 d(15.9)
7.54 d(15.9)
3.84 s
6.48 d(15.9)
7.51 d(15.9)
3.80 s
OMe
Malonic acid
–CH2–
3.34 s
3.33 s
Glucose A
1
2
3
4
5.71 d(8.0)
4.05 dd(8.0, 8.9)
3.62 t*(8.5)
3.42 m
5.65 d(7.0)
4.05 dd(7.0, 7.9)
3.55 m
5.65 d(7.6)
4.00 t*(8.3)
3.58 t*(9.5)
3.38 m
5.59 d(7.6)
3.94 t*(8.2)
3.64 t*(8.9)
3.33 m
3.31 m
5
6a
6b
4.00 m
4.26 m
4.37 m
3.90 m
4.29 m
4.47 brd(12.5)
3.97 dd(6.1, 11.9)
4.23 m
4.31 m
3.46 m
3.54 m
3.69 brd(11.6)
Glucose B
1
2
5.19 d(8.0)
3.55 m
3.42 m
5.20 d(6.7)
3.53 m
3.20–3.90
5.03 d(7.6)
3.48 dd(7.6, 8.3)
3.33 m
5.09 d(7.6)
3.44 dd(7.6, 8.9)
3.35 m
3
4
5
6a
6b
3.26 m
3.28 m
4.03 m
4.39 brd(10.4)
3.24 m
3.48
3.45–3.78
3.28 m
3.40–3.50
3.62–3.73
]
4.35 m
4.35 m
]
]
Glucose C
1
2
3
4
4.34 d(8.0)
2.98 dd(8.0, 8.5)
3.14 t*(8.9)
3.06 t*(9.2)
3.26 m
4.32 d(7.3)
2.95 t*(8.3)
3.11 t*(8.6)
3.03 t*(9.2)
3.26 m
4.28 d(8.0)
2.94 t*(8.6)
3.07 t*(8.9)
3.01 t*(9.2)
3.24–3.90
4.34 d(7.7)
3.04 dd(7.7, 8.9)
3.18 m
3.10–3.37
5
6a
6b
3.25–3.90
3.20–3.90
]
]
]
]
]
]
Xylose
1
2
3
4
5.20 d(8.2)
4.83 dd(8.2, 8.8)
3.71 m
3.42 m
3.20–3.90
5.19 d(7.0)
4.80 t*(8.9)
3.69 m
3.40 m
3.20–3.90
5.15 d(7.7)
4.78 dd(7.7, 8.9)
3.68 t*(9.5)
3.18 m
4.80 d(8.0)
3.14 m
3.31 t*(8.5)
3.01 t*(9.2)
3.54 m
5a
5b
3.22–3.75
]
3.66 m
Coupling constants (J in Hz) in parentheses.
s = singlet, brs = broad singlet, t* = distorted triplet, m = multiplet, dd = double doublet.
Baxter, 1999; Honda and Saito, 2002). For further confir-
mation of the identity between the structures of pigment 3
and demalonyl pigment 1, demalonyl pigment 1 was pre-
pared by treatment of pigment 1 with 1 N HCl solution (Sai-
to and Harborne, 1992; Saito et al., 2007), and compared
with pigment 3 directly. The chromatographic and spectro-
scopic data of demalonyl pigment 1 are shown in Table 1.
These data including the analyses of mass and NMR spectra
of demalonyl pigment 1 and pigment 3 revealed that both
pigments were quite identical with each other.

