6-Hydroxypelargonidin glycosides in the orange-red flowers of Alstroemeria

Article abstract of DOI:10.1016/S0031-9422(02)00683-0

Two 6-hydroxypelargonidin glycosides were isolated from the orange-red flowers of Alstroemeria cultivars, and determined to be 6-hydroxypelargonidin 3-O-(β-D-glucopyranoside) and 3-O-[6-O-(α-L-rhamnopyranosyl)-β-D-glucopyranoside], respectively, by chemic

Full text of DOI:10.1016/S0031-9422(02)00683-0

Phytochemistry 62 (2003) 1239–1242
www.elsevier.com/locate/phytochem
6-Hydroxypelargonidin glycosides in the orange–red flowers
of Alstroemeria
Fumi Tatsuzawaa,*, Norio Saitob, Naho Muratac, Koichi Shinodac, Atsushi Shigiharad,
Toshio Hondad
aHokkaido Junior College, Takushoku University, Fukagawa, Hokkaido 074-8585, Japan
bChemical Laboratory, Meiji-Gakuin University, Totsuka, Yokohama, Japan
cNational Agricultural Research Center for Hokkaido Region, Sapporo, Hokkaido, Japan
dFaculty of Pharmaceutical Sciences, Hoshi University, Shinagawa, Tokyo, Japan
Received 25 July 2002; received in revised form 31 October 2002
Abstract
Two 6-hydroxypelargonidin glycosides were isolated from the orange–red flowers of Alstroemeria cultivars, and determined to be
6-hydroxypelargonidin 3-O-(b-d-glucopyranoside) and 3-O-[6-O-(a-l-rhamnopyranosyl)-b-d-glucopyranoside], respectively, by
chemical and spectroscopic methods. In addition, five known anthocyanidin glycosides, 6-hydroxycyanidin 3-malonylglucoside, 6-
hydroxycyanidin 3-rutinoside, cyanidin 3-malonylglucoside, cyanidin 3-rutinoside and pelargonidin 3-rutinoside were identified in
the flowers.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Alstroemeriaceae; Alstroemeria cultivars; Flower colour; Anthocyanin; 6-Hydroxypelargonidin (aurantinidin); 6-Hydroxypelargonidin 3-
rutinoside; 6-Hydroxypelargonidin 3-glucoside
1. Introduction
occurrence of 6-hydroxypelargonidin was found only in
Impatiens aurantiaca (Balsaminaceae; Clevenger, 1964)
and no report on the occurrence of its glycoside has
appeared to date. Therefore, this finding is the first
report on the presence of 6-hydroxypelargonidin glyco-
sides in plants. The occurrence of 6-hydroxy-
pelargonidin in plants will provide important
information for the biogenesis of anthocyanins, and
also for the breeding of flowers with unprecedented
colors.
Alstroemeria species are native to subtropical and
temperate South America, and their hybrid cultivars are
grown as popular ornamental plants with attractive
flowers. 6-Hydroxycyanidin glycosides were isolated
and identified from the red flowers of Alstroemeria cul-
tivars (Saito et al., 1985), whereas 6-hydroxydelphinidin
glycosides were found in purple–red flowers (Saito et al.,
1988). Anthocyanins, namely 6-hydroxycyanidin 3-gluco-
side, four 3-rutinosides of cyanidin, delphinidin,
6-hydroxycyanidin and 6-hydroxydelphinidin, and a
3-malonylglucoside of 6-hydroxycyanidin, were also
found in Alstroemeria cultivars (Saito et al., 1985, 1988;
Tatsuzawa et al., 2001), in addition to 3-malonylgluco-
sides of cyanidin and delphinidin (Nørbæk et al., 1996,
1998). In this paper, two anthocyanins, 6-hydroxy-
pelargonidin 3-rutinoside 1 and 3-glucoside 2, were iso-
lated and identified from the orange-red cultivars of
Alstroemeria. To the best of our knowledge, the
2. Results and discussion
In a survey of three orange-red cultivars ‘Oreiju’,
‘Mayprista’ and ‘Spotty-red’ of Alstroemeria by HPLC
analysis, seven anthocyanins, 6-hydroxycyanidin 3-ruti-
noside (11.3–13.5% determined by HPLC analysis),
pigment 1 (32.7–64.0%), pigment 2 (0–0.3%), cyanidin
3-rutinoside (20.2–30.4%), 6-hydroxycyanidin 3-mal-
onylglucoside (0–1.2%), pelargonidin 3-rutinoside (0–
21.3%) and cyanidin 3-malonylglucoside (0–1.0%) were
observed in the flower extracts as the main anthocyanins.
