Acylated cyanidin 3-sambubioside-5-glucosides from the purple-violet flowers of Matthiola longipetala subsp. bicornis (Sm) P. W. Ball. (Brassicaceae)

Article abstract of DOI:10.1016/j.phytol.2014.03.011

A novel acylated cyanidin 3-sambubioside-5-glucoside was isolated from the purple-violet flowers of Matthiola longipetala subsp. bicornis (Sm) P. W. Ball. (family: Brassicaceae), and determined to be cyanidin 3-O-[2-O-(2-O-(trans- feruloyl)-β-xylopyranosyl)-6-O-(trans-feruloyl)-β-glucopyranoside] -5-O-[6-O-(malonyl)-β-glucopyranoside] by chemical and spectroscopic methods. In addition, two known acylated cyanidin 3-sambubioside-5-glucosides, cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-β-xylopyranosyl)-6-O-(trans- feruloyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside] and cyanidin 3-O-[2-O-(β-xylopyranosyl)-6-O-(trans-feruloyl)-β- glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside] were identified in the flowers.

Full text of DOI:10.1016/j.phytol.2014.03.011

G Model
PHYTOL 688 1–5
Phytochemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
1
2
3
4
Acylated cyanidin 3-sambubioside-5-glucosides from the
purple-violet flowers of Matthiola longipetala subsp. bicornis
(Sm) P. W. Ball. (Brassicaceae)
*
Q1 Fumi Tatsuzawa
5
6
Laboratory of Olericultural and Floricultural Science, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan
A R T I C L E I N F O
A B S T R A C T
Article history:
A novel acylated cyanidin 3-sambubioside-5-glucoside was isolated from the purple-violet flowers of
Received 12 February 2014
Received in revised form 13 March 2014
Accepted 25 March 2014
Available online xxx
Matthiola longipetala subsp. bicornis (Sm) P. W. Ball. (family: Brassicaceae), and determined to be
cyanidin 3-O-[2-O-(2-O-(trans-feruloyl)-
O-[6-O-(malonyl)- -glucopyranoside] by chemical and spectroscopic methods. In addition, two known
acylated cyanidin 3-sambubioside-5-glucosides, cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)- -xylopyr-
anosyl)-6-O-(trans-feruloyl)- -glucopyranoside]-5-O-[6-O-(malonyl)- -glucopyranoside] and cyanidin
3-O-[2-O-( -xylopyranosyl)-6-O-(trans-feruloyl)- -glucopyranoside]-5-O-[6-O-(malonyl)- -glucopyr-
anoside] were identified in the flowers.
b-xylopyranosyl)-6-O-(trans-feruloyl)-b-glucopyranoside]-5-
b
b
b
b
Keywords:
b
b
b
Matthiola longipetala subsp. bicornis (Sm) P.
W. Ball.
ß 2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Brassicaceae
Anthocyanin
Purple-violet flower color
Tri-acylated cyanidin 3-sambubioside-5-
glucoside
7
8
1. Introduction
2012a), Moricandia ramburii (Tatsuzawa et al., 2012c), Moricandia 27
arvensis (Tatsuzawa et al., 2013), Orychophragmus violaceus (Honda 28
et al., 2005), and Raphanus sativus (Tatsuzawa et al., 2008b). In this 29
study, the structure elucidation of a new acylated cyanidin 3- 30
sambubioside-5-glucoside together with known ones from the 31
purple-violet flowers of M. longipetala subsp. bicornis was reported. 32
9
Matthiola longipetala subsp. bicornis is an ornamental plant with
deliciously scented purple-violet flowers, which open in the
evening (common name: Night Scented Stocks). In the limited
flavonoid studies reported for this species, flavonol glycosides have
been characterized from other subsp. livida (Marzouk et al., 2008).
Moreover, floral anthocyanins from the genus Matthiola have only
been characterized from the flowers of M. incana (Saito et al., 1995,
1996; Tatsuzawa et al., 2012a) and not from the flowers of M.
longipetala.
Recently, the ornamental plants in the Brassicaceae are known
as the source of complicated acylated anthocyanins in the flowers
of Arabis blepharophylla (Ito et al., 2013), Aubrieta ꢀ cultorum
(Tatsuzawa et al., 2012b), Cheiranthus cheiri, Lobularia maritima,
Lunaria annua (Tatsuzawa et al., 2006, 2007, 2010), Heliophila
coronopifolia (Saito et al., 2011), Hesperis matronalis (Tatsuzawa,
2012), Iberis umbellata (Saito et al., 2008), Ionopsidium acaule
(Tatsuzawa et al., 2014), Malcolmia maritima (Tatsuzawa et al.,
2008a), Matthiola incana (Saito et al., 1995, 1996; Tatsuzawa et al.,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
2. Results and discussion
33
More than 10 anthocyanins were detected in a methanol–acetic 34
acid–water (MAW) (4:1:5, v/v/v, 100 mL) extract from dried 35
purple-violet flowers of Matthiola longipetala subsp. bicornis (1 g) 36
by high performance liquid chromatography (HPLC) analysis 37
(Fig. 1). The percentage of major three pigments 1–3, calculated 38
as the total anthocyanin content by HPLC vis peak area at 530 nm, 39
was 40.1%, 30.4%, and 10.1%, respectively.
