Purification and antiradical properties of the structural unit of betalains

Article abstract of DOI:10.1021/np200950n

Betalamic acid [4-(2-oxoethylidene)-1,2,3,4-tetrahydropyridine- 2,6-dicarboxylic acid] is a naturally occurring compound that is normally found condensed with amino acids, amines, cyclo-DOPA, and cyclo-DOPA derivatives to form the betalains. Betalains are

Full text of DOI:10.1021/np200950n

Article
pubs.acs.org/jnp
Purification and Antiradical Properties of the Structural Unit of
Betalains
Fernando Gandía-Herrero,* Josefa Escribano, and Francisco García-Carmona
Departamento de Bioquímica y Biología Molecular A, Unidad Docente de Biología, Facultad de Veterinaria, Universidad de Murcia,
E-30100 Espinardo, Murcia, Spain
ABSTRACT: Betalamic acid [4-(2-oxoethylidene)-1,2,3,4-tetrahydropyridine-
2,6-dicarboxylic acid] is a naturally occurring compound that is normally found
condensed with amino acids, amines, cyclo-DOPA, and cyclo-DOPA derivatives to
form the betalains. Betalains are the pigments responsible for the yellow to violet
color of the fruits and flowers of plants belonging to the order Caryophyllales.
Betalamic acid is the structural feature common to all of these pigments and
contains the electron resonance system responsible for the spectroscopic
properties. Betalamic acid was purified by chromatography and identified by
UV−vis spectrophotometry and ESI mass spectrometry. The antioxidant and free
radical scavenging capacities of betalamic acid were assessed using the FRAP and
ABTS·+ radical assays. A pKa of 6.8 was found for the deprotonation equilibrium involved in the nucleophilic activity of betalamic
acid; this pKa explains the observed pH effect on the free radical scavenging capacity of these pigments.
etalains are water-soluble nitrogen-containing pigments
that are present in the fruits and flowers of most plant
B
species belonging to the order Caryophyllales.1 These
compounds are also present in fungi, such as Amanita2 and
Hygrocybe.3 More than 50 natural betalains have been
described4 and are divided into the violet betacyanins and the
yellow betaxanthins. These exhibit absorption maxima around
536 and 480 nm, respectively. The structural identification of
betalains was first performed in the early 1960s. Wyler et al.5
identified the betacyanin betanidin as the immonium derivative
of betalamic acid condensed with cyclo-DOPA. Piattelli et al.6
characterized indicaxanthin, the first betaxanthin identified, as
the immonium derivative of betalamic acid condensed with L-
proline.
Figure 1. General structures of betalamic acid (A), betaxanthins (B),
and betacyanins (C). R1 is any lateral residue present in amines or
amino acids. R2 is −OH (betanidin), glucose (betanin), or a complex
sugar residue.
Other studies have revealed the existence of betaxanthins that
are derived from different amino acids and amines and the
existence of complex betacyanins that have sugar moieties
incorporated into their structures.1 Betalamic acid is the pivotal
molecule for the formation of the great variety of the betalain
pigments. Figure 1 shows the structures of betalamic acid and
its derivatives.
Although the biosynthetic pathway of betalains was partially
elucidated in 1981,7 reactions involving pigment transformation
were not characterized until recently.8 The formation of
betalamic acid starts from the amino acid tyrosine, which is
hydroxylated by the tyrosinase enzyme to form 3,4-dihydrox-
yphenylalanine (DOPA).9 DOPA is then transformed into a
4,5-secodopa intermediate by the enzyme dioxygenase.10
Betalains can be found in a variety of species and tissues, but
red beet (Beta vulgaris) roots and the fruits of cacti belonging to
the genus Opuntia (mainly Opuntia ficus indica) are the best
known edible sources of betacyanins and betaxanthins. The
color is primarily due to the betacyanin betanin in beet root11
and to the betaxanthin indicaxanthin in Opuntia fruits.12
Betanin-containing beet root extracts are used to give a pink or
violet color to foods and beverages as the additive 73.40 in the
21 CFR section of the Food and Drug Administration (FDA)
in the United States and under the E-162 code in the European
Union. New colorants containing betaxanthins have also been
proposed.13 Betalamic acid in its free form has been reported to
be a natural component of the fruits of Opuntia ficus indica.14
The first investigations demonstrating the radical scavenging
capacity of betalains were performed with the pigments present
in beet root.15 Subsequent research revealed the existence of an
intrinsic activity present in all betalains that is modulated by
structural factors.16−19 Furthermore, very low concentrations of
dietary betanin have been shown to inhibit skin and liver tumor
formation in mice20 and to protect mice from the effects of γ
radiation.21 In humans, the plasma concentrations of betalain
Received: December 6, 2011
Published: May 29, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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after ingestion are sufficiently high that this compound is
incorporated into LDL and red blood cells, where it can protect
the cells from oxidative damage and hemolysis.22,23 There has
been no prior evidence of the possible biological activity of free
betalamic acid.