* Corresponding author. Tel. +81-164-23-4111; fax: +81-164-23-
4411.
0031-9422/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0031-9422(02)00683-0

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F. Tatsuzawa et al. / Phytochemistry 62 (2003) 1239–1242
These pigments were isolated from the flowers of the
three cultivars with MAW (MeOH–HOAc–H2O, 4:1:5)
solvent, and purified using Diaion HP-20 column chro-
matography (CC), prep. HPLC and TLC. The struc-
tures of the known anthocyanins were identified on the
basis of TLC, HPLC and spectral analysis with authen-
tic anthocyanins as shown in Sections 3.4. and 3.5. (Har-
borne, 1967; Saito et al., 1988; Torskangerpoll et al.,
1999; Tatsuzawa et al., 2001). However, the structures
of pigments 1 and 2 could not be determined at this
stage, since they exhibited unusual short lmax (493 and
493 nm) values in the visible region (Harborne, 1967).
Upon acid hydrolysis of pigments 1 and 2, only one
anthocyanidin, 6-hydroxypelargonidin was obtained,
and glucose and rhamnose were detected for pigment 1,
and only glucose for pigment 2 as their sugar compo-
nents. On addition of AlCl3 this anthocyanidin did not
exhibit a bathochromic shift, indicating that it lacks a
vicinal hydroxyl group in its B-ring (see Section 3.4.).
Moreover, the aglycone showed an lmax of 500 nm in
0.1% HCl–MeOH in the visible region, at shorter
wavelength than that (520 nm) of pelargonidin (see
Section 3.4). On TLC analysis of Forestal (HOAc–HCl–
H2O, 30:3:10) the aglycone showed a smaller Rf value
(0.46) than that (0.70) of pelargonidin, but closely rela-
ted to the value (0.41) of cyanidin (see Section 3.4.). The
FAB mass measurement of the aglycone gave a mole-
cular ion [M]+ at m/z 287, in agreement with the mass
calculated for 6-hydroxypelargonidin (C15H11O6). The
elemental component was unambiguously confirmed by
HR-FABMS(see Section 3.4.1.). Based on these spec-
tral data, the anthocyanidin was presumed to be
6-hydroxypelargonidin. To confirm the structure of the
anthocyanidin, its 500 MHz 1H NMR spectra were
obtained for CF3CO2D–DMSO-d6 (1:9) solution, and
analyzed. The chemical shifts of four aromatic protons
in the B-ring were readily assigned by 2D COSY spec-
tral analysis (see Section 3.4.). Signals at 8.88 (1H, s)
and 7.27 (1H, s) ppm were assigned H-4 and H-8,
respectively, based on previous work, where those sig-
nals of pelargonidin 3,5-diglucoside were observed at
8.83 and 7.21 ppm (Toki et al., 1995). However, the
signal of H-6 (6.97 ppm of pelargonidin 3,5-diglucoside)
was not observed in the spectrum suggesting the pre-
sence of a hydroxyl group at the 6-position. Therefore,
this anthocyanidin was determined to be 6-hydroxy-
pelargonidin.
