Maqui Berry

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Phytochemical Analysis
Phytochem. Anal. 17: 8–14 (2006)
Published online 13 October 2005 in Wiley InterScience ( DOI: 10.1002/pca.872
Anthocyanins in Berries of Maqui [Aristotelia chilensis (Mol.)
1 Laboratorio de Nutrición y Bromatología, Facultad de Farmacia, Universidad de Salamanca, Campus Miguel de Unamuno s/n, E-37007 Salamanca,
2 Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Santiago de Chile, Chile
Received 14 February 2005; Revised 11 May 2005; Accepted 13 May 2005
The anthocyanin composition of berries of Maqui [Aristotelia chilensis (Mol.) Stuntz] was determined by HPLC with photodiode
array and MS detection. Eight pigments corresponding to the 3-glucosides, 3,5-diglucosides, 3-sambubiosides and 3-
sambubioside-5-glucosides of delphinidin and cyanidin were identified, the principal anthocyanin being delphinidin 3-
sambubioside-5-glucoside (34% of total anthocyanins). The average total anthocyanin content was 137.6 ± 0.4 mg/100 g of
fresh fruit (211.9 ± 0.6 mg/100 g of dry fruit). The relative high anthocyanin content and the important presence of polar
polyglycosylated derivatives makes the fruits of A. chilensis an interesting source of anthocyanin extracts for food and phar-
maceutical uses. Copyright © 2005 John Wiley & Sons, Ltd.
Keywords: Quantitative HPLC; anthocyanins, sambubiosides, delphinidin, cyanidin; berries; Aristotelia chilensis; Maqui.
attention has been paid to polyphenols and especially
the anthocyanins present in the berries, not only for
Maqui [Aristotelia chilensis (Mol.) Stuntz] is a native
their use as natural colorants, but also for their poten-
South America evergreen shrub that grows in dense
tial beneficial effects on human health, including
thickets and can reach 3–5 m in height. It is a
suggestions that they be used as dietary supplements
dioecious plant that belongs to the family Elaeo-
in functional food products (Du et al., 2004). Potential
carpaceae and produces small edible purple/black
effects include anti-oxidant (Heinonen et al., 1998;
berries, about 6 mm in diameter, that are eaten fresh
Prior et al., 1998; Connor et al., 2002; Miranda-
or used for juice, jams or wine-making. The plant pre-
Rottmann et al., 2002) and anti-atherogenic activities
fers slightly acidic, moderately fertile and well-drained
(Miranda-Rottmann et al., 2002), inhibition of HIV
soils. It grows rapidly with adequate moisture and
virus (Andersen and Helland, 1995), prevention of
readily colonises abandoned, burned or overexploited
visual problems (Morazzoni and Bombardelli, 1996;
soils, thus protecting them from erosion. The intense
Sparrow et al., 2003) and activity against some types of
red colour of the aqueous extract of its fruit is due to
human leukaemia and human colon carcinoma (Kamei
the presence of anthocyanin pigments causing it to be
et al., 1995; Katsube et al., 2003).
used as a natural dye. The leaves and fruits are astrin-
The development of techniques such as HPLC
gent and have been used in Chilean folk medicine as
coupled to PAD and MS detectors has allowed rapid
anti-diarrhoeic, anti-inflammatory, anti-haemorrhagic
advances in the identification of the anthocyanin
and febrifuge. (Hoffmann et al., 1992).
composition of many plants (Giusti et al., 1999; Da
Previous reports concerning the chemical composi-
Costa et al., 2000; De Pascual-Teresa and Rivas-
tion of A. chilensis showed the presence of indole and
Gonzalo, 2003). However, the anthocyanin composition
quinoline alkaloids (Silva, 1992; Kan et al., 1997), as
of the berries of A. chilensis has been scarcely studied.
well as high levels of polyphenols that have been
According to our knowledge, only two reports exist
suggested to be responsible for the high in vitro anti-
concerning the tentative identification of some mono-
oxidant activity exhibited by the juice of the fruit
and di-glucosides of delphinidin, cyanidin, malvidin
(Miranda-Rottmann et al., 2002). Recently, particular
and petunidin, and these were based on TLC (Mazza
and Miniati, 1993) and spectrophotometric charac-
teristics (Díaz et al., 1984) for which no further
* Correspondence to: J. C. Rivas-Gonzalo, Laboratorio de Nutrición
y Bromatología, Facultad de Farmacia, Universidad de Salamanca,
confirmation has been made. The aim of the present
Campus Miguel de Unamuno s/n, E-37007 Salamanca, Spain.
work was to revise and update information about the
E-mail: [email protected]
anthocyanin composition of the berries of A. chilensis
Contract/grant sponsor: Plan Nacional I+D and FEDER program of
UE; Contract/grant number: AGL 2002-0167.
using modern HPLC-coupled techniques.