The high solubility of betalains in water means that they can
be extracted from vegetable sources in buffered aqueous
solutions or purified water.15,24 However, varying amounts of
miscible organic solvents, such as EtOH,25 MeOH,26 and
acetone,27 are typically added to the extraction solution. Other
methods for pigment extraction include the application of
pulsed electric fields to fresh sections of vegetable material.28
Betalamic acid can be synthesized in vitro using a process that
involves multiple steps but gives low yields.29 This compound
can also be obtained from the degradation of betanin
(betanidin-5-O-β-glucoside) by alkaline hydrolysis.30 In this
case, the presence of the other degradation product, cyclo-
DOPA-glucoside, drives the reverse reaction, yielding the
original pigment.31 Further acidification of the hydrolysis
medium and EtOAc-mediated extraction of the resulting
mixture, followed by solvent evaporation and residue filtration,
yield betalamic acid.32 Despite low yields, this method has been
successfully used to produce stable betaxanthins.
Betalains exhibit high antioxidant activity and free radical
scavenging capacity. However, the activity of the structural unit
common to all of these pigments has not been evaluated, and a
suitable procedure to obtain and purify this compound in
sufficient amounts has not been developed.
Figure 2. Purification of betalamic acid. (A) Purification protocol A,
described in the Experimental Section, was followed after the injection
of 100 μL of 250 μM betanin hydrolysis medium. (B) Scaled-up
purification protocol B was followed after the injection of 10 mL of
250 μM betanin hydrolysis medium. The absorbance was followed at λ
= 424 nm (−) and λ = 280 nm (---). The addition of solvent B (%) is
also shown (···).
RESULTS AND DISCUSSION
■
Betalamic Acid Purification. Betalamic acid may be
obtained through the alkaline hydrolysis of betanin by
increasing the solution pH above 11.0. Degradation may be
followed by a change in color from violet (betanin, λm= 536
nm) to yellow (betalamic acid, λm = 424 nm). However, the
presence of any amino acid or amine, including cyclo-DOPA-
glucoside, in solution with betalamic acid will drive
condensation at moderate pH values via a Schiff reaction,
which yields the corresponding imines.33
The aim of the protocol described herein was to separate
betalamic acid from any free amine present in the hydrolysis
medium to prevent the formation of imines via the Schiff
reaction and to obtain pure betalamic acid.
Betalamic Acid Characterization. The betalamic acid
obtained was analyzed by HPLC following a suitable
protocol.33 A typical chromatogram obtained for the analysis
of the fractions purified by ionic exchange is shown in Figure
3A. The peak corresponding to betalamic acid had a retention
time of tR = 14.59 min. This peak was the only one found using
a photodiode detector, thus demonstrating the high degree of
purification obtained. The maximum wavelength of the
absorbance spectrum was λm = 407 nm in the presence of
MeCN under the HPLC analysis conditions. Figure 3B shows
the UV−visible spectrum for betalamic acid in H2O; this
spectrum exhibited a maximum at a wavelength of λm = 424
nm. The molar absorption coefficient of betalamic acid was
calculated by a procedure based on the basic hydrolysis of
betanin to yield betalamic acid using betanin solutions of
known concentrations as a reference (ε536 = 65 000
M−1·cm−1).35 The value determined for the betalamic acid
coefficient was ε = 27 000 M−1·cm−1 at a wavelength of λ = 424
nm in H2O. This value is consistent with the data in the
literature obtained by acid hydrolysis and amino acid
quantification (ε = 24 000 M−1·cm−1 at λ = 424 nm).36
The acid obtained was also analyzed by ESIMS. Sample
ionization was performed in the positive mode, and the masses
were detected in the m/z range 50−600. Under these
conditions, an m/z of 212 was obtained for the parent ion,
which corresponds to the expected protonated form of
betalamic acid [M − H]+. This m/z value confirmed the
proposed structure of the purified molecule. Two main
Hence, the Schiff reaction must be limited to obtain
betalamic acid in its free form.34 In our study, betalamic acid
was chromatographically purified from the amine moiety of the
betalain structure before the reaction could occur, obviating the
need to acidify the reaction medium. Anionic exchange
chromatography was performed on a trimethyl ammonium
matrix with sample injection at the hydrolysis pH (>11.0). A
phosphate buffer with a low ionic strength was used as the
mobile phase after the sample was injected, followed by a
gradient of NaCl to promote betalamic acid elution. Betalamic
acid interacts with the cationic matrix at pH 6.0, most likely due
to the two deprotonated carboxylic acid groups in the molecule.