rhamnose. The detailed structure of anthocyanin 1 was
elucidated based on measurements of its 1H NMR
spectra including 2D COSY and negative difference
NOE (DIFNOE) spectral techniques. Six aromatic pro-
ton signals of 6-hydroxypelargonidin were assigned
1
based on an analysis of its H–1H COSY spectrum, as
shown in Table 1. The signals of two anomeric protons
of the sugars appeared at 5.39 ppm (d, J=7.7Hz, H-1 of
glucose) and 4.56 ppm (s, H-1 of rhamnose). Based on
the observed coupling constants, the glucose had a b-d-
pyranose form and the rhamnose had a a-l-pyranose
form. The linkage between 3-OH of 6-hydroxy-
pelargonidin and C-10 of glucose was confirmed by
analysis of its DIFNOE spectra (Fig. 1). The methylene
protons in the glucose moiety were shifted to rather low
field (3.94 and 3.44 ppm), indicating that the 6-OH of
glucose is linked to the rhamnose unit. This linkage was
Table 1
NMR spectral data for 6-hydroxypelagonidin glycosides from the
flowers of Alstroemeria (500 and 125.78 MHz, in CF3CO2D–DMSO-
d6 (1:9), TMSas internal standard, J Hz in parentheses)
Pigment 1
ꢀC
Pigment 2
ꢀH
ꢀH
6-Hydroxypelargonidin
2
164.5
144.6
133.9
113.4
141.1
134.6
172.5
94.7
3
4
8.93 s
9.02 s
7.28 s
4a
5
6
7
8
7.17 s
9
150.2
120.0
134.5
117.1
160.7
117.1
134.5
10
20
30
40
50
60
8.57 d (8.9)
7.08 d (8.9)
8.49 d (9.2)
7.07 d (9.2)
7.08 d (8.9)
8.57 d (8.9)
7.07 d (9.2)
8.49 d (9.2)
Glucose
1
2
3
4
5
6
102.6
73.5
76.7
70.1
76.4
66.8
5.39 d (7.7)
3.49 m
3.43 m
5.35 d (ca.7)
3.20–3.75
3.20–3.75
3.20–3.75
3.20–3.75
3.20–3.75
3.26 t (8.9)
3.70 m
3.49 m, 3.94 brd (10.7)
2.1. Pigment 1
Rhamnose
1
The FAB mass spectrum of pigment 1 showed a
molecular ion [M]+ at m/z 595 (C27H31O15). The ele-
mental components of anthocyanin 1 were confirmed by
HR-FABMS(see in Section 3.4.2.). The results indi-
cated that this pigment is composed of 6-hydro-
xypelargonidin with one molecule each of glucose and
101.1
70.7
71.1
72.3
68.9
18.3
4.56 s
3.66 brs
3.49 m
2
3
4
3.19 t (9.2)
3.43 m
1.09 d (6.1)
5
CH3

F. Tatsuzawa et al. / Phytochemistry 62 (2003) 1239–1242
1241
further confirmed by detection of free rutinose in the
3. Experimental
H2O2 degradation product of pigment 1 (Harborne,
1984). Thus, the structure of pigment 1 was determined
to be 6-hydroxypelargonidin 3-O-[6-O-(a-l-rhamno-
pyranosyl)-b-d-glucopyranoside], which is a new antho-
cyanin. This structure was also confirmed by analyses of
3.1. General procedures
TLC was carried out on plastic coated cellulose sheets
(Merck) using seven mobile phases: BAW (n-BuOH–
HOAc–H2O, 4:1:2), BuH (n-BuOH–2N HCl, 1:1), 1%
HCl and AHW (HOAc–HCl–H2O, 15:3:82) for antho-
cyanins, and BAW, EAA (EtOAc–HOAc–H2O, 3:1:1),
ETN (EtOH–NH4OH–H2O, 16:1:3) and EFW (EtOAc–
HCOOH–H2O, 5:2:1) for organic acids and sugars.
Analytical HPLC was performed on a LC-10A system
(Shimadzu), using a Waters C18 (4.61ꢀ250 mm) col-
1
its 13C NMR and H–13C COSY spectra (Table 1). In
the 13C NMR spectra, the chemical shift (134.6 ppm) of
C-6 was apparently shifted to lower field than that
(104.1 ppm) of pelargonidin 3,5-diglucoside (Toki et al.,
1995), indicating oxygenation at C-6.
