Anthocyanins from flowers of Cichorium intybus

Article abstract of DOI:10.1016/S0031-9422(02)00055-9

From the blue perianth segments of Cichorium intybus we isolated four anthocyanins. The pigments were identified as delphinidin 3,5-di-O-(6-O-malonyl-β-D-glucoside) and delphinidin 3-O-(6-O-malonyl-β-D-glucoside)-5-O-β-D-glucoside and the known compounds

Full text of DOI:10.1016/S0031-9422(02)00055-9

Phytochemistry 60 (2002) 357–359
www.elsevier.com/locate/phytochem
Anthocyanins from flowers of Cichorium intybus
Rikke Nørbæka,*, Karina Nielsena, Tadao Kondob
aDanish Institute of Agricultural Sciences, Department of Horticulture, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark
bGraduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
Received 14 November 2001; received in revised form 4 February 2002
Abstract
From the blue perianth segments of Cichorium intybus we isolated four anthocyanins. The pigments were identified as delphinidin
3,5-di-O-(6-O-malonyl-b-d-glucoside) and delphinidin 3-O-(6-O-malonyl-b-d-glucoside)-5-O-b-d-glucoside and the known com-
pounds were delphinidin 3-O-b-d-glucoside-5-O-(6-O-malonyl-b-d-glucoside) and delphinidin 3,5-di-O-b-d-glucoside. In addition
3-O-p-coumaroyl quinic acid has been identified. # 2002 Published by Elsevier Science Ltd.
1
Keywords: Cichorium intybus; Compositae; Flower pigments; Malonated anthocyanins; Quinic acid derivative; FABMS; H NMR; DIF NOE
1. Introduction
2. Results and discussion
Chicory, a typical Mediterranean plant indigenous to
Europe, Western Asia, Egypt and North America, varies
in perianth colour from white, red to blue and the
flowering period is from June to September (Fernald,
1950; Clapham et al., 1962). From lmax measured on the
flower extract it has been suggested that delphinidin
glycosides are present, but the pigments were not further
identified (Proctor and Creasy, 1969). Subsequently,
from blue perianth segments of Cichorium intybus, the
presence of delphinidin 3,5-di-O-malonylglucoside has
been suggested by FAB–MS measurement but the posi-
tions of sugars and malonic acid moieties has not been
clarified (Takeda et al., 1986). From a leaf extract, cya-
nidin 3-O-(6-O-malonyl-b-d-glucoside) (Timberlake et
al., 1971; Bridle et al., 1984), quercetin 3-O-b-d-glucoside
(Saleh et al., 1975), several coumarin and cinnamic acid
derivatives such as chicoric acid (Scarpati and Oriente,
1958), caffeic acid and chlorogenic acid (Trease, 1969)
have been found. However, in the flowers no other
compound with UV-absorption besides anthocyanins
have yet been studied.
Blue perianth segments of C. intybus were extracted with
aqueous acetonitrile containing 0.5% TFA. The HPLC
chromatogram showed four peaks of anthocyanin and
one peak of UV-absorbing substance. The anthocyanins
were isolated by column chromatography on Amberlite
XAD-7 with subsequent preparative HPLC. Pigment 1,
2, 3 and 4 were obtained as dark red TFA salts.
The HR FAB–MS spectrum of 1 showed a molecular
ion at m/z 799.1561, in good agreement with the mass
calculated for C33O23H+35 to be comprised of delphinidin,
and two malonylhexoses and that of 2 gave a molecular
ion peak at m/z 713.1566 (C33O20H+33) corresponding to
monomalonyl dihexosyl delphinidin. Partial hydrolysis
of 1 gave the products corresponding to 2, 3 and 4
(Scheme), and after 60 min hydrolysis the remaining
products were 4 and delphinidin. These results strongly
suggested that 1 is monomalonated compound of 3 and
2 is an isomer of 3.
