Triacylated peonidin 3-sophoroside-5-glucosides from the purple flowers of Moricandia ramburii Webb.

Article abstract of DOI:10.1016/j.phytochem.2011.12.019

Triacylated peonidin 3-sophoroside-5-glucosides were isolated from the purple flowers of Moricandia ramburii Webb. (Family: Brassicaceae), and determined to be peonidin 3-O-[2-O-(2-O-(trans-feruloyl)-glucosyl)-6-O-(trans-p- coumaroyl)-glucoside]-5-O-[6-O-(malonyl)-glucoside] (1), peonidin 3-O-[2-O-(2-O-(trans-feruloyl)-glucosyl)-6-O-(cis-p-coumaroyl)-glucoside] -5-O-[6-O-(malonyl)-glucoside] (2) and peonidin 3-O-[2-O-(2-O-(trans-sinapoyl)- glucosyl)-6-O-(trans-p-coumaroyl)-glucoside]-5-O-[6-O-(malonyl)-glucoside] (3), respectively, by chemical and spectroscopic methods. In addition, one known acylated cyanidin glycoside, cyanidin 3-O-[2-O-(2-O-(trans-feruloyl)-glucosyl)- 6-O-(trans-p-coumaroyl)-glucoside]-5-O-[6-O-(malonyl)-glucoside] (4), was also identified in the flowers. Peonidin glycosides have not been reported hitherto in floral tissues in to Brassicaceae.

Full text of DOI:10.1016/j.phytochem.2011.12.019

Phytochemistry 76 (2012) 73–77
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Triacylated peonidin 3-sophoroside-5-glucosides from the purple flowers
of Moricandia ramburii Webb.
a
b
c
a
Fumi Tatsuzawa a, , Kanako Ito , Hiroki Muraoka , Toshiharu Namauo , Kazuhisa Kato ,
⇑
Yoshihito Takahata a, Satoshi Ogawa b
a Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan
b Faculty of Engineering, Iwate University, Morioka, Iwate 020-8551, Japan
c Center for Regional Collaboration in Research and Education, Iwate University, Morioka, Iwate 020-8551, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 July 2011
Received in revised form 11 November 2011
Available online 20 January 2012
Triacylated peonidin 3-sophoroside-5-glucosides were isolated from the purple flowers of Moricandia
ramburii Webb. (Family: Brassicaceae), and determined to be peonidin 3-O-[2-O-(2-O-(trans-feruloyl)-
glucosyl)-6-O-(trans-p-coumaroyl)-glucoside]-5-O-[6-O-(malonyl)-glucoside] (1), peonidin 3-O-[2-O-(2-
O-(trans-feruloyl)-glucosyl)-6-O-(cis-p-coumaroyl)-glucoside]-5-O-[6-O-(malonyl)-glucoside] (2) and
peonidin 3-O-[2-O-(2-O-(trans-sinapoyl)-glucosyl)-6-O-(trans-p-coumaroyl)-glucoside]-5-O-[6-O-(malo-
nyl)-glucoside] (3), respectively, by chemical and spectroscopic methods. In addition, one known
acylated cyanidin glycoside, cyanidin 3-O-[2-O-(2-O-(trans-feruloyl)-glucosyl)-6-O-(trans-p-couma-
royl)-glucoside]-5-O-[6-O-(malonyl)-glucoside] (4), was also identified in the flowers. Peonidin
glycosides have not been reported hitherto in floral tissues in to Brassicaceae.
Keywords:
Moricandia ramburii Webb.