40
The major pigments were extracted from the purple-violet 41
flowers with 5% HOAc, followed by isolation using Diaion HP-20 42
(Mitsubishi Chemical’s Ion Exchange Resins, aromatic type 43
adsorbent) column chromatography (CC), preparative HPLC and 44
thin layer chromatography (TLC) (Tatsuzawa et al., 2012a).
45
Acid hydrolysis of 1–3 yielded cyanidin as their anthocyanidin 46
(Harborne, 1984), while also indicating glucose, xylose, hydro- 47
xycinnamic acid and malonic acid moieties. Moreover, trans-ferulic 48
*
Tel.: +81 19 621 6145; fax: +81 19 621 6145.
E-mail address: fumi@iwate-u.ac.jp
http://dx.doi.org/10.1016/j.phytol.2014.03.011
1874-3900/ß 2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Please cite this article in press as: Tatsuzawa, F., Acylated cyanidin 3-sambubioside-5-glucosides from the purple-violet flowers of
Matthiola longipetala subsp. bicornis (Sm) P. W. Ball. (Brassicaceae). Phytochem. Lett. (2014), http://dx.doi.org/10.1016/
j.phytol.2014.03.011

G Model
PHYTOL 688 1–5
2
F. Tatsuzawa / Phytochemistry Letters xxx (2014) xxx–xxx
the NOESY spectrum, the weak correlations between H-6a,b of Glc
A and H- 6.30) of ferulic acid (I) and H-2 of Xyl and H- 6.52)
96
97
a
(
d
a
(
d
of ferulic acid (II) were observed, establishing the acylation acid at
C-6 OH (Glc A) and C-2 OH (Xyl) as ferulic acid (I) and ferulic acid
(II), respectively.
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
In the HMBC spectrum, the correlations between the anomeric
proton of Glc A and C-3 carbon (
anomeric proton of Glc B and C-5 carbon (
anomeric proton of Xyl and C-2 carbon ( 77.3) of Glc A, methylene
protons of Glc A and COOH carbon ( 166.8) of ferulic acid (I),
methylene protons of Glc B and COOH carbon ( 167.0) of malonic
acid, methine proton of H-2 of Xyl and COOH carbon ( 166.3) of
ferulic acid (II), and methine proton of H-2 of Glc A and C-1 carbon
101.4) of Xyl were observed, establishing the glycosylation or
d
144.5) of cyanidin, the
d
154.9) of cyanidin, the
d
d
d
d
Fig. 1. HPLC profile (530 nm) and structure of acylated anthocyanins isolated from
the purple-violet flowers of Matthiola longipetala subsp. bicornis. Observed NOEs are
indicated by arrows. Observed HMBCs are indicated by dotted arrows. 1: pigment 1,
R15H, 2: pigment 2, R15OCH3.
(
d
acylation groups at C-3 OH (cyanidin), C-5 OH (cyanidin), C-2 OH
(Glc A), COOH (ferulic acid (I)), COOH (malonic acid), COOH (ferulic
acid (II)), and C-1 OH (Xyl) as Glc A, Glc B, Xyl, Glc A, Glc B, Xyl, and
Glc A, respectively (Fig. 1). Consequently, the structure of pigment
1 was determined to be cyanidin 3-O-[2-O-(2-O-(trans-feruloyl)-
49
50
51
52
53
54
55
56
57
58
59
60
61
62
acid, trans-sinapic acid and trans-ferulic acid, and trans-ferulic acid
were detected in the hydrolysates of 1, 2, and 3 by TLC,
respectively, with malonic acid found in all pigments (Harborne,
1984).
Alkaline hydrolysis of 1–3 yielded cyanidin 3-sambubioside-5-
glucoside. The deacyl anthocyanin structures tentatively identified
via co-TLC and co-HPLC with authentic cyanidin 3-sambubioside-
5-glucoside (see Section 4.4.1), prepared from Matthiola incana
(Tatsuzawa et al., 2012a) by alkaline hydrolysis.
b
-xylosyl)-6-O-(trans-feruloyl)-b-glucoside]-5-O-[6-O-(malo-
nyl)- -glucoside], which is a new anthocyanin in plants (Andersen
b
and Jordheim, 2006; Harborne and Baxter, 1999; Honda and Saito,
2002; Veitch and Grayer, 2008, 2011).
