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One-Step Synthesis of Betalains Using a Novel Betalamic Acid

Derivatized Support
Juana Cabanes, Fernando Gandía-Herrero, Josefa Escribano, Francisco García-Carmona,
and Mercedes Jiménez-Atiénzar*
Departamento de Bioquı ́mica y Biologı ́a Molecular A, Unidad Docente de Biologı ́a, Facultad de Veterinaria, Regional Campus of
International Excellence “Campus Mare Nostrum”, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain

ABSTRACT: Betalains are plant pigments with high antioxidant and cancer chemopreventive properties used by the food
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industry as safe colorants. Betalains are restricted to species of the order Caryophyllales, and difficulty in obtaining individual
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molecules has limited their structural identification and application. This study was designed to develop a betalamic acid
derivatized support generated from a primary amine polymer. The novel material presents color properties of a
pseudobetaxanthin, and it is stable for at least 6 months. The bond formed can be displaced at mild conditions by the
addition of amines in aqueous solutions over a broad pH range and at 25 °C. This releases the betalamic acid while forming the
corresponding pigment. This one-step procedure significantly simplifies the process of obtaining semisynthetic betalains, and it is
optimized here for the formation of betaxanthins and betacyanins derived from tyramine, dopamine, pyrrolidine, and indoline.
The new method makes access to single betalains available to the entire scientific community and could stimulate research and
applications in the field.
KEYWORDS: betalamic acid, derivatization, pigments, support, synthesis

■ INTRODUCTION
Betalains are the natural pigments that bestow coloration to
pigments yields energy conversion efficiencies of up to 2.7%,
above that of natural photosynthesis.12,13 Other potential
plants of the order Caryophyllales. Betalains are yellow applications of betalains are their use in microscopy as a new
(betaxanthins) or violet (betacyanins) conjugates of the probe for live cell imaging, based on their fluorescent
structural unit betalamic acid (1) with free amines or properties,14,15 and as a sensor for colorimetric assays.16
indoline-derived compounds, respectively (Figure 1).1 The The promising bioactive and applied potential of betalains
presence of both types of pigments generates the wide variety contrasts with the limited number of available tools developed
of shades ranging from pale yellow to violet including the to obtain individual molecules. The synthesis of standards is of
orange, red, and pink colors characteristic of the flowers, fruits, importance in food technology research, in pigment identi-
and roots of betalain-containing plants. Notable among the fication from natural sources and in bioactivities character-
edible sources are red beet roots (Beta vulgaris) and the fruits of ization. Betalamic acid (1) is the structural unit of all betalains
cacti belonging to the genus Opuntia, but betalains are also and can be used as the starting point to obtain betalains
present in the ulluco tubers (Ullucus tuberosus) or the berries through a Schiff condensation reaction with free amine groups
from Rivina humilis among others.2−5 In addition, betalains are (betaxanthins) or indoline-containing structures (betacyanins)
used by the food industry to give color to foods as the additive (Figure 1).1 Betalamic acid can be synthesized chemically in a
73.40 in the 21 CFR section of the U.S. Food and Drug procedure that involves multiple steps and gives low yields.17 A
Administration (FDA) and under the E-162 code in the feasible alternative is to get the acid from the degradation
European Union. products of natural betanin (betanidin-5-O-β-glucoside), but
In recent years, strong bioactive properties have been the presence of the other degradation product, cyclo-DOPA-
described for betalains. These include a high antioxidant and glucoside, reverses the reaction to the starting compound.18,19
free radical scavenging capacity present in all pigments, which is To date, the technology for the formation of betalains involves
modulated by structural factors.6,7 The characterized antiradical betalamic acid extraction in ethyl acetate and further
activity of betalains may explain their proven potential in the condensation with amines, which results in very low yields,
chemoprevention of cancer using different cell lines.8,9 The or a combined procedure for betanin degradation and betalains
inhibition of the growth of cancer cells is dose-dependent, and synthesis, which implies an immediate purification to avoid the
it has been demonstrated with both plant extracts and purified reversal of the reaction.20,21
betalains. In addition, experiments with model animals have A betalamic acid derivatized support is developed here for
shown that very low concentrations of dietary pigments inhibit the first time. It is based on an imine formation reaction among
the formation of tumors in vivo, showing a potent cancer
chemopreventive activity.10,11 Received: January 29, 2014
Other applications of individual betalains come from their Revised: April 1, 2014
use in dye-sensitized solar cells for solar energy conversion due Accepted: April 1, 2014
to their redox capacity to transfer electrons. The use of pure Published: April 1, 2014

