Article

pubs.acs.org/JAFC

Betalain Profile, Phenolic Content, and Color Characterization of

Different Parts and Varieties of Opuntia ficus-indica
María Jesús Cejudo-Bastante,† Makhlouf Chaalal,§ Hayette Louaileche,§ Juan Parrado,#
and Francisco J. Heredia*,†
†
Food Color and Quality Laboraty, Departament of Nutrition and Food Science, Faculty of Pharmacy, University of Sevilla, 41012
Sevilla, Spain
§
Laboratoire de Biochimie Appliquée, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, 06000 Bejaia, Algeria
#
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Seville, 41012 Seville, Spain

ABSTRACT: Three different varieties of Opuntia ficus-indica (R, red; Y, yellow; RY, red-yellow) have been considered in this
study. Attention was focused on differential tristimulus colorimetry and on the analysis of individual betalains (HPLC-DAD-ESI-
ToF-MS) and phenolic content, scarcely previously reported in these kinds of samples. The importance of this research stems
from the elucidation of the parts and varieties of cactus pear more optimal for use as natural colorants and sources of phenolics
and betalains. Thus, the RY pulp was appropriate to obtain colorants with high color intensity (C*ab = 66.5), whereas the whole
Y fruit and R pulp reached powerful and stable yellow and red colors, respectively (C*ab/hab, 57.1/84.7 and 61.1°/81.8°). This
choice was also based on the visually appreciable differences (ΔE*ab > 5) among samples, mainly quantitative (%Δ2L, %Δ2C). In
addition, seeds of all Opuntia varieties showed significantly (p < 0.05) similar phenolic content (around 23.3 mg/g) and color
characteristics.
KEYWORDS: Opuntia ficus-indica, differential tristimulus colorimetry, betalains, phenolics, HPLC-DAD-ESI-ToF-MS

■ INTRODUCTION
In the past few years, consumers have become more conscious
of the importance of healthy and natural food both in the
manufacture or in consumption. One part of this reality is the
use of natural products as colorants. In this way, there is a large
extent of natural colorants, extracted from different natural raw
materials, rich in compounds such as anthocyanins, carotenoids,
and chlorophylls. Apart from those, there is a group of chemical
compounds that it is still poorly investigated, betalains.
Betalains are water-soluble compounds present in a restricted
number of families of the plant order Caryophyllales and of the
genus Amanita of the Basidiomycetes.1 Numerous advanta-
geous on consuming betalain-rich food have been reported,
such as antioxidant activity, antiviral, anti-inflammatory, and
anticarcinogenic effects.2,3 Betalains are structured in two
chemical families (betacyanins and betaxanthin). The common
moiety of both chemical families corresponds to betalamic acid,
and the nature of the additional residue determines the pigment
classification as betacyanin (hydroxycinnamic acid derivatives
or sugars) or betaxanthin (amines or amino acids),4 with
maximum absorptions around 540 and 480 nm, respectively.2,5
There are manifold species and varieties used as natural food
colorants. In that way, although the most studied and widely
distributed source of betalains is the red beet (Beta vulgaris
L.),6−8 betalains have been also studied in other raw materials.
Some examples are the Swiss chard vegetable,9 flowers
(Gomphrena globosa L. and Bougainvillea sp.),10 and fruits
such as pitaya11 and cactus pear (Opuntia sp.).12−16
Opuntia ficus-indica is a type of cactus that provides edible
prickly pears. This kind of cactus is widely cultivated in the
world due to its adaptability to difficult growing conditions
(arid and semiarid zones).17 Recently, the study of cactus pear
has become more and more prominent because of its high
content of betalains and other phenolic compounds. In fact,
those compounds have been previously studied on this matter
in different edible and nonedible parts of prickly pears such as
pulps,14 skins,16 and seeds.18,19 These research studies have
been focused on the general chemical characterization,12,20 the
total content of both phenolic and betalains,21−23 and the
antioxidant activity determination.24 However, only a few
studies have been developed about individual betalains in that
raw material.13,14,25 Moreover, although this fruit has a potential
enterprise projection as natural colorant, hitherto, scarce
advanced colorimetric studies have been developed.12,21,26
Therefore, there are still many aspects to be elucidated about
the repercussion on the color of O. ficus-indica when it is added
as safe colorant.
Therefore, the aim of this research was to carry out an
extensive chemical and colorimetric characterization of O. f icus-
indica [L.] Mill cultivated in Algeria. Different parts of three
cactus pear varieties were taken into account, which have not
been previously considered in conjunction. Our interest was
focused on the study of the phenolic content and individual
betalain profile and also on colorimetric characteristics by
applying differential tristimulus colorimetry, scarcely reported
in the literature. Therefore, the objective is not only the
chemical characterization of different parts of prickly pears but
Received: May 23, 2014
Revised: July 23, 2014
Accepted: July 30, 2014
Published: July 30, 2014