2.2. Pigment 2
119
The structures of 1–3 were confirmed based on the analyses of
The molecular ion [M]+ of 2 was observed at m/z 1211
(C56H59O30), indicating a cyanidin structure with two molecules of
glucose, and one molecule each of ferulic acid, sinapic acid and
malonic acid. The elemental components of pigment 2 were
confirmed by measuring its high resolution FAB-MS (see Section
4.4.3).
The 1H NMR spectrum of 2 was similar to that of 1, with 2
containing a sinapic acid moiety (II) instead of a ferulic acid moiety
(I) (Table 1). The two aromatic proton signals of sinapic acid (II)
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
their 1H (400 MHz), 13C (100 MHz) and 2D (COSY, NOESY, 1H–13
C
HMQC and 1H–13C HMBC) NMR spectra in DMSO-d6-CF3COOD
(9:1), as well as their fast atom bombardment mass spectra
(FABMS).
63
2.1. Pigment 1
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
The molecular ion [M]+ of 1 was observed at m/z 1181
(C55H57O29), indicating a cyanidin structure with two molecules
each of glucose and ferulic acid, and one molecule each of xylose
and malonic acid. The elemental components of pigment 1 were
confirmed by measuring its high resolution FAB-MS (see Section
4.4.2).
The chemical shifts of the 12 aromatic protons of the cyanidin
and ferulic acid moieties with their coupling constants were
assigned as shown in Table 1. Six protons were assigned to the two
methoxyl groups of ferulic acids. Two sets of two pairs of doublet
resonances, assigned to the four olefinic proton signals of the
ferulic acid moieties, indicated trans configuration for the acids
based on their coupling constants (J = 15.8 Hz each) (Table 1). The
chemical shifts of the sugar moiety protons were observed in the
were observed at
6.54 (d, J = 15.8 Hz, H-
a trans-sinapic acid moiety establishing 2 as cyanidin 3-O-[2-O-(2-
O-(trans-sinapoyl)- -xylopyranosyl)-6-O-(trans-feruloyl)- -glu-
copyranoside]-5-O-[6-O-(malonyl)- -glucopyranoside], which is
d
7.00 (H-2 and H-6). The olefinic proton signals
[d
a
) and 7.58(d, J = 15.8 Hz, H- )] indicating
b
b
b
b
a new anthocyanin in species M. longipetala, although this pigment
has been found in Matthiola incana (family: Brassicaceae) (Saito
et al., 1995; Tatsuzawa et al., 2012a). This structure was also
confirmed by analysis of its 13C, including 1H–13C HMQC and
1H–13C HMBC NMR spectra (Table 1, Fig. 1).
2.3. Pigment 3
139
region of
at 5.70 (d, J = 7.6 Hz, Glc A), 5.18 (d, J = 7.3 Hz, Glc B), and 5.15 (d,
J = 8.0 Hz, Xyl). Based on the observed coupling constants (Table 1),
these three sugars were assumed to be in the -pyranose forms.
d
5.70–3.21, with the three anomeric proton resonances
The molecular ion [M]+ of 3 was observed at m/z 1005
(C45H49O26), indicating a cyanidin structure with two molecules of
glucose, and one molecule each of ferulic acid and malonic acid.
The elemental components of pigment 3 were confirmed by
measuring its high resolution FAB-MS (see Section 4.4.4).
The 1H NMR spectrum of pigment 3 was similar to that of
pigment 1 except for the absence of the signals corresponding to
ferulic acid (II). The linkages and/or positions of the attachments of
the sugar and acyl groups in this pigment were confirmed by using
2D COSY and NOESY experiments. Therefore, the structure of
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
d
b
The linkages and/or positions of the attachments of the sugar and
acyl groups were determined based on 2D COSY and NOESY
experiments.
By application of the NOESY experiment, NOEs between H-1 of
Glc A and H-4 (
d
8.75) of cyanidin, H-1 of Glc B and H-6 (
d 6.97) of
cyanidin, and H-2 (
d
4.04) of Glc A and H-1 of Xyl were observed
(Fig. 1), supporting glycosylation of the C-3 and C-5 cyanidin
hydroxyl groups with Glc A and Glc B, respectively, while also
indicating a sambubiose structure between Glc A and Xyl.
