7-Polyacylated delphinidin 3,7-diglucosides from the blue flowers of Leschenaultia cv. Violet Lena

Article abstract of DOI:10.1016/j.phytochem.2006.11.012

The triacyl anthocyanins, Leschenaultia blue anthocyanins 1 and 2 (LBAs 1 and 2) were isolated from the blue flowers of Leschenaultia R. Br. cv. Violet Lena (Goodeniaceae), in which LBA 1 was present as a dominant pigment. The structure of LBA 1 was elucidated to be delphinidin 3-O-[6-O-(malonyl)-β-d-glucopyranoside]-7-O-[6-O-(4-O-(6-O-(4-O-(β-d-glucopyranosyl)-trans-caffeoyl)-β-d-glucopyranosyl)-trans-caffeoyl)-β-d-glucopyranoside] by application of chemical and spectroscopic methods. Since LAB 2 was isolated in small amount, its structure was tentatively assigned as either delphinidin 3-(malonylglucoside)-7-[(glucosyl-p-coumaroyl)-(glucosylcaffeoyl)-glucoside] or delphinidin 3-(malonyl-glucoside)-7-[(glucosyl-caffeoyl)(glucosyl-p-coumaroyl)-glucoside]. This is the first report of the occurrence of 7-polyacylated anthocyanins in the family of Goodeniaceae, although others have been found in the families of the Ranunculaceae, Campanulaceae, and Compositae. Moreover, delphinidin 3-glycoside-7-di-(glucosylcaffeoyl)-glucoside has been reported only in the flowers of Platycodon grandiflorum (Campanulaceae). From a chemotaxonomical viewpoint, the Goodeniaceae may be closely related to the Campanulaceae.

Full text of DOI:10.1016/j.phytochem.2006.11.012

PHYTOCHEMISTRY
Phytochemistry 68 (2007) 673–679
www.elsevier.com/locate/phytochem
7
-Polyacylated delphinidin 3,7-diglucosides from the blue flowers
of Leschenaultia cv. Violet Lena
a
b
c
a
a,
*
Norio Saito , Fumi Tatsuzawa , Yoshikazu Yazaki , Atsushi Shigihara , Toshio Honda
a
Faculty of Pharmaceutical Sciences, Hoshi University, Shinagawa, Tokyo 142-8501, Japan
Experimental Farm, Faculty of Horticulture, Minami-Kyushu University, Takanabe, Miyazaki 884, Japan
b
c
Department of Chemical Engineering, Monash University, Clayton, Vic. 3800, Australia
Received 10 August 2006; received in revised form 27 October 2006
Available online 18 December 2006
Abstract
The triacyl anthocyanins, Leschenaultia blue anthocyanins 1 and 2 (LBAs 1 and 2) were isolated from the blue flowers of Leschenaul-
tia R. Br. cv. Violet Lena (Goodeniaceae), in which LBA 1 was present as a dominant pigment. The structure of LBA 1 was elucidated to
be delphinidin 3-O-[6-O-(malonyl)-b-D-glucopyranoside]-7-O-[6-O-(4-O-(6-O-(4-O-(b-D-glucopyranosyl)-trans-caffeoyl)-b-D-glucopyr-
anosyl)-trans-caffeoyl)-b-D-glucopyranoside] by application of chemical and spectroscopic methods. Since LAB 2 was isolated in small
amount, its structure was tentatively assigned as either delphinidin 3-(malonylglucoside)-7-[(glucosyl-p-coumaroyl)-(glucosylcaffeoyl)-
glucoside] or delphinidin 3-(malonyl-glucoside)-7-[(glucosyl-caffeoyl)(glucosyl-p-coumaroyl)-glucoside]. This is the first report of the
occurrence of 7-polyacylated anthocyanins in the family of Goodeniaceae, although others have been found in the families of the Ran-
unculaceae, Campanulaceae, and Compositae. Moreover, delphinidin 3-glycoside-7-di-(glucosylcaffeoyl)-glucoside has been reported
only in the flowers of Platycodon grandiflorum (Campanulaceae). From a chemotaxonomical viewpoint, the Goodeniaceae may be closely
related to the Campanulaceae.