1
Analysis of the H NMR spectrum of 1 agreed well
with the MS data of the presence of delphinidin and two
malonylated hexosyl residues (Table 1). The assignment of
1
1
In this paper we report on the identification of four
anthocyanins and one quinic acid derivative from blue
perianth segments of C. intybus.
the two hexoses was done by H HOHAHA spectra, H
COSY and 1H TCOSY. All vicinal coupling constants (J)
of both sugars were 7.8–9.8 Hz, including two anomeric
protons at ꢀ 5.44 (d, J=7.8 Hz, glucoside A) and ꢀ 5.14
(d, J=7.8 Hz, glucoside B). Therefore, both sugar
units were determined to be b-d-glucopyranoside. The
6-methylene protons of glucoside A and B are lowfield
* Corresponding author. Tel.: +45-639-04302; fax: +45-639-
04395.
E-mail address: rikke.norbaek@agrsci.dk (R. Nørbæk).
0031-9422/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0031-9422(02)00055-9

358
R. Nørbæk et al. / Phytochemistry 60 (2002) 357–359
Table 1
1H NMR spectral data of two novel acylated anthocyanins from
Cichorium intybus (in CD3OD, containing 10% TFA-d)
1
2a
Delphinidin
4
8.56 s
8.67 s
6
6.71 d (1.8)
6.69 br d
7.43 s
6.84 d (1.8)
6.80 br d
7.57 s
8
20
60
7.43 s
7.57 s
Glucoside A
100
5.32 d (7.8)
5.25 d (7.8)
200
3.55 dd (7.8, 9.0)
3.43 t (9.0)
3.24 t (9.0)
3.56 dd (7.8, 9.0)
3.39 t (9.0)
3.24 t (9.0)
300
400
500
3.72 ddd (2.2,7.7,9.0)
4.24 dd (2.2,12.0)
4.14 dd (7.7, 12.0)
3.71 ddd (2.4,7.0,9.0)
4.25 dd (2.4, 12.0)
4.15 dd (7.0, 12.0)
6a00
6b00
Glucoside B
1000
5.02 d (7.8)
4.98 d (8.4)
2000
3.52 dd (7.8, 9.0)
3.40 t (9.0)
3.29 t (9.0)
3.49 dd (8.4, 9.0)
3.35 t (9.0)
3.25 t (9.0)
3000
4000
5000
3.62 ddd (1.8, 7.3, 9.0)
4.32 dd (1.8, 12.0)
4.16 dd (7.3, 12.0)
3.38 ddd (2.2, 6.2, 9.0)
3.75 dd (2.2, 12.0)
3.54 dd (6.2, 12.0)
Scheme 1.
6a000
6b000
Coupling constants J (in Hz) in parentheses.
The methylene protons of malonic acid disappeared by displacement of
delphinidin 3,5-di-O-b–d-glucoside, respectively (Nør-
bæk and Kondo, 1999; Nørbæk et al., 1996) by com-
parison of retention time on HPLC and H NMR with
authentic samples.
Also 3-O-p-coumaroylquinic acid (Loponen et al.,
1998; Ossipov et al., 1996) was isolated as a major UV-
absorbing product in Cichorium intybus for the first time.
deuterium.
a
By irradiation of H-100 of glucoside A at 0 ꢀC in CD3OD con-
taining 10% TFA-d, a strong negative NOE was observed at H-4 of
delphinidin. By irradiation of H-1000 of glucoside B, a strong negative
NOE was observed at H-6 of the nucleus. A weak negative NOE was
also observed between the same anomeric proton and H-4.
1
shifted by ca. 0.6 ppm more than 4 (Nørbæk and Kondo,
1999). Therefore, the malonyl groups were esterified to the
6-OH of each glucoside (Kondo et al., 1989, 1990). The
positions of the glucosidic linkages were determined by
1H NOESY. NOE was observed between H-4 (ꢀ 8.68)
and the anomeric proton (H-100). Also, NOE between
H-6 (ꢀ 6.83, d, J=1.8 Hz) and H-1000 (glucoside B) was
observed, but no NOE was found between H-8 (ꢀ 6.81,
br d) and H-1000. Thus, 1 is delphinidin 3,5-di-O-(6-O-
malonyl-b-d-glucoside) (Scheme 1).
