Structure of anthocyanin from the blue petals of Phacelia campanularia and its blue flower color development

Article abstract of DOI:10.1016/j.phytochem.2005.12.024

The dicaffeoyl anthocyanin, phacelianin, was isolated from blue petals of Phacelia campanularia. Its structure was determined to be 3-O-(6-O-(4′-O- (6-O-(4′-O-β-D-glucopyranosyl-(E)-caffeoyl)-β-D-glucopyranosyl) -(E)-caffeoyl)-β-D-glucopyranosyl)-5-O-(6-O

Full text of DOI:10.1016/j.phytochem.2005.12.024

PHYTOCHEMISTRY
Phytochemistry 67 (2006) 622–629
www.elsevier.com/locate/phytochem
Structure of anthocyanin from the blue petals of
Phacelia campanularia and its blue flower color development
a
b
c
d,
*
Mihoko Mori , Tadao Kondo , Kenjiro Toki , Kumi Yoshida
a
Graduate School of Human Informatics, Nagoya University, Chikusa, Nagoya 464-8601, Japan
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
b
c
Laboratory of Floriculture, Minami-Kyusyu University, Takanabe, Miyazaki 844-0003, Japan
Graduate School of Information Science, Nagoya University, Chikusa, Nagoya 464-8601, Japan
d
Received 21 September 2005; received in revised form 16 November 2005
Available online 7 February 2006
Abstract
The dicaffeoyl anthocyanin, phacelianin, was isolated from blue petals of Phacelia campanularia. Its structure was determined to be
3-O-(6-O-(40-O-(6-O-(40-O-b-D-glucopyranosyl-(E)-caffeoyl)-b-D-glucopyranosyl)-(E)-caffeoyl)-b-D-glucopyranosyl)-5-O-(6-O-malonyl-
b-D-glucopyranosyl)delphinidin. The CD of the blue petals of the phacelia showed a strong negative Cotton effect and that of the
suspension of the colored protoplasts was the same, indicating that the chromophores of phacelianin may stack intermolecularly in
an anti-clockwise stacking manner in the blue-colored vacuoles. In a weakly acidic aqueous solution, phacelianin displayed the same blue
color and negative Cotton effect in CD as those of the petals. However, blue-black colored precipitates gradually formed without metal
ions. A very small amount of Al3+ or Fe3+ may be required to stabilize the blue solution. Phacelianin may take both an inter- and intra-
molecular stacking form and shows the blue petal color by molecular association and the co-existence of a small amount of metal ions.
We also isolated a major anthocyanin from the blue petals of Evolvulus pilosus and revised the structure identical to phacelianin.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Phacelia campanularia; Evolvulus pilosus; Hydrophyllaceae; Convolvulaceae; Phacelianin; Diacylated anthocyanin; Blue flower color develo-
pment; Circular dichroism
1. Introduction
dated (Goto, 1987; Goto and Kondo, 1991). Now, many
hundreds of polyacylated anthocyanins are known, and a
fair percentage of them is from blue petals; therefore, poly-
acylated anthocyanins may contribute greatly to blue
flower color development. Although the stability and blue
color development of polyacylated anthocyanins are
expected to be established with the intramolecular stacking
of aromatic acyl residues to the anthocyanidin nucleus
(Goto and Kondo, 1991; Yoshida et al., 1992, 2000; Brouil-
lard and Dangles, 1994), their stacking structure and the
mechanism of intramolecular charge-transfer, which is
thought to derive from the bathochromic shift in the
absorption spectrum, are obscure.
Most flower colors, especially blue colors, are due to
anthocyanins (Goto and Kondo, 1991; Brouillard and
Dangles, 1994). However, little is known about the mecha-
nism of blue color development of anthocyanins; the blue
dayflower (Kondo et al., 1992) and the blue cornflower
(Kondo et al., 1994, 1998) acquire their color by a supra-
molecular metal complex, and the blue morning glory by
an increase in vacuolar pH to 7.7 (Yoshida et al., 1995).
Since Goto et al. (1982) reported the structure of gentiodel-
phin from the blue petals of Gentiana makinoi, many poly-
acylated anthocyanins, which contain two or more
aromatic acyl residues, were isolated and structurally eluci-
Recently, we have been focusing our research on flower
color development in colored cells and established several
methodologies such as the simultaneous measurement of
the absorption spectrum of a single cell (Yoshida et al.,
*
Corresponding author. Fax: +81 52 789 5638.