ꢀ
2006 Elsevier Ltd. All rights reserved.
Keywords: Leschenaultia R. Br. cv. Violet Lena; Goodeniaceae; Blue flower color; Acylated anthocyanins; Delphinidin 3-malonylglucoside-7-di-(gluco-
sylcaffeoyl)-glucoside
1
. Introduction
been no report of anthocyanins in flowers of the genus
Leschenaultia. In extending our acylated anthocyanin study
(Honda and Saito, 2002), we thus decided to elucidate the
structures of anthocyanin pigments of the blue flowers of
Leschenaultia cv. Violet Lena, whose anthocyanins were
anticipated to be heavily acylated with molecules of
hydroxycinnamic acids and malonic acid.
In this paper, we report the structure elucidation of two
acylated delphinidin 3,7-diglucosides (Leschenaultia blue
anthocyanins 1 and 2, abbreviated as LBAs 1 and 2) in
the blue flowers of Leschenaultia cv. Violet Lena.
The Leschenaultia species, in the family of the Gooden-
iaceae, are native to Australia. There are 21 known species
in Western Australia, where the aboriginal people have
described the blue Leschenaultia (Leschenaultia biloba)
flower as ‘‘the floor of the sky’’. In 1999, one (N.S.) of
the authors came across the beautiful blue flowers of
L. biloba in the parks and gardens of Western Australia
during his sabbatical leave. Since then, he had been inter-
ested in the chemical structures of anthocyanin pigments
of L. biloba flowers. In Japan, Leschenaultia R. Br. cv. Vio-
let Lena (L. biloba cv. Sky Blue · L. formosa cv. Star Blas-
ter) is grown as a popular greenhouse shrub and Japanese
people admire its blue flower color. There has, however,
2. Results and discussion
Two major and five minor anthocyanin peaks were
*
observed in the 5% acetic acid extract from the blue flow-
Corresponding author. Tel.: +81 3 5498 5791; fax: +81 3 3787 0036.
E-mail address: honda@hoshi.ac.jp (T. Honda).
ers of Leschenaultia R. Br. cv. Violet Lena using high
0
031-9422/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.11.012

674
N. Saito et al. / Phytochemistry 68 (2007) 673–679
performance liquid chromatography (HPLC) with moni-
toring at 530 nm. The proportions were 57.5% (LBA 1)
and 12.3% (LBA 2), based on a percentage of the total
absorbance of the anthocyanin peaks. The two anthocya-
nins, LBAs 1 and 2, were then extracted from the dried
flowers with 5% aqueous acetic acid and purified using
Diaion HP-20 (Mitsubishi Chemical’s Ion Exchange Res-
ins) column chromatography (CC), preparative PC,
HPLC and TLC, according to the procedures described
previously (Tatsuzawa et al., 2004; Honda et al., 2005)
to yield 1 and 2 in approximately 30 mg and 3 mg
amounts, respectively. However, the other minor pigments
could not be obtained in pure form due to their small
amounts. The chromatographic and spectroscopic proper-
ties of LBAs 1 and 2 are summarized in Table 1.
Acid hydrolysis of LBAs 1 and 2 resulted in delphinidin,
glucose, caffeic acid and malonic acid. In addition, p-cou-
maric acid was detected in the hydrolysate of LBA 2. Alka-
line hydrolysis of LBA 1 resulted in one deacylated
anthocyanin, glycosylcaffeic acid and caffeic acid. On the
other hand, similar treatment of LBA 2 resulted in only
the same deacylated anthocyanin as that from LBA 1; how-
ever, other compounds such as glycosyl hydroxycinnamic
acids, caffeic acid, p-coumaric acid and malonic acid, could
not be detected due to the small amounts of LBA 2
available.
b-glucopyranose form from their observed J values. By
application of NOESY experiments, the long range NOEs
between H-1 of Glu A and H-4 (d 8.95) of delphinidin, and
also between H-1 of Glu B and H-8 (d 7.24) of delphinidin
were observed (Fig. 1), suggesting that OH-3 and OH-7 of
delphinidin are glycosylated with Glu A and Glu B, respec-
tively. Therefore, the deacylanthocyanin was determined
to be delphinidin 3-O-b-D-glucopyranoside-7-O-b-D-
glucopyranoside.
The FAB mass spectrum of glycosylcaffeic acid gave a
+
molecule ion [M + 1]
at 343 m/z (calc. for
C H O = 343.095) indicating that the glycosylcaffeic
1
5
19
9
acid is composed of caffeic acid with a molecule of glucose.