© 2014 American Chemical Society 8491 dx.doi.org/10.1021/jf502465g | J. Agric. Food Chem. 2014, 62, 8491−8499
Journal of Agricultural and Food Chemistry Article

Table 1. Mean CIELAB Parameters and Mean Values of Total Polyphenols (TP) Concentration Corrected for Ascorbic Acid
(and Standard Deviations) of Different Parts of the Fruit of the Three Varieties: Influence of the Tested Factors As Assessed by
Univariate Statistical Analysisa
statistical main effects
by part of fruit by variety
L* C*ab hab TP L* C*ab hab TP L* C*ab hab TP
YS 97.7 ± 0.7 4.4 ± 0.6 92.5 ± 1.8 3.8 ± 0.5 S/W * * * * Y/R
YW 88.7 ± 0.9 57.1 ± 3.2 84.7 ± 0.1 13.3 ± 1.1 Y S/P * * * * S Y/RY
YP 84.8 ± 7.8 54.5 ± 0.4 85.2 ± 1.1 8.1 ± 0.5 W/P * R/RY
RS 95.7 ± 2.8 6.2 ± 2.1 90.9 ± 4.1 4.1 ± 0.4 S/W * * * * Y/R * *
RW 88.7 ± 1.4 54.9 ± 2.1 83.4 ± 0.1 6.6 ± 1.5 R S/P * * * * W Y/RY * *
RP 80.8 ± 1.7 61.1 ± 0.8 81.8 ± 0.3 11.8 ± 1.3 W/P * * * * R/RY *
RYS 94.9 ± 3.5 7.7 ± 2.9 88.7 ± 2.6 4.0 ± 0.1 S/W * * * Y/R * * *
RYW 89.3 ± 0.2 62.5 ± 3.7 83.7 ± 0.1 8.4 ± 1.0 RY S/P * * * * P Y/RY * * *
RYP 88.9 ± 0.9 66.5 ± 0.1 83.1 ± 0.4 12.9 ± 1.4 W/P * R/RY * * *
a
Asterisks denote significant differences by pairs according to Tukey test (p < 0.05). S, seeds; P, pulp; W, whole fruit; R, red variety; Y, yellow
variety; RY, red-yellow variety.

its practical application as a natural colorant by deepening the length glass cells and distilled water as reference. The CIELAB
study of differential colorimetry and betalain profile. parameters (L*, a*, b*, C*ab, and hab) were determined by using the