E-mail address: yoshidak@is.nagoya-u.ac.jp (K. Yoshida).
0031-9422/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2005.12.024

M. Mori et al. / Phytochemistry 67 (2006) 622–629
623
2003a,b) and vacuolar pH (Yoshida et al., 1995, 2003a,b),
and the reproduction of cell color by mixing components
in colored cells (Yoshida et al., 1995, 2003a,b). In this
report, we describe the structure of a newly isolated polya-
cylated anthocyanin, phacelianin (1), in the blue petals of
Phacelia campanularia. An interesting mechanism for blue
color development of 1 is also discussed. We revised the
structure of the major anthocyanin in blue petals of Evolv-
ulus pilosus to be the same as 1.
2. Results and discussion
2.1. Isolation and structure of anthocyanins in petals of
P. campanularia
Blue petals of P. campanularia were extracted with aque-
ous acetonitrile (CH3CN) containing trifluoroacetic acid
(TFA), and the extract was analyzed by HPLC. As shown
in Fig. 1, the composition of the petal extract is relatively
simple, containing only two anthocyanins (1 and 2) with
a small amount of a UV-absorbing compound (3). The
results of photodiode array detection-HPLC indicated that
1 and 2 may have the same chromophore and two aromatic
acyl residues. Compound 3 may not be a flavonoid but a
cinnamic acid derivative. The components from the petals
(300 g) were purified according to our general procedure
(Yoshida et al., 1992, 1996, 2002, 2003b) to give 1
(170 mg), 2 (24 mg) and 3 (24 mg).
Scheme 1. Structure of 1 and 2.
ester of 1 being very similar in behavior to malonylshisonin
(Kondo et al., 1989; Yoshida et al., 1997). These data
strongly suggest that 2 is a demalonylated pigment from 1.
By 1D- and 2D TOCSY experiments (Kondo et al.,
1990), the signals of all sugar residues of 1 were assigned
so that all were b-glucopyranosides (Table 1). The linkage
of glc-1 to the 3 position and glc-2 to the 5 position of the
delphinidin nucleus was confirmed by the NOEs between
the anomeric proton of glc-1 and H-4, and that of glc-2
and H-6, respectively (Fig. 2). The methylene protons of
the sugar residues (glc-1, 2 and 3) showing a downfield shift
indicated that the 6-positions of glc-1, 2 and 3 were acyl-
ated. To determine the linkage of the acyl groups, an
HMBC experiment was conducted (Fig. 2). One of the E-
caffeoyl residues (C-1) was esterified to the 6-OH of the
glc-1 because of the correlation between the a proton of
C-1 and H-6 of glc-1, and the other caffeoyl residue (C-2)
was connected to 6-OH of the glc-3 because of the correla-
tion between the a proton of C-2 and H-6 of glc-3 (Fig. 2).
Therefore, the substituted position of the malonic acid was
determined to be the 6-OH of glc-2. This was confirmed
from the correlation between H-6 of glc-2 and the 13C sig-
nal of 168.5 ppm assigned to be the carbonyl carbon of the
malonyl residue.
The structures of 1 and 2 were determined by FABMS
and various 1D and 2D NMR experiments combined with
degradation reactions (see Scheme 1). FABMS of 1 gave a
molecular ion peak at m/z = 1361. The 1H NMR spectrum
of 1 showed the existence of a delphinidin nucleus, four
sugars and two E-caffeoyl residues (Table 1). The molecu-
1
lar weight was larger by 86 Da than that estimated by H
NMR analysis, strongly suggesting that the remaining res-
idue is malonyl group. The molecular ion peak of 2 was
1275, which was 86 mass units smaller than that of 1, cor-
1
responding to a loss of a malonyl residue. The H NMR
spectrum of 2 was very similar to that of 1, but only the sig-
nals of one sugar residue (glc-2) were different. Further-
more, acidic methanol treatment of 1 gave 2 via methyl
Fig. 1. HPLC chromatograms (detection: 280 nm) of the extract from the blue petals of Phacelia campanularia (left) and the spectra of each peak obtained
with 3D-detection HPLC (right).