Brassicaceae
Purple flower color
Acylated peonidin 3-sophoroside-5-
glucoside
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
purple flowers of M. ramburii by high performance liquid chroma-
tography (HPLC) UV–Vis analysis (Fig. 1). The percentage of pig-
Moricandia DC. (Violet Cabbage) species (Brassicaceae) are native
to the Mediterranean region, and cultivated as ornamental plants
with purple, violet and white flowers in Europe and America. In the
limited flavonoid work reported for this genus, flavonol glycosides
have been characterized (Braham et al., 2005). To date, 17 acylated
3-sophoroside-5-glucosides of pelargonidin and cyanidin isolated
from 2 genera and 45 acylated 3-sambubioside-5-glucosides of
pelargonidin, cyanidin and delphinidin isolated from 7 genera, have
been unambiguously identified from flowers of Brassicaceae (Honda
et al., 2005; Saito et al., 1995a,b, 1996, 2008, 2011; Tatsuzawa et al.,
2006, 2007, 2008a,b, 2010). Distribution of 3-sophoroside-5-gluco-
sides of anthocyanidin is, however, restricted to the flowers of the
genera Raphanus and Iberis (Saito et al., 2008; Tatsuzawa et al.,
2008b), while the occurrence of peonidin has not been reported in
flowers of Brassicaceae species. In this paper, the structure elucida-
tion of three new triacylated peonidin 3-sophoroside-5-glucosides
from the purple flowers of Moricandia ramburii, is reported.
ments 1–4, calculated as the total anthocyanin content by HPLC
vis peak area at 530 nm, was 35.9%, 6.2%, 12.9% and 19.2% respec-
tively, with the MAW extract also containing numerous small pig-
ment peaks.
The four pigments (1–4) were extracted from the purple flowers
with 5% AcOH, followed by isolation using Diaion HP-20 column
chromatography, preparative HPLC and thin layer chromatography
(Tatsuzawa et al., 2010).
Acid hydrolysis of pigments 1–3 and 4 yielded peonidin and
cyanidin as their anthocyanidins, respectively (Harborne, 1984),
while also affording glucose, hydroxycinnamic acid and malonic
acid moieties. Moreover, trans-p-coumaric acid and trans-ferulic
acid, cis-p-coumaric acid and trans-ferulic acid, and trans-p-couma-
ric acid and trans-sinapic acid were detected in the hydrolysates of
1 and 4, 2, and 3 by TLC, respectively, with malonic acid found in all
pigments (Harborne, 1984).
Alkaline hydrolysis of 1–3 yielded peonidin 3-sophoroside-5-
glucoside, and alkaline hydrolysis of 4 yielded cyanidin 3-sophoro-
side-5-glucoside. The deacyl anthocyanin structures were
tentatively identified via co-TLC and co-HPLC with authentic
peonidin 3-sophoroside-5-glucoside and cyanidin 3-sophoroside-
5-glucoside, with the latter being prepared from Ipomoea tricolor
(Kondo et al., 1987) and Ipomoea purpurea (Saito et al.,1995b),
respectively, by alkaline hydrolysis.
2. Results and discussion
Four major anthocyanin pigments (1–4) were found in the
methanol–acetic acid–water (MAW) (4:1:5, v/v/v) extracts from
The structures of 1–4 were confirmed based on the analyses of
⇑
Corresponding author. Tel./fax: +81 19 621 6145.
E-mail address: fumi@iwate-u.ac.jp (F. Tatsuzawa).
their 1H (500 MHz), 13C (126 MHz) and 2D (COSY, NOESY, 1H–13
C
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.12.019

74
F. Tatsuzawa et al. / Phytochemistry 76 (2012) 73–77
served, establishing acylation group at C-6 OH (Glc A and Glc B)
and C-2 OH (Glc C) as p-coumaric acid, malonic acid and ferulic
acid, respectively. Consequently, the structure of pigment 1 was
determined to be peonidin 3-O-[2-O-(2-O-(trans-feruloyl)-gluco-
syl)-6-O-(trans-p-coumaroyl)-glucoside]-5-O-[6-O-(malonyl)-glu-
coside], which is a new anthocyanin in plants (Andersen and
Jordheim, 2006; Harborne and Baxter, 1999; Honda and Saito,
2002). This structure was also confirmed by analysis of its 13C,
1H–13C HMQC and 1H–13C HMBC NMR spectra. (Table 1, Fig. 1).
