Anthocyanins acylated with gallic acid from chenille plant,
Acalypha hispida

Bergitte Reiersena, Bernard T. Kiremireb, Robert Byamukamab, Øyvind M. Andersena,*
aDepartment of Chemistry, University of Bergen, Allégt. 41, N-5007 Bergen, Norway
bDepartment of Chemistry, Makerere University, PO Box 7062, Kampala, Uganda

Received 9 May 2003; accepted 15 July 2003
Abstract

Three anthocyanins were isolated from the red flowers of chenille plant, Acalypha hispida Burm. (Euphorbiaceae) by a combi-
nation of chromatographic techniques. Their structures were elucidated mainly by homo- and heteronuclear nuclear magnetic

resonance spectroscopy and electrospray mass spectrometry, and supported with complete assignments of 13C NMR resonances.
The novel pigment, cyanidin 3-O-(200-galloyl-600-O-a-rhamnopyranosyl-b-galactopyranoside) (5%), contains the disaccharide robi-
noside. The other anthocyanins were identified as cyanidin 3-O-(200-galloyl-b-galactopyranoside) (85%), and cyanidin 3-O-b-galac-
topyranoside (5%). Anthocyanins acylated with gallic acid have previously been identified in species from the families

Nymphaeaceae and Aceraceae, and tentatively in Abrus precatorius (Leguminosae).
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Acalypha hispida; Euphorbiaceae; Flowers; Anthocyanins; Robinoside; Cyanidin 3-O-(200-galloyl-600-O-a-rhamnopyranosyl-b-galacto-
pyranoside); 13C NMR; Chemotaxonomy
1. Introduction

The genus Acalypha (Euphorbiaceae) contains about
430 species of evergreen shrubs and trees, and annuals,
from tropical and subtropical regions. Reports on
anthocyanin identification from this genus have hitherto
been restricted to the principal anthocyanin of Acalypha
hispida, tentatively identified as cyanidin 3-arabino-
sylglucoside (Bailoni et al., 1998). Collectively from
other genera in the family Euphorbiaceae the 3-rutino-
sides of cyanidin, delphinidin and pelargonidin, the 3-
glucosides of cyanidin and pelargonidin and cyanidin 3-
galactoside have been reported (Asen, 1958; Stewart et
al., 1979; Del V. Galarza et al., 1983). Capsules con-
taining anthocyanins from Euphorbia splendens for the
treatment of blood circulation disorders have been
patented (Fujii et al., 1987).
During our survey of the anthocyanin content of

plants from tropical regions, we found that the major
anthocyanin of flowers of the horticultural important
chenille plant, A. hispida Burm., was cyanidin 3-O-(200-
galloyl-b-galactopyranoside), and not cyanidin 3-arabi-
nosylglucoside as reported by Bailoni et al. (1998). In
this paper we present the isolation and structure eluci-
dation of three anthocyanins from flowers of Acalypha
hispida including a novel anthocyanin containing the
disaccharide robinose acylated with gallic acid.
2. Results and discussion

The HPLC chromatogram of the crude extract of A.
hispida showed one major, 2, and two minor anthocya-
nins, 1 and 3 (Table 1). The pigments in the extract were
purified by partition against ethyl acetate followed by
Amberlite XAD-7 column chromatography, and iso-
lated as three separated bands by Sephadex LH-20
chromatography. The isolated anthocyanins were
checked for homogeneity by analytical HPLC.
The visible part of the UV–vis spectra of 1 and 2

(Table 1) were in accordance with cyanidin 3-/peonidin 3-
glycosides (Andersen, 1985). The UV–vis spectrum of 2
taken on-line during HPLC showed a visible maximum
at 523 nm with A440/A523 and A280/A523 of 28 and 99%,
0031-9422/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/S0031-9422(03)00494-1
Phytochemistry 64 (2003) 867–871

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* Corresponding author. Tel.: +47-55-583460; fax: +47-55-

583490.

E-mail address: oyvind.anderson@kj.uib.no (O. M. Andersen).