■
MATERIALS AND METHODS
Chemicals and Solvents. Methanol of analytical or HPLC grade
was purchased from J. T. Baker (Baker Mallinckrodt, Mexico), and
formic acid and Folin−Ciocalteu reagent were supplied by Sigma-
Aldrich (St. Louis, MO, USA). HPLC grade water was obtained by a
Milli-Q plus water purification system (Millipore Corp., Bedford, MA,
USA). Sodium ascorbate and L-ascorbic acid were from Panreac
(Barcelona, Spain). Betanin was supplied by Cymit Quimica S.L.
(Barcelona, Spain).
Samples. Three different cultivars of prickly pear fruits (O. f icus-
indica [L.] Mill.) with diverse physical qualities have been selected for
this study. Those cactus pear varieties were typically cultivated in the
area of Bousselam (Setif, Algeria). Fully ripe cactus pears were
collected in August from different points of the plant and in various
parts of the parcel. Samples were selected on the basis of their color
(both pulp and skin) and the shape and presence of cladode spines (R,
red, ovoid and cladode spineless; Y, yellow, elongate with cladode
spines; RY, red-yellow, ovoid with cladode spines). Fruits were
harvested at a desirable maturity and in good sanitary conditions (R, Y,
and RY: pH, 6.14, 5.86 and 5.95; titratable acidity, 0.10, 0.11, and 0.09;
°Brix, 12.83, 11.55, and 14.22, respectively).
Fruits were carefully washed and manually peeled. The seeds were
removed from the pulp and washed with distilled water. Three
different parts of each variety was studied, corresponding to the edible
part of the fruit: seeds, pulp, and whole fruit (seeds + pulp). They were
separately lyophilized (Christ, Alpha 1-4 LD plus, Germany), ground
with a crusher (IKA A 11B, Germany), and passed through a 500 μm
sieve.
Preparation of Extracts. Lyophilized samples (1 g) were added to
3 mL of methanol/water (50:50) containing 50 mM sodium ascorbate
(for avoiding possible oxidations). Subsequently, samples were stirred
at 225 rpm for 10 min in darkness. Afterward, samples were
centrifuged at 12000g at 10 °C for 5 min, separating supernatants from
the plant tissue. To achieve the complete discoloration of the plant
material, the residues was rinsed once more with the extraction
solution and finally with 100% methanol. The extracts were then
concentrated in a vacuum (30 °C) until around 3 mL to remove
methanol, adding purified water until a total of 4 mL. The mean pH
values of the resulting extracts were similar among varieties, as follows:
seeds, 5.83; pulp, 6.20; and whole fruit, 6.04. All experiments were
carried out in triplicate.
Colorimetric Measurements. A Hewlett-Packard UV−vis
HP8452 spectrophotometer (Palo Alto, CA, USA) was used to carry
out color measurements. The whole visible spectrum (380−770 nm)
was recorded at constant intervals (Δλ = 2 nm) using 2 mm path
original software CromaLab27 following the Commission Internatio-
nale de L’Eclariage’s recommendations,28 the CIE 1976 10° Standard
Observer and the Standard Illuminant D65. Euclidean distances
between two points in the three-dimensional space define by L*, a*,
and b* were used for calculating color differences (ΔE*ab of CIELAB):
ΔE*ab = [(ΔL*)2 + (Δa*)2 + (Δb*)2]1/2. Moreover, the relative
contribution of the three color attributes with respect to Δ2E*ab that
constitutes the total CIELAB color difference was also calculated,
expressed as percentage of the quadratic increases of lightness, chroma,
and hue.29
Total Phenolic (TP) Content. TP content was determined using a
modification of the Folin−Ciocalteu method.30 Absorbance was
measured at 765 nm, and the results were expressed as milligrams of
gallic acid per liter (mg GAE/L). Subsequently, total phenolic content
was expressed as milligrams per gram of dry weight (DW). To
eliminate the contribution of ascorbic acid to the measurement of
absorbance in the Folin−Ciocalteu assay, a correction factor was
calculated,31 quantifying also the ascorbic acid present in each
sample.32 Subsequently, the impact of ascorbic acid in terms of
milligrams of GAE per liter was deduced from the spectrophotometri-
cally determined total polyphenol values.
HPLC-DAD-ESI-ToF-MS Analysis of Betalains. HPLC separa-
tion, identification, and semiquantification of betalains were performed
in an Agilent 1200 chromatographic system equipped with a
quaternary pump, an UV−vis diode array detector, an automatic
injector, and ChemStation software (Palo Alto, CA, USA). Prior to
direct injection, the samples were filtered through a 0.45 μm nylon
filter (E0034, Análisis Vı ́nicos, Spain). All analyses were made in
triplicate.
The betalains’ identification was carried out following the method
proposed by Castellanos-Santiago and Yahia,14 using 1% formic acid in
water (v/v, eluent A) and methanol (eluent B). Betalains were
separated in a Zorbax C18 column (250 × 4.6 mm, 5 μm particle size)
maintained at 25 °C, at a flow rate of 1 mL/min. The injection of the
volume for all extracts was 20 μL. Betalain compounds were separated
starting with 100% A, followed by a linear gradient from 0 to 10% B in
20 min, then a linear gradient from 10 to 30% B in 10 min, and from
30 to 100% B in 5 min. To re-establish the initial conditions, a linear
gradient from 100% B to 100% A was used during 5 min. UV−vis
spectra were recorded from 200 to 800 nm with a bandwidth of 1.0
nm. Betacyanins and betaxanthins were monitored at 535 and 482 nm,
respectively. The identification of each chromatographic peak was
tentatively assigned by their visible spectral characteristics in
comparison with standard and retention times according to the
method proposed by Castellanos-Santiago and Yahia.14 Semi-