The structure of this glycosylcaffeic acid was elucidated to
be 4-O-(b-D-glucopyranosyl)-trans-caffeic acid by the anal-
1
ysis of its H NMR spectra (Section 4.4.2). Its linkage
between glucose and caffeic acid moieties was confirmed
by NOE experiments. Strong NOEs were observed between
the H-1 signal (d 4.78, d, J = 7.3 Hz) of glucose and the H-
5 resonance (d 7.11, d, J = 8.2 Hz) of caffeic acid by irradi-
ation of H-1 (Glu) and H-5 (caffeic acid), respectively. Fur-
thermore, this structure was confirmed by the analyses of
TLC and HPLC with the authentic samples obtained from
Cineraria and Evolvulus anthocyanins (Toki et al., 1994,
1995).
2.2. LBA 1
2
.1. Deacylanthocyanin and glycosylcaffeic acid from LBA 1
+
The molecular ion [M] of LBA 1 was observed at m/z
1361 (calc. for C H O , 1361.325) using FABMS, indi-
The FAB mass spectrum of deacylanthocyanin (deacy-
6
0
65 36
+
lated LBA 1) gave a molecular ion [M] at 627 m/z (calc.
for C H O , 627.156 m/z), indicating that the deacy-
cating that LBA 1 is composed of delphinidin with four
molecules of glucose, two molecules of caffeic acid and
one molecule of malonic acid. The elemental components
were confirmed by measuring its high-resolution; FABMS;
calc. for C H O : 1361.3256. Found: 1361.3285. The
2
7
31 17
lanthocyanin is composed of delphinidin with two mole-
cules of glucose. The chromatographic and spectroscopic
data of the deacylanthocyanin are shown in Table 1. The
6
0
65 36
1
H NMR spectrum (500 MHz in CF CO D–CD OD,
structure of LBA 1 was further elucidated on the basis of
3
2
3
1
13
1
:9) of deacylanthocyanin exhibited five aromatic protons
analyses on its H and C NMR spectra [500 MHz for
1
13
being identified with those of delphinidin moieties (Toki
et al., 1994), (Table 2). The anomeric protons of sugar moi-
eties were assigned at d 5.38 (d, J = 7.9 Hz, H-1 of Glu A)
and d 5.19 (d, J = 7.4 Hz, H-1 of Glu B), and both glucose
moieties of Glu A and Glu B were determined to be in the
H and 125.78 MHz for C spectra in CF CO D–CD OD
3 2 3
(1:9) or DCl–DMSO-d (1:9)], including 2D COSY, 2D
6
NOESY, HMQC and HMBC spectra.
The chemical shifts of 11 aromatic protons of delphini-
din and caffeic acid moieties with their coupling constants
Table 1
Chromatographic and spectroscopic data for Leschenaultia blue anthocyanins of Leschenaultia cv. Violet Lena*
Anthocyanins**
R
values (·100)
Spectral data in 0.1% HCl–MeOH
max (nm)
HPLC
FABMS***
f
+
BAW BuHCl 1% HCl AHW
k
E
acyl/Emax (%)
E
440/Emax (%) AlCl
3
R
t
(min) [M ]
LBA 1
LBA 2
Demalonyl LBA 1
Deacyl LBAa 1 and 2
5
5
4
6
0
0
0
0
3
4
1
4
17
20
12
18
551, (318), 285 129
548, (308), 284 198
554, (317), 285 143
16
19
16
21
+
+
+
+
23.7
25.0
22.3
7.7
1361
1345
1275
627
539, 278
–
LBA 2: delphinidin 3-malonylglucoside-7-glucosylcaffeoylglucosyl-p-coumaroylglucoside or delphinidin 3-malonylglucoside-7-glucosyl-p-cou-
maroylglucosylcaffeoylglucoside; ***(calc. for C60 35: 1345.331).
Demalonyl LBA 1: delphinidin 3-glucoside-7-glucosylcaffeoylglucosylcaffeoylglucoside; ***(calc. for C57
65
H O
63
H O33: 1275.324).
Deacyl LBAa 1 and 2: delphinidin 3,7-diglucoside; ***(calc. for C27
H
31
O
17: 627.156).
*
The composition of the solvents is given in Section 4.1.
*
*
65
LBA 1: delphinidin 3-malonylglucoside-7-glucosylcaffeoylglucosylcaffeoylglucoside; ***(calc. for C60H O36: 1361.325).