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respectively, indicating no 5-sugar substituents and the
presence of an aromatic acyl group. The relative high
mobility of 2 in the TLC system and the long retention
time (HPLC) compared to pigment 1 (Table 1), con-
firmed aromatic acylation. Pigment 1 co-chromato-
graphed (HPLC and TLC) with cyanidin 3-galactoside
from Vaccinium vites-idae (Andersen, 1985). The struc-
tures of 1 and 2 were elucidated by one- and two-
dimensional NMR results (Tables 2 and 3) to be cyani-
din 3-O-b-galactopyranoside and cyanidin 3-O-(200-gal-
loyl-b-galactopyranoside), respectively. Their structures
were confirmed by electrospray ESI-MS data (Table 1).
The UV–vis spectrum of 3 was very similar to the

corresponding spectrum of 2, indicating an anthocyani-
din with two oxygen-functions on the B-ring (Table 1)
(Andersen, 1985). This pattern was confirmed by the 3H
AMX system at � 8.10 (dd, 8.8 Hz, 2.2 Hz, H-60), 7.90
(d, 2.2 Hz, H-20) and 6.89 (d, 8.8 Hz, H-50). In the
downfield region additional signals were observed at �
9.04 (H-4), and a 2H AX-system at � 6.94 (H-8) and �
6.90 (H-6) (Table 2). This latter system was influenced
by a 5-bonding zig-zag coupling between H-4 and H-8
(J=0.9 Hz). The chemical shifts for the corresponding
Table 1

Chromatographic (HPLC) and spectral (UV–vis and MS) data recorded for the anthocyanins, 1–3, isolated from flowers of Acalypha hispida
Compound
 On-line HPLC
 ES-MS
lvis-max (nm)
 lUV-max (nm)
 AUV-max/Avis-max (%)
 A440/Avis-max (%)
 tR (min)
 M+ m/z
 A+ m/z
1
 520
 280
 73
 30
 13.24
 449
 287
2
 523
 280
 99
 28
 14.45
 601
 287
3
 523
 280
 98
 28
 14.80
 747
 287
Table 2
1H NMR spectral data for the anthocyanins, 1–3, isolated from flow-

ers of Acalypha hispida dissolved in CD3OD:CF3COOD (20:1) at 25 �C
1 � (ppm), J (Hz)
 2 � (ppm), J (Hz)
 3 � (ppm), J (Hz)
Aglycone
4
 9.11 d 0.9a
 9.07 s
 9.04 d 0.9a
6
 6.74 d 2.0
 6.71 d 2.0
 6.75 d 2.0
8
 6.98 dd 2.0, 0.9a
 6.87 s br
 6.94 dd 2.0, 0.9a
20
 8.17 d 2.4
 7.84 d 1.8
 7.90 d 2.2
50
 7.11 d 8.8
 6.82 d 8.8
 6.89 d 8.8
60
 8.34 dd 8.8, 2.4
 8.02 dd 8.8, 1.8
 8.10 dd 8.8, 2.2
3-O-�-Galactopyranoside

100
 5.34 d 7.7
 5.64 d 8.1
 5.67 d 7.9
200
 4.08 m
 5.76 t br
 5.78 dd 9.9, 7.9
300
 3.76 dd 9.9, 3.3
 4.05 m
 4.07 dd 9.9, 3.3
400
 4.04 t 3.3
 4.14 m
 4.09 dd 3.3, 0.9
500
 3.89 m
 4.03 m
 4.23 m
6A00
 3.87 m
 3.99 m
 4.00 m
6B00
 3.87 m
 3.94 m
 3.84 m
600-O-�-Rhamnopyranosyl
1000
 4.78 d 1.8
2000
 3.93 dd 3.5, 1.8
3000
 3.76 dd 9.4, 3.5
4000
 3.46 t 9.4
5000
 3.71 dd 9.4, 6.4
6000
 1.36 d 6.4
200-O-Galloyl
20000/60000
 7.10 s
 7.08 s
See Fig. 1 for pigment identification.
a The coupling constant is determined by Gauss multiplication.
Table 3
13C NMR spectral data for the anthocyanins, 1–3, isolated from