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M. Mori et al. / Phytochemistry 67 (2006) 622–629
Table 1
Assignment of the 1H and 13C NMR spectra of 1 and 2 (1H: 600 MHz,13C: 150 MHz, 23 ꢁC, 10% TFA d-CD3OD)
1
2
1
2
1H d (ppm)
J (Hz)
1H d (ppm)
J (Hz)
13C d (ppm)
13C d (ppm)
2
164.3
146.2
132.8
156.4
106.2
168.7
97.6
164.1
146.3
133.2
156.6
105.9
168.9
97.6
3
4
8.70 s
7.00 d
6.76 d
8.66 s
7.00 d
6.68 d
5
6
2.0
2.0
2.0
2.0
7
8
9
156.3
112.8
156.3
112.7
10
10
119.7
113.3
147.5
146.4
119.6
113.3
147.4
146.1
20,60
30,50
40
7.64 s
6.91 d
7.57 s
6.98 d
C-1
1
130.9
117.5
148.7
148.5
118.6
121.5
117.3
146.5
168.8
130.8
116.8
148.6
148.5
119.1
122.3
117.2
146.4
168.7
2
2.0
2.0
3
4
5
7.03 d
6.75 dd
6.37 d
7.31 d
8.5
7.03 d
6.69 dd
6.35 d
7.27 d
8.5
6
8.5, 2.0
16.0
8.5, 2.0
16.0
a
b
16.0
16.0
C@O
C-2
1
130.4
115.3
148.3
148.7
117.2
122.0
117.2
145.8
168.5
130.4
115.3
148.3
148.7
117.2
122.0
117.2
145.8
168.5
2
6.81 d
2.0
6.79 d
2.0
3
4
5
6.84 d
6.43 dd
6.17 d
7.25 d
8.5
6.82 d
6.36 dd
6.15 d
7.20 d
8.0
6
8.5, 2.0
16.0
8.0, 2.0
16.0
a
b
16.0
16.0
C@O
glc-1
1
5.36 d
8.0
5.37 d
3.90 dd
3.64 t
8.0
102.6
74.1
78.3
72.7
75.4
65.0
102.8
74.1
78.3
72.6
75.5
64.8
2
3.90 dd
3.63 t
9.0, 8.0
9.0
9.0, 8.0
9.0
3
4
3.54 dd
4.03 td
4.58 dd
4.53 dd
9.5, 9.0
9.5, 2.5
11.5, 9.5
11.5, 2.5
3.56 t
9.0
5
4.01 td
4.64 dd
4.48 dd
9.0, 3.0
11.5, 9.0
11.5, 3.0
6a
6b
glc-2
1
5.09 d
7.5
5.12 d
7.5
103.4
74.9
78.1
70.9
76.1
65.1
103.5
74.9
78.0
71.4
79.2
62.8
2
3.78 dd
3.53 t
9.0, 7.5
9.0
3.78 dd
3.55 t
9.0, 7.5
9.0
3
4
3.42 t
9.0
3.37 t
9.0
5
3.73 ddd
4.53 dd
4.23 dd
9.0, 6.5, 2.5
11.5, 2.5
11.5, 6.5
3.61 ddd
4.01 dd
3.64 dd
9.0, 7.0, 1.5
11.5, 1.5
11.5, 7.0
6a
6b
glc-3
1
4.92 d
7.5
4.92 d
7.5
103.2
74.7
77.7
72.4
75.5
65.4
103.2
74.7
77.8
72.5
75.5
65.4
2
3.57 dd
3.56 dd
3.40 dd
3.84 td
4.67 dd
4.21 dd
8.5, 7.5
9.0, 8.5
9.5, 9.0
9.5, 2.0
11.5, 2.0
11.5, 9.5
3.58 dd
3.55 t
9.0, 7.5
9.0
3
4
3.39 dd
3.82 td
4.65 dd
4.19 dd
9.5, 9.0
9.5, 2.0
11.5, 2.0
11.5, 9.5
5
6a
6b
glc-4
1
4.90 d
8.5
4.91 d
7.5
102.6
74.7
77.8
71.2
77.6
62.4
102.5
74.7
77.5
71.1
78.0
62.3
2
3.54 t
8.5
3.53 dd
3.57 t
9.0, 7.5
9.0
3
3.55 dd
3.44 dd
3.56 m
3.89 dd
3.74 dd
9.0, 8.5
9.5, 9.0
4
3.47 dd
3.58 m
3.89 dd
3.75 dd
9.5, 9.0
5
6a
6b
12.5, 2.5
12.5, 5.0
12.5, 2.0
12.5, 5.0
Malonyl
C@O (carboxylate)
C@O (carboxylic acid)
168.5
170.6
The connections between glc-3 to 4-OH of C-1 and glc-4
to 4-OH of C-2 were strongly indicated by the NOEs
between the anomeric proton of glc-3 to 5-H of C-1 and
that of glc-4 to 5-H of C-2 (Fig. 2). However, the latter con-
nection was not confirmed by the HMBC experiments
because it was difficult to differentiate the 13C signals of