N. Saito et al. / Phytochemistry 68 (2007) 673–679
675
Table 2
NMR spectroscopic data of Leschenaultia blue anthocyanin 1
a
b
a
LBA 1
Demalonyl LBA 1
Deacyl LBA 1
1
3
1
1
1
C d (ppm)
H d (ppm)
H d (ppm)
H d (ppm)
Delphinidin
2
3
4
5
6
7
8
9
163.1
146.2
133.0
155.8
105.6
166.9
94.5
157.3
113.6
119.9
113.6
147.8
147.3
147.8
113.6
8.52 s
8.65 s
8.95 s
6.74 d (2.2)
6.98 d (2.2)
7.28 brs
7.38 brs
6.84 brs
7.24 brs
1
1
2
3
4
5
6
0
0
0
0
0
0
0
7.79 s
7.88 s
7.85 s
7.85 s
7.79 s
7.88 s
Caffeic acid (I)
1
2
3
4
5
6
a
b
129.8
118.7
147.7
148.6
116.0
121.4
118.4
144.2
168.0
6.91 d (1.9)
7.18 brs
6.66 d (8.6)
6.79 d (8.6)
6.95 brd (8.6)
6.53 d (15.9)
7.58 d (15.9)
6.79 dd (1.9, 8.6)
6.52 d (15.9)
7.58 d (15.9)
2
CO H
Caffeic acid (II)
1
2
3
4
5
6
a
b
130.2
114.9
147.9
148.2
116.5
121.4
118.4
144.2
168.0
6.75 d (2.1)
7.02 brs
6.53 d (8.2)
6.66 d (8.3)
6.90 m
6.27 d (16.2)
7.02 d (16.2)
6.29 dd (2.1, 8.2)
6.08 d (15.9)
6.93 d (15.9)
CO
2
H
Malonic acid
–
COOH
COOH
–
CH
2
40–50
169.1
170.3
3.58–3.63
Glucose A
1
2
3
4
5
102.9
74.5
77.7
71.1
76.2
65.7
5.39 d (7.7)
5.33 d (8.0)
3.75 m
5.38 d (7.9)
3.74 dd (7.9, 9.2)
3.52–3.58
3.46 brt (9.6)
3.57–3.65
3.73 dd (6.2, 12.2)
3.97 dd (2.2, 12.2)
3.78 dd (7.7, 9.0)
3.62 brt (9.0)
3.58 dd (8.9, 9.0)
3.92 m
4.52 dd (7.6, 10.9)
4.76 brd (10.9)
9
>
>
=
3
.15–3.65
>
>
;
6
6
a
b
3.82 d (11.3)
Glucose B
1
2
3
4
5
101.8
74.2
77.6
74.3
74.7
66.0
5.24 d (8.0)
3.73 dd (8.0, 9.2)
3.59 m
3.37 dd (9.2, 9.5)
4.02 ddd (2.5, 9.5, 11.9)
4.34 dd (9.5, 11.9)
4.73 dd (1.9, 11.9)
5.24 d (8.0)
3.53 m
5.19 d (7.4)
3.54 m
3.52–3.55
3.41 brt (9.5)
3.58–3.65
3.72 dd (6.1, 12.2)
3.94 dd (1.9, 12.2)
9
>
=
3.15–3.65
>
;
6a
4.25 m
4.62 d (11.3)
6
b
Glucose C
1
2
101.8
73.9
4.91 d (7.9)
3.60 dd (7.9, 8.7)
4.92 m d (8.0)
3.46 m
(
continued on next page)

676
N. Saito et al. / Phytochemistry 68 (2007) 673–679
Table 2 (continued)
a
b
a
LBA 1
Demalonyl LBA 1
Deacyl LBA 1
1
3
1
1
1
C d (ppm)
H d (ppm)
H d (ppm)
H d (ppm)
9
=
3
4
5
6
6
77.4
71.1
75.9
66.0
3.47 dd (8.7, 9.2)
3.48 dd (9.2, 9.5)
3.57 m
3.94 m
4.76 brd (10.9)
3.15–3.65
;
a
b
3.95 m
4.62 brd (11.0)
Glucose D
1
2
3
4
5
102.2
74.7
77.1
73.1
75.9
61.9
4.53 d (7.7)
3.44 dd (7.7, 9.6)
3.71 dd (7.7, 9.6)
3.63 m
3.46 m
3.72 m
4.49 d (7.4)
3.32 m
9
{ >
>
=
3
.15–3.65
>
>
;
6
6
a
b
3.94 m
3.98 m
TMS as an internal standards.
a
CD
CD
3
OD/CF
OD/DMSO-d
3
CO
2
D = 9:1.
