F. Tatsuzawa et al. / Phytochemistry Letters 7 (2014) 69–76
75
3.2. Plant materials
3.4.2. Alkaline hydrolyses
Alkaline hydrolyses of pigments 1–4 (ca. 0.5 mg, each) were
carried out with 2 N NaOH solution (1 ml) using a degassed
syringe stirring for 15 min. Then, cyanidin 3-sambubioside-5-
glucoside, 4-O-glucosyl-p-coumaric acid, ferulic acid and malonic
acid were obtained from 1 to 3 as their hydrolysates and cyanidin
Seeds of pale-violet flowers of I. acaule were purchased from
Takii Co. Ltd (Kyoto). Seeds were sown in August, 2009 and plants
were grown in a greenhouse of Iwate University. Flowers with a
pale violet color [Violet 85D by Royal Horticultural Society
R.H.S) Color Chart and b*(ꢂ4.79)/a*(7.70) = ꢂ0.62, L* = 79.42 by
X
(
3-(3 -glucosylsambubioside)-5-glucoside, 4-O-glucosyl-p-cou-
a SE 2000 Spectro Color Meter (Nppon Denshoku Industries Co.,
Ltd.)] were collected in winter to spring in 2010. Spectral
absorption of flowers were directly measured on intact petals
using a recording spectrophotometer operating as a double-
beam instrument (Type: MPS-2450) (Saito, 1967; Yokoi and
Saito, 1973). The petals (3 mm in its diameter and four petals per
a flower) were trimmed from flowers (about 10,000 flowers) by
hand and dried by air for 1 day at 45 8C. Then they were kept at
ꢂ20 8C until used.
maric acid, ferulic acid and malonic acid were obtained from 4
as its hydrolysate. These compounds were confirmed by direct
comparison of TLC, UV–vis and/or HPLC with the authentic
samples which were obtained from anthocyanins of Malcolmia
and Lobularia by alkaline hydrolysis (Tatsuzawa et al., 2007,
2008a).
3.4.2.1. Deacyl anthocyanin of pigments 1–3 (cyanidin 3-sambubio-
side-5-glucoside). UV–vis (in 0.1% HCl–MeOH): lmax 527, 278 nm,
E
440/E527 (%) = 13, AlCl
3
shift + TLC: (R
f
-values) BAW 0.28, BuHCl
(min) 13.1.
3
.3. Isolation and purification of anthocyanins
0.04, 1% HCl 0.24, AHW 0.50; HPLC: R
t
X
Dried flowers (ca. 50 g) of I. acaule were immersed in 5% HOAc
3.4.2.2. Deacyl anthocyanin of pigment 4, cyanidin 3-(3 -glucosyl-
(
5 l) at room temperature for 5 h and extracted. Five anthocyanin
sambubioside-5-glucoside). UV–vis (in 0.1% HCl–MeOH):
278 nm, E440/E526 (%) = 14, AlCl shift + TLC: (R -values) BAW 0.18,
BuHCl 0.03, 1% HCl 0.34, AHW 0.53; HPLC: R
lmax 526,
pigments was isolated and purified by Diaion HP-20 (Mitsubishi
Chemical’s Ion Exchange Resins) column (90 ꢀ 150 mm) chroma-
tography, paper chromatography and preparative HPLC from the
extract as described previously (Tatsuzawa et al., 2006, 2007). The
purified five anthocyanins were obtained from the flowers as
follows; pigment 1 (ca. 3 mg), pigment 2 (ca. 3 mg), pigment 3 (ca.
3
f
t
(min) 13.7.
f
3.4.2.3. 4-O-Glucosyl-p-coumaric acid. TLC: (R -values) BAW 0.76,
t
EAA 0.82, EFW 0.79, HPLC: R (min) 8.0.
1
5 mg), pigment 4 (ca. 9 mg) and pigment A (ca. 2 mg).
3.4.3. Pigment 1
Dark red powder; for UV–vis (in 0.1% HCl–MeOH):
315sh, 294, 283 nm, E315/E530 (%) = 127, E440/E530 (%) = 10, AlCl
shift + TLC: (R -values) BAW 0.41, BuHCl 0.12, 1% HCl 0.10, AHW
t
0.37, HPLC: R (min) 34.9, H NMR spectrum, see Table 1; HR-
lmax 530,
3
.4. Chemical and spectroscopic analyses of purified anthocyanins
3
f
1
The identification of anthocyanins was carried out by standard
procedures (Harborne, 1984).
65
FABMS calc. for C60H O33: 1313.3408. Found: 1313.3376.