Five characteristic downfield shifted proton signals were
pigment 3 was determined to be cyanidin 3-O-[2-O-(
anosyl)-6-O-(trans-feruloyl)- -glucopyranoside]-5-O-[6-O-(malo-
nyl)- -glucopyranoside], which is a new anthocyanin in species
b-xylopyr-
b
b
Matthiola longipetala, although this pigment has been found in
Lunaria annua and Matthiola incana (family: Brassicaceae) (Tatsu-
zawa et al., 2006, 2012a). This structure was also confirmed by
analysis of its 13C, including 1H–13C HMQC and 1H–13C HMBC NMR
spectra (Table 1, Fig. 1).
assigned to the methylene protons of Glc A (
6a and b) and Glc B ( 3.98 and 4.40, H-6a and b), and to a methine
proton ( 4.67, t, J = 8.7 Hz, H-2) of Xyl, indicating acylation of C-6
OH (Glc A and Glc B) and C-2 OH (Xyl) with three acid molecules. In
d 4.22 and 4.41, H-
d
d
Please cite this article in press as: Tatsuzawa, F., Acylated cyanidin 3-sambubioside-5-glucosides from the purple-violet flowers of
Matthiola longipetala subsp. bicornis (Sm) P. W. Ball. (Brassicaceae). Phytochem. Lett. (2014), http://dx.doi.org/10.1016/
j.phytol.2014.03.011

G Model
PHYTOL 688 1–5
F. Tatsuzawa / Phytochemistry Letters xxx (2014) xxx–xxx
3
Table 1
NMR spectroscopic data of anthocyanins from the flowers of Matthiola longipetala in DMSO-d6/CF3COOD (9:1).
1
2
3
1H
d(ppm)
13C
d(ppm)
1H
d(ppm)
13C
d(ppm)
1H
d(ppm)
13C
d(ppm)
Cyanidin
2
162.3
144.5
131.5
155.2
104.6
167.4
96.2
162.5
144.7
130.6
155.4
104.6
167.8
96.4
162.6
144.7
131.7
155.1
104.9
167.6
96.4
3
4
8.75
6.97
7.03
s
8.47
6.97
7.01
s
8.79
6.98
7.06
s
5
6
brs
brs
brs
brs
brs
brs
7
8
9
154.9
111.6
119.7
117.5
146.7
155.5
116.7
128.8
155.1
111.9
119.9
117.4
146.9
155.8
116.8
129.0
155.4
111.8
119.8
117.9
146.6
155.5
116.9
128.4
10
10
20
30
40
50
60
8.01
d(2.3)
7.99
d(2.2)
8.06
d(2.2)
7.07
8.50
d(8.8)
7.06
8.47
d(8.7)
7.08
8.40
d(8.8)
dd(2.3, 8.8)
dd(2.2, 8.7)
dd(2.2, 8.8)
Glucose A
1
5.70
4.04
3.63
3.44
3.97
4.22
4.41
d(7.6)
98.0
77.3
77.2
70.7
73.6
63.2
5.68
4.03
3.63
3.46
3.96
4.20
4.42
d(7.2)
t(7.8)
98.2
77.7
77.4
71.1
73.8
63.5
5.72
4.04
3.78
3.50
4.04
4.27
4.50
d(7.6)
98.6
80.7
76.8
70.0
74.1
63.4
2
t(8.2)
t(8.0)
3
t(8.8)
t(8.9)
t(9.0)
4
t(9.0)
m
t(9.3)
5
ddd(2.4, 7.2, 10.2)
dd(6.8, 11.7)
m
m
m
6a
6b
dd(6.8, 11.5)
brd(11.5)
dd(7.0, 11.9)
dd(1.8, 11.9)
Glucose B
1
5.18
3.51
3.44
3.21
3.78
3.98
4.40
d(7.3)
t(8.4)
t(9.0)
t(9.3)
m
101.7
73.3
75.7
69.7
74.3
64.1
5.16
3.54
3.43
3.22
3.77
3.90
4.38
d(8.3)
t(8.5)
m
101.9
73.5
76.0
70.0
74.5
64.3
5.18
3.57
3.43
3.25
3.78
3.96
4.43
d(7.6)
102.0
73.4
76.0
69.9
74.5
64.3
2
t(8.3)
3
t(9.0)
4
t(9.2)
m
t(9.3)
5
m
6a
6b
m
m
dd(6.6, 11.7)
dd(1.2, 11.7)
brd(10.5)
brd(11.0)
Xylose
1
5.15
4.67
3.41
3.44
3.90
3.21
d(8.0)
t(8.7)
m
101.4
73.9
74.8
70.1
66.4
5.14
4.66
3.43
3.46
3.90
3.22
d(8.6)
t(8.4)
m
101.5
74.2
75.0
70.3
66.6
4.75
3.05
3.18
3.27
3.57
3.00
d(7.8)
t(8.0)
t(8.8)
m
104.9
74.5
76.8
69.7
66.3
2
3
4
m
m
5a
5b
m
m
m
t(10.0)
m
t(11.0)
Ferulic acid (I)
1
126.1
111.8
147.9
149.5
115.6
123.0
114.1
145.1
55.7
126.3
111.8
148.1
149.7
116.0
123.1
114.3
145.7
55.9