/CF CO
b
3
6
3
2
D = 4:2:1.
OH
HO
respectively. These results indicated that the three OH-6
groups of Glu A, B and C were acylated with two mole-
cules of caffeic acid and one molecule of malonic acid,
respectively. By analysis of the NOESY spectrum, long
range NOEs between H-1 of Glu A and H-4 (d 8.52) of del-
phinidin, H-1 of Glu B and H-8 (d 6.98) of delphinidin, H-
1 of Glu C and H-5 (d 6.66) of caffeic acid I, and H-1 of
Glu D and H-5 (d 6.53) of caffeic acid II were observed
(Fig. 1). These data indicated that the OH-3 and OH-7
groups of delphinidin were glycosylated with Glu A and
Glu B, respectively, while the OH-4s of caffeic acid I and
II were glycosylated with Glu C and Glu D, respectively.
Furthermore, rather weak long range NOEs were observed
between H-5 (d 4.02) of Glu B and H-5 (d 6.66) of caffeic
acid I, and also between H-5 of Glu B and H-6 (d 6.79) of
caffeic acid I. These results supported the view that the
OH-6 of Glu B was esterified with the COOH group of caf-
feic acid I (Fig.1). Unfortunately, the linkages between Glu
A and malonic acid, as well as between Glu C and caffeic
acid II, could not be confirmed by IDNOE, NOESY and
HMBC experiments. In order to determine the bonding
position of malonic acid in LBA 1, demalonyl LBA 1
was prepared according to the procedure described previ-
ously (Saito et al., 1994). The structure determination of
demalonyl LBA 1 is described in the following Section
2.3. The results revealed that OH-6 of Glu A was not acyl-
ated in demalonyl LBA 1, but it was acylated with malonic
acid in LBA 1. Then, OH-6 of Glu C is acylated with caf-
feic acid II, and Glu D is terminal of this long substituent
group in the molecule. Consequently, the structure of LBA
1 was elucidated to be delphinidin 3-O-[6-O-(malonyl)-b-D-
glucopyranoside]-7-O-[6-O-(4-O-(6-O-(4-O-(b-D-glucopyr-
anosyl)-trans-caffeoyl)-b-D-glucopyranosyl)-trans-caffeoyl)-
b-D-glucopyranoside], which is a new anthocyanin in plants.
OH
Glu-B
O
O
+
O
O
HO
O
OH
OH
O
HO
HO
O
OH
OH
Glu-A
O
I
OH
HO
O
HO
OH
O
O
O
2
OH
O
O
Glu-C
II
HO
OH
HO
O
O
OH
OH
Glu-D
1
1
2
: Leschenaultia blue anthocyanin 1 (LBA 1)
: Demalonyl LBA 1
Observed NOE's are indicated by arrows.
Fig. 1. 1: Leschenaultia blue anthocyanin 1 (LBA 1). 2: Demalonyl LBA 1
Observed NOE’s are indicated by arrows.
were assigned by the analysis of its 2D COSY spectrum as
shown in Table 2. The H NMR spectrum exhibited four
1
olefinic proton signals of caffeic acid with large coupling
constants (J = 15.9 Hz and 15.9 Hz). These two caffeic
acids were determined to be in the trans configuration.
The chemical shifts of the sugar moieties were observed
in the region of d 5.39 – 3.37, where the four anomeric pro-
tons resonated at d 5.39 (d, J = 7.7 Hz, Glu A-H1), d 5.24
(
d, J = 8.0 Hz, Glu B-H1), d 4.91 (d, J = 7.9 Hz, Glu C-
H1), and d 4.53 (d, J = 7.7 Hz, Glu D-H1), respectively.
Based on the observed coupling constants (Table 2), these
four sugars were assumed to be in the b-pyranose forms.