Two different column volumes were used to evaluate the
possibility of scaling up the purification process. Figure 2 shows
two typical elution profiles for the purification of betalamic acid.
Figure 2A shows the elution process for a 1 mL column
(protocol A), and Figure 2B shows the data obtained for a 5
mL column (protocol B). The yields obtained were 86% of the
starting material, as determined by spectrophotometry. This is a
significant improvement over the previous protocols where the
separation of betalamic acid from the amine moiety of the
betalain pigment was achieved with low yields by EtOAc
extraction after acidification.32
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Figure 3. Analysis of betalamic acid. (A) High-performance liquid
chromatography analysis of a betalamic acid sample after anionic
exchange purification. A volume of 20 μL of 80 μM betalamic acid was
injected. (B) UV−visible spectrum of a 25 μM betalamic acid solution
in H2O at 25 °C.
Figure 4. Free radical scavenging activity of betalamic acid. (A)
Decreases in the absorbance at λ = 734 nm reflect the depletion of the
ABTS•+ radical after the addition of increasing concentrations of
daughter ions of m/z 102 and 166 were obtained, with the
latter most likely corresponding to the loss of one carboxylic
acid group.
○
●
betalamic acid ( ) or Trolox ( ). The starting concentration of
ABTS•+ in the assay was 60 μM in a reaction medium containing 50
mM Na3PO4 buffer, pH 7.0. (B) Effect of pH on the free radical
scavenging activity of betalamic acid. The starting concentration of
Free Radical Scavenging Activity of Betalamic Acid.
The free radical scavenging capacity of betalamic acid was
evaluated by determining the effect of this compound on
solutions of the stable colored radical ABTS•+ [2,2′-azino-bis(3-
ethylbenzthiazoline-6-sulfonic acid)]. The ABTS•+ decoloriza-
tion assay was performed by following the decrease in the
absorbance at a wavelength of λ = 734 nm.15 The effect of
betalamic acid was compared with that of Trolox (6-hydroxy-
2,5,7,8-tetramethylchromane-2-carboxylic acid), a potent water-
soluble antiradical derived from vitamin E that is commonly
used as a reference.
Figure 4A shows the effects of the addition of six different
concentrations of betalamic acid and Trolox to ABTS•+ radical
solutions at pH 7.0. Increasing concentrations of both
molecules were correlated with decreases in the absorbance
due to the presence of the radical. The response to betalamic
acid was higher. The effect of betalamic acid on the ABTS•+
radical relative to the effect of Trolox could be determined from
the linear response shown. The ratio of the slopes obtained for
the two compounds reflected the amount of Trolox needed to
achieve the same effect as that produced by betalamic acid. The
observed TEAC (Trolox equivalent antiradical capacity) value
of betalamic acid was 2.7 0.2. This value is equivalent to that
of the antioxidant resveratrol (TEAC = 2.7),39 which is present
in grapes, and is close to the values determined for the betalain
pigments with the simplest structures.19
ABTS•+ in the assay medium was 60 μM. Betalamic acid ( ) and
○
●
Trolox ( ) were added at final concentrations of 50 μM to 50 mM
■
buffered solutions with the selected pH values. Purified H2O ( ) was
used in the corresponding control samples.
ABTS•+ radical assay to evaluate the effect of the protonation
state of betalamic acid on its free radical scavenging capacity.
NaOAc was used as a buffer in solutions with pH values ranging
from 3.5 to 5.5, and Na3PO4 was used for solutions with pH
values from 5.5 to 8.5. No difference was observed in the
activities measured in the two buffers at pH 5.5. As shown in
Figure 4B, pH values above pH 5.5 were associated with a
notable increase in the radical scavenging capacity of betalamic
acid, with only basal scavenging activity observed at pH values
below 5.5. In any case, the activity at more acidic pH values was
higher than that exhibited by Trolox.