Copyright © 2005 John Wiley & Sons, Ltd.
Phytochem. Anal. 17: 8–14 (2006)
DOI: 10.1002.pca

5% acetic acid (solvent A) and methanol (solvent B)
initially at 90:10 (A:B), increased to 85:15 over 20 min,
Chemicals and standards. Delphinidin-3-glucoside
followed by isocratic elution at 85:15 for 10 min,
and cyanidin-3-glucoside were purchased from
increased to 82:18 over 5 min, then to 60:40 18% B
Polyphenols Labs (Sandnes, Norway). D(+)-glucose,
over 10 min, and finally to 0:100 over 15 min. The flow
α-L-rhamnose and
rate was 3 mL/min and detection was at 520 nm.
D(+)-mannose were purchased from Sigma-Aldrich
Fractions containing anthocyanins were collected in an
(Steinhein, Germany), and D(+)-xylose was from
autosampler, concentrated under vacuum, redissolved
Panreac Química (Barcelona, Spain). Solvents used
in 0.1 M hydrochloric acid and freeze-dried. The purity
were HPLC-grade and purchased from Merck
of the anthocyanins obtained was assessed by HPLC-
(Darmstadt, Germany). All other chemicals were
analytical grade and were supplied by Panreac
HPLC-PAD-MS analysis. A Hewlett-Packard (Agilent
Technologies Inc, Palo Alto, CA, USA) model 1100
Plant material. Wild fruits of Aristotelia chilensis
system equipped with a quaternary pump, a PAD and
(Mol.) Stuntz were picked at maturity in the University
a Spherisorb (Waters Corporation, Milford, Mas-
Campus of Santiago, Chile. The taxonomic identity
sachusetts, USA) ODS2 column (150 × 4.6 mm; 3 µm),
was confirmed by comparing with the authenticated
thermostated at 35°C, was employed. The mobile phase
herbarium specimen (SQF 17092) at Santiago Uni-
consisted of 0.1% trifluoroacetic acid (solvent C) and
versity herbaria. Fruits were weighed, freeze-dried and
acetonitrile (solvent D), with initial isocratic elution at
stored in dark glass containers until required for
90:10 (C:D) for 5 min, increased to 85:15 over 15 min,
followed by isocratic elution at 85:15 for 5 min, and
finally increased to 65:35 over 15 min. The flow rate
Extraction of fruit material. The desiccated samples
was 0.5 mL/min. Eluent flow from the column passed
were homogenised in methanol containing 0.1% of
first through the PAD and then to the probe of the MS.
concentrated hydrochloric acid, kept overnight at 3–
Mass spectrometry was performed using a Finnigan
5°C, and then filtered through a Büchner funnel under
LCQ (Thermoquest, San José, CA, USA) equipped
vacuum. The solid residue was washed exhaustively
with an API source and employing an electrospray
with methanol, the filtrates obtained were centrifuged
ionisation (ESI) interface. Both auxiliary and sheath
(4000 g; 15 min; 2°C) and the solid residue further
gases were a mixture of nitrogen and helium. The
treated using the same protocol until all of the
capillary temperature was 180°C and the capillary
coloured material was completely extracted. All of the
voltage 3 V. The MS detector was programmed to
methanolic extracts were combined, a small volume of
perform two consecutive scans: a full scan in the range
water was added and the extract concentrated (30°C)
m/z 120–1500, and an MS2 scan of the most abundant
by rotary evaporation to remove the methanol. The
ion using a relative collision energy of 20%. Spectra
aqueous extract obtained was washed with n-hexane to
were recorded in the positive ion mode.
remove lipids and further purified by CC over a mixed
Quantification of anthocyanins was performed
stationary phase composed of 20% Polyclar AT (PVP)
from the peak areas recorded at 520 nm by reference
and 80% silica gel prepared as previously described
to a calibration curve obtained with a standard of
(Escribano-Bailón et al., 2002). The extract was
delphinidin-3-glucoside. Berry extracts were analysed
carefully added to the column, which was washed with
in triplicate.
water to remove sugars and acids, and then eluted
with methanol containing 0.1% hydrochloric acid in
Acid hydrolysis. Isolated anthocyanins were dissolved
order to obtain the anthocyanins. After adding a little
in 6 M hydrochloric acid and heated to 100°C in screw-
water, the methanol was removed from the eluate in a
cap test tubes for 40 min. Subsequently, the extract
rotary evaporator and the aqueous extract adjusted
was cooled, concentrated under vacuum, and the
with water to a known volume for further analysis.