M. Mori et al. / Phytochemistry 67 (2006) 622–629
625
In the survey of polyacylated anthocyanins in blue
flower petals, we found that the HPLC data of the major
pigment of the blue petals of E. pilosus were identical to
those of 1. However, Toki et al. (1994) reported a different
structure for the petal anthocyanin. Therefore, we isolated
anthocyanin from the blue petals of E. pilosus and com-
pared it with 1 and the authentic pigment. The HPLC data
1
and H NMR spectral data were identical to those of 1,
resulting in the structure of the pigment in E. pilosus and
P. campanularia being identical to 1.
The structure of 3 was deduced to be 3-O-(E)-caffeoyl-
quinic acid (chlorogenic acid) by comparison with an
authentic sample.
2.2. VIS absorption spectra and CD of petals and colored
cells of P. campanularia
To help understand how pigment molecules are present
in a living petal cell, we measured the VIS absorption spec-
tra and CD of a living petal and colored cells. The CD can
provide very important information on molecular associa-
tion, especially the stacking manner of the chromophores
(Goto and Kondo, 1991; Kondo et al., 1992). Thus, the
CD recording of intact flower petals were also conducted
by Hoshino (1986). However, CD measurement of intact
petals has some difficulties; the colorless intercellular space
filled with air causes diffuse reflections following low sensi-
tivity with a noisy spectrum. To overcome these problems,
we applied a single cell measurement using the micro-
spectrophotometric method for recording the VIS spec-
trum. As another solution to reduce scattered reflection,
we evacuated the petals, filling the intercellular space with
water. The obtained petals became more transparent, and
the VIS spectrum and CD measurements could then be
conducted more noiselessly with higher sensitivity. We also
purified the blue-colored cells and CD of the gathered cells
was recorded in a quartz cuvette.
Fig. 3 shows the VIS absorption spectra and CD of fresh
petals and colored cells from the blue petals of P. campanu-
laria. The absorption spectrum of the petals showed three
kmax around 638, 578 and 546 nm, coinciding with that
of a single protoplast. In the CD, a negative Cotton effect
was observed at the absorption maximum at the VIS
region, and there was no difference between the CD of
the evacuated petal and the suspension of the colored pro-
toplasts (Fig. 3). The negative Cotton effect strongly sug-
gests chiral self-association of the anthocyanidin nuclei of
1 in an anti-clockwise stacking manner. Until now, many
polyacylated anthocyanins have been isolated and their
CD spectra recorded. However, no pigment showed such
a CD with Cotton effects. Generally, aromatic acyl residues
of polyacylated anthocyanins are stacked from both sides
of the anthocyanidin nucleus to stabilize the chromoph-
ores, preventing hydration of the nucleus. Only simple
anthocyanins without aromatic acyl residues (Hoshino
et al., 1981a,b; Goto et al., 1987) or with one acyl residue
(Yoshida et al., 1991; Kondo et al., 1992; Kondo et al.,
Fig. 2. NOE and HMBC correlation of 1.
the 3 and 4 positions of C-2. Therefore, we conducted alka-
line hydrolysis of 1. Reaction of 1 in aq. NaOH-methanol
under an argon (Ar) atmosphere gave a mixture of 4-O-glu-
cosyl-(E)-caffeic acid (6) and methyl 4-O-glucosyl-(E)-caff-
eate (7). The structure was confirmed by NMR, MS
experiment and comparison with authentic sample (Goto
et al., 1981). The combined yield of 6 and 7 was quantita-
tive (195%) to 1; therefore, the linkage of glc-3 and 4 was
determined to be at the 4-OH of caffeoyl residues, C-1
and C-2, respectively. Thus, 1 was deduced to be 3-O-(6-
O-(40-O-(6-O-(40-O-b-D-glucopyranosyl-(E)-caffeoyl)-b-D-
glucopyranosyl)-(E)-caffeoyl)-b-D-glucopyranosyl)-5-O-(6-
O-malonylb-D-glucopyranosyl)delphinidin.