This marked dependence of the measured activity on pH is
similar to that described for the betalain molecules, for which
the effect of pH has been studied.19 The similarity of the pH
curves suggests that there exists a protonation equilibrium
involving the betalamic acid moiety of the pigments, which was
not demonstrated for the free acid. The protonation
equilibrium for betalamic acid may result from the secondary
amine group, which is conjugated via resonance with the
hydroxy group that participates in the keto−enol tautomeric
equilibrium of the formyl group. In this case, deprotonation
pH Effect and Characterization of the Deprotonation
Equilibrium. Solutions with different pHs were used in the
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would yield a form with a higher nucleophilic capacity, which
may explain the observed effect of pH on the free radical
scavenging capacity. In addition, the nucleophilic capacity of
the deprotonated form of betalamic acid would promote its
nucleophilic attack of other molecules such as quinones,
yielding adducts.38,39 To observe this phenomenon, a stable 4-
methylcatecholquinone was prepared from 4-methylcatechol
using the catecholase activity of tyrosinase. After completion of
the reaction, the medium was filtered to remove the catalyst.
Figure 5A shows the evolution of 4-methylcatecholquinone
betalamic acid, and the existence of lag periods in the formation
of the adduct was observed.
The diagram below shows the reactions involved in the
nucleophilic attack by betalamic acid of the quinones formed
from 4-methylcatechol by tyrosinase:
where M is 4-methylcatechol, Q represents the corresponding
quinone, BH is the protonated form of betalamic acid, B−
corresponds to the deprotonated form of the acid, and A is the
adduct formed by the reaction of 4-methylcatecholquinone and
betalamic acid. v0 represents the formation rate of quinone Q
from 4-methylcatechol. k and k′ are the reaction constants for
the nucleophilic attack of quinone Q by the deprotonated and
protonated forms of betalamic acid, respectively.
The lag period in the reaction was determined at each pH,
revealing a low capacity for nucleophilic attack at acidic pH
values. The adduct formation reaction proceeds without a lag
phase at higher pH values (results not shown). The apparent
reaction constant (kapp) at each pH value may be calculated as
the inverse of the lag period time (L) according to the
following equations derived from the deprotonation equili-
brium of betalamic acid and the diagram above using the
stationary-state kinetic approximation:
[B−][H+]
[BH]
Ka =
[B−] + [BH] = [B0]
dQ
= v0 − (k[B−] + k′[BH])[Q] = 0
dt
v0t = [Q] + [A]
where Ka is the equilibrium constant for betalamic acid
deprotonation and [B0] is the total concentration of the acid.
The appearance of the adduct and the apparent constant for its
formation (kapp) may be written as a function of [H+] as
Figure 5. Nucleophilic attack of 4-methylcatecholquinone by
betalamic acid. (A) Spectroscopic changes in a betalamic acid solution
(45 μM) after the addition of filtered 4-methylcatecholquinone at a
final concentration of 200 μM. The assay was performed in 50 mM
Na3PO4 buffer at pH 7.0, and the spectra were recorded at 1 min
intervals for 10 min. Inset: Differential spectra derived from the
previous scans, subtracting the first recording from the rest. (B) Effect
of pH on the nucleophilic capacity of betalamic acid measured as the
apparent reaction constant (kapp) associated with the attack of 4-
methylcatecholquinone. The medium contained 100 μM betalamic
acid, 200 μM 4-methylcatechol, and tyrosinase (13 units/mL) as a
catalyst in 50 mM buffered solution at the indicated pH. The reactions
were followed at 530 nm. In the absence of the quinone, there was no
detectable reaction.
v0
[A] = v0t −
Ka[B0]
Ka + [H+]
[H+][B0]
k
+ k′
Ka + [H+]
Ka[B0]
Ka + [H+]
[H+][B0]
Ka + [H+]
1
L
kapp
=
= k
+ k′
At low pH values, the apparent constant for adduct
formation can be simplified as kapp = k′[B0], whereas at high
pH, kapp = k[B0]. Figure 5B presents the values determined for
the apparent constant kapp as a function of pH. As observed, the
reaction constant was higher at high pH values than at low pH
values. Thus, the curve shape was significantly affected by the
deprotonation equilibrium, which is closely related to the
higher free radical scavenging capacity observed when the pH
was increased, as shown in Figure 4B. The nonlinear fitting of
the data shown in Figure 5B to the kapp equation gave values for
the reaction constants of k (0.0371 0.0005 min−1·μM−1) and
over time in an assay with betalamic acid in an aqueous solution
at pH 7.0. The appearance of the new product was observed as
an increase in the absorbance spectrum centered at λ = 530 nm.