2.2. Pigment 2
ꢁ
umn at 40 C with a flow rate of 1 ml/min and mon-
The chromatographic and spectral data of pigment 2
are shown in Section 3.4. The FAB mass spectrum of
pigment 2 showed a molecular ion [M]+ at m/z 449
corresponding to the presumed molecular formula
C21H21O11 (449.1083). Unfortunately, the HR-FABMS
data for pigment 2 was not measured because of the
small amount of the pigment 2 obtained. The structure
of pigment 2 was elucidated in a manner similar to that
used for pigment 1. The six aromatic protons in the
aglycone were assigned as shown in Table 1, and these
values were identical with those of pigment 1. The pro-
ton signals of the glucose moiety appeared in the region
of 5.35–3.20 ppm, and an anomeric proton signal was
observed at 5.35 ppm (d, J=ca. 7 Hz). By HPLC ana-
lysis, the Rt (min) of pigment 2 is identical with that of
6-hydroxypelargonidin 3-glucoside, obtained by partial
acid hydrolysis of pigment 1. Therefore, pigment 2 is
determined to be 6-hydroxypelargonidin 3-O-(b-d-glu-
coside), which is a new naturally occurring anthocyanin.
itoring at 530 nm. The eluant was applied as a linear
gradient from 20 to 85% solvent B (1.5% H3PO4, 20%
HOAc, 25% MeCN in H2O) in solvent A (1.5% H3PO4
in H2O). UV–vis spectra were recorded on a MPS-2400
(Shimadzu) in 0.1% HCl–MeOH (from 200 to 700 nm),
whereas FAB mass spectra were obtained in the positive
ion mode using the magic bullet. NMR spectra were
acquired at 500 MHz for 1H spectra and at 125.78 MHz
for 13C spectra in DMSO-d6–CF3COOD (9:1). Chemi-
cal shifts are reported relative to a TMSint. standard
(d) and coupling constants are in Hz.
3.2. Plant materials
The orange-red flowers of Alstroemeria cultivars
‘Oreiju’, ‘Mayprista’ and ‘Spotty-red’ were purchased
from the Takii nursery (Japan). The flowers exhibited a
red color (Red 43A by R.H.S. color chart and its chro-
maticity values, b*/a*=0.75–0.97) and the freshꢁflowers
ꢁ
were dried overnight at 37 C and stored at 10 C until
needed.
3.3. Isolation of anthocyanins
Dried flowers (50 g) of Alstroemeria cultivars were
immersed overnight in 10% HOAc–MeOH (1:l) at room
temp. The extract was concd (100 ml) and purified by
TLC (BAW, 4:1:2) and prep. HPLC by previous proce-
dures (Tatsuzawa et al., 2001). Prep. HPLC utilised a
Waters C18 (191ꢀ150 mm) column at 40 ꢁC with a flow
rate of 4 ml/min monitoring at 485 nm for anthocya-
nins. The solvent systems used were as follows: linear
gradient elution for 15 min from 40 to 60% solvent B in
solvent A. Fractions (about 50 ml) were transferred to a
DIAION HP-20 column. Anthocyanins were eluted
with 5% HOAc–MeOH, evapn residues were dissolved
in a small volume of 5% HOAc–EtOH followed by
addition of excess Et2O, and then dried to give pigment
powders, 6-hydroxypelargonidin 3-rutinoside 1 (5 mg)
and 6-hydroxypelargonidin 3-glucoside 2 (2 mg). In
addition, other five known pigments (ca. 1–3 mg) were
also obtained.
Fig. 1. 6-Hydroxypelargonidin glycosides from the orange-red flowers
of Alstroemeria. 1: pigment 1, 2: pigment 2. Observed NOE corre-
lations are indicated by arrows.