The structure of 2 was determined by using the same MS
and NMR techniques (Table 1) to be 3-O-(6-O-(40-O-(6-O-
(40-O-b-D-glucopyranosyl-(E)-caffeoyl)-b-D-glucopyranosyl)-
(E)-caffeoyl)-b-D-glucopyranosyl)-5-O-b-D-glucopyranosyl-
delphinidin.
By NOESY and ROESY experiments long range NOEs
between the anomeric proton of glc-4 and the protons at 20
and 60 of the delphinidin nucleus were observed (Fig. 2)
Furthermore, the protons at 2, 5 and 6 positions in the
both caffeoyl residues (C-1 and C-2) shifted toward 0.16–
0.61 ppm upfield as compared with the corresponding pro-
tons of methyl 4-O-glucosyl-(E)-caffeate (7) (Table 1 and
data in Section 3.2.2). These data indicate that the caffeoyl
residues of 1 and 2 stack to the anthocyanidin nucleus
intramolecularly in TFA d-CD3OD just as the same as gen-
tiodelphin (Yoshida et al., 1991, 2000). Since intramolecu-
lar stacking of acyl residues of polyacylated anthocyanins
to the anthocyanidin nucleus becomes stronger in neutral
aqueous condition than that in acidic methanol (Yoshida
et al., 1991, 2000), the caffeoyl residues of phacelianin (1)
and its demalonylated pigment (2) should stack to the del-
phinidin nucleus in the petal cells.

626
M. Mori et al. / Phytochemistry 67 (2006) 622–629
Fig. 3. VIS absorption spectra (lower) and CD (upper) of the blue petal
and suspension of the blue cells of phacelia. -----: petal, ——: suspension
of protoplasts.
Fig. 4. Reproduction of the blue color of the protoplast of Phacelia
campanularia by mixing 1 (100 lM) with/without Al3+ in buffered solution
(100 mM acetate buffer, pH 5.5), path length, 1.0 mm.
: suspension of
protoplasts, ——: without Al3+ : 0.02 eq. Al3+, -----: 1 eq. Al3+
,
.
1994; Kondo et al., 2001) are supposed to be able to stack
together with an anthocyanidin nuclei. Therefore, the Cot-
ton effect of the blue petal and cells is very interesting and
important information on blue color development.
the same as that of petals and colored protoplasts, was
obtained and after 24 h, no precipitates were formed. Com-
pound 2 showed the same behavior and color development
as 1. Since the bathochromic shift of anthocyanins by addi-
tion of trivalent metal ions are well known (Harborne,
1958; Bayer, 1958; Bayer et al., 1966; Takeda et al., 1990;
Kondo et al., 1994; Dangles et al., 1994; Yoshida et al.,
2003a; Kondo et al., 2005) metal complexation of Al3+
and/or Fe3+ may involve in the blue coloration of phacelia
petals in some extent.
In conclusion, phacelianin (1) was the first structural
elucidated polyacylated anthocyanin that takes two kinds
of molecular association, self-association and intramolecu-
lar stacking. In the colored vacuole, the anthocyanidin
nuclei of 1 stacked each other in an anticlockwise stacking
manner, and intramolecular caffeoyl residues stacked to the
other side of the nucleus. Furthermore, a small amount of
trivalent metal ions may solubilize 1 in weakly acidic vacu-
oles without any co-pigments and the blue petal color is
developed.
2.3. Reproduction of blue color with combined petal
components
To clarify the mechanism of the blue flower color devel-
opment of P. campanularia, we first analyzed the compo-
nents of colored cells. The HPLC detected at 280 nm
showed that only 1 with a small amount of 2 and 3 existed
in the colored cells. The content of metals was analyzed,
and Mg, Al and Fe were detected. The molar ratio of
Mg, Al and Fe to the combined anthocyanins (1 and 2)
was 0.2–0.5 eq., 0.01–0.02 eq. and 0.01–0.02 eq.,
respectively.