2.2. Pigment 2
The molecular ion [M]+ of 2 was observed at m/z 1195.3142
(C56H59O29) indicating a peonidin structure with three molecules
of glucose, and one molecule each of p-coumaric acid, ferulic acid
and malonic acid, similar to 1. The 1H NMR spectrum of 2 was sim-
ilar to that of 1, except for the signals of the p-coumaric acid moi-
ety (Table 1). The olefinic protons of 2 were shifted upfield to dH
5.63 and 6.34 (d, J = 12.9 Hz each) in comparison with those of 1,
establishing 2 as peonidin 3-O-[2-O-(2-O-(trans-feruloyl)-gluco-
syl)-6-O-(cis-p-coumaroyl)-glucoside]-5-O-[6-O-(malonyl)-gluco-
side], which is a new anthocyanin in plants. This structure was
also confirmed by analysis of its 13C, including 1H–13C HMQC
and 1H–13C HMBC NMR spectra. (Table 1).
Fig. 1. HPLC profile (530 nm) and structure of acylated anthocyanins isolated from
the purple flowers of Moricandia ramburii. Observed main NOEs are indicated by
arrows. Observed HMBCs of 1 are indicated by dotted arrows. 1: R1 = CH3, R2 = trans,
R
R
3 = H; 2: R1 = CH3, R2 = cis, R3 = H; 3: R1 = CH3, R2 = trans, R3 = OCH3; 4: R1 = H,
2 = trans, R3 = H.
2.3. Pigment 3
HMQC and 1H–13C HMBC) NMR spectra in DMSO-d6-CF3COOD
(9:1), as well as their fast atom bombardment mass spectra
(FABMS).
The molecular ion [M]+ of 3 was observed at m/z 1225.3224
(C57H61O30), indicating a peonidin structure with three molecules
of glucose, and one molecule each of p-coumaric acid, sinapic acid
and malonic acid. The 1H NMR spectrum of 3 was similar to that of
1, with 3 containing a sinapic acid moiety instead of a ferulic acid
moiety (Table 1). The two aromatic proton signals of sinapic acid
(II) were observed at dH 6.76 (H-2 and H-6). The olefinic proton
2.1. Pigment 1
The molecular ion [M]+ of 1 was observed at m/z 1195.3160
(C56H59O29), indicating a peonidin structure with three molecules
of glucose, and one molecule each of p-coumaric acid, ferulic acid
and malonic acid.
signals [dH 6.31 (d, J = 16.0 Hz, H-a) and (d, J = 16.0 Hz, H-b)]
indicating a trans-sinapic acid moiety establishing 3 as peonidin
3-O-[2-O-(2-O-(trans-sinapoyl)-glucosyl)-6-O-(trans-p-coumaroyl)-
glucoside]-5-O-[6-O-(malonyl)-glucoside], which is a new antho-
cyanin in plants. This structure was also confirmed by analysis of
its 13C, including 1H–13C HMQC and 1H–13C HMBC NMR spectra.
(Table 1).
The chemical shifts of the 13 aromatic protons of the peonidin,
p-coumaric acid and ferulic acid moieties, together with their cou-
pling constants, were assigned as shown in Table 1. Six protons
were assigned to the two methoxyl groups of peonidin and ferulic
acid. Two sets of two pairs of doublet resonances, assigned to the
four olefinic proton signals of the p-coumaric and ferulic acid moi-
eties, indicated a trans configuration for the acids based on their
coupling constants (J = 16.0 Hz each) (Table 1). The chemical shifts
of the sugar moiety protons were observed in the region of dH
5.74–3.17, with the three anomeric proton resonances at dH 5.74
(d, J = 7.5 Hz, Glc A), 5.14 (d, J = 7.5 Hz, Glc B), and 5.15 (d,
J = 8.5 Hz, Glc C). Based on the observed coupling constants (Ta-
ble 1), these three sugars were assumed to be in their b-pyranose
forms. The linkages and/or positions of the attachments of the su-
gar and acyl groups were determined based on 2D COSY and
NOESY experiments.