125.7
111.8
148.0
149.6
115.7
123.2
114.3
145.7
55.8
2
7.03
brs
6.95
brs
7.06
brs
3
4
5
6.74
6.95
6.30
7.40
3.74
d(8.0)
6.73
6.91
6.27
7.42
3.72
d(8.3)
brd(8.3)
d(15.8)
d(15.8)
s
6.76
6.97
6.33
7.42
3.76
d(8.3)
brd(8.3)
d(15.8)
d(15.8)
s
6
a
b
dd(1.4, 8.0)
d(15.8)
d(15.8)
s
—OCH3
COOH
166.8
167.1
166.9
Ferulic or Sinapic acid (II)
1
125.6
111.6
148.2
149.5
115.8
123.2
115.4
145.5
55.9
125.8
106.6
148.6
138.9
148.6
106.6
115.8
145.7
56.5
2
7.32
d(1.4)
7.00
s
3
4
5
6.85
7.16
6.52
7.59
3.87
d(8.0)
6
a
b
dd(1.4, 8.0)
d(15.8)
d(15.8)
s
7.00
6.54
7.58
3.83
3.83
s
d(15.8)
d(15.8)
—OCH3
—OCH3
COOH
s
s
56.6
166.3
166.5
Malonic acid
—CH2
3.39
s
41.2
167.0
168.2
3.36
s
41.5
167.2
168.5
3.41
s
41.1
167.1
168.3
COOH
COOH
Coupling constants (J in Hz) in parentheses.
Please cite this article in press as: Tatsuzawa, F., Acylated cyanidin 3-sambubioside-5-glucosides from the purple-violet flowers of
Matthiola longipetala subsp. bicornis (Sm) P. W. Ball. (Brassicaceae). Phytochem. Lett. (2014), http://dx.doi.org/10.1016/
j.phytol.2014.03.011

G Model
PHYTOL 688 1–5
4
F. Tatsuzawa / Phytochemistry Letters xxx (2014) xxx–xxx
158
3. Concluding remarks
ion mode using 1:1 mixture of dithiothreitol and 3-nitrobenzyl
220
221
222
223
224
225
alcohol as a matrix. 1H (400 MHz) and 13C (100 MHz) NMR spectra
were measured on a JEOL JNM-AL-400 NMR spectrometer (JEOL)
using CF3COOD-DMSO-d6 (1:9) as a solvent. Chemical shifts are
reported on the d-scale from tetramethylsilane as the internal
standard, and coupling constants (J) are in Hz.
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
A new acylated cyanidin glycoside was isolated from the
purple-violet flowers of M. longipetala subsp. bicornis. From the
chemotaxonomical point of view, there are two C-3 OH glycosidic
patterns for anthocyanidins found in the flowers of the Brassica-
ceae, namely acylated 3-sambubioside from Arabis blepharophylla,
Aubrieta x cultorum, Cheiranthus cheiri, Heliophila coronopifolia,
Hesperis matronalis, Ionopsidium acaule, Lobularia maritima, Lunaria
annua, Malcolmia maritima, M. incana and Orychophragmus
violaceus (Honda et al., 2005; Ito et al., 2013; Saito et al., 1995,
1996, 2011; Tatsuzawa, 2012; Tatsuzawa et al., 2006, 2007, 2008a,
2010, 2012a,b, 2014) and acylated 3-sophoroside from Iberis
umbellata, Moricandia arvensis, Moricandia ramburii and Raphanus
sativus (Saito et al., 2008; Tatsuzawa et al., 2008b, 2012c, 2013).
Therefore, the floral anthocyanins of M. longipetala subsp. bicornis
are grouped into the former pattern.
The FAB mass spectrum of an unknown anthocyanin from the
purple-violet flower cultivars of M. incana (Saito et al., 1995,
anthocyanin C) exhibited its molecular ion at 1181 m/z,
corresponding to C55H57O29. This unknown anthocyanin C is
identical with pigment 1 which has been isolated and identified
as a major anthocyanin in the flowers of M. longipetala subsp.
bicornis in the present study. In addition, the distribution of the
pigments 2 and 3 from the flowers of M. longipetala subsp.