© 2014 American Chemical Society 3776 dx.doi.org/10.1021/jf500506y | J. Agric. Food Chem. 2014, 62, 3776−3782
Journal of Agricultural and Food Chemistry Article

Figure 1. Structures for betalamic acid (1), the structural unit of betalains, and the derived pigments tyramine−betaxanthin (2), dopamine−
betaxanthin (3), pyrrolidine−betaxanthin (4), and indoline−betacyanin (5).

betalamic acid and the amine groups present in the matrix starting capacity of synthesis. Then, the dopamine betaxanthin
surface, which can be displaced at mild conditions by the synthesis was performed with the support at different times
addition of amines. The novel material allows a one-step throughout 6 months. Measurements were performed in triplicate,
procedure for the formation of betaxanthins and betacyanins, and mean and standard deviations were plotted. Relative synthetic
activity was determined as the percentage of activity remaining after
thus significantly simplifying the process of obtaining semi- storage with respect to controls. Errors associated with the results
synthetic betalains. provided were calculated on the basis of the residual standard

■ MATERIALS AND METHODS

Chemicals and Reagents. Red beet juice concentrate (B-50-WS)
deviations. The stability of the support was also studied by color
assessment analysis. Color determinations of the immobilized
betalamic acid support were made at 25 °C using a V-650
spectrophotometer equipped with an ISV-722 integrating sphere
was purchased from CHR Hansen (Madrid, Spain). It is a liquid (Jasco Corp., Tokyo, Japan). The color parameters corresponded to
formulation obtained by squeezing out, concentrating, and pasteuriz- the uniform CIELAB space (L*, a*, b*, C*, and h°) and were directly
ing the juice of beetroots, B. vulgaris. The starting matrix was Lewatit obtained from the apparatus software Spectra Manager version 2.07.22
VP OC 1065 (surface area = 50 m2/g), obtained from Sigma (Madrid, Spectroscopy. A V-630 spectrophotometer (Jasco Corp., Tokyo,
Spain). Solvents were from Merck (Madrid, Spain). HPLC grade Japan), attached to a Tectron thermostatic bath (JP Selecta, Barcelona,
acetonitrile was purchased from Labscan Ltd. (Dublin, Ireland). Other Spain), was used for absorbance spectroscopy. For the quantitation of
chemicals and reagents were obtained from Sigma (Madrid, Spain). betalains, pigment concentration was evaluated by taking a molar
Distilled water was purified using a Milli-Q system (Millipore, Bedford, extinction coefficient of ε = 48000/M/cm at 480 nm for betaxanthins
MA, USA). and ε = 71000/M/cm at 524 nm for indoline−betacyanin.7,23
Development of the Betalamic Acid Support. Red beet juice Measurements were made in water at 25 °C.
concentrate was filtered by a 10 kDa ultrafiltration step (QuixStand HPLC Analysis. A Shimadzu LC-20AD apparatus (Kyoto, Japan)
System, GE Healthcare, Milwaukee, WI, USA). Betanin from this equipped with an SPD-M20A photodiode array detector (PDA) was
filtered solution was used as starting material. Basic hydrolysis (pH used for analytical HPLC separations. Reversed phase chromatography
11.4) of betanin in the presence of the starting matrix released was performed with a 250 × 4.6 mm Luna C-18(2) column packed
betalamic acid (30 min, at room temperature) (1), which was then with 5 μm particles (Phenomenex, Torrance, CA, USA). Gradients
condensed with the primary amine present in the inorganic matrix were formed with two solvents, A and B. Solvent A was H2O with
after reaching pH 5.0. This bound betalamic acid to the surface of the 0.05% trifluoroacetic acid (TFA); solvent B was acetonitrile with
support, which was then washed three times with 20 mM sodium 0.05% TFA. A linear gradient was performed from 5 to 35% B for 35
acetate buffer, pH 5.0. The binding process was carried out in nitrogen min. The flow rate was 1 mL/min, operated at 25 °C. Injection volume
atmosphere. was 20 μL.24 All assays were performed in triplicate, and mean and
Semisynthesis of Betalains. For standard assays, the betalamic standard deviations were plotted. Errors associated with the results
acid derivatized support (0.16 g) was incubated in a final volume of 0.5 provided were calculated on the basis of the residual standard
mL with different amines, at the specified concentration, in 20 mM deviations.
sodium acetate buffer, pH 5.0, or in 20 mM sodium phosphate buffer, Scanning Electron Microscopy (SEM). Particle morphology was
pH 8.0, for tyramine. The reaction mixture for pyrrolidine− evaluated by SEM. Powders were attached to pieces of double-sided
betaxanthin (4) and indoline−betacyanin (5) syntheses also contained adhesive tape mounted on SEM stubs, coated with gold under vacuum
1.36 M sodium chloride to facilitate the release of the pigments from using a SEM coating system by Bio-Rad Polaron Division and
the support surface into the reaction medium. The formation of the examined with a JSM-6100 (JEOL, Tokyo, Japan) scanning electron
corresponding betaxanthin or betacyanin was revealed by a character- microscope operated at 15 kV. Images were analyzed using the open
istic deep yellow or violet color, respectively, in the reaction medium. source image analysis software ImageJ (National Institutes of Health,
After the indicated reaction time, an aliquot of 100 μL was removed NIH, Bethesda, MD, USA). Briefly, images were calibrated and, after
for HPLC analysis. To analyze the formation of betalains with time, that, an interactive measurement of the spheres was carried out.
the reaction was scaled up to a final volume of 8 mL. The evolution of Micrographs were obtained with an Inca Oxford Image capture system
the reaction was monitored by HPLC analysis of the aliquots extracted (Oxford Instruments, Abingdon, UK).