Using the same NMR experiments 2 gave the similar
NMR data as that of 1 but only showed lowfield shifted
methylene protons (H-6a00 at ꢀ 4.37 and H-6b00 at ꢀ 4.27),
of the glucoside A. Thus, 2 is delphinidin 3-O-(6-O-
malonyl-b-d-glucoside)-5-O-b-d-glucoside (Scheme 1).
The presence of 1 in perianth segments of C. intybus
and of 2 in flowers of Labiatae have previously been
suggested by Takeda et al. (1986) and Saito and Har-
borne (1992), respectively. Therefore, this is the first
time that the glucosidic linkages and the acyl positions
have been determined.
3. Experimental
3.1. Plant material
The blue perianth segments of Cichorium intybus were
collected in September from plants growing wild around
the area Aarslev, Denmark. The plant material was
identified by N. Jacobsen, The Royal Veterinary and
Agricultural University, Dept. of Ecology, Botanical
Section, Copenhagen, Denmark.
3.2. Spectroscopic analysis
UV-visible spectra were recorded in MeOH contain-
ing 0.1% HCl. FAB–MS (JMS-700) spectra were
obtained by using glycerol–HCl as a matrix. Further-
more MALDI-TOF (Voyager DE-PRO) were measured
by using cyano-4-hydroxycinnamic acid in H2O:CH3CN
(1:1) as a matrix. H NMR was measured in CD3OD
containing 10% TFA-d by 600 MHz (JNM alpha 600,
Jeol) with internal standard CD2HOD (3.326 ppm). 2D
1
Structures of 3 and 4 were established as delphinidin
3-O-b-d-glucoside-5-O-(6-O-malonyl-b-d-glucoside) and

R. Nørbæk et al. / Phytochemistry 60 (2002) 357–359
359
Acknowledgements
spectra were obtained using a pulse sequence supplied
from Jeol.
We thank Niels Poulsen, Danish Institute of Agri-
cultural Sciences, for collecting the plant material of
Cichorium intybus, Berta Goncalves, University of Tras-
os-Montes e Alto Douro, for helping with isolation
of the pigments, and Sin-ichi Oyama, Chemical Instru-
ment Center, Nagoya Univesity, for taking of the MS
data.
3.3. HPLC analysis
Analytical ODS-HPLC was carried out by using
reversed phase column (4.6fꢁ250 mm, Develosil ODS-
ꢀ
HG-5, Nomura Chemicals) at 40 C, detected at lmax
530 nm. A linear gradient from 0 to 30% aq. CH3CN
containing 0.5% TFA during 60 min with a flow rate of
1 ml minꢂ1 was used.
References
3.4. Isolation of anthocyanins
Bridle, P., Thomas, Loeffler, R.S., Timberlake, C.F., Self, R., 1984.
Cyanidin 3-malonylglucoside in Cichorium intybus. Phytochemistry
23, 2968–2969.
Freeze-dried perianth segments of Cichorium intybus
(50 g) were extracted with 50% aq. CH3CN containing
0.5% trifluoroacetic acid (TFA) at room temp. for 1 h.
The conc. extract was adsorbed on an Amberlite XAD-7
column, washed with 0.5% TFA aq. soln and then
eluted stepwise from 7 to 25% aq. CH3CN containing
0.5% TFA. For further purification, the crude antho-
cyanins were sepd on prep. ODS-HPLC (20fꢁ250 mm,
Develosil ODS-HG-5, Nomura Chemicals) using step-
wise elution (8–25% aq. CH3CN containing 0.5% TFA)
at a flow rate of 5 ml minꢂ1. The pigment frs. were conc.
Clapham, A.R., Tutin, T.G., Warburg, E.F., 1962. Flora of the British
Isles. Cambridge University Press, Cambridge.
Fernald, M.L., 1950. Gray’s Manual of Botany. American Book
Company, New York.