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F. Tatsuzawa et al. / Phytochemistry 69 (2008) 1029–1036
Table 3
Table 3 (continued)
13C NMR spectroscopic data (d) of anthocyanins isolated from Malcolmia
maritima (125.78 MHz, DMSO-d6–CF3COOD, d values in ppm)
Pigment 1
Pigment 3
Pigment 4
Xylose
Pigment 1
Pigment 3
Pigment 4
1
2
3
4
5
101.4
72.1
83.7
–
101.4
72.2
83.7
77.2
60.9
103.9
73.0
87.4
70.4
61.3
Cyanidin
2
3
4
5
6
7
8
162.3
144.4
131.3
155.0
105.1
167.4
96.1
162.3
144.4
131.7
155.2
105.0
167.7
96.3
162.4
144.6
132.2
155.2
104.1
167.5
96.1
56.4
2.4. Pigment 2
9
155.1
111.4
119.7
117.6
146.7
155.5
116.7
128.7
155.3
111.7
119.8
117.7
146.7
155.5
116.7
128.7
155.3
111.5
119.6
117.8
146.4
155.3
116.8
128.2
The molecular ion [M]+ of pigment 2 was observed at
m/z 1343 (C61H67O34) which was identical with that of pig-
ment 1 supporting that pigment 2 is composed of cyanidin
with three molecules of glucose, one molecule each of
xylose, sinapic acid, p-coumaric acid and malonic acid.
The elemental components were also confirmed by measur-
ing its HRMS, and the data obtained were summarized in
Section 4.4. The 1H NMR spectrum of pigment 2 was sim-
ilar to that of pigment 1 except for signals of p-coumaric
acid moiety as shown in Table 2. Particularly, the chemical
shifts of the olefinic protons were shifted to a higher mag-
netic field at d 5.69 and 6.46 with smaller coupling con-
stants (J = 12.8 and 12.8 Hz) in comparison with those (d
6.25, J = 15.9 Hz, and d 7.35, J = 15.9 Hz) of pigment 1.
Therefore, the configuration of p-coumaric acid was con-
firmed to be in the cis-form, and the structure of pigment
2 was determined to be cyanidin 3-O-[2-O-(2-O-(trans-sina-
poyl)-3-O-(b-D-glucopyranosyl)-b-D-xylopyranosyl)-6-O-
(cis-p-coumaroyl)-b-D-glucopyranoside]-5-O-[6-O-(malonyl)-
b-D-glucopyranoside], which is a new anthocyanin in plants.
The cis-configuration was unambiguously confirmed by
comparison of TLC and HPLC (Table 1) with an authentic
sample prepared from its trans-derivative (pigment 1) as
shown in Section 4 (Saito and Harborne, 1992).
10
10
20
30
40
50
60
p-Coumaric acid
1
2,6
3,5
4
7a
8b
9
125.0
130.5
115.7
160.0
113.8
145.4
166.9
125.1
130.7
116.0
160.1
113.8
145.4
166.9
Sinapic acid
1
2,6
3,5
4
7a
8a
9
125.0
106.5
148.3
138.5
114.3
145.2
166.1
56.4
125.1
106.6
148.3
138.6
115.8
145.4
166.1
56.4
OMe
Malonic acid
–CH2–
COO
41.3
167.0
168.1
COO
Glucose A
1
2
3
4
5
6
97.8
77.2
76.6
–
73.3
64.6
97.9
77.9
77.3
70.8
73.5
63.2
98.9
80.5
77.0
69.2
73.1
60.5
3. Concluding remarks
As already described (Tatsuzawa et al., 2006), there are
two glycosidic patterns in the Cruciferae at OH-3 of antho-
cyanidins, 3-sambubioside and 3-sophoroside. In this study,
it is demonstrated that an additional new 3-glycosidic pat-
tern is found in Malcolmia maritima, cyanidin 3-(3X-glucos-
ylsambubioside)-5-glucoside. This glycosidic pattern in M.
maritima is comparable to the former pattern of 3-sambu-
bioside; however, it is noteworthy that this pigment has
novel glycosidic pattern, 3X-glucosylsambubioside, in
which sumbubioside is xylosyl(1–2)glucose. The observed
glycosidic pattern seems to be unique based on the consid-
eration of previous observations. This is the first report of
the occurrence of this trisaccharide in conjunction with
anthocyanins (Andersen and Jordheim, 2006) or flavonoids
(Williams, 2006). From a chemotaxonomical point of view,
this result is very interesting in the classification of M. mari-
tima in the Cruciferae, and the glycosylation of the trisac-
Glucose B
1
2
3
4
5
6
101.9
73.4
–
–
73.3
64.2
102.5
77.7
76.1
73.9
73.5
61.4
101.5
77.1
75.9
69.6
77.6
61.3
Glucose C
1
2
3
4
5
6
103.2
73.3
75.8
70.3
–
103.3
73.5
76.7
70.3
–
104.0
73.9
76.2
–
–
61.3
61.3
61.1

F. Tatsuzawa et al. / Phytochemistry 69 (2008) 1029–1036
1035
charide at OH-3 of anthocyanin indicated a advanced char-
acteristic in comparison with that of the sambubiose.