The maximum could be detected better by subtracting the
initial recording from the rest (differential spectra; Figure 5A,
inset). This wavelength was used to follow the capacity of
betalamic acid to attack the quinone as a function of pH. The
compound 4-methylcatecholquinone was produced in the
reaction media at different pH values in the presence of
k′ (0.0016
0.0003 min−1·μM−1); the corresponding
dissociation constant of betalamic acid was Ka = (0.1438
0.0105) × 10−6 M. Thus, the pKa value for betalamic acid
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coefficient for betalamic acid was determined using an end-point
method by performing a set of betanin degradation experiments. The
spectra of betanin solutions of known concentrations were recorded
with the above instrument, and then the solutions were submitted to
basic hydrolysis using ammonia at a final concentration of 1.2 M. The
process was monitored spectrophotometrically for 30 min. Spectra
were taken at 2 min intervals with a scan speed of 2000 nm·min−1. The
resulting betalamic acid solution corresponded to the initial pigment
concentration, and the molar absorption coefficient at the wavelength
corresponding to the absorbance maximum was calculated. Enzyme
assays were also performed with the above-mentioned spectropho-
tometer using mushroom tyrosinase (EC 1.14.18.1), which was
purchased from Sigma. The compound 4-methylcatecholquinone was
prepared from 3.6 mM 4-methylcatechol with the addition of 130
units/mL tyrosinase. After the reaction ended, the quinones were
filtered through Biomax-10 membranes (Millipore) to remove the
catalyst. Filtered 4-methylcatecholquinone was used at a final
concentration of 200 μM. Betalamic acid reaction assays were
performed in 50 mM Na3PO4 buffer, pH 7.0. Other conditions are
described in the text and figure legends. The reactions were followed
by recording spectra at 1 min intervals with a scan speed of 2000
nm·min−1 or by measuring the absorbance at 530 nm. The lag period
was calculated as the intercept on the abscissa obtained by
extrapolation of the linear part of the product accumulation curve.
No detectable evolution was detected in the absence of the quinone.
Measurements were performed in triplicate, and the mean values and
standard deviations were plotted. In each case, the errors associated
with the results correspond to the residual standard deviations. Kinetic
data analysis was performed by nonlinear regression fitting44 using
SigmaPlot Scientific Graphing for Windows version 8.0 (2001, SPSS
Inc.). A Shimadzu LC-10A apparatus (Kyoto, Japan) equipped with a
SPD-M10A photodiode array detector (PDA) was used for analytical
HPLC separations. Reversed-phase chromatography was performed
with a 250 × 4.6 mm Kromasil 100 C-18 column packed with 5 μm
particles (Teknokroma, Barcelona, Spain). Gradients were formed
with two He-degassed solvents. Solvent A was H2O containing 0.05%
TFA, and solvent B was MeCN containing 0.05% TFA. A linear
gradient was performed for 25 min from 0% B to 35% B. The flow rate
was 1 mL·min−1, and the operating temperature was 25 °C. The
injection volume was 20 μL. An Agilent VL 1100 apparatus with an
LC/MSD trap (Agilent Technologies, Palo Alto, CA, USA) was used
for the HPLC-ESIMS analysis. The elution conditions were as
described above using a Zorbax SB-C18 (30 × 2.1 mm, 3.5 μm)
column (Agilent Technologies) with a flow rate of 0.3 mL min−1. The
vaporizer temperature was 350 °C, and the voltage was maintained at
3.5 kV. The sheath gas was N2 at 45 psi. Samples were ionized in the
positive mode. The ion monitoring mode was full scan in the 50−600
m/z range. The electron multiplier voltage for detection was 1350 V.
All chemicals and reagents were obtained from Sigma (St. Louis, MO,
USA). HPLC-grade MeCN was purchased from Labscan Ltd. (Dublin,
Ireland). Distilled H2O was purified using a Milli-Q system (Millipore,
Bedford, MA, USA). Betanin was extracted and purified from red beet
roots.33
deprotonation was pKa = 6.8. This pKa explains the behavior of
betalamic acid as a function of pH in terms of reactivity and
antiradical activity. The strong antiradical capacity of betanin
and its pH dependence have been explained previously in terms
of betanin’s electron donor capacity,40 whereas in flavonoids,
deprotonation also generates a more active phenolate
anion.41,42
Antioxidant Activity. The antioxidant activity of free
betalamic acid was evaluated through the direct reduction of
Fe(III) to Fe(II). The ferric reducing antioxidant power
(FRAP) assay was used, as described by Benzie and Strain,43 to
spectrophotometrically monitor the reduction at λ = 593 nm.