■
at different times. For pH studies the reaction was carried out in 20
mM sodium acetate buffer (pH 3.5−5.5) or in 20 mM sodium
phosphate buffer (pH 6.0−7.0). The buffer used for pH values >7.5 RESULTS AND DISCUSSION
was 20 mM sodium borate. Development of a Betalamic Acid Derivatized
Stability of the Betalamic Acid Support. The stability of the Support. Betalamic acid (1) is the structural and bioactive
novel support was investigated by studying its capacity to synthesize
betaxanthins as a function of storage time. Freshly made betalamic acid unit of plant pigments betalains and the starting point for their
support (0.16 g) was incubated with 1.35 mM dopamine in 20 mM formation both in vivo and in vitro. The production of
sodium acetate buffer, pH 5.0. An aliquot was taken after 40 min of individual compounds implies a purification of the acid, which
reaction to analyze dopamine−betaxanthin (3) formation by HPLC. leads to multistep procedures with low yields.17,20,21 The aim of
The betaxanthin concentration value obtained was considered as the this work is the development of a novel support derivatized
3777 dx.doi.org/10.1021/jf500506y | J. Agric. Food Chem. 2014, 62, 3776−3782
Journal of Agricultural and Food Chemistry Article

Figure 2. Schematic overview for the betalamic acid derivatized support and the surface reaction leading to pigment synthesis, exemplified for
dopamine-derived betaxanthin (3): (A) starting material containing primary amine groups; (B) betalamic acid derivatized support; (C) amine attack
to betalamic acid; (D) synthesis and concomitant release of the pigment.