By analysis of the 2D COSY spectrum, six characteristic
proton signals, shifted to lower magnetic field, were
assigned as methylene protons of Glu A (d 4.52 and
2.3. Demalonyl LBA 1
4
.76, H-6a and -6b), Glu B (d 4.34 and 4.73, H-6a
Hydrolysis of LBA 1 (ca. 15 mg) with 1 N HCl at room
and -6b) and Glu C (d 3.94 and 4.76, H-6a and -6b),
temperature for 18 days, followed by separation with HPLC

N. Saito et al. / Phytochemistry 68 (2007) 673–679
677
gave its demalonylanthocyanin (demalonyl LBA 1, ca.
mg). The chromatographic and spectroscopic data of
aromatic acids at 7-glycosyl residues, has been reported
only in the three families of the Ranunculaceae, Campanul-
aceae, and Compositae (Honda and Saito, 2002). From a
chemotaxonomical point of view, since Leschenaultia blue
anthocyanins LBAs 1 and 2 were found to be 7-polyacy-
lated anthocyanins, the family of Goodeniaceae, to which
Leschenaultia belongs, should be added to the above three
families. Furthermore, the structure of LBA 1 is similar to
platyconin isolated from the blue-violet flowers of Platyc-
odon grandiflorum in the family of Campanulaceae (Saito
et al., 1971; Goto et al., 1983). The difference in the struc-
tures is only at the residue of 3-malonylglucoside for LBA
1 and of 3-rutinoside for platyconin. These results may be
chemotaxonomically important as they suggest that the
Goodeniaceae are closely related to the Campanulaceae.
According to DNA sequence studies, this is apparently also
the case (Angiosperm Phylogeny Group, 1998).
7
demalonyl LBA 1 are shown in Table 1. The FAB mass spec-
trum of demalonyl LBA 1 exhibited a molecular ion at 1275
m/z in agreement with the mass C H O (1275.324). The
5
7
63 33
elemental components were also confirmed by measuring its
high-resolution FABMS; calc. for C H O : 1275.3252.
5
7
63 33
1
Found: 1275.3264. The H NMR spectrum of demalonyl
LBA 1 showed the presence of one molecule of delphinidin,
two molecules of caffeic acid and four molecules of glucose
1
(
Table 2). By analyses of its H NMR spectrum, as well as its
2
D COSY and NOESY spectra, it was revealed that the pro-
ton chemical shifts of demalonyl LBA 1 were almost in
agreement with those of LBA 1 except for the proton signals
of Glu A and malonic acid moieties (Table 2). In particular,
upfield shifts of methylene protons (d 3.82, 3.65–3.15 H-6b
and 6a) of Glu A in demalonyl LBA 1 were observed in com-
parison to those (d 4.52, 4.76 H-6a and 6b) of LBA 1. There-
fore, the OH-6 of Glu A in demalonyl LBA 1 was free of
malonic acid. Other proton signals of demalonyl LBA 1
were assigned in the process described in LBA 1 (Table 2).
Therefore, demalonyl LBA 1 was determined to be
delphinidin 3-O-[b-D-glucopyranoside]-7-O-[6-O-(4-O-(6-
O-(4-O-(b-D-glucopyranosyl)-trans-caffeoyl)-b-D-glucopyr-
anosyl)-trans-caffeoyl)-b-D-glucopyranoside], which has not
been described before.
The flowers of Leschenaultia cv. Violet Lena exhibited a
blue color (Blue 99C by RHS Colour Chart) with chroma-
*
*
ticity value b /a = ꢀ1.95. As shown in Fig. 2, the visible
absorption curve of intact flower petals gave k at 542,
max
579, and 630 nm and was similar to that of Platycodon gran-
diflorum having kmax at 538, 573, and 615 nm (Saito, 1967).
This indicates that the blue flower color of L. cv. Violet
Lena is very likely due to the formation of intramolecular
co-pigmentation between delphinidin and hydroxycinnamic
acids (Honda and Saito, 2002). However, the visible absorp-
tion spectra of LBA 1 showed kmax at 537–540, 573–576,
and 613–616 nm in the phosphate–citrate buffer (MacIlv-
ains) at pH 6 and 7 (Fig. 2) which were not the same as
the kmax observed for fresh flower petals (Fig. 2). The differ-
ence in the kmax between L. cv. Violet Lena and LBA 1 in
buffer solutions strongly indicates that an additional bluing
factor (e.g. intermolecular co-pigmentation) may be
involved in the blue Leschenaultia flowers as is the case
for the blue flower of Ceanothus papillosus (Bloor, 1997).