Using Fe(II) (FeSO4) solutions with known concentrations, a
calibration curve was established to estimate the number of
electrons involved in the reduction. Figure 6 shows the signal
Figure 6. Antioxidant activity of betalamic acid measured by the FRAP
assay. The change in the absorbance at 593 nm reflects the formation
of the colored TPTZ and Fe(II) complex after the reduction of Fe(III)
○
by betalamic acid ( ). The TPTZ concentration was 741 μM, and the
initial Fe(III) concentration was 1.48 mM in NaOAc buffer, pH 3.6. A
Fe(II) standard curve determined under the same conditions was used
●
as a reference ( ).
obtained for the ferric reduction by betalamic acid relative to
the signal obtained for the calibration curve. Both slopes were
calculated, and on the basis of the relation between these
slopes, the number of electrons involved was determined to be
two. Each betalamic acid molecule was able to directly reduce
two Fe(III) ions to Fe(II).
For the first time, betalamic acid has been obtained and
purified using a chromatographic method that yields sufficient
material to characterize the properties of this compound. The
results demonstrate that the extraordinary antiradical capacity
of betalains is linked to its extended conjugate system. The
results obtained in this study also explain the pH dependence of
the antiradical capacity of betalamic acid. The pure structural
unit of betalains might have applications in the food and
pharmaceutical industries in its free form or as a starting
material to obtain existing or new betalains.
Betalamic Acid Purification. Betalamic acid was purified by
̈
anionic exchange chromatography after betanin hydrolysis in an Akta
purifier apparatus (General Electric Healthcare, Milwaukee, WI, USA).
The equipment was operated with a PC running Unikorn software
version 3.00. The purification solvents used were 20 mM Na3PO4
buffer, pH 6.0 (solvent A), and the same buffer, containing 2 M NaCl
(solvent B). Protocol A: A 25 × 7 mm, 1 mL Q-Sepharose Fast Flow
column (cross-linked agarose with quaternary ammonium as the
exchanger group, 90 μm particle size) was purchased from General
Electric Healthcare. After sample injection, the elution process was as
follows: 0% B from the beginning to 7.0 mL, followed by a linear
gradient from 0% B to 35% B over 20 mL. One-milliliter fractions were
collected. The injection volume was 100 μL of hydrolysis medium (at
the hydrolysis pH), and the flow rate was 1.0 mL·min−1. Protocol B: A
25 × 16 mm, 5 mL Q-Sepharose Fast Flow column (General Electric
Healthcare) was used for scaled-up purification. The elution process
was as follows: 0% B from the beginning to 65 mL, followed by a linear
EXPERIMENTAL SECTION
■
General Experimental Procedures. A Uvikon 940 spectropho-
tometer (Kontron Instruments, Zurich, Switzerland) attached to a
Tectron thermostatic bath (JP Selecta, Barcelona, Spain) was used for
absorbance spectroscopy. Measurements were taken in H2O at 25 °C.
The molar absorption coefficient at 536 nm, ε = 65 000 M−1·cm−1, was
used for the quantification of betanin.35,36 The molar absorption
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gradient from 0% B to 35% B over 100 mL. The injection volume was
10 mL of hydrolysis medium. Two-milliliter fractions were collected,
and the flow rate was 1.0 mL min−1.
(10) Sasaki, N.; Abe, Y.; Goda, Y.; Adachi, T.; Kasahara, K.; Ozeki, Y.
Plant Cell Physiol. 2009, 50, 1012−1016.
(11) Piattelli, M.; Minale, L.; Prota, G. Phytochemistry 1965, 4, 121−
125.
Free Radical Scavenging Activity. The antiradical capacity of
betalamic acid was evaluated by following its effect on the stable free
radical ABTS•+. The decolorization of the ABTS•+ solutions was
monitored spectrophotometrically at λ = 734 nm.15 The ABTS•+
radical was prepared using 2 mM ABTS solutions and peroxidase (88
units/L commercial horseradish peroxidase type VI, purchased from
Sigma) in the presence of H2O2 (45 μM) in 12 mM NaOAc buffer, pH
5.0. The reaction was diluted by 2/3 by the addition of samples, and
the reactions were performed in 53 mM Na3PO4 buffer, pH 7.0. Other
conditions are as specified in the text. Measurements of the
absorbances of the 96-well plates were performed after 24 h
incubations at 20 °C in a Synergy HT plate reader (Bio-Tek
Instruments, Winooski, VT, USA). All measurements were performed
in duplicate, and the mean values were plotted. The final volume in
each well was 300 μL (calculated path length = 0.87 cm), and the
sample volume added was 20 μL. Detector linearity under the assay
conditions was confirmed (r = 0.9993).