flowers of Acalypha hispida dissolved in CD3OD:CF3COOD (20:1) at

25 �C
1 � (ppm)
 2 � (ppm)
 3 � (ppm)
Aglycone
2
 164.82
 163.93
 164.32
3
 145.92
 145.11
 145.34
4
 137.00
 136.29
 135.77
5
 159.35
 159.20
 158.85
6
 103.39
 103.30
 103.40
7
 170.44
 170.41
 170.37
8
 95.09
 95.08
 95.12
9
 157.87
 157.46
 157.90
10
 113.50
 113.08
 113.09
10
 121.47
 120.77
 121.09
20
 118.48
 117.67
 117.80
30
 147.50
 147.31
 147.49
40
 155.91
 155.72
 156.17
50
 117.45
 117.30
 117.39
60
 128.07
 128.47
 128.68
3-O-�-Galactopyranoside

100
 104.41
 102.14
 101.98
200
 71.94
 73.07
 72.98
300
 74.98
 73.04
 72.92
400
 70.16
 70.30
 70.65
500
 77.73
 78.00
 76.46
600
 62.37
 62.31
 68.01
600-O-�-Rhamnopyranosyl
1000
 99.38
2000
 71.80
3000
 72.52
4000
 73.98
5000
 69.88
6000
 17.80
200-O-Galloyl
10000
 120.96
 121.21
20000/60000
 110.48
 110.48
30000/50000
 146.29
 146.36
40000
 140.00
 140.03
C=O
 167.76
 167.97
See Fig. 1 for pigment identification.
868 B. Reiersen et al. / Phytochemistry 64 (2003) 867–871



aglycone carbons and the quarternary carbons were
assigned by the HSQC and HMBC NMR spectra,
respectively, in accordance with the anthocyanidin cya-
nidin (Table 3). The procedure used for assignments of
the 13C NMR signals of cyanidin has previously been
reported by Andersen et al. (1991).
The two-dimensional TOCSY NMR spectrum of 3

was in agreement with two sugar units. Starting from
the anomeric proton at � 5.67 and the two 600-sugar
protons we could, through the crosspeaks in the DQF-
COSY spectrum supported by cross-peaks in the HSQC
spectrum assign all the seven sugar protons (Table 2).
The corresponding sugar carbons (Table 3) were
assigned by their cross-peaks in the HSQC spectrum.
The five non-anomeric carbons had chemical shift
values from � 68.0 to � 76.5 (Table 3), indicating a hex-
ose with a pyranose form (Markham and Chari, 1982).
The chemical shifts and the coupling constants (Tables 2
and 3) were in accord with a substituted b-galactopyr-
anoside. A cross-peak at � 5.67/145.44 in the HMBC
spectrum showed that this sugar unit was connected to
the 3-position of the aglycone.
From the TOCSY spectrum it was observed that one

of the proton signals of the second sugar unit was at
high-field (� 1.36). This 3H-doublet (J=6.4 Hz) is typi-
cal for a rhamnose moiety. By using the DQF-COSY
and the HSQC spectra, it was possible to assign all the
chemical shifts and the 1H–1H coupling constants for
the rhamnopyranosyl moiety in accordance with a a-
rhamnopyranosyl (Tables 2 and 3). Confirmed by the
downfield shift of C-600 (� 68.01) and the cross-peak at �
68.01/4.78 between C-600 and H-l000 in the HMBC-spec-
trum, the rhamnosyl moiety was found to be connected
to the galactosyl 6-position. Thus the sugar moiety is
determined as the disaccharide robinose.
The UV–vis spectrum of 3 showed higher absorbances