with betalamic acid suitable for the synthesis of betalains. The

basis of the proposed process is summarized in Figure 2.
First, betalamic acid is obtained in the presence of a resin that
contains free primary amine groups. Given the reactivity of the
aldehyde group of the acid, it was proposed that an imine
would be formed with the amine of the support, thus causing
the immobilization of betalamic acid to the resin. Betalamic acid
was obtained by basic hydrolysis of purified betanin, the main
pigment present in red beet root (B. vulgaris) extracts by the
addition of ammonia.21 Hydrolysis was carried out in the
presence of the easily available cross-linked polystyrene resin
VP OC 1065 (Lewatit), which contains primary amine groups,
at room temperature and under nitrogen atmosphere. Under Figure 3. Spectroscopic and microscopic characterization of the
these experimental conditions betalamic acid was released from betalamic acid derivatized support: (A) UV−vis spectrum for the solid
matrix showing the characteristic spectrum for betaxanthins (spectrum
betanin. Neutralization with acetic acid was carried out after was obtained with an integrating sphere after the subtraction of the
hydrolysis to promote the reaction of betalamic acid with the spectrum for the basal absorbance of the starting matrix); (B) SEM
amines through a Schiff condensation reaction. This involved a image obtained for the spheres that constitute the novel matrix (scale
change of color of the resin spheres from pale yellow to deep bar = 500 μm); (C) closer SEM view showing the surface of the
yellow, indicating the derivatization with betalamic acid. The spheres (scale bar = 5 μm).
resin was washed with 20 mM sodium acetate buffer, pH 5.0,
until the washing solution was colorless. It was then allowed to
shows that the surface porosity of the spheres was not affected
dry at room temperature.
by the process (Figure 3C).
The binding reaction of betalamic acid to the matrix was
Thus, a matrix derivatized with betalamic acid was obtained
confirmed by UV−vis spectrophotometry using an integrating for the first time. It is proposed that this support could be used
sphere. This allowed the analysis of the color changes in the for betalains synthesis simply by adding the selected amine. The
solid matrix. Figure 3A shows the spectrum obtained for the reaction of betalamic with the amine would lead to the release
resin after betalamic acid reaction. The maximum wavelength of the pigment, as shown graphically in Figure 2.
was determined as λmax 486 nm. Both the shape and maximum Semisynthesis of Betalains. To test the concept of easy
wavelength corresponded to the typical results of a betaxanthin, synthesis of betalains through the novel betalamic acid
the yellow betalains formed by condensation of betalamic acid derivatized support, the resin was used for the production of
with simple amines.7,23 Thus, the reaction between betalamic different pigments. Four amines were selected as models to
acid and the amine groups of the resin generated a solid demonstrate this application: tyramine, dopamine, pyrrolidine,
support with the spectroscopic properties of a pseudobeta- and indoline. The choice of tyramine and dopamine was based
xanthin. The novel support was further characterized by SEM on the bioactive potential of their corresponding betaxanthins.
to analyze the effect of the treatment on the particle surface. Tyramine−betaxanthin (2) and dopamine−betaxanthin (3)
Figure 3B shows a SEM microphotograph of the resin after possess high antioxidant and free radical scavenging activities
betalamic acid immobilization. Particle analysis and sphericity due to the presence of phenolic hydroxyl groups (Figure 1).25
were analogous to the results obtained for the resin without They are natural pigments, also known by the trivial names
betalamic acid, indicating that the morphology of the resin was miraxanthin III, and miraxanthin V, respectively, which were
not altered by the immobilization process. Analysis of the originally described in flowers of Mirabilis jalapa but are also
particle morphology shows that the resin spheres possess a present in the species Celosia argentea and B. vulgaris among
mean particle diameter of 0.59 ± 0.08 mm. SEM analysis also others.26−28 Structurally, these betaxanthins are derived from
3778 dx.doi.org/10.1021/jf500506y | J. Agric. Food Chem. 2014, 62, 3776−3782
Journal of Agricultural and Food Chemistry Article