2
.4. LBA 2
The chromatographic and spectroscopic properties of
LBA 2 are shown in Table 1. As mentioned before, LBA 2
gave delphinidin, glucose, caffeic acid, p-coumaric acid,
and malonic acid by acid hydrolysis. Furthermore, delphin-
idin 3,7-diglucoside was obtained from the alkaline hydroly-
sate as its deacyl anthocyanin. The FAB mass spectrum of
+
LBA 2 gave its molecular ion [M] at 1345 m/z (calcd for
+
C H O 1345.330) and at 1259 m/z (M -malonyl),
6
0
65 35
indicating that LBA 2 is composed of delphinidin with four
molecules of glucose and one molecule each of caffeic acid,
p-coumaric acid and malonic acid. Because of the small
amounts obtained, no further structural studies of LBA 2
could be carried out. However, since both LBA 1 and 2 coex-
ist in the flower petals, it is very likely that the substitution
pattern of LBA 2 would be similar to that of LBA 1. Based
on this assumption and the above chromatographic and
spectroscopic data, a possible structure of LBA 2 would
be either delphinidin 3-(malonylglucoside)-7-[(glucosyl-
p-coumaroyl)(glucosylcaffeoyl)-glucoside] or delphinidin
4. Experimentals
4.1. General procedures
TLC was carried out on plastic coated cellulose sheets
(Merck) using eight mobile phases: BAW (n-BuOH–
HOAc–H O, 4:1:2), BuHCl (n-BuOH–2 N HCl, 1:1, upper
2
layer), AHW (HOAc–HCl–H O, 15:3:82), 1% HCl for
2
anthocyanins, and BAW (4:1:5), EAA (EtOAc–HOAc–
H O, 3:1:1), ETN (EtOH–NH OH–H O, 16:1:3) and
2
4
2
3
-(malonylglucoside)-7-[(glucosylcaffeoyl)(glucosyl-p-cou-
EFW (EtOAc–HCOOH–H O, 5:2:1) for sugars and
2
maroyl)-glucoside], either of which is a new anthocyanin in
plants.
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
30 nm. The eluant was applied as a linear gradient elution
for 40 min from 20% to 85% solvent B (1.5% H PO , 20%
3
. Concluding remarks
5
To date, the distribution of 7-polyacylated anthocya-
3
4
nins, which are acylated with more than two molecules of
HOAc, 25% MeCN in H O) in solvent A (1.5% H PO in
2 3 4

678
N. Saito et al. / Phytochemistry 68 (2007) 673–679
Fig. 2. Visible absorption spectra of LBA 1 and blue petal-tissue of Leschenaultia.
H O). UV–Vis spectra were recorded on a MPS-2400
Diaion HP-20 resin (Mitsubishi Chemical’s Ion Exchange
Resins) column (90 / · 150 mm), on which acylated antho-
cyanins were adsorbed. Then, the column was thoroughly
2
(
Shimadzu) in 0.1% HCl–MeOH (from 200 to 700 nm).
Spectral absorption of the blue flower was directly
measured on the intact petals using a recording spectro-
photometer operated as a double-beam instrument (Type
MPS-2400) (Saito, 1967; Yokoi and Saito, 1973).
High resolution FAB mass (FABMS) spectra were
obtained in the positive ion mode using the magic bullet
washed with H O (2000 mL) and eluted with 5% HOAc–
2
MeOH (500 mL) to recover the anthocyanins. After con-
centration, the eluates were separated and purified with
paper chromatography (PC) using BAW. The separated
pigments were further purified with 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 monitor-
ing at 530 nm for anthocyanins. The solvent used was as
follows: a linear gradient elution for 15 min from 50% to
60% solvent B in solvent A. Fractions were transferred to
a Diaion HP-20 column, on which pigments were
adsorbed. Anthocyanin pigments were eluted with 5%
(
5:1 mixture of dithiothreitol and dithioerythritol) as a
1
matrix. NMR spectra were determined at 500 MHz for H
spectra and at 125.78 MHz for C spectra in CF COOD–
1
3
3
CD OD (1:9), DCl–DMSO-d (1:9) and DCl–CD OD
3
6
3
(
1:9). Chemical shifts are reported relative to a TMS inter-
nal standard (d), and coupling constants are in Hz.
4
.2. Plant materials
HOAc–MeOH followed by addition of excess Et O, and
2
then dried. The entire process was repeated for a second
100 g dried flowers to yield a total of dark blue-violet color
powders, LBA 1 (ca. 30 mg) and LBA 2 (ca. 3 mg).