By referring to the above-mentioned data, we tried to
reproduce the same blue color of phacelia petals by mixing
petal components in various pH aqueous solutions. As
shown in Fig. 4, 1 (5 · 10ꢀ4 M) in aqueous solution at
pH 5.5–6.0 gave the same VIS spectrum and CD as those
of petals. However, the stability of the blue solution was
low at this pH and after 24 h, blue precipitates were
formed. By the addition of Al3+ or Fe3+ (>1/3 eq. to 1),
a blue solution with different VIS spectra and CD from
that of petals and protoplasts was obtained. The addition
of an excessive amount of Mg2+ (1–10 eq.) to 1 in buffer
(pH 5.5) affects neither the spectrum nor the stability of
the solution. However, by the addition of a small amount
of Al3+ or Fe3+ to 1 (<0.02 eq.), a stable blue solution,
3. Experimental
3.1. General
UV–Vis spectra and reflection spectra were recorded
on a JASCO V-560 spectrometer. The absorption spectra
of the colored cells were measured with an inverted

M. Mori et al. / Phytochemistry 67 (2006) 622–629
627
microscope (IX70, OLYMPUS) equipped with a micro-
3.3. Alkaline hydrolysis of 1
spectrophotometer (MCPD-7000, Photal). Circular
dichroism (CD) was measured with a JASCO J-720 spec-
trometer. NMR spectra were obtained with a JEOL
ECA-500 (1H: 500 MHz, 13C: 125 MHz) and JNM-
A600 spectrometer (1H: 600 MHz, 13C: 150 MHz) in a
5-mm B tube at variable temperature using 5–10%
TFA d-CD3OD as a solvent. Chemical shifts were
recorded as parts per million (ppm) with the CD2HOD
resonance as a standard. FABMS data were recorded
on a JEOL JMS-700 using glycerol–HCl as a matrix.
Analytical and preparative HPLC were conducted
according to our procedure (Yoshida et al., 1992, 1996,
2002, 2003b) using an ODS-column (Develosil ODS-
HG-5 2.0 mm B · 250 mm, 4.6 mm B · 250 mm and
20 mm B · 250 mm, Nomura Chemical). HPLC was per-
formed at 40 ꢁC with 30-min linear gradient elution from
10% to 30% aq. CH3CN solution containing 0.5% TFA,
or isocratic elution with various concentrations of
CH3CN aq. solution containing 0.5% TFA. Elemental
analysis was conducted with a Perkin–Elmer Optima
2100 DV ICP-AES instrument.
Compound 1 (TFA salt, 5.7 mg, 3.8 mmol) was dis-
solved in methanol (1 mL) and 0.1 M aq. NaOH (2 mL)
under an argon atmosphere and stood at 0 ꢁC for 1 h. A
small portion of 6 M aq. HCl (0.2 mL) was added to the
reaction mixture, and the mixture afforded preparative
HPLC to give 2.1 mg of delphin (5, 74%), 1.0 mg of 4-O-
b-glucopyranosylcaffeate (6, 72%) and 1.7 mg of methyl
4-O-b-glucopyranosylcaffeate (7, 123%), respectively.
3.3.1. 4-O-Glucosyl-(E)-caffeic acid (6)
1H NMR (500 MHz, CD3OD): 3.41 (1H, dd, 9.5, 8.0,
glc-4), 3.45 (1H, ddd, 9.5, 5.0, 1.5, glc-5), 3.48 (1H, dd,
9.5, 8.0, glc-3), 3.52 (1H, dd, 9.0, 7.5, glc-2), 3.72 (1H, dd,
12.0, 5.0, glc-6a), 3.90 (1H, dd, 12.0, 1.5, glc-6b), 4.85
(1H, d, 7.5, glc-1), 6.31 (1H, d, 16.0, caf-a), 7.03 (1H, dd,
8.5, 2.5, caf-6), 7.10 (1H, d, 2.5, caf-2), 7.19 (1H, d, 8.5,
caf-5), 7.55 (1H, d, 16.0, caf-b).