around 280 nm (A280/Avis-max=98%) than those of 1,
cyanidin 3-galactoside, (A280/Avis-max=73%), indicating
the presence of an aromatic acyl group. The CAPT
spectrum of 3 showed in addition to the corresponding
signals of 1, four positive and one negative carbon sig-
nals in the aromatic region (Table 3). Two of the signals
(� 146.36 and 110.48) represented each two carbon
atoms. The latter negative CAPT signal (C20000/C60000) was
correlated (revealed by the HSQC spectrum) with a 2H
singlet at � 7.08. In the HMBC spectrum this 2H singlet
showed 3J(CH) responses to C40000 (� 7.08/140.03) and
COO (� 7.08/167.97) of a galloyl (3,4,5-trihydroxy-
benzoyl) moiety. C10000 and C30000/C50000 were thereafter
assigned by the weaker 2J(CH) responses to the same
singlet (� 7.08/121.21 and (� 7.08/146.36, respectively).
The cross-peak at � 5.78/167.97 in the HMBC spectrum
between H-200 and the carboxylic carbon showed that
the galloyl moiety was connected to C-200 on the galac-
tose ring. This linkage was also confirmed by the pro-
nounced downfield shift of H-200 (1.7 ppm), and that
H-100 and H-300 of 3 were ca 0.3 ppm more downfield
than the corresponding signals of 1 (Table 2). Similarly,
C-200 was deshielded (1 ppm) and C-100 and C-300 were
shielded (3.4 and 2.1 ppm, respectively) compared to the
corresponding signals of 1 (Table 3). A molecular ion at
m/z 747, and a fragment ion at m/z 287 in the ESI-MS
spectrum of 3 corresponding to cyanidin, confirmed the
identity of 3 to be cyanidin 3-O-(200-O-galloyl-600-O-a-
rhamnopyranosyl-b-galactopyranoside).
The relative proportions of 1–3 are 5, 85 and 5%,

respectively. Thus, galloylated anthocyanins constitute
90% of the total anthocyanin content of A. hispida.
Even though acylation of anthocyanins with aromatic
acids of the cinnamoyl type has widespread occurrence,
similar acylation with gallic acid is much more restric-
ted; it has been identified in Aceraceae as cyanidin 3-(600-
galloylglucoside) in Dipteronia sinensis and several Acer
taxa, as cyanidin 3-(00-galloylrutinoside) in some Acer
taxa (Ji et al., 1992a,b; Fossen and Andersen, 1999a),
and as cyanidin 3-(200,300-digalloylglucoside) from red
leaves of Acer platanoides (Fossen and Andersen,
1999a). In Nymphaeaceae the 3-(200-galloylgalactosides)
and 3-O-(200-O-galloyl-600-O-acetyl-b-galactopyrano-
sides) of delphinidin and cyanidin have been identified
(Strack et al., 1992; Fossen and Andersen, 1997, 2001;
Fossen et al., 1998), as well as the 30-O-(200-O-galloyl-b-
galactopyranoside) and 30-O-(200-O-galloyl-600-O-acetyl-
b-galactopyranoside) of delphinidin in blue flowers of
Nymphaéa caerulea (Fossen and Andersen, 1999b). In
addition, delphinidin (coumarylgalloyl)-glucoside has
been tentatively identified in the seed coat of Abrus pre-
Fig. 1. The structures of the anthocyanins in flower extract of Acaly-

pha hispida. 1=cyanidin 3-O-b-galactopyranoside, 2=cyanidin 3-O-

(200-O-galloyl-b-galactopyranoside), and 3=cyanidin 3-O-(200-O-gal-

loyl-600-O-a-rhamnopyranosyl-b-galactopyranoside).
B. Reiersen et al. / Phytochemistry 64 (2003) 867–871 869



catorius (Legumiosae) (Karawya et al., 1981). Thus, the
major pigment of A. hispida, cyanidin 3-O-(200-galloyl-b-
galactopyranoside) (2) which has previously been iso-
lated from Victoria amazonica leaves (Strack et al.,
1992), and the novel cyanidin 3-O-(200-O-galloyl-600-O-a-
rhamnopyranosyl-b-galactopyranoside) (3) may have
chemotaxonomic significance.
3. Experimental