2.4. Pigment 4
The molecular ion [M]+ of 4 was observed at m/z 1181.2991
(C55H57O29), indicating a cyanidin structure with three molecules
of glucose, and one molecule each of p-coumaric acid, ferulic acid
and malonic acid. The 1H NMR spectrum of 4 was similar to that
of 1, with 4 containing a cyanidin moiety instead of a peonidin
moiety, establishing (Table 1),
4 as cyanidin 3-O-[2-O-(2-O-
(trans-feruloyl)-glucosyl)-6-O-(trans-p-coumaroyl)-glucoside]-5-
O-[6-O-(malonyl)-glucoside], which has been found in Iberis
umbellata (Family: Brassicaceae) (Saito et al., 2008). This structure
By application of a NOESY experiment, NOEs between H-1 of Glc
A and H-4 (dH 8.73) of peonidin, H-1 of Glc B and H-6 (dH 6.94) of
peonidin, and H-2 (dH 4.04) of Glc A and H-1 of Glc C were observed
(Fig. 1), supporting glycosylation of the C-3 and C-5 peonidin hy-
droxyl groups with Glc A and Glc B, respectively, while also indi-
cating a sophorose structure between Glc A and Glc C.
was also confirmed by analysis of its 13C, 1H–13C HMQC and 1H–13
HMBC NMR spectra (Table 1).
C
3. Concluding remarks
Five characteristic downfield shifted proton signals were as-
signed to the methylene protons of Glc A (dH 4.12 and 4.35, H-6a
and b) and Glc B (dH 4.06 and 4.37, H-6a and b), and to a methine
proton (dH 4.68, t, J = 8.8 Hz, H-2) of Glc C, indicating acylation of C-
6 OH (Glc A and Glc B) and C-2 OH (Glc C) with three acid mole-
cules. In the NOESY spectrum, the correlations between H-6a,b of
Three new acylated peonidin glycosides were isolated along
with one known acylated cyanidin glycoside from the purple
flowers of M. ramburii. From a chemotaxonomical point of view,
this report is the first to describe the distribution of peonidin gly-
cosides in the flowers of Brassicaceae (Harborne, 1967; Timberlake
and Bridle, 1975; Hrazdina, 1982; Harborne and Grayer, 1988;
Strack and Wray, 1994; Andersen and Jordheim, 2006). Moreover,
Glc A and H-
a,b of p-coumaric acid, H-6a,b of Glc B and -CH2- of
malonic acid, and H-2 of Glc C and H-
a,b of sinapic acid were ob-

F. Tatsuzawa et al. / Phytochemistry 76 (2012) 73–77
75
Table 1
1H and 13C NMR spectroscopic data of acylated anthocyanins from the flowers of Moricandia ramburii in DMSO-d6/CF3CO2D (9:1).
1
2
3
4
dH
dC
dH
dC
dH
dC
dH
dC
Anthocyanidin
2
3
4
5
6
7
8
162.4
144.5
132.7
155.3
105.0
168.0
96.8
161.7
144.3
131.8
155.0
103.9
168.3
96.3
162.4
144.5
132.7
155.2
105.0
168.4
96.8
162.8
144.8
131.1
155.3
104.9
167.1
96.4
8.73 s
6.94 s
6.93 s
8.53 s
8.74 s
8.69 s
6.81 brs
6.72 brs
6.95 brs
6.92 brs
6.94 d(1.5)
6.89 d(1.5)
9
155.4
111.9
119.9
114.5
148.8
156.3
117.2
129.6
56.3
155.1
111.9
119.8
113.9
148.9
156.1
116.9
129.4
56.0
155.4