bicornis has been reported in the violet, purple-violet, and red-
purple flower cultivars of M. incana (Saito et al., 1995; Tatsuzawa
et al., 2012a). The distribution of 28 acylated cyanidin 3-
sambubioside-5-glucosides has been reported from petals of 10
genera of the Brassicaceae (Honda et al., 2005; Ito et al., 2013;
Saito et al., 1995, 1996, 2011; Tatsuzawa, 2012; Tatsuzawa et al.,
2006, 2007, 2010, 2012a ,b, 2014). Among them, the distribution
4.2. Plant materials
226
Seeds of purple-violet flowers of M. longipetala subsp. bicornis
were purchased from Thompson & Morgan. Ltd (UK). Seeds were
sown in October, 2012 and plants were grown in a greenhouse of
Iwate University. Flowers with a purple-violet color [Purple-
Violet N80B by Royal Horticultural Society (R.H.S) Color Chart
227
228
229
230
231
232
233
234
235
236
and b*(ꢁ8.03)/a*(13.41) = ꢁ0.60, L* = 62.29 by
a CM-700d
Spectro Color Meter (Konica-Minolta Co., Ltd.)] were collected
in spring in 2013. The petals were trimmed from flowers by hand
and dried by air for 1 day at 45 8C. Then they were kept at ꢁ20 8C
until used.
4.3. Isolation and purification of anthocyanins
237
Dried flowers (ca. 100 g) of M. longipetala subsp. bicornis were
immersed in 5% HOAc (10 l) at room temperature for 5 h and
extracted. Three anthocyanin pigments was isolated and purified
by Diaion HP-20 (Mitsubishi Chemical’s Ion Exchange Resins)
column (90 mm ꢀ 150 mm) chromatography, paper chromatogra-
phy and preparative HPLC from the extract as described previously
(Tatsuzawa et al., 2006, 2007). The purified three anthocyanins
were obtained from the flowers as follows; pigment 1 (ca. 35 mg),
pigment 2 (ca. 23 mg), and pigment 3 (ca. 12 mg).
238
239
240
241
242
243
244
245
246
of
(trans-feruloyl)-
(1) and cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-
(trans-feruloyl)- -glucoside]-5-O-[6-O-(malonyl)-
cyanidin
3-O-[2-O-(2-O-(trans-feruloyl)-
-glucoside]-5-O-[6-O-(malonyl)-
-xylosyl)-6-O-
-glucoside]
b
-xylosyl)-6-O-
4.4. Chemical and spectroscopic analyses of purified anthocyanins
247
b
b
-glucoside]
b
Acid hydrolyses of pigments 1–3 (ca. 0.5 mg, each) were carried
out with 2 N HCl (1 ml) at 100 8C for 2 h, and resulted in cyanidin,
glucose, xylose, ferulic acid and malonic acid. Moreover, sinapic
acid was detected in the hydrolysates of 1. These compounds were
confirmed by direct comparison of TLC and/or HPLC with the
authentic samples.
By alkaline hydrolysis, pigments 1–3 yielded cyanidin 3-
sambubioside-5-glucoside as their deacyl anthocyanin. The deacyl
anthocyanin structure was identified in direct comparison by the
analyses of co-TLC and co-HPLC with authentic cyanidin 3-
sambubioside-5-glucoside which was prepared from Lunaria
annua (Tatsuzawa et al., 2006).
248
249
250
251
252
253
254
255
256
257
258
259
b
b
(2) has only been reported in the genus Matthiola.
194
195
4. Experimental
4.1. General procedures
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
TLC was carried out on plastic coated cellulose sheets (Merck)
using eight mobile phases: BAW (n-BuOH-HOAc-H2O, 4:1:2, v/v/v),
BuHCl (n-BuOH-2 N HCl, 1:1, v/v, upper layer), AHW (HOAc-HCl-
H2O, 15:3:82, v/v/v), 1% HCl for anthocyanins, Forestal (HOAc-HCl-
H2O, 30:3:10, v/v/v) for anthocyanidin and BAW, EAA (EtOAc-
HOAc-H2O, 3:1:1, v/v/v), ETN (EtOH-NH4OH-H2O, 16:1:3, v/v/v)
and EFW (EtOAc-HCOOH-H2O, 5:2:1, v/v/v) for sugars and organic
acids 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 mm ꢀ 250 mm) column at 40 8C 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) with 5 min of re-equilibration at 20% solvent
B, for anthocyanins, anthocyanidins and hydroxycinnamic acids
(method 1). The other eluant for malonic acid was applied as an
isocratic elution of solvent A for 10 min and monitoring at 210 nm
(Tatsuzawa et al., 2013b) (method 2).
4.4.1. Deacyl anthocyanin of pigments 1–3 (cyanidin 3-
sambubioside-5-glucoside)
260
261
262
263
264
UV–VIS (in 0.1% HCl–MeOH): lmax 527, 278 nm, E440/E527
(%) = 13, AlCl3 shift + TLC: (Rf-values) BAW 0.28, BuHCl 0.05, 1% HCl
0.45, AHW 0.67; HPLC: Rt (min) 13.2.