Antioxidant Capacity. The antioxidant power of betalamic acid
was characterized by the reduction of Fe(III) to Fe(II). The method
used to evaluate FRAP was performed as described by Benzie and
Strain.43 Briefly, FeCl3 solutions at a final concentration of 1.48 mM in
223 mM NaOAc buffer, pH 3.6, were used. The reduction of Fe(III)
to Fe(II) was observed through the addition of the agent 2,4,6-tris(2-
pyridyl)-s-triazine (TPTZ) at a final concentration of 741 μM; this
compound forms a colored complex with Fe(II). The reduction
reaction was followed spectrophotometrically at λ = 593 nm (Uvikon
940 spectrophotometer). All measurements were performed in
duplicate, and the mean values and standard deviations were plotted.
(12) Butera, D.; Tesoriere, L.; Di Gaudio, F.; Bongiorno, A.; Allegra,
M.; Pintaudi, A. M.; Kohen, R.; Livrea, M. A. J. Agric. Food Chem.
2002, 50, 6895−6901.
́ ́
(13) Gandía-Herrero, F.; Jimenez-Atienzar, M.; Cabanes, J.; García-
Carmona, F.; Escribano, J. J. Agric. Food Chem. 2010, 58, 10646−
10652.
(14) Stintzing, F. C.; Schieber, A.; Carle, R. Planta Med. 1999, 65,
632−5.
(15) Escribano, J.; Pedreno, M. A.; García-Carmona, F.; Munoz, R.
̃
̃
Phytochem. Anal. 1998, 9, 124−127.
(16) Pavlov, A.; Kovatcheva, P.; Georgiev, V.; Koleva, I.; Ilieva, M. Z.
Naturforsch. 2002, 57c, 640−644.
(17) Cai, Y.; Sun, M.; Corke, H. J. Agric. Food Chem. 2003, 51, 2288−
2294.
(18) Gandía-Herrero, F.; Escribano, J.; García-Carmona, F. J. Nat.
Prod. 2009, 72, 1142−1146.
(19) Gandía-Herrero, F.; Escribano, J.; García-Carmona, F. Planta
2010, 232, 449−460.
(20) Kapadia, G. J.; Azuine, M. A.; Sridhar, R.; Okuda, Y.; Tsuruta,
A.; Ichiishi, E.; Mukainake, T.; Takasaki, M.; Konoshima, T.; Nishino,
H.; Tokuda, H. Pharmacol. Res. 2003, 47, 141−148.
(21) Lu, X.; Wang, Y.; Zhang, Z. Eur. J. Pharmacol. 2009, 615, 223−
227.
(22) Tesoriere, L.; Butera, D.; D’Arpa, D.; Di Gaudio, F.; Allegra, M.;
Gentile, C.; Livrea, M. A. Free Radical Res. 2003, 37, 689−696.
(23) Tesoriere, L.; Butera, D.; Allegra, M.; Fazzari, M.; Livrea, M. A.
J. Agric. Food Chem. 2005, 53, 1266−1270.
(24) Kujala, T. S.; Vienola, M. S.; Klika, K. D.; Loponen, J. M.;
Pihlaja, K. Eur. Food Res. Technol. 2002, 214, 505−510.
(25) Wybraniec, S.; Platzner, I.; Geresh, S.; Gottlieb, H. E.;
Haimberg, M.; Mogilnitzki, M.; Mizrahi, Y. Phytochemistry 2001, 58,
1209−1212.
AUTHOR INFORMATION
Corresponding Author
*Phone: +34 868 884786. Fax: +34 868 884147. E-mail:
fgandia@um.es.
■
(26) Cai, Y.; Sun, M.; Corke, H. J. Agric. Food Chem. 2001, 49, 1971−
1978.
Notes
The authors declare no competing financial interest.
(27) Martínez-Parra, J.; Munoz, R. J. Agric. Food Chem. 1997, 45,
̃
2984−2988.
ACKNOWLEDGMENTS
This work was supported by the Ministerio de Ciencia e
(28) Fincan, M.; De Vito, F.; Dejmek, P. J. Food Eng. 2004, 64, 381−
■
388.