primary amines and possess open structures. In contrast,

pyrrolidine is a secondary amine, and the pigment derived (4)
possesses a ring attached to the betalamic acid moiety while
maintaining properties of a betaxanthin (Figure 1). Indoline is
also a secondary amine and possesses the same ring of
pyrrolidine plus an additional aromatic ring (Figure 1). This
additional electron resonance system promotes a bathochromic
shift in the absorbance spectrum, which makes the derived
betalain violet. The indoline-derived pigment (5) is the
simplest molecule with betacyanin properties and, thus, the
betacyanin defining structure.7
When colorless solutions containing the individual model
amines were added to the novel support, the solutions turned
yellow in the case of tyramine, dopamine, and pyrrolidine and
purple in the case of indoline. In contact with the support, the
amines yielded the corresponding pigments through a Schiff
condensation reaction with the aldehyde group of betalamic
acid, thus releasing the synthesized betalain as postulated in
Figure 2C. All pigments were characterized by HPLC-DAD,
confirming the expected identity and the absence of additional
coloring molecules. Retention times for tyramine-derived (2)
and dopamine-derived (3) betaxanthins were 18.3 and 16.5
min, respectively, whereas for pyrrolidine-derived (4) and
indoline-derived (5) betalains retention times were 15.2 and
23.6 min, respectively. The optimal experimental conditions for
the novel synthesis on support were investigated by measuring
the pigment generation by HPLC-DAD analysis of the
solutions. Figure 4 shows the evolution of the synthesis
reaction with time for each amine assayed at 25 °C. The Figure 4. Time course for the evolution of pigment formation in
reaction media containing the betalamic acid derivatized support at 25
temperature of 4 °C was also assayed in this study under the
°C and different amines; (A) tyramine, 0.85 M; (B) dopamine, 1.35
same conditions (data not shown). At this temperature the M; (C) pyrrolidine, 0.6 M; (D) indoline, at saturating concentration.
concentration of product obtained was lower than at 25 °C in In panels C and D, results are shown in the presence (●) and in the
all cases. Analysis of the time courses obtained at 25 °C absence (○) of 1.36 M NaCl. Experimental conditions are detailed
determined the optimal times of reaction at this temperature, under Materials and Methods.
which varied depending on the amine used. The highest
pyrrolidine-derived pigment (4) concentration was reached betalains from the resin surface into the solution (Figure
after only 4 min of reaction. For the rest of the compounds the 4C,D). This increased the amount of pigment obtained from
estimated optimum reaction times were 20, 40, and 60 min for the reaction on the novel material. An optimal concentration of
indoline-derived (5), dopamine-derived (3), and tyramine- 1.36 M NaCl was determined, with values below this causing a
derived (2) betalains, respectively. This variation may result lower pigment release.
from the different affinities of betalamic acid toward the amine The effect of pH was also investigated in the pigment
groups of the support and the amines added in solution. This synthesis from the derivatized support. Figure 5 shows the
apparent affinity was greater for pyrrolidine. The existence of a results obtained when the pH of the amine-containing solution
maximum in all of the time courses presented indicates the was varied from 3.5 to 9.0. Only in the case of dopamine was
existence of two processes taking place at the surface of the the range limited to 6.5 to avoid the catecholic substructure
spheres. One is the synthesis and concomitant release of the oxidation at higher pH values. Tyramine−betaxanthin (2)
pigments as discussed, causing the appearance of betalains in synthesis reached a maximum at pH 8.0 (Figure 5A), which was
the medium immediately after the addition of the amines. The then selected for standard assays with this amine. However, as
other is responsible for withdrawing the pigments obtained can be seen for the rest of amines, the pH variation did not
from the medium after their formation, so causing the decrease significantly affect the synthesis. A pH of 5.0 was preferred to
of concentration after synthesis in the solution. Pigment more alkaline values because of the higher stability of the
stability in the medium was confirmed by repetitive analysis of compounds.29,30
the pigment concentration after separation from the matrix, Betalain synthesis was also dependent on the amine
with no appreciable loss of the betalains (results not shown). concentration. As shown in Figure 6, an increase in the
Pigment concentration decreased only after the maximum concentrations of the amines resulted in a general increase in
times described above in the presence of the matrix, thus the formation and release of the derived betalains to the
indicating an adsorption phenomenon. The nature of the solution. However, the substrate concentration effect depended
adsorption process might be unspecific, but an electrostatic on the nature of each amine. Increasing concentrations of
contribution was investigated. The addition of sodium chloride tyramine (Figure 6A) and dopamine (Figure 6B) led to a
to the reaction mixture in the case of the pyrrolidine-derived higher level of the derived betaxanthins in the solution.
betaxanthin (4) and the indoline-derived betacyanin (5) However, their concentrations could not be raised further
mitigated the electrostatic contribution of the positive charge because of the relatively low solubility of these compounds. In
of the pigments and facilitated the release of the resulting the case of pyrrolidine (Figure 6C), betaxanthin production
3779 dx.doi.org/10.1021/jf500506y | J. Agric. Food Chem. 2014, 62, 3776−3782
Journal of Agricultural and Food Chemistry Article