4.4.2. Pigment 1
265
266
267
268
269
270
Dark red powder; for UV–VIS (in 0.1% HCl–MeOH): lmax
530,327,296,284 nm, E327/E530 (%) = 117, E440/E530 (%) = 15, AlCl3
shift + TLC: (Rf-values) BAW 0.71, BuHCl 0.54, 1% HCl 0.32, AHW
0.72, HPLC: Rt (min) 34.9, NMR spectrum, see Table 1; HR-FABMS
calc. for C55H57O29: 1181.2986. Found: 1181.3014.
4.4.3. Pigment 2
271
272
273
274
275
276
UV–VIS spectra were recorded on UV–Vis Multi Purpose
Spectrophotometer (MPS-2450, Shimadzu) in 0.1% HCl–MeOH
(from 200 to 700 nm).
High resolution FAB mass (FABMS) spectra were determined on
a JEOL JMS-700 Mass spectrometer (JEOL) operating in the positive
Dark red powder; for UV–VIS (in 0.1% HCl–MeOH): lmax
530,324,298,284 nm, E324/E530 (%) = 132, E440/E530 (%) = 16, AlCl3
shift + TLC: (Rf-values) BAW 0.68, BuHCl 0.49, 1% HCl 0.36, AHW
0.74, HPLC: Rt (min) 30.4, NMR spectrum, see Table 1; HR-FABMS
calc. for C56H59O30: 1211.3091. Found: 1211.3065.
Please cite this article in press as: Tatsuzawa, F., Acylated cyanidin 3-sambubioside-5-glucosides from the purple-violet flowers of
Matthiola longipetala subsp. bicornis (Sm) P. W. Ball. (Brassicaceae). Phytochem. Lett. (2014), http://dx.doi.org/10.1016/
j.phytol.2014.03.011

G Model
PHYTOL 688 1–5
F. Tatsuzawa / Phytochemistry Letters xxx (2014) xxx–xxx
5
Saito, N., Tatsuzawa, F., Suenaga, E., Toki, K., Shinoda, K., Shigihara, A., Honda, T., 313
2008. Tetra-acylated cyanidin 3-sophoroside-5-glucosides from the flowers of 314
277
278
279
280
281
282
4.4.4. Pigment 3
Dark red powder; for UV–VIS (in 0.1% HCl–MeOH): lmax
530,327,295,281 nm, E327/E530 (%) = 88, E440/E530 (%) = 13, AlCl3
shift + TLC: (Rf-values) BAW 0.48, BuHCl 0.22, 1% HCl 0.35, AHW
0.69, HPLC: Rt (min) 33.2, NMR spectra, see Table 1; HR-FABMS
calc. for C45H49O26: 1005.2512. Found: 1005.2555.
Iberis umbellata L. (Cruciferae). Phytochemistry 69, 3139–3150.
315
Saito, N., Tatsuzawa, F., Toki, K., Shinoda, K., Shigihara, A., Honda, T., 2011. The blue 316
anthocyanin pigments from the blue flowers of Heliophila coronopifolia L. 317
(Brassicaceae). Phytochemistry 72, 2219–2229.
318
Tatsuzawa, F., Saito, N., Shinoda, K., Shigihara, A., Honda, T., 2006. Acylated cyanidin 319
3-sambubioside-5-glucosides in three garden plants of the Cruciferae. Phyto- 320
chemistry 67, 1287–1295.
321
Tatsuzawa, F., Toki, K., Saito, N., Shinoda, K., Shigihara, A., Honda, T., 2007. Four 322
acylated cyanidin 3-sambubioside-5-glucosides from the purple-violet flowers 323
283
Acknowledgement
of Lobularia maritima. Heterocycles 71, 1117–1125.
324
Tatsuzawa, F., Saito, N., Toki, K., Shinoda, K., Shigihara, A., Honda, T., 2008a. 325
284
285
286
Author thanks Mr. James Hall (Iwate University) for his careful
revision of the manuscript. This work was supported in part by a
Grant-in Aid for Science Research (C) [No. 25450037] from the
Triacylated cyanidin 3-(3X-glucosylsambubioside)-5-glucosides from the flow- 326
ers of Malcolmia maritima. Phytochemistry 69, 1029–1036.
327
Tatsuzawa, F., Toki, K., Saito, N., Shinoda, K., Shigihara, A., Honda, T., 2008b. 328
Anthocyanin occurrence in the root peels, petioles and flowers of red radish 329
287 Q2 Japan Society for the Promotion of Science (JSPS).
(Raphanus sativus L.). Dyes Pigm. 79, 83–88.