112.0
119.9
114.1
148.8
156.3
117.2
129.6
56.3
155.7
114.1
120.0
117.5
146.9
156.3
117.3
128.8
10
10
20
7.95 d(2.0)
7.86 d(1.9)
7.94 d(1.9)
7.91 d(2.2)
30
40
50
7.13 d(9.0)
8.30 dd(2.0, 9.0)
3.93 s
7.17 d(8.8)
8.25 dd(1.9, 8.8)
3.96 s
7.13 d(8.8)
8.29 dd(1.9, 8.8)
3.93 s
7.11 d(9.0)
8.32 dd(2.2, 9.0)
60
–OCH3
p-Coumaric acid (I)
1
125.4
130.7
116.0
160.3
114.1
145.5
167.1
125.6
132.5
114.9
159.3
115.0
143.3
166.5
125.4
130.7
116.0
160.3
114.1
145.6
167.1
125.3
130.7
116.0
160.2
114.7
145.4
166.4
2,6
3,5
4
a
b
7.23 d(8.5)
6.68 d(8.5)
7.16 d(8.8)
6.41 d(8.8)
7.22 d(8.5)
6.68 d(8.5)
7.23 d(8.5)
6.67 d(8.5)
6.16 d(16.0)
7.28 d(16.0)
5.63 d(12.9)
6.34 d(12.9)
6.16 d(16.1)
7.27 d(16.1)
6.39 d(15.8)
7.29 d(15.8)
COOH
Ferulic acid or Sinapic acid (II)
1
2
3
4
5
6
a
b
126.0
111.4
148.4
149.8
115.9
123.5
115.2
145.3
166.2
56.1
125.9
111.3
148.3
149.8
115.9
123.4
115.0
144.3
166.1
55.8
124.9
106.5
148.5
138.8
148.5
106.5
115.6
145.5
166.2
56.5
126.3
111.6
148.4
149.8
116.2
123.4
115.5
145.3
167.3
56.1
7.06 brs
7.01 brs
6.76 s
7.20 d(1.3)
6.71 d(8.0)
6.70 d(8.5)
6.77 d(8.5)
6.98 brd(8.0)
6.25 d(16.0)
7.35 d(16.0)
6.86 brd(8.5)
6.22 d(15.8)
7.31 d(15.8)
6.76 s
6.31 d(16.0)
7.34 d(16.0)
7.03 dd(1.3, 8.5)
6.18 d(15.8)
7.47 d(15.8)
COOH
–OCH3
–OCH3
3.75 s
3.74 s
3.74 s
3.74 s
3.80 s
56.5
Malonic acid
–CH2–
COOH
3.30 s
41.5
167.3
168.4
3.42 s
41.7
167.4
167.9
3.30 s
41.6
167.3
168.0
3.31 s
41.5
167.7
168.0
COOH
Glucose (A)
1
2
3
4
5
6a
6b
5.74 d(7.5)
4.04 t(7.8)
3.72 t(8.5)
3.44 t(9.0)
3.95 m
98.3
78.3
75.1
71.0
74.1
63.5
5.71 d(7.9)
4.07 t(8.5)
3.71 t(9.2)
3.31 t(8.8)
3.90 ddd(2.2, 8.2, 10.4)
4.39 dd(7.6, 11.5)
4.24 brd(11.5)
97.4
77.9
75.9
70.4
74.7
63.3
5.75 d(7.6)
4.05 t(8.2)
3.72 m
3.42 t(9.5)
3.95 m
98.2
78.3
75.8
70.8
74.1
63.5
5.65 d(7.6)
4.13 t(8.0)
3.64 t(9.2)
3.37 t(9.5)
3.95 m
98.8
77.8
76.7
70.9
74.0
63.5
4.21 dd(7.5, 12.0)
4.35 brd(12.0)
4.21 dd(7.3, 12.0)
4.35 m
4.22 dd(7.3, 12.5)
4.36 brd(12.5)
Glucose (B)
1
2
3
4
5
6a
6b
5.14 d(7.5)
3.56 t(8.5)
3.42 t(9.0)
3.27 t(9.5)
3.78 m
102.3
73.5
74.7
69.9
74.7
64.4
5.15 d(7.6)
3.55 t(8.5)
3.44 t(8.8)
3.28 t(9.1)
3.81 ddd(2.6, 6.5, 9.1)
4.31 dd(7.6, 11.5)
4.46 brd(11.5)
101.2
73.4
75.9
70.4
74.6
64.4
5.14 d(7.3)
3.56 t(8.4)
3.42 t(9.5)
3.27 t(9.3)
3.78 m
102.2
73.5
76.1
69.9
74.7
64.4
5.14 d(7.6)
3.54 t(8.5)
3.39 t(9.1)
3.24 t(9.2)
3.75 m
101.0
73.5
76.1
69.9
74.6
64.4
4.06 dd(7.0, 12.0)
4.37 brd(12.0)
4.06 dd(5.9, 11.8)
4.37 m
4.03 dd(6.2, 11.9)
4.38 brd(11.9)
Glucose (C)
1
2
3
4
5
6a
6b
5.15 d(8.5)
4.68 t(8.8)
3.40 t(7.7)
3.18 t(8.0)
3.17 m
99.2
74.2
75.1
70.8
77.8
61.6
5.19 d(8.2)
4.66 t(8.8)
3.38 t(8.8)
3.16 t(9.5)
3.17 m
99.0
74.3
74.9
70.9
77.9
61.6
5.15 d(7.9)
4.69 t(8.8)
3.40 t(9.8)
3.19 t(9.8)
3.16 m