significantly simplifies the previous methodology.20,21 Betalamic

acid obtained from red beet roots was previously used to obtain
small amounts of betalain standards for pigment identification
by its addition to amines after a process of hydrolysis and
immediate partitioning steps with ethyl acetate followed by
concentration and re-extraction prior to the synthesis
reaction.20 The combination of the hydrolysis and imine
formation reactions in one merged procedure allowed higher
amounts of betalains to be obtained but still required
immediate purification steps with anionic exchange chromatog-
raphy to avoid reversal of the reaction.21 The methodology
described here avoids the immediate purification steps and
yields stable betalains in minutes, thus simplifying the process
to obtain individual pigments. Figure 7 shows the use of the
novel betalamic acid derivatized support as a matrix in
chromatographic cartridges to speed the recovery of the
pigments. The result is shown after the addition of the selected
amines at the corresponding optimum concentration, pH, and
reaction times determined above.
Stability of the Betalamic Acid Derivatized Support.
The stability of betalamic acid after the resin derivatization was
investigated by studying its capacity to synthesize betaxanthins
as a function of storage time. As a control, a freshly prepared
derivatized support was used to obtain dopamine−betaxanthin
(3) as described above. The amount of betaxanthin obtained
was considered to be the starting capacity of synthesis of the
Figure 5. pH effect on the synthesis of betalains using the betalamic support. The synthesis reaction was repeated with the support
acid derivatized support and different amines: (A) tyramine, 0.85 M; at different times over 6 months. The support was stored at 4
(B) dopamine, 1.35 M; (C) pyrrolidine, 0.6 M; (D) indoline, at °C in the absence of light. The results obtained for dopamine−
saturating concentration. The reaction was carried out in 20 mM betaxanthin (Figure 8) indicate that the resin retained
sodium acetate buffer (pH 3.5−5.5), 20 mM sodium phosphate buffer approximately 50% of its potential after 6 months of storage.
(pH 6.0−7.0), or 20 mM sodium borate buffer (pH 8.0−9.0). The loss of synthetic activity was pronounced during the first
month, and from then on it remained practically stable in the
grew with increasing concentrations of the starting amine up to production of the betaxanthin.
a maximum at 0.6 M. The scarce solubility of indoline in In addition, the stability of the derivatized support was
aqueous media prevented the study of the concentration effect, evaluated by color assessment analysis of the resin. The changes
and this amine was always added to the reaction mixture at in the appearance of the resin were quantitated by the results of
saturating concentration under the assay conditions. the color CIELAB parameters, determined by spectropho-
From the above results it can be concluded that the tometry using an integrating sphere. Data were collected
optimization of the parameters which affect the novel one-step immediately after derivatization with betalamic acid. These
method for the easy synthesis of betalains should be made for results obtained for the freshly made support were compared to
each individual compound. Once optimized, the method allows those found after 6 months of storage (Table 1). As can be
a routine process to obtain individual compounds that seen, the a* and b* values decreased, indicating a loss in the red

Figure 6. Effect of amine concentration on the formation of the betaxanthins derived from (A) tyramine, (B) dopamine, and (C) pyrrolidine. The
reaction medium contained the derivatized support and the amine, at the specified concentration, in 20 mM sodium phosphate buffer, pH 8.0
(tyramine), or in 20 mM sodium acetate buffer, pH 5.0 (dopamine and pyrrolidine).

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Journal of Agricultural and Food Chemistry Article

and yellow colors, respectively. This is probably due to a limited

degradation of the pseudobetaxanthin formed, which possesses
a maximum wavelength of λmax 486 nm. This contributed to the
change in hue angle (h°) and chroma (C*). The L* parameter
was moderately increased, indicating that the color of the resin
became lighter after the storage time. In conclusion, the color
(a*, b*, h°), its intensity (C*), and the lightness (L*) of the
derivatized support changed slightly after 6 months of storage,
as confirmed by the value of ΔE*. This parameter accounts for
the color variation calculated with the differences between L*,
a*, and b* of the sample stored and the initial conditions. As
can be seen, it was only slightly varied, indicating the stability of
the betalamic acid resin.
The present work develops for the first time a support
derivatized with the structural unit of betalains, betalamic acid.
The novel material containing this natural molecule presents
color properties of a pseudobetaxanthin and allows a procedure
for the easy synthesis of individual betalains. The methodology
is based on the addition of amines, which provokes the
production of the corresponding derived pigments with the
concomitant release from the matrix. The procedure is
described in detail and provides a one-step method for the
synthesis of betalains, thus considerably simplifying the
previous methodologies. This tool opens up new perspectives
in the research on betalains and may benefit current and future
Figure 7. Application of the betalamic acid derivatized support to the applications of the pigments.

■
synthesis of betalains in chromatographic cartridges. Solutions
containing the individual pigments are obtained after the synthesis
of (A) tyramine−betaxanthin (2), (B) dopamine−betaxanthin (3), AUTHOR INFORMATION
(C) indoline-derived betacyanin (5), and (D) pyrrolidine−betaxanthin Corresponding Author
(4) under the optimized conditions.
*(M.J.-A.) Phone: +34 868 884767. Fax: +34 868 884147. E-
mail: [email protected].
Funding
This work was supported by the Ministerio de Ciencia e
Innovación (MICINN, FEDER, Spain, Project AGL2011-
25023) and by Fundación Séneca, Agencia de Ciencia y
Tecnologı ́a de la Región de Murcia (Plan Regional de Ciencia y
Tecnologı ́a 2007/2010, Programa de Ayudas a Grupos de
Excelencia de la Región de Murcia). F.G.-H. has a contract with
the Programa Ramón y Cajal (MICINN, FEDER, Spain).
Notes
The authors declare no competing financial interest.

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