330
Tatsuzawa, F., Usuki, R., Toki, K., Saito, N., Shinoda, K., Shigihara, A., Honda, T., 2010. 331
Acylated pelargonidin 3-sambubioside-5-glucosides from the red-purple flow- 332
288
References
ers of Lobularia maritima. J. Jpn. Soc. Hortic. Sci. 79, 84–90.
333
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
Andersen, Ø.M., Jordheim, M., 2006. The anthocyanins. In: Andersen, Ø.M., Mark-
ham, K.R. (Eds.), Flavonoids: Chemistry, Biochemistry and Applications. CRC
Press, Boca Raton, pp. 471–551.
Harborne, J.B., 1984. Phytochemical Methods, 2nd ed. Chapman & Hall, London.
Harborne, J.B., Baxter, H., 1999. The Handbook of Natural Flavonoids, vol. 2. John
Wiley & Sons, Chichester.
Honda, T., Saito, N., 2002. Recent progress in the chemistry of polyacylated antho-
cyanins 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.
Ito, S., Kato, K., Tatsuzawa, F., 2013. Acylated cyanidin 3-sambubioside-5-glucosides
in the red-purple flowers of Arabis blepharophylla Hook. & Arn. (Brassicaceae).
Biochem. Syst. Ecol. 48, 65–69.
Marzouk, M.M., Kawashty, S.A., Ibrahium, L.F., Saleh, N.A.M., Al-Nowaihi, A.-S.M.,
2008. Two new kaempferol glycosides from Matthiola longipetala (subsp. livida)
(Delile) Maire and carcinogenic evaluation of its extract. Nat. Prod. Commun. 3,
1325–1328.
Saito, N., Tatsuzawa, F., Nishiyama, A., Yokoi, M., Shigihara, A., Honda, T., 1995.
Acylated cyanidin 3-sambubioside-5-glucoside in Matthiola incana. Phyto-
chemistry 38, 1027–1032.
Saito, N., Tatsuzawa, F., Hongo, A., Win, K.W., Yokoi, M., Shigihara, A., Honda, T.,
1996. Acylated pelargonidin 3-sambubioside-5-glucosides in Matthiola incana.
Phytochemistry 41, 1613–1620.
Tatsuzawa, F., 2012. Acylated cyanidin 3-sambubioside-5-glucosides in the 334
purple flowers of Hesperis matronalis L. (Brassicaceae). Biochem. Syst. Ecol. 335
44, 374–379.
336
Tatsuzawa, F., Saito, N., Toki, K., Shinoda, K., Honda, T., 2012a. Flower colors and 337
their anthocyanins in Matthiola incana cultivars (Brassicaceae). J. Jpn. Soc. 338
Hortic. Sci. 81, 91–100.
339
Tatsuzawa, F., Aiba, Y., Morino, T., Saito, N., Shinoda, K., Kato, K., Toki, K., Honda, T., 340
2012b. Copigmentation with acylated anthocyanin and kaempferol glycoside in 341
violet and purple flower cultivars of Aubrieta x cultorum (Brassicaceae). J. Jpn. 342
Soc. Hortic. Sci. 81, 275–284.
343
Tatsuzawa, F., Ito, K., Muraoka, H., Namauo, T., Kato, K., Takahata, Y., Ogawa, S., 344
2012c. Triacylated peonidin 3-sophoroside-5-glucosides from the purple flow- 345
ers of Moricandia ramburii Webb. Phytochemistry 76, 73–77.
346
Tatsuzawa, F., Ito, S., Sato, M., Muraoka, H., Kato, K., Takahata, Y., Ogawa, S., 2013. 347
Tetra-acylated cyanidin 3-sophoroside-5-glucoside from the purple-violet 348
flowers of Moricandia arvensis (L.) DC (Brassicaceae). Phytochem. Lett. 6, 349
170–173.
350
Tatsuzawa, F., Takahashi, N., Kato, K., Shinoda, K., Saito, N., Honda, T., 2014. Acylated 351
cyanidin glycosides from the pale-violet flowers of Ionopsidium acaule (Desf.) 352
Rchb. (Brassicaceae). Phytochem. Lett. 7, 69–76.
353
Veitch, N.C., Grayer, R.J., 2008. Flavonoids and their glycosides, including antho- 354
cyanins. Nat. Prod. Rep. 25, 555–611.
Veitch, N.C., Grayer, R.J., 2011. Flavonoids and their glycosides, including antho- 356
cyanins. Nat. Prod. Rep. 28, 1626–1695.
355
357
Please cite this article in press as: Tatsuzawa, F., Acylated cyanidin 3-sambubioside-5-glucosides from the purple-violet flowers of
Matthiola longipetala subsp. bicornis (Sm) P. W. Ball. (Brassicaceae). Phytochem. Lett. (2014), http://dx.doi.org/10.1016/
j.phytol.2014.03.011