99.2
74.2
75.1
71.0
77.8
61.6
5.15 d(7.6)
4.65 t(9.0)
3.32 m
3.16 t(8.8)
3.04 m
100.1
74.4
75.1
71.0
77.8
61.6
3.44 dd(7.0, 11.0)
3.70 brd(11.0)
3.46 dd(7.3, 11.7)
3.72 m
3.46 m
3.71 brd(10.7)
3.42 m
3.66 m

76
F. Tatsuzawa et al. / Phytochemistry 76 (2012) 73–77
there are two C-3 OH glycosidic patterns for anthocyanidins found
in the flowers of Brassicaceae species, namely acylated 3-sophoro-
side from Iberis umbellata and Raphanus sativus (Saito et al., 2008;
Tatsuzawa et al., 2008b) and acylated 3-sambubioside from Chei-
ranthus cheiri, Heliophila coronopifolia, Lobularia maritima, Lunaria
annua, Malcolmia maritima, Matthiola incana and Orychophragmus
violaceus (Honda et al., 2005; Saito et al., 1995a, 1996, 2011;
Tatsuzawa et al., 2006, 2007, 2008a, 2010). Therefore, the floral
anthocyanins of M. ramburii are grouped into the former pattern.
sorbed. The column was thoroughly washed with H2O (2L) and
eluted with 5% AcOH:MeOH (500 mL, 5:95, v/v) to recover the
anthocyanins. After concentration, the residue was separated and
purified with paper chromatography (PC) using BAW. The sepa-
rated pigments were further purified by TLC (15% AcOH) and prep.
HPLC. With the latter 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 pigments were
adsorbed. Anthocyanin pigments were eluted with 5% AcOH–
MeOH followed by addition of excess of Et2O, and then dried.
The purified pigments from the flowers were obtained as follows;
pigment 1 (ca. 95 mg), pigment 2 (ca. 13 mg), pigment 3 (ca.
23 mg) and pigment 4 (ca. 55 mg).
4. Experimental
4.1. General procedures
TLC was carried out on plastic coated cellulose sheets (Merck)
using eight mobile phases: BAW (n-BuOH–AcOH–H2O, 4:1:2, v/v/
v), BuHCl (n-BuOH-2N HCl, 1:1, v/v, upper layer), AHW (AcOH–
HCl–H2O, 15:3:82, v/v/v), 1% HCl for anthocyanins, Forestal
(AcOH–HCl–H2O, 30:3:10, v/v/v) for anthocyanidin and BAW,
EAA (EtOAc–AcOH–H2O, 3:1:1, v/v/v), ETN (EtOH–NH4OH–H2O,
16:1:3, v/v/v) and EFW (EtOAc–HCO2H–H2O, 5:2:1, v/v/v) for sug-
ars 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 ꢀ 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% solvent
B (1.5% H3PO4, 20% AcOH, 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., 2009) (method 2).
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 (FABMS) spectra were determined on
a JEOL JMS-700 Mass spectrometer operating in the positive ion
mode using 1:1 mixture of dithiothreitol and 3-nitrobenzyl alcohol
as a matrix. 1H (500 MHz) and 13C (126 MHz) NMR spectra were
measured on a Bruker Avance III 500 MHz NMR spectrometer using
CF3COOD-DMSO-d6 (1:9) as a solvent. Chemical shifts are reported
relative to DMSO (2.5 ppm), and coupling constants (J) are in Hz.