residue dissolved in 1 mL of water. An aliquot (100 µL)
of the solution was analysed by HPLC-PAD for
Fractionation of anthocyanins. The purified berry
identification of the hydrolysed anthocyanidins. The
extract was submitted to semi-preparative HPLC using
remaining portion was loaded onto a Waters C18
a Waters (Milford, MA USA) model 600 pump and a
Sep-Pak cartridge and eluted with water, concentrated
Ultracarb ODS20 column (250 × 10 mm i.d.; 5 µm)
under vacuum, and the residue recovered in a
(Phenomenex, Torrance, CA, USA). The aqueous
minimum volume of water for HPTLC analysis of free
extract was concentrated at the head of the column
and ultrapure (100%) water was passed through the
column for 15 min at a flow rate of 3 mL/min. The
Analysis of sugars. HPTLC was carried out using
column was eluted with a mobile phase consisting of
silica gel 60 layers (5 × 5 cm; Merck) according to the
Copyright © 2005 John Wiley & Sons, Ltd.
Phytochem. Anal. 17: 8–14 (2006)
DOI: 10.1002.pca

Table 1
HPLC retention time (R ), spectral characteristics, protonated molecular ion and
main MS2 fragments of the anthocyanins detected in berries of Aristotelia chilensis
Molecular ion
Fragment ions in
at 440 nm
[M+] (m/z)
MS2 (m/z)
465, 597, 303
465, 303
449, 581, 287
449, 287
a Peak numbering as in Fig. 1: for the proposed identities of compounds see Fig. 4.
Identification of anthocyanins
Figure 1 shows the HPLC profile of the anthocyanins
present in an extract of fruits of Aristotelia chilensis.
Data (retention time, λ
in the visible region, molecu-
lar ion and main fragments observed in MS2) obtained
for the anthocyanin peaks in the HPLC-PAD-MS ana-
lysis are summarised in Table 1.
Peaks 5 and 7 were readily identified as being
associated with delphinidin-3-glucoside and cyanidin-
3-glucoside, respectively, by comparison with antho-
cyanin standards; this was confirmed by UV–vis and
MS characteristics (Table 1). Furthermore, the nature
of the substituting sugar (glucose) was confirmed by
HPTLC after isolation of the compounds and acid
In determining the structures of the remaining
anthocyanins, UV–vis spectra and HPLC retention
characteristics were first considered. Compounds asso-
ciated with peaks 1, 2 and 4, showing λ
at 275, 350
and 524 nm (Fig. 2), similar to that of peak 5, were re-
lated to delphinidin derivatives. Similarly, peaks 3 and
6 (λ
at 275 and 516 nm as for peak 7) were assigned
as cyanidin-derived anthocyanins. The greater polarity
(earlier elution) of the unknown compounds in relation
to the corresponding 3-glucosides (peaks 5 and 7) sug-
Figure 1
HPLC chromatogram recorded at 520 nm show-
gested that they were polyglycosylated anthocyanins.
ing the anthocyanin profile of an extract of Maqui fruits
Peak 4 showed a molecular ion at m/z 597 that pro-
(Aristotelia chilensis). The proposed identities of compounds
duced a unique MS2 fragment at m/z 303 (delphinidin)
associated with the peaks shown are given in Fig. 4. (For
(Fig. 3). The direct loss of 294 amu (−[162 + 132] ) indi-
chromatographic protocol see Experimental section.)
cated the separation of a disaccharide (hexose +
pentose) residue (Giusti et al., 1999; Alcalde-Eon et al.,
2004) otherwise fragments corresponding to the
procedure previously described (Di Paola-Naranjo et al.,
sequential loss of the individual sugar residues would
2004). The identification of sugars was performed
have been observed in the MS2 spectrum. This
by comparison with standards of D(+)-glucose, D(+)-
supposition is also supported by the existence of a
galactose, L(+)-arabinose, α-L-rhamnose, D(+)-mannose
shoulder around 440 nm in the visible spectrum of the
and D(+)-xylose.
peak (Fig. 2), which is characteristic of 3-glycosylated
Copyright © 2005 John Wiley & Sons, Ltd.