3.4.1. Acid hydrolyses
3.4.4. Pigment 2
Acid hydrolyses of pigments 1–4 (ca. 0.5 mg, each) were
Dark red powder; for UV–vis (in 0.1% HCl–MeOH):
315sh, 295, 283 nm, E315/E530 (%) = 122, E440/E530 (%) = 12, AlCl
shift + TLC: (R -values) BAW 0.74, BuHCl 0.33, 1% HCl 0.27, AHW
0.64, HPLC: R
lmax 530,
carried out with 2 N HCl (1 ml) at 100 8C for 2 h, and resulted in
cyanidin, glucose, xylose, p-coumaric acid, ferulic acid and
malonic acid. These compounds were confirmed by direct
comparison of TLC and/or HPLC with the authentic samples
3
f
1
t
(min) 27.0, H NMR spectrum, see Table 1; HR-
65
FABMS calc. for C60H O33: 1313.3408. Found: 1313.3422.
(
Honda et al., 2005).
Partial acid hydrolysis of pigment 3 (ca. 0.5 mg) was carried out
3.4.5. Pigment 3
with 2 N HCl (0.5 ml) and hydrolyzed by heating in a water bath
ca. 90 8C) for 10 min. Two intermediary pigment products were
detected in the hydrolysates as the main products by HPLC
analysis, and isolated from the hydrolysates. These pigment
products were identified by comparison with cyanidin 3-O-[2-O-
Dark red powder; for UV–vis (in 0.1% HCl-MeOH):
315sh, 296, 284sh nm, E315/E533 (%) = 146, E440/E533 (%) = 11, AlCl
shift + TLC: (R -values) BAW 0.74, BuHCl 0.38, 1% HCl 0.10, AHW
0.40, HPLC: R
lmax 533,
(
3
f
1
13
t
(min) 37.1, H and C NMR spectra, see Table 1; HR-
73
FABMS calc. for C70H O36: 1489.3882. Found: 1489.3885.
(2-O-(trans-feruloyl)-xylosyl)-6-O-(4-O-glucosyl-trans-p-coumar-
oyl)-glucoside]-5-O-glucoside and cyanidin 3-O-[2-O-(2-O-(trans-
feruloyl)-xylosyl)-6-O-(trans-p-coumaroyl)-glucoside]-5-O-gluco-
side which were obtained from Lobularia purple-violet flowers
described previously (Tatsuzawa et al., 2007). The data of two
intermediary pigment products are shown in Sections 3.4.1.1 and
3.4.6. Pigment 4
Dark red powder; for UV–vis (in 0.1% HCl–MeOH):
317sh, 295, 285sh nm, E317/E532 (%) = 159, E440/E532 (%) = 14,
AlCl shift + TLC: (R -values) BAW 0.62, BuHCl 0.20, 1% HCl 0.36,
AHW 0.60, HPLC: R
Table 1; HR-FABMS calc. for C76
651.4465.
lmax 532,
3
f
1
13
t
(min) 37.1, H and C NMR spectra, see
41: 1651.4410. Found:
3
.4.1.2.
83
H O
1
3
.4.1.1. Cyanidin 3-O-[2-O-(2-O-(trans-feruloyl)-xylosyl)-6-O-(4-O-
glucosyl-trans-p-coumaroyl)-glucoside]-5-O-glucoside. UV–vis (in
.1% HCl–MeOH): max 532, 318sh, 298sh, 279 nm,
532(%) = 95, E440/E532 (%) = 13, AlCl
3.4.7. Pigment A
0
E
l
E
318
/
For the identification of pigment A isolated from the flowers of I.
acaule, we used a purified anthocyanin, cyanidin 3-[2-(xylosyl)-6-
(trans-p-coumaryl)-glucoside]-5-[6-(malonyl)-glucoside],
obtained from the flowers of Lunnaria annua as a comparative
standard (Tatsuzawa et al., 2006), and analyzed by the methods of
TLC, HPLC, UV–vis and HR-FAB mass spectra. As the results, we
were able to determine the structure A to be cyanidin 3-O-[2-O-
(xylosyl)-6-O-(trans-p-coumaroyl)-glucoside]-5-O-[6-O-(malo-
nyl)-glucoside] as follows.
3
shift + TLC: (R
.43, BuHCl 0.19, 1% HCl 0.30, AHW 0.61, HPLC: R
f
-values) BAW
0
t
(min) 25.5.
3
.4.1.2. Cyanidin
3-O-[2-O-(2-O-(trans-feruloyl)-xylosyl)-6-O-
(
trans-p-coumaroyl)-glucoside]-5-O-glucoside. UV–vis (in 0.1%
HCl–MeOH): max 531, 320, 298, 281 nm, E320/E531 (%) = 123,
l
E440/E531 (%) = 12, AlCl
3
shift + TLC: (R
f
-values) BAW 0.67, BuHCl
t
(min) 31.9.
0
.48, 1% HCl 0.20, AHW 0.51, HPLC: R