4.4. Analyses of anthocyanins
The identification of anthocyanins was carried out by standard
procedures and both alkaline and acid hydrolyses (Harborne 1984).
Acid hydrolysis of pigments (ca. 1 mg each) was carried out with
2N HCl (1 mL) at 100 °C for 1 h. Alkaline hydrolysis of pigments
(ca. 1 mg each) was carried out with 2N NaOH solution (1 mL) un-
der degassed syringe allowed to stand for 15 min. The solution was
next acidified with 2N HCl (1.1 mL) and evaporated in vacuo to
dryness. The data of TLC (Rf values), HPLC (Rt-min, method 1),
UV–Vis (kmax), and FABMS spectra are shown in Section 4.4.1.–
4.4.4.
4.4.1. Pigment 1
Dark purple-red powder; UV–Vis (in 0.1% HCl-MeOH): kmax 533,
318, 295, 283 nm, Eacyl/Emax = 109%, E440/Emax = 13%, AlCl3 shift 0;
TLC: Rf-values (100ꢀ) BAW 61, BuHCl 84, 1% HCl 33, AHW 78;
HPLC: Rt (min) 39.8; for 1H and 13C NMR spectroscopic data, see Ta-
ble 1; HR-FABMS calc. for
1195.3160.
C56H59O29: 1195.3142. Found:
4.4.2. Pigment 2
Dark purple-red powders; UV–Vis: kmax 538, 320, 295, 283 nm,
Eacyl/Emax = 87%, E440/Emax = 14%, AlCl3 shift 0; TLC: Rf-values
(100ꢀ) BAW 55, BuHCl 85, 1% HCl 41, AHW 82; HPLC: Rt (min)
31.9; for 1H and 13C NMR spectroscopic data, see Table 1; HR-FAB-
MS calc. for C56H59O29: 1195.3142. Found: 1195.3105.
4.2. Plant materials
4.4.3. Pigment 3
The seeds of M. ramburii Webb. (accession number: MOR-RAM-
1) were obtained from Prof. T. Nishio at the Tohoku University
Brassica Seed Bank, and grown in greenhouses at the Laboratory
of Olericultural and Floricultural Science, Faculty of Agriculture,
Iwate University. The flowers exhibited a purple coloration [purple
78B by Royal Horticultural Society color chart and chromaticity va-
lue, b⁄(ꢁ29.36)/a⁄(78.66)]. Flowers were collected from winter to
spring seasons in Iwate, Japan and dried overnight at 40 °C, and
kept in a refrigerator at 4 °C. Chromaticity values were recorded
on a NR-3000 handy colorimeter (Nippon Denshoku Industries
Co., Ltd.).
Dark purple-red powders; UV–Vis: kmax 533, 318, 296, 283 nm,
Eacyl/Emax = 107%, E440/Emax = 12%, AlCl3 shift 0; TLC: Rf-values
(100ꢀ) BAW 53, BuHCl 78, 1% HCl 36, AHW 86; HPLC: Rt (min)
39.0; for 1H and 13C NMR spectroscopic data, see Table 1; HR-FAB-
MS calc. for C57H61O30: 1225.3248. Found: 1225.3224.
4.4.4. Pigment 4
Dark purple-red powders; UV–Vis: kmax 530, 318, 296, 284 nm,
Eacyl/Emax = 112%, E440/Emax = 12%, AlCl3 shift 0; TLC: Rf-values
(100ꢀ) BAW 53, BuHCl 83, 1% HCl 28, AHW 71; HPLC: Rt (min)
35.3; for 1H and 13C NMR spectroscopic data, see Table 1; HR-FAB-
MS calc. for C55H57O29: 1181.2986. Found: 1181.2991.
4.3. Isolation of anthocyanin
Dried flowers (100 g) of M. ramburii were immersed in AcOH–
H2O (3L; AcOH–H2O, 1:19) at room temperature for 5 h. and ex-
tracted. The extract was passed through a Diaion HP-20 (Mitsubi-
shi Chemical’s Ion Exchange Resins, aromatic type adsorbent)
column (90 ꢀ 150 mm), on which acylated anthocyanins were ad-
4.5. Analyses of hydrolysates (deacylanthocyanins, anthocyanidins,
sugar and acids)
The identification of hydrolysates by alkaline and acid, was car-
ried out by standard procedures (Harborne, 1984).