3.3.2. Methyl 4-O-glucosyl-(E)-caffeate (7)
1H NMR (500 MHz, CD3OD): 3.41 (1H, dd, 9.0, 8.0,
glc-4), 3.45 (1H, ddd, 9.0, 5.0, 1.5, glc-5), 3.48 (1H, dd,
9.0, 8.0, glc-3), 3.52 (1H, dd, 9.0, 7.5, glc-2), 3.72 (1H, dd,
12.0, 5.0, glc-6a), 3.76 (3H, s, OCH3), 3.90 (1H, dd, 12.0,
1.5, glc-6b), 4.86 (1H, d, 7.5, glc-1), 6.35 (1H, d, 16.0,
caf-a), 7.04 (1H, dd, 8.5, 2.5, caf-6), 7.10 (1H, d, 2.5, caf-
2), 7.19 (1H, d, 8.5, caf-5), 7.57 (1H, d, 16.0, caf-b).
3.2. Isolation of phacelianin (1) and demalonylphacelianin
(2)
3.2.1. Isolation of 1 and 2 from P. campanularia
Blue petals (300 g) of P. campanularia cultivated at the
Nagoya University Farm were extracted with 2.6 L of
35% aq. CH3CN solution containing 0.5% TFA. The
extract was evaporated under reduced pressure to half vol-
ume, and the condensed extract was purified with an
Amberlite XAD-7 column. The column was eluted with a
stepwise gradient elution from 0.5% TFA–H2O to 20%
aq. CH3CN solution containing 0.5% TFA. The 15% aq.
CH3CN fraction was evaporated to give a crude anthocya-
nin. Further purification was conducted by a preparative
ODS-HPLC eluted with 5–16% aq. CH3CN solution con-
taining 0.5% TFA. Compounds 1 (170 mg) and 2 (24 mg)
were obtained from the 16% aq. CH3CN fraction as
dark-red amorphous TFA salts with 3 (10% aq. CH3CN
fraction, 24 mg).
3.4. Acid hydrolysis of 1
Compound 1 (TFA salt, 1.0 mg) was dissolved in 1%
HCl–methanol (1 mL) and stood at 40 ꢁC for 3 days. The
reaction mixture was analyzed with HPLC (column:
2 mm B, solvent: 16% aq. CH3CN solution containing
0.5% TFA) repeatedly.
3.5. Preparation of protoplasts
The preparation of colored protoplasts was conducted
using
a slightly modified method (Yoshida et al.,
2003a,b). Fresh petals (0.5 g) were cut at a thickness of
1 mm and incubated in a medium (0.6 M mannitol,
20 mM Tris–AcOH, pH 6.0) containing 0.2% (w/v) Mac-
erozyme R-10 (Yakult) and 2.0% (w/v) Cellulase ONO-
ZUKA R-10 (Yakult) at 30 ꢁC for 80 min. The reaction
mixture was filtered through Miracloth (Calbiochem),
and the obtained filtrate was washed 3 times. The obtained
protoplast mixture was further purified by sucrose density
gradient centrifugation. The protoplast mixture was sus-
pended in 2.5 mL of buffer (1.0 M sucrose, 50 mM Mes–
Tris pH 6.0). The suspension was overlaid on 2.5 mL of
0.6 M sucrose and 0.65 M mannitol gradient in 50 mM
Mes–Tris, pH 6.0 and centrifuged at 320g for 10 min.
The blue-colored cells obtained at the interface between
0.6 M sucrose and 0.65 M mannitol were collected and
washed.
3.2.2. Isolation of 1 from blue petals of E. pilosus
Blue petals (150 g) of E. pilosus cultivated at the Nagoya
University Farm were extracted with 1.5 L of 50% aq.
CH3CN solution containing 1.0% TFA. The extract was
evaporated under reduced pressure to one-third volume,
and the condensed extract was purified with an Amberlite
XAD-7 column. The column was eluted with a stepwise
gradient elution from 1.0% TFA–H2O to 50% aq. CH3CN
solution containing 1.0% TFA. In the 15–20% aq. CH3CN
fraction, phacelianin (1), demalonylphacelianin (2), chloro-
genic acid (3), and 3,5-dicaffeoylquinic acid (4) were eluted.
Further purification was conducted by repeated prepara-
tive ODS-HPLC to give 1 (53 mg) and 2 (19 mg) as dark-
red amorphous TFA salts with 3 (7.6 mg) and 4 (73 mg).

628
M. Mori et al. / Phytochemistry 67 (2006) 622–629
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3.6. VIS spectrum and CD measurement of petals and
colored cells
The reflection spectrum of a fresh petal was recorded
according to a previous report (Yoshida et al., 2003a,b).