Phytochem. Anal. 17: 8–14 (2006)
DOI: 10.1002.pca

were made for peak 6. This compound showed a
molecular ion at m/z 581 that produced a unique
MS2 fragment at m/z 287 (cyanidin), suggesting its
tentative identity as cyanidin-3-sambubioside, a major
pigment in elderberry (Sambucus nigra L.) (see Mazza
and Miniati, 1993 and references therein) and other
Sambucus species (Chiarlo et al., 1978; Johansen et al.,
1991; Inami et al., 1996). Cyanidin 3-sambubioside
has also been identified in red currant (Ribes rubrum
L.; Mazza and Miniati, 1993) and fruits of Viburnum
(Kim et al., 2003). Both cyanidin- and
delphinidin-3-sambubiosides have been found together
in the berries of Vaccinium padifolium (Cabrita and
Andersen, 1999) and V. myrtillus (Du et al., 2004),
petals of Roselle (Hibiscus sabdariffa L.; Pouget et al.,
Figure 2
UV–vis spectra of compounds associated with
1990) and purple pods of Pisum sativum L. (Mazza
peaks 2 (delphinidin 3,5-diglucoside; dotted line) and 4
(delphinidin 3-sambubioside; solid line) in the chromatogram
and Miniati, 1993). The sambubioside nature of
shown in Fig. 1. The arrow indicates the shoulder around
compounds 4 and 6 is consistent with their relative
440 nm that is characteristic of anthocyanidin-3-glycosides.
retention times in the HPLC by eluting before the corre-
sponding 3-glucoside derivatives (i.e. delphinidin 3-
glucoside, peak 5, and cyanidin 3-glucoside, peak 7)
anthocyanidins (Santos-Buelga et al., 2003). Thus,
owing to their greater polarity (Santos-Buelga et al.,
peak 4 would correspond to a delphinidin-3-
diglycoside. In order to identify the sugar residues,
A molecular ion at m/z 759 was found for peak 1
the compound was isolated by semi-preparative
that produced MS2 fragments at m/z 597 (M+-162, loss
HPLC and submitted to acid hydrolysis. Analysis by
of a hexose residue), 465 (M+-[162+132], loss of hexose
HPTLC revealed glucose and xylose as released sugars.
+ pentose) and 303 (delphinidin) (Fig. 5). Such MS2
Since sambubiose (i.e. 2-O-β-D-xylosyl-D-glucose) is
fragments require different substitution positions for
the xylosylglucoside most frequently found as an
each sugar residue on the aglycone. According to the
anthocyanin substituent (Mazza and Miniati, 1993),
literature (Mazza and Miniati, 1993; Rivas-Gonzalo,
peak 4 was tentatively assigned as delphinidin 3-
2003), no multi-substituted anthocyanins have been
sambubioside (Fig. 4).
identified that lack a sugar residue at position 3, and
Similar observations to those described for com-
the preferred location for a second sugar would be at
pound 4, regarding fragmentation pattern, absorption
position 5 rather than at 7 or 4′. The absence of a
spectrum and sugar identification (glucose and xylose)
shoulder around 440 nm in the visible spectrum
Figure 3
Molecular ion (m/z 597) and MS2 fragments of compound associated with peak 4 (Fig. 1).
Copyright © 2005 John Wiley & Sons, Ltd.
Phytochem. Anal. 17: 8–14 (2006)
DOI: 10.1002.pca

Figure 4
Suggested identities of the anthocyanins in Maqui fruits. Peak numbers are those shown in the chromatogram of Fig. 1.
Glc, glucose; Xyl, xylose.
Figure 5
Molecular ion (m/z 759) and MS2 fragments of compound associated with peak 1 (Fig. 1).
(Fig. 2), as observed for anthocyanidin 3,5-diglucosides
anthocyanin revealed glucose and xylose as substitut-
(Hebrero et al., 1989), would support the existence of
ing sugars. Thus, the compound associated with peak
substitution at positions 3 and 5 of the aglycone.
1 was identified as delphinidin 3-sambubioside-
HPTLC analysis following acid hydrolysis of the isolated
5-glucoside. This anthocyanin has been previously
Copyright © 2005 John Wiley & Sons, Ltd.
Phytochem. Anal. 17: 8–14 (2006)
DOI: 10.1002.pca

described in purple pods of P. sativum L. (Mazza and
logical that cyanidin-derived anthocyanins (peaks 3a,
Miniati, 1993).