Regarding the acylations, the anthocyanins of M. mari-
tima exhibited common patterns like the anthocyanins in
the Cruciferae, in which OH-6 of glu A and OH-2 of glu
C or xylose in these pigments were acylated with hydroxy-
cinnamic acids, and also OH-6 of glu B was acylated with a
malonic acid (Giusti et al., 1998; Igarashi et al., 1990; Mori
et al., 2006; Otsuki et al., 2002). These acylations with
hydroxycinnamic acids in anthocyanin pigments are also
responsible for the bluing effect and also for stability of
the flower color of M. maritima (Honda and Saito, 2002).
ity value, b*(5.70)/a*(12.36)]. Flowers and stems were
collected from winter to spring seasons in Japan and dried
overnight at 40 ꢁC, and kept in a refrigerator at about 4 ꢁC.
The chromaticity values were recorded on a SE-2000 Spectro
Color Meter (Nippon Denshoku Industries Co., Ltd.).
4.3. Isolation of anthocyanins
Dried flowers (ca. 60 g) of Malcolmia maritima were
immersed in 5%HOAc–H2O (3L; HOAc–H2O, 1:19) at
room temp. for 5 h and extracted. The extract was passed
through a Diaion HP-20 (Mitsubishi Chemical’s Ion
Exchange Resins) column (90/ · 150 mm), on which acyl-
ated anthocyanins were adsorbed. Then, the column was
thoroughly washed with H2O (2 L) and eluted with 5%
HOAc–MeOH (500 mL) to recover the anthocyanins.
After concentration, the eluates were separated and puri-
fied with paper chromatography (PC) using BAW. The sep-
arated pigments 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
4 mL/min and monitoring at 530 nm. The solvent used
was as follows: a linear gradient elution for 16 min from
60% to 80 % solvent B in solvent A. Each fraction was
transformed to a Diaion HP-20 column, on which pig-
ments were adsorbed. Anthocyanin pigments were eluted
with 5% HOAc–MeOH followed by addition of excess of
Et2O, and then dried. The purified pigments from the flow-
ers were obtained as follows; pigment 1 (ca. 10 mg), pig-
ment 2 (ca. 3 mg) and pigment 3 (ca. 1 mg).
4. Experimental
4.1. General procedures
TLC was carried out on plastic coated cellulose sheets
(Merck) using eight 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, 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 phthalate spray
reagent (Harborne, 1984).
Analytical HPLC was performed on a LC 10A system
(Shimadzu), using a Waters C18 (4.6 / · 250 mm) column
at 40ꢁC with a flow rate of 1 mL/min and monitoring at
530 nm. 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).
Dried stems (ca. 100 g) of M. maritima was immersed in
5% HOAc–H2O (5 L) at room temp. for 5 h. The extract
was purified by the same process as for the dried flowers
as described above. The pigments were obtained as follows;
pigment 1 (ca. 1 mg), pigment 3 (ca. 20 mg) and pigment 4
(ca. 5 mg).
UV–Vis spectra were recorded on UV–Vis Multi Pur-
pose Spectrophotometer (MPS-2450, Shimadzu) in 0.1%
HCl-MeOH (from 200 to 700 nm).
High resolution FAB mass (FABMS) 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
CF3COO–DMSO-d6 (1:9). Chemical shifts are reported rel-
ative to a TMS internal standard (d), and coupling con-
stants are in Hz.
4.4. Analyses of anthocyanins
The identification of anthocyanins was carried out by
standard procedures involving deacylation with acid, and
both alkaline and acid hydrolyses (Harborne, 1984). The
data of TLC (Rf value), HPLC (Rt-min), UV–VIS (kmax),
and FABMS spectra are shown in Table 1 and also
described in Sections 4.4.1.– 4.4.4.