CD of the petal was recorded with a pre-evacuated tissue.
A fresh petal was cut into squares of ca. 10 mm · 10 mm,
and the pieces were put into a flask with water. The flask
was evacuated several times, then, the intercellular space
of the petal tissue was filled with water and the transpar-
ency of the petal increased. The petal was fixed on a quartz
plate and CD was measured (400–800 nm). The suspension
of colored cells in a medium (0.6 M mannitol, 20 mM Tris–
AcOH, pH 6.0) was poured into a quartz cuvette (path
length, 1 mm) and the VIS absorption spectrum and CD
were recorded.
3.7. Analysis of components in colored protoplasts
Hoshino, T., 1986. Circular dichroic measurements on anthocyanins in
intact flower petals. Phytochemistry 25, 829–832.
An aliquot quantity of the suspension of colored pro-
toplasts was diluted with water containing 0.5% TFA and
analyzed with HPLC. The amount of 1 was calculated
directly by the calibration curve of standard solutions.
Another aliquot quantity of the suspension of colored pro-
toplasts was diluted with 0.5% (w/v) aq. HNO3, and Al,
Mg and Fe were analyzed by ICP-AES under standard
operating conditions.
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Kondo, T., Tamura, H., Yoshida, K., Goto, T., 1989. Structure of
malonylshisonin,
a genuine pigment in purple leaves of Perilla
ocimoides L. var. crispa Benth. Agric. Biol. Chem. 53, 797–800.
Kondo, T., Ueda, M., Goto, T., 1990. Structure of ternatin B1, a
pentaacylated anthocyanin substituted on the B-ring asymmetrically
with two long chains. Tetrahedron 46, 4749–4756.
3.8. Reproduction of petal color
Kondo, T., Yoshida, K., Nakagawa, A., Kawai, T., Tamura, T., Goto, T.,
1992. Structural basis of blue-colour development in flower petals:
structure determination of commelinin from Commelina communis.
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Kondo, T., Ueda, M., Tamura, H., Yoshida, K., Isobe, M., Goto, T.,
1994. Composition of protocyanin, a self-assembled supramolecular
pigment form the blue cornflower Centaurea cyanus. Angew. Chem.,
Int. Ed. Engl. 33, 978–979.
Compound 1 (TFA salt) was dissolved in water at a con-
centration of 5 · 10ꢀ2 M. Individual solutions were diluted
to 5 · 10ꢀ4 M with 0.1 M acetate buffer (pH 5–5.5) or
0.1 M phosphate buffer (pH 6–8) with/without Mg2+
,
Al3+, Fe3+, and 3. UV–Vis spectra and CD were measured
in a quartz cell (path length: 1 mm) at 25 ꢁC.
Kondo, T., Ueda, M., Isobe, M., 1998. A new molecular mechanism of
blue color development with protocyanin, a supramolecular pigment
from cornflower, Centaurea cyanus. Tetrahedron Lett. 49, 8307–8310.
Kondo, T., Oyama, K., Yoshida, K., 2001. Chiral molecular recognition
Acknowledgements
on formation of
complex pigment from blue flower of Salvia patens. Angew. Chem.,
Int. Ed. 40, 894–897.
a metalloanthocyanin: a supramolecular metal
We are grateful to Ms. N. Yoshino of Nagoya
University for the cultivation of plant materials. This work
was financially supported by the Ministry of Education,
Science, Sports, Culture, Technology, Japan ((B)
No. 16370021The, 21st Century COE Program No.
14COEB01-00, Creative Scientific Research No.
16GS0206 and Scientific Research on Priority Areas
No. 17035041).
Kondo, T., Toyama-Kato, Y., Yoshida, K., 2005. Essential structure of
co-pigment for blue sepal-color development of hydrangea. Tetrahe-
dron Lett., 6645–6649.
Takeda, K., Yamashita, T., Takahashi, A., Timberlake, C.F., 1990. Stable
blue complexes of anthocyanin-aluminium-3-p-coumaroyl- or 3-caf-
feoyl-quinic acid involved in the blueing of Hydrangea flower.
Phytochemistry 29, 1089–1091.
Toki, K., Saito, N., Kawano, K., Lu, T.S., Shigihara, A., Honda, T., 1994.
An acylated delphinidin glycoside in the blue flowers of Evolvulus
pilosus. Phytochemistry 36, 609–612.
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