3b, 6 and 7) elute later than their delphinidin-derived
Peak 2 showed a molecular ion at m/z 627 yielding
counterparts (peaks 1, 2, 4 and 5) showing the same
MS2 fragments at m/z 465 and 303 (delphinidin), re-
glycosylation pattern.
sulting from the sequential loss of two hexose residues
Among the identified pigments, only delphinidin 3-
located at different positions on the anthocyanidin.
glucoside and cyanidin 3-glucoside have been previ-
Glucose was the only sugar released after acid hydro-
ously described in Aristotelia chilensis fruits (Díaz
lysis as identified by HPTLC. Following similar reason-
et al., 1984). However, the other pigments identified
ing as for the previous anthocyanins, the compound
by the these authors were not detected in our extracts,
associated with peak 2 was identified as delphinidin 3-
although this could be due to the existence of differ-
glucoside-5-glucoside. This identity was confirmed by
ent Maqui varieties. However, considering that the
comparison of its retention time and spectral charac-
identifications made in earlier reports were performed
teristics with those of the same compound previously
using paper chromatography and spectroscopic tech-
identified in our laboratory in hybrid grapes (Hebrero
niques, and that they have not been confirmed later,
et al., 1989). The number of sugar residues in com-
we have employed HPLC-PAD-MS techniques to
pounds 1 and 2 explains their greater polarity (earlier
provide more precise information about compound
elution) compared with delphinidin 3-glucoside (peak
5). The early elution of peak 2 compared with
delphinidin 3-sambubioside (peak 4) is also logical
based on the presence of two hexose moieties versus
Content and distribution of anthocyanins
one hexose and one pentose.
in Maqui fruits
The tailing character of peak 3 suggested that it was
not pure. Even though the UV–vis spectrum did not
The measured concentrations (expressed as
change across the peak, mass detection revealed the
delphinidin 3-glucoside equivalents) of the different
co-elution of two compounds (3a and 3b). The com-
anthocyanins identified in the fruits of A. chilensis
pound associated with peak 3a showed a molecular ion
are shown in Table 2. Delphinidin derivatives (73%)
at m/z 743 and MS2 fragments at m/z 581 (M+-162,
predominated over those derived from cyanidin (37%),
loss of a hexose residue), 449 ([M+-[162 + 132], loss of
with delphinidin-3-sambubioside-5-glucoside as the
hexose + pentose moieties) and 287 (cyanidin). This
major anthocyanin (34% of total anthocyanins). The
fragmentation pattern is similar to that observed for
average total anthocyanin content was 137.6 ± 0.4 mg/
compound 1 and, furthermore, xylose and glucose
100 g of fresh fruit (211.9 ± 0.6 mg/100 g dry weight),
were detected after acid hydrolysis of the isolated
which is similar to those concentrations found in other
anthocyanin. Thus, peak 3a was identified as being
berries considered to be good sources of anthocyanins
associated with cyanidin 3-sambubioside-5-glucoside.
(Mazza and Miniati, 1993; Clifford, 2000). A prominent
This anthocyanin has been found in Sambucus species
feature of the anthocyanin composition of Maqui is
(Brønnum-Hansen and Hansen, 1983; Mazza and
that its biosynthetic pathway is largely channelled
Miniati, 1993; Inami et al., 1996) and purple pods of
towards the formation of polyglycosylated derivatives
P. sativum L. (Mazza and Miniati, 1993). Furthermore,
that are highly polar and water-soluble. These charac-
different acylated derivatives of the compound have
teristics are attractive for extraction and potential use
been described in various Brassicaceae species, e.g.
as food colorants, as well as for pharmacological uses.
seedlings of Sinapis alba (Takeda et al., 1988), flowers
of Matthiola incana (Saito et al., 1995) and leaves and
Table 2
Contents (expressed in equivalents of delphinidin
stems of Arabidopsis thaliana (Bloor and Abrahams,
3-glucoside) and proposed identities of the anthocyanins
detected in the berries of Aristotelia chilensis
Fragmentation of the compound associated with
peak 3b (molecular ion at m/z 611) yielded fragments
at m/z 449 [M+-162] and 287 (cyanidin), and sugar
(mg/100 g)
analysis (glucose) was similar to that obtained for peak
2, thus suggesting its identity as cyanidin 3-glucoside-
46.4 ± 0.1
5-glucoside. The identification of the compound asso-
23.7 ± 0.2
ciated with peak 3b was confirmed by comparison with
18.7 ± 0.2
standard material and data from our laboratory
14.2 ± 0.1
(Hebrero et al., 1989). The elution of compounds 3a
17.1 ± 0.2
and 3b in relation to cyanidin-3-sambubioside and
8.9 ± 0.04
cyanidin-3-glucoside is coherent with their expected
8.6 ± 0.05
polarity based on the sugar substituents. It is also
Total anthocyanins
137.6 ± 0.4
Copyright © 2005 John Wiley & Sons, Ltd.
Phytochem. Anal. 17: 8–14 (2006)
DOI: 10.1002.pca

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Copyright © 2005 John Wiley & Sons, Ltd.
Phytochem. Anal. 17: 8–14 (2006)
DOI: 10.1002.pca