(29) Buchi, G.; Fliri, H.; Shapiro, R. J. Org. Chem. 1977, 42, 2192−
̈
Innovacion
́
(MICINN, FEDER, Spain) (Projects AGL2010-
2194.
17938 and AGL2011-25023) and by the Programa de Ayudas a
Grupos de Excelencia de la Region de Murcia, de la Fundacion
Seneca, Agencia de Ciencia y Tecnologia de la Region
Murcia (Plan Regional de Ciencia y Tecnologia 2007/2010).
F.G.-H. has a contract with the “Programa Ramon y Cajal”
(30) Wyler, H.; Wilcox, M. E.; Dreiding, A. S. Helv. Chim. Acta 1965,
48, 361−366.
́
́
́
́
́
de
(31) Huang, A. S.; von Elbe, J. H. J. Food Sci. 1985, 50, 1115−1120.
(32) Schliemann, W.; Kobayashi, N.; Strack, D. Plant Physiol. 1999,
119, 1217−1232.
́
́
(MICINN, FEDER, Spain).
(33) Gandía-Herrero, F.; García-Carmona, F.; Escribano, J.
Phytochem. Anal. 2006, 17, 262−269.
REFERENCES
́
(34) Escribano-Cebrian, J.; García-Carmona, F.; Gandía-Herrero, F.
■
European Patent ES 2,349,522, 2009.
(35) Schwartz, S. J.; von Elbe, J. H. J. Agric. Food Chem. 1980, 28,
540−543.
(1) Moreno, D. A.; García-Viguera, C.; Gil, J. I.; Gil-Izquierdo, A.
Phytochem. Rev. 2008, 7, 261−280.
(2) Musso, H. Tetrahedron 1979, 35, 2843−2853.
(36) Trezzini, G. F.; Zryd, J.-P. Phytochemistry 1991, 30, 1901−1904.
̈
(3) von Ardenne, R.; Dopp, H.; Musso, H.; Steiglich, W. Z.
̈
(37) Kerem, Z.; Regev-Shoshani, G.; Flaishman, M. A.; Sivan, L. J.
Nat. Prod. 2003, 66, 1270−1272.
Naturforsch. 1974, 29c, 637−639.
(4) Svenson, J.; Smallfield, B. M.; Joyce, N. I.; Sanson, C. E.; Perry,
N. B. J. Agric. Food Chem. 2008, 56, 7730−7737.
(5) Wyler, H.; Mabry, T. J.; Dreiding, A. S. Helv. Chim. Acta 1963, 46,
1745−1748.
(38) Valero, E.; Escribano, J.; García-Carmona, F. Phytochemistry
1988, 27, 2055−2061.
(39) García-Carmona, F.; Valero., E.; Cabanes, J. Phytochemistry
1988, 27, 1961−1964.
(6) Piattelli, M.; Minale, L.; Prota, G. Tetrahedron 1964, 20, 2325−
2329.
́
́
(40) Gliszczynska-Swigło, A.; Szymusiak, H.; Malinowska, P. Food
Addit. Contam. 2006, 23, 1079−1087.
(7) Piattelli, M. The Biochemistry of Plants; Conn, E. E., Ed.;
Academic Press Inc.: New York, 1981; Vol. 7, pp 557−575.
(8) Gandía-Herrero, F.; Escribano, J.; García-Carmona, F. Plant
Physiol. 2005, 138, 421−432.
(9) Gandía-Herrero, F.; García-Carmona, F.; Escribano, J. J. Agric.
Food Chem. 2004, 52, 609−615.
(41) Madsen, H. L.; Andersen, C. M.; Jørgensen, L. V.; Skibsted, L.
H. Eur. Food Res. Technol. 2000, 211, 240−246.
́
́
(42) Muzolf, M.; Szymusiak, H.; Gliszczynska-Swigło, A.; Rietjens, I.
M. C. M.; Tyrakowska, B. E. J. Agric. Food Chem. 2008, 56, 816−823.
(43) Benzie, I. F. F.; Strain, J. J. Anal. Biochem. 1996, 239, 70−76.
1035
dx.doi.org/10.1021/np200950n | J. Nat. Prod. 2012, 75, 1030−1036

Journal of Natural Products
Article
(44) Marquardt, D. W. J. Soc. Ind. Appl. Math. 1963, 11, 431−441.
1036
dx.doi.org/10.1021/np200950n | J. Nat. Prod. 2012, 75, 1030−1036