The plants of Leschenaultia cv. Violet Lena were
purchased from Daiichi-engei (Tokyo), and grown in green-
houses in Hokkaido, Japan (Department of Agro-Environ-
mental Science, Hokkaido Junior College, Takushoku
University, Fukagawa, Hokkaido). Flowers were collected
during winter seasons in Japan. The flowers exhibited a blue
color (Blue 99C by Royal Horticultural Society Color Chart
4.4. Analyses of anthocyanins
The identification of anthocyanins was carried out by
standard procedures involving deacylation with acid, and
hydrolysis with alkaline and acid (Harborne, 1984; Tera-
hara et al., 1990; Saito and Harborne, 1992). The data of
TLC (R values), HPLC (R -min), UV–VIS (kmax), and
*
*
and its chromaticity value b (ꢀ41.7)/a (21.3) = ꢀ1.95 by
Minolta CM-2002); they were dried at 40 ꢁC overnight
and stored at ꢀ10 ꢁC in the refrigerator until needed.
f
t
FABMS spectra are shown in Table 1 and are also
described in Sections 4.4.1–4.4.3.
4
.3. Isolation of anthocyanins
Leschenaultia blue anthocyanin 1 (1). Dark blue-violet
powders; for UV–VIS, see Table 1; for TLC, see Table 1;
Dried flowers (ca. 100 g) of Leschenaultia cv. Violet
1
13
Lena were immersed in 5% HOAc–H O (2 L) at room
for H and C NMR spectra, see Table 2; HR-FABMS
calc. for C H O : 1361.3256. Found: 1361.3285.
2
temperature overnight. The extract was passed through a
6
0
65 36

N. Saito et al. / Phytochemistry 68 (2007) 673–679
679
Leschenaultia blue anthocyanin 2 (2). Dark blue-violet
powders; UV–VIS: kmax 548, (307), 284 nm, Eacyl/Emax
vacuo, the concentrated residues were dissolved in a small
volume of 5% HOAc–MeOH followed by addition of
(
%) = 198, E440/Emax (%) = 19, +AlCl = +shift, TLC:
excess Et O, from which solids were then dried in vacuo
3
2
R -values BAW 0.05, BuHCl 0.00, 1% HCl 0.04, AHW
to give demalonyl LBA 1 powder (ca. 7 mg).
f
1
0
.20, HPLC: R (min) 25.0; FABMS calc. for C H O :
For UV–VIS, see Table 1; for TLC, see Table 1; for H
t
60 65 35
1
345.331. Found m/z 1345.5.
spectrum, see Table 2; HR-FABMS calc. for C H O :
5
7
63 33
1275.3252. Found: 1275.3264.
4
.4.1. Deacylanthocyanin, caffeic acid, and 4-O-glucosyl-
caffeic acid
Acid hydrolysis of LBAs 1 and 2 (ca. 1 mg each) was
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carried out with 2 N HCl (10 mL) at 100 ꢁC for 1 h, with
the hydrolysates analyzed using of TLC and HPLC relative
to authentic standard compounds. LBA 1 (ca. 15 mg) was
dissolved in 2 N NaOH (10 ml) under a degassed syringe
and allowed to stand for 15 min. The solution was next
acidified with 2 N HCl and evaporated in vacuo to dryness.
The residue was dissolved in 1% HCl–MeOH and applied
on TLC (BAW) to yields deacylanthocyanin LBA1 (ca.
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1
405.
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mg), caffeic acid (ca. 1 mg), and 4-O-glucosylcaffeic acid
2
181–2184.
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1
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4
.4.2. 4-O-Glucopyranosyl-trans-caffeic acid
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f
BuHCl 0.46, 1% HCl 0.33 and 0.72, AHW 0.55 and 0.99,
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C H O , 342.095), H NMR (500 MHz, DCl–DMSO-d
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5
18
9
6
3
015.
(
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H-2), 7.11 (d, J = 8.2 Hz, H-5), 7.08 (dd, J = 1.9 and
.2 Hz, H-6), 7.46 (d, J = 15.9 Hz, H-a), 6.32 (d. J = 15.9
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8
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.27 (H-4 and H-5), 3.47 (m, H-6a), 3.72 (d, J = 11.6 Hz,
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4
.4.3. Demalonyl LBA 1
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2
tion (20 ml) and allowed to stand at room temperature for
8 days. At this point, demalonylated LBA 1 was formed in
1
its solution. Demalonylated LBA 1 was then absorbed on
the resin column of Diaion HP-20 and was eluted with
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5
% HOAc–MeOH from the column. After evaporation in