4.2. Plant materials
4.4.1. Pigment 1
Dark purple-red powder; for UV–Vis and TLC, see
Table 1; for H and C NMR spectra, see Tables 2 and
3; HR-FABMS calc. for C61H67O34: 1343.3514. Found:
1343.3481.
1
13
The seeds and seedling of Malcolmia maritima (L.) R. Br.
were purchased from Unwins Seeds Ltd. (Histon, Cam-
bridge, UK) and Sakata Co. Ltd (Yokohama, Japan), and
grown in greenhouses at the Experimental farm of Mina-
mikyushu University. The flowers exhibited from violet [Vio-
let 87B by Royal Horticultural Society color chart and
chromaticity value, b*(ꢀ36.48)/a*(36.42)] to a purple-violet
[Purple-Violet 81B, b*(ꢀ27.46)/a*(46.07)]. The stems exhib-
ited a purple [Purple 77A by RHS color chart and chromatic-
4.4.2. Pigment 2
Dark purple-red powder; for UV–Vis and TLC, see
1
13
Table 1; for H and C NMR spectra, see Tables 2 and
3; HR-FABMS calc. for C61H67O34: 1343.3514. Found:
1343.3494.

1036
F. Tatsuzawa et al. / Phytochemistry 69 (2008) 1029–1036
4.4.3. Pigment 3 (demalonyl pigment 1)
Dark purple-red powder; for UV–Vis and TLC, see
Table 1; for H and C NMR spectra, see Tables 2 and
3; HR-FABMS calc. for C58H65O31: 1257.3510. Found:
1257.3523.
with authentic cyanidin glycosides. Four intermediary glu-
cosides were found in these partial hydrolysates, and iden-
tified to be 3-glucoside (Rt; 15.9 min by HPLC, TLC;
AHW 0.10, BAW 0.15), 5-glucoside (Rt; 16.8, TLC;
AHW 0.12, BAW 0.25), 3,5-diglucoside (Rt;13.3, TLC;
AHW 0.22, BAW 0.07), and 3-sambubioside-5-glucoside
(Rt;12.6, TLC; AHW 0.58, BAW 0.05) of cyanidin as well
as deacylanthocyanin (Rt; 13.2, TLC; AHW 0.64, BAW
0.03).
1
13
4.4.4. Pigment 4 and deacyl pigments 1, 2 and 3
Dark red powder; for UV–Vis and TLC, see Table 1; for
1H and C NMR spectra, see Tables 2 and 3; HR-FAB-
13
MS calc. for C38H49O25: 905.2563. Found: 905.2550.
4.4.5. Deacylanthocyanin
References
Pigments 1 and 3 (ca.10 mg) were dissolved in 2 N
NaOH (1 mL) under a degassed syringe and allowed to
stand for 15 min. Then the solution was sufficiently acidi-
fied with 2 N HCl and evaporated in vacuo to dryness.
The residue was dissolved in 1% HCl–MeOH and applied
on TLC (BAW) to yield a deacylanthocyanin (ca. 5 mg).
The characteristic properties of deacylanthocyanin are
shown in Table 1.
Andersen, Ø.M., Jordheim, M., 2006. The anthocyanins. In: Andersen,
Ø.M., Markham, K.R. (Eds.), Flavonoids: Chemistry, Biochemistry
and Aplications. CRC Press, Boca Raton, pp. 471–551.
Giusti, M.M., Ghanadan, H., Wrolstad, R.E., 1998. Elucidation of the
structure and conformation of red radish (Raphanus sativus) anthocy-
anins using one- and two-dimensional nuclear magnetic resonance
techniques. J. Agric. Food Chem. 46, 4858–4863.
Harborne, J.B., 1984. Phytochemical Methods, 2nd ed. Chapman and
Hall, London.
Harborne, J.B., Baxter, H., 1999. The Handbook of Natural Flavonoids,
vol. 2. John Wiley & Sons, Chichester.