3.1. Isolation of pigments

Whole flowers of A. hispida (=A. sanderi, A. sanderi-
ana, chenille plant, red-hot cat tail) were collected in
Kampala (Uganda) in February 2002. Voucher speci-
mens are deposited at Department of Chemistry,
Makerere University (Byamukama No. 10). The frozen
flowers (ca 77 g) were extracted for 2 h in 450 ml
methanol containing H2O (10%, v/v) and CF3COOH,
TFA (1%, v/v). After filtration and repeated extraction
(250 ml), the combined extracts were concentrated and
purified by partition against ethyl acetate before appli-
cation on an Amberlite XAD-7 column. After washing
the column with H2O, the anthocyanins were eluted by
methanol containing 1% TFA. The anthocyanins were
isolated into three bands on a Sephadex LH-20 column
(100 � 5.0 cm, Pharmacia) using H2O–MeOH–TFA
(first 80:19:1 and then 70:29:1, v/v) as eluent. Analytical
HPLC was performed on an HP-1050 module system
(Hewlett-Packard) using an ODS Hypersil column (20
� 0.5 cm, 5 mm). The elution consisted of a linear gra-
dient from 10% B to 100% B during the first 17 min,
isocratic elution using 100% B for the next 4 min fol-
lowed by a linear gradient back to 10% B during 1 min.
The flow rate was 0.75mlmin�1, and aliquots of 10 ml were
injected. TLC was carried out on microcrystalline cellulose
(Art. 5565, DC-Plastikfolien, Cellulose F, Merck) using
the solvent HCO2H–conc. HCl–H2O; 1:1:2 v/v. The RF-
values for 1–3 are 0.50, 0.56 and 0.68, respectively.

3.2. Spectroscopy

UV–vis absorption spectra were recorded on-line
during HPLC over the wavelength range 240–600 nm in
steps of 2 nm. Relative amounts of each anthocyanin
are reported as percentages of total peak area in HPLC
chromatograms based on absorptions recorded for
every second nm between 500 and 540 nm.
The NMR experiments on 1–3 were obtained at

600.13 MHz and 150.90 MHz for 1H and 13C respec-
tively, on a Bruker DRX-600 instrument equipped with
a multinuclear inverse probe for the 1D 1H and the 2D
Heteronuclear Single Quantum Coherence (1H–13C
HSQC), Heteronuclear Multiple Bond Correlations
(1H–13C HMBC), Double Quantum Filtered Corre-
lation Spectroscopy (1H–1H DQF-COSY) and Total
Correlation Spectroscopy (1H–1H TOCSY) experi-
ments. The 13C 1D CAPT experiment, which was per-
formed only on pigment 3, was executed on a 1H/13C
BBO probe. Sample temperatures were stabilised at
25 �C. The deuteriomethyl 13C signal and the residual
1H signal of the solvent (CD3OD:CF3CO2D; 20:1, v/v)
were used as secondary references (� 39.0 and � 3.4 from
TMS, respectively). Mass spectral data on 1–3 were
achieved by a LCMS system (Waters 2690 HPLC-sys-
tem connected to Micromass LCZ mass spectrometer)
with electrospray ionization in positive mode (ESP+).
The following ion optics were used: Capillary 3 kV,
cone 30 and 60 V, and extractor 7 V. The source block
temperature was 120 �C and the desolvation tempera-
ture was 150 C. The electrospray probe-flow was adjus-
ted to 100 ml/min. Continuous mass spectra were
recorded over the range m/z 150–800 with scan time 1 s
and interscan delay 0.1 s.
Acknowledgements

The authors are grateful to Dr. Rune Slimestad
(Polyphenols Laboratories AS) for the electrospray MS
spectra.
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B. Reiersen et al. / Phytochemistry 64 (2003) 867–871 871


	Anthocyanins acylated with gallic acid from chenille plant, Acalypha hispida
	Introduction
	Results and discussion
	Experimental
	Isolation of pigments
	Spectroscopy

	Acknowledgements
	References