Kondo, T., Tamura, H., Yoshida, K., Goto, T., 1989. Structure of
malonylshisonin, a genuine pigment in purple leaves of Perilla oci-
moides 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.
Loponen, J., Ossipov, V., Lempa, K., Haukioja, E., Pihlaja, K., 1998.
Concentrations and among-compound correlations of individual
phenolics in white birch leaves under air pollution stress. Chemos-
pere 37, 1445–1456.
ꢀ
to dryness in vacuo and stored at ꢂ80 C as TFA salts;
1 (10 mg), 2 (5 mg), 3 (7 mg), 4 (8 mg).
Nørbæk, R., Christensen, L.P., Bojesen, G., Brandt, K., 1996.
Anthocyanins in Chilean species of Alstroemeria. Phytochemistry
42, 97–100.
3.5. Partial acid hydrolysis
Nørbæk, R., Kondo, T., 1999. Further anthocyanins from flowers of
Crocus antalyensis (Iridaceae). Phytochemistry 50, 325–328.
Ossipov, V., Nurmi, K., Loponen, J., Haukioja, E., Pihlaja, K., 1996.
High-performance liquid chromatographic separation and identifi-
cation of phenolic compounds from leaves of Betula pubescens and
Betula pendula. J. Chromatogr. A. 721, 59–68.
Hydrolysis of anthocyanin 1 was carried out in 1 M
HCl at 100 ꢀC. At suitable time intervals (2, 5, 10, 20, 40
and 60 min) samples were taken out, cooled in an icebath
and analyzed by HPLC.
Proctor, J.T.A., Creasy, L.L., 1969. An anthocyanin-decolorizing sys-
tem in florets of Cichorium intybus. Phytochemistry 8, 1401–1403.
Saito, N., Harborne, J.B., 1992. Correlations between anthocyanin
type, pollinator and flower colour in the Labiatae. Phytochemistry
31, 3009–3015.
3.6. Physical data of isolated compounds
1: Rt (min) of 25.6, UV-vis (0.1% HCl–MeOH): 270,
532nm. 2: Rt (min) of 19.0, UV-vis 271, 533 nm, Rt
(min) of 3: 21.4, UV-vis of 3: 271, 535 nm, Rt (min) of 4:
14.2, UV-vis of 4: 269, 532 nm.
Saleh, M.R.I., Metwally, A.M., Amer, M.M.A., 1975. Isolation of a fla-
vonoid substance from Cichorium pumilum Jacq. Pharmazie 30, 404.
Scarpati, M.L., Oriente, G., 1958. Chicoric acid (dicaffeyltartic acid)-
its isolation from chicory (Chicorium intybus) and synthesis. Tetra-
hedron 4, 43–48.
1: lmax 538 (" 27800) , 345, 274 nm (in 1% HCl–
MeOH).
NMR and MS data for trans-3-p-coumaroylquinic
Takeda, K., Harborne, J.B., Self, R., 1986. Identification and dis-
tribution of malonated anthocyanins in plants of the Compositae.
Phytochemistry 25, 1337–1342.
1
acid. H NMR: ꢀ 7.41 (1H, d, J=16.2Hz, H- b), ꢀ 7.23
Timberlake, C.F., Bridle, P., Tanchev, S.S., 1971. Some unusual
anthocyanins occurring naturally or as artefacts. Phytochemistry 10,
165–169.
(2H, d, J=8.4 Hz, H-20, H-60), ꢀ 6.57 (2H, d, J=8.4 Hz,
H-30, H-50), ꢀ 6.11 (1H, d, J=16.2Hz, H- a), ꢀ 5.10 (1H,
m, H-3), ꢀ 3.96 (1H, m, H-5), ꢀ 3.50 (1H, m, H-4), ꢀ 2–
03–1.81 (4H, m, H-2, H-2). MS: 338 [M]+.
Trease, G.E., 1969. A Text Book of Pharmacognosy, 9th edn.
Bailliere, Tindall and Cassell, London.