4.4.6. Demalonyl pigment 1
Pigment 1 (ca. 5 mg) was dissolved in 1 N HCl solution
(2 ml) and allowed to stand at room temperature for 2
weeks. At this point, demalonylated pigment 1 was formed
in its solution (Saito and Harborne, 1992; Saito et al.,
2007). Demalonylated pigment 1 was then absorbed on
the resin column of Diaion HP-20 and eluted with 5%
HOAc–MeOH from the column. After evaporation of the
solvent in vacuo, the concentrated residue was dissolved
in a small volume of 5% HOAc–MeOH followed by addi-
tion of excess Et2O, from which solids were then dried in
vacuo to give a demalonyl pigment 1 powder (ca. 3 mg).
The characteristic properties of demalonyl pigment 1 are
shown in Table 1.
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.
Igarashi, K., Abe, S., Satoh, J., 1990. Effects of Atsumi-kabu (Red turnip,
Brassica campestris L.) anthocyanin on serum cholesterol levels in
cholesterol-fed rats. Agric. Biol. Chem. 54, 171–175.
Mori, M., Nakagawa, S., Maeshima, M., Niikura, S., Yoshida, K., 2006.
Anthocyanins from the rhizome of Raphanus sativus, and change in the
composition during maturation. Heterocycles 69, 239–251.
Otsuki, T., Matsufuji, H., Takeda, M., Toyoda, M., Goda, Y., 2002.
Acylated anthocyanins from red radish (Raphanus sativus L.). Phyto-
chemistry 60, 79–87.
Saito, N., Harborne, J.B., 1992. Correlation between anthocyanin type,
pollinator and flower color in the Labiatae. Phytochemistry 31, 3009–
3015.
Saito, N., Tatsuzawa, F., Nishiyama, A., Yokoi, M., Shigihara, A.,
Honda, T., 1995. Acylated cyanidin 3-sambubioside-5-glucoside in
Matthiola incana. Phytochemistry 38, 1027–1032.
Saito, N., Tatsuzawa, F., Hongo, A., Win, K.W., Yokoi, M., Shigihara,
A., Honda, T., 1996. Acylated pelargonidin 3-sambubioside-5-gluco-
side in Matthiola incana. Phytochemistry 41, 1613–1630.
Saito, N., Tatsuzawa, F., Yazaki, Y., Shigihara, A., Honda, T., 2007. 7-
Polyacylated delphinidin 3,7-diglucosides from the blue flowers of
Leschenaultia cv. Violet Lena. Phytochemistry 68, 673–679.
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.
4.4.7. Isomerization of pigment 1 by sunlight
Pigment 1 (ca. 1 mg) was dissolved in 5% HOAc–MeOH
(2 ml), and exposed for sunlight for half an hours to trans-
form it into the cis-isomer by about 50% (Saito and Har-
borne, 1992). The isomer was isolated and purified by
TLC and HPLC. After this process, the purified isomer
of pigment 1 (ca. 0.5 mg) was obtained: UV–Vis in 0.1%
HCl–MeOH 535, 316, (298), 279 nm; TLC (Rf · 100)
BAW 13, BuHCl 18, 1% HCl 57, AHW 86; HPLC (Rt
(min)) 25.0; FAB mass [M]+ 1343.
4.4.8. Partial hydrolysates of deacylanthocyanin
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 maritime. Heterocycles 71, 1117–
1135.
Williams, C.A., 2006. Flavone and flavonol O-glycosides. In: Andersen,
Ø.M., Markham, K.R. (Eds.), Flavonoids: Chemistry, Biochemistry
and Applications. CRC Press, Boca Raton, pp. 749–856.
Three kinds of acid hydrolysis for deacylanthocyanin
were performed as following. Three 1 N HCl solutions of
deacylanthocyanin (containing ca. 1 mg in 5 mL 1 N HCl
each) were hydrolyzed by heating on a water bath (80–
100 ꢁC) for 1, 5, and 10 min, respectively. These three par-
tial hydrolysates were quickly analyzed by TLC and HPLC