F. Tatsuzawa et al. / Phytochemistry 76 (2012) 73–77
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AlCl3 shift 0; TLC: Rf-values (100ꢀ) BAW 29, BuHCl 13, 1% HCl 50,
AHW 82; HPLC (method 1): Rt (min) 15.8.
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4.5.2. Cyanidin 3-sophoroside-5-glucoside
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4.5.3. Peonidin
UV–Vis: kmax 537, 275 nm, E440/Emax = 32%, AlCl3 shift 0; TLC: Rf-
values (100ꢀ) Forestal 60; HPLC(method 1): Rt (min) 32.6.
4.5.4. Cyanidin
UV–Vis: kmax 536, 273 nm, E440/Emax = 44%, AlCl3 shift +; TLC: Rf-
values (100ꢀ) Forestal 42; HPLC (method 1): Rt (min) 25.3.
Saito, N., Tatsuzawa, F., Nishiyama, A., Yokoi, M., Shigihara, A., Honda, T., 1995a.
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4.5.5. Glucose
TLC: Rf-values (100ꢀ) BAW 24, EAA 18, ETN 62, EFW 49; Color
(AHP) Brown.
Saito, N., Tatsuzawa, F., Hongo, A., Win, K.W., Yokoi, M., Shigihara, A., Honda, T.,
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4.5.6. p-Coumaric acid
TLC: Rf-values (100ꢀ) BAW 91, EAA 96, EFW 85; Color (under
UV) Violet; HPLC (method 1): Rt (min) 17.2 (trans), 16.2 (cis).
4.5.7. Ferulic acid
TLC: Rf-values (100ꢀ) BAW 87, EAA 92, EFW 78; Color (under
anthocyanin pigments from the blue flowers of Heliophila coronopifolia
L
UV) Bright Blue; HPLC (method 1): Rt (min) 20.6.
(Brassicaceae). Phytochemistry 72, 2219–2229.
Strack, D., Wray, V., 1994. The Flavonoids, Advances in Research since 1986. In:
Harborne, J.B. (Ed.). Chapman and Hall, London, p. 1.
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.
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.
4.5.8. Sinapic acid
TLC: Rf-values (100ꢀ) BAW 83, EAA 88, EFW 73; Color (under
UV) Bright Blue; HPLC (method 1): Rt (min) 22.1.
4.5.9. Malonic acid
Tatsuzawa, F., Saito, N., Toki, K., Shinoda, K., Shigihara, A., Honda, T., 2008a.
Triacylated cyanidin 3-(3X-glucosylsambubioside)-5-glucosides from the
flowers of Malcolmia maritima. Phytochemistry 69, 1033–1040.
Tatsuzawa, F., Toki, K., Saito, N., Shinoda, K., Shigihara, A., Honda, T., 2008b.
Anthocyanin occurrence in the root peels, Petioles and flowers of red radish
(Raphanus sativus L.). Dyes Pigm. 79, 83–88.
Tatsuzawa, F., Saito, N., Mikanagi, Y., Shinoda, K., Toki, K., Shigihara, A., Honda, T.,
2009. An unusual acylated malvidin 3-glucoside from flowers of Impatiens
textori Miq. (Balsaminaceae). Phytochemistry 70, 672–674.
HPLC (method 2): Rt (min) 4.1.
Acknowledgement
This work was supported in part by a Grant-in-Aid for Science
Research (C) [No. 22580024, to F.T.] from the Japan Society for
the Promotion of Science (JSPS).
Tatsuzawa, F., Usuki, R., Toki, K., Saito, N., Shinoda, K., Shigihara, A., Honda, T., 2010.
Acylated pelargonidin 3-sambubioside-5-glucosides from the red-purple
flowers of Lobularia maritima. J. Jpn. Soc. Hort. Sci. 79, 84–90.
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