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F. Tatsuzawa et al. / Phytochemistry 62 (2003) 1239–1242
3.4. Analysis of anthocyanins
3.5. Reference of anthocyanins
Characterization of pigments were carried out by
using TLC and HPLC, and UV–vis, FABMSand NMR
spectral data are shown in Table 1. Furthermore, pro-
ducts of the pigments 1 and 2 were analyzed by TLC
after acid hydrolysis, partial acid hydrolysis (2% HCl–
MeOH) and H2O2 degradation (Harborne, 1984). 6-
Hydroxypelargonidin 3-glucoside and 6-hydroxy-
pelargonidin were detected and confirmed in the partial
hydrolysis-products of pigment 1, and obtained as pur-
Cyanidin, cyanidin 3-glucoside, cyanidin 3-rutinoside,
cyanidin 3-malonylglucoside, 6-hydroxycyanidin, 6-
hydroxycyanidin 3-glucoside, 6-hydroxycyanidin 3-ruti-
noside and 6-hydroxycyanidin 3-malonylglucoside were
isolated from the red flowers of Alstroemeria cultivars
(Saito et al., 1985, 1988; Tatsuzawa et al., 2001). Pelar-
gonidin, pelargonidin 3-glucoside and pelargonidin 3-
rutinoside were isolated from the orange-red flowers of
Turipa cultivars (Harborne, 1967; Torskangerpoll et al.,
1999). These pigments were fully identified by spectro-
scopic and chemical methods.
1
ified pigment powders. The UV–vis and H NMR spec-
tra of both pigment products were measured, which
confirmed their structures as shown in Table 1 and this
section. The sugars involved in pigments 1 and 2 were
confirmed to be rutinose in pigment 1 (BAW=0.24,
EAA=0.20, ETN=0.59, EFW=0.12), and glucose in
pigment
2 (BAW=0.29, EAA=0.26, ETN=0.65,
References
EFW=20), respectively, by TLC analysis after the
H2O2 degradation of both pigments.
Clevenger, S., 1964. A new anthocyanidin in Impatiens. Canadian
Journal of Biochemistry 42, 154–155.
Harborne, J.B., 1967. Comparative Biochemistry of the Flavonoids.
Academic Press, London and New York.
3.4.1. 6-Hydroxypelargonidin
UV: lmax 500, 272 nm, E440/Emax (%)=37, AlCl3 shift
0, 1H NMR; ꢀ 8.88(1H, s, H-4), 8.50 (2H, d, J=8.9 Hz, H-
20 and 60), 7.27 (1H, s, H-8), 7.12 (2H, d, J=8.9 Hz, H-30
and 50), TLC: Rf-values BAW 0.66, BuHCl 0.69, 1% HCl
0.01, AHW 0.07, Forestal 0.46, HPLC: Rt (min) 23.58.
HR-FAB mass calc. of C15H11O6: 287.0556. Found:
287.0563.
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and Hall, London.
Nørbæk, R., Christensen, L.P., Bojesen, G., Brandt, K., 1996.
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glycosides from the flowers of Alstroemeria. Phytochemistry 24,
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3.4.2. Pigment 1 (6-hydroxypelargonidin 3-rutinoside)
UV: lmax 493, 271 nm, E440/Emax (%)=38, AlCl3
shift 0, TLC: Rf-values BAW 0.34, BuHCl 0.18, 1%
HCl 0.16, AHW 0.35, HPLC: Rt (min) 16.97. HR-FAB
mass calc. for C27H31O15: 595.1663. Found: 595.1635.
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Hydroxyanthocyanidin glycosides in the flowers of Alstroemeria.
Phytochemistry 27, 1399–1401.
Tatsuzawa, F., Murata, N., Shinoda, K., Saito, N., Shigihara, A.,
Honda, T., 2001. 6-Hydroxycyanidin 3-malonylglucoside from
flowers of Alstroemeria ‘Tiara’. Heterocycles 55, 1195–1199.
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pigments of tulips. Phytochemistry 52, 1687–1692.
3.4.3. Pigment 2 (6-hydroxypelargonidin 3-glucoside)
UV: lmax 493, 270 nm, E440/Emax (%)=41, AlCl3
shift 0, TLC: Rf-values BAW 0.28, BuHCl 0.16, 1%
HCl 0.05, AHW 0.17, HPLC: Rt (min) 15.10.