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Journal of Agricultural and Food Chemistry
Figure 1. Representation of Opuntia f icus-indica extracts obtained from different parts of the fruit of three varieties in the (a*b*) plane (1) and
representation of their L* (lightness) values (2). S, seeds; P, pulp; W, whole fruit; R, red variety; Y, yellow variety; RY, red-yellow variety.
quantification was done on the basis of the mean areas of individual
betalains.
The identity of individual betalains was confirmed by mass
spectrometry. The separation was performed in a Dionex Ultimate
3000RS U-HPLC (Thermo Fisher Scientific, Waltham, MA, USA),
using the column and mobile phase above indicated but with a
postcolumn split of 0.4 mL/min. The mass spectra was obtained using
a microToF-QII high-resolution time-of-flight mass spectrometer
(UHR-ToF) with Q-ToF geometry (Bruker Daltonics, Bremen,
Germany) equipped with an electrospray ionization (ESI) source.
The instrument was operated in positive ion mode using a scan range
from m/z 50 to 1200. Nitrogen was used as the dry gas at a flow rate of
8 mL/min with nebulizing (1.2 bar), and the nebulized temperature
was set at 200 °C. Mass spectra were acquired in MS full scan mode,
and data were used to perform multiple target screening using Target
Analysis 1.2 software (Bruker Daltonics). Instrument control was
performed using Bruker Daltonics HyStar 3.2. The accurate mass data
of the molecular ions were processed through the software Data
Analysis (Bruker Daltonics). The accepted accuracy threshold for
confirmation of elemental compositions has been established at 5 ppm.
Statistical Analysis. Statistical analysis was carried out by using
Statistica version 8.0 software.33 Univariate analyses of variance
(Tukey test and ANOVA) and correlation analysis were applied to
discriminate among the means of chemical data by “variety” and by
“part of the fruit” factors.
■
RESULTS AND DISCUSSION
Colorimetric Characteristics. The color was objectively
calculated by tristimulus colorimetry, and the values of L*,
C*ab, and hab of each sample are shown in Table 1. The color
points represented in the (a*b*) color diagram as well as the
lightness of the sample extracts appear in Figure 1.
8493
Article
The different locations of the diverse parts of Opuntia
varieties could permit the objective establishment of the
chromatic characteristics of the fruits. As can be seen, two
different groups are formed: seeds (S), on the one hand, and
pulp (P) and whole fruit (W), on the other hand. Regardless of
the “variety”, P and W were located in the first quadrant
(positive values of a* and b*) of the (a*−b*) plane, whereas S
was situated between the first and second quadrants (positive
values of b*). It can be noted that, although seed extracts
showed intense yellowish tonalities (values of hue close to 90°),
their chromatic intensity was very low (values of C*ab between
4 and 8) (Table 1). However, P and W had a very intense
orange-yellow color (values of C*ab and hab around 60° and
82°, respectively) (Table 1). Pulp extracts of the different
varieties were clearly separated in the space according to both
b* and a* parameters, whereas the whole fruits were
discriminated only on the basis of b* (Figure 1). Moreover, a
separation “by variety” of these samples was observed (Figure
1). Thus, the red-yellow (RY) variety showed the highest values
of b*, whereas a* is the predominant parameter in the red (R)
variety. With regard to the lightness, seeds and pulp (especially
RP) were noted to occupy opposite positions, keeping the
whole fruit extracts in an intermediate stage.
Among “variety” and “part of the fruit” factors, it was largely
the latter the most influenced color (Table 1). When ANOVA
was applied to the set of data to draw attention to the possible
significant differences according to “variety” and “part of the
fruit” (n = 27 and n = 9, respectively; data not shown), only the
latter factor showed significance. For a better understanding, a
comparison by pairs using the Tukey test was applied (Table
1). Thus, when the “part of the fruit” factor was considered, the
dx.doi.org/10.1021/jf502465g | J. Agric. Food Chem. 2014, 62, 8491−8499
Journal of Agricultural and Food Chemistry
Figure 2. Color variation (ΔE*ab) by pairs in relation to the part of the fruit (a) and variety (b) factors. S, seeds; P, pulp; W, whole fruit; R, red
variety; Y, yellow variety; RY, red-yellow variety. Dotted line represents the value of ΔE*ab appreciable to the human eye.
major significant variations on CIELAB parameters were
assigned to the R variety. Clearly, as could be expected, S
significantly differed from P and W for all Opuntia varieties in
all colorimetric parameters. Moreover, due to the absence of
significance (p > 0.05) in C*ab and hab between P and W
extracts in Y and RY varieties, both pulp and whole fruit for
both varieties could lend the same color to foods and they
could be used indistinctly as natural yellow colorants. However,
the values of chroma in P extracts of R variety were significantly
higher when compared with W, with a considerably strong red
tonality. Seeds could contribute to those differences between W
and P because of their low values of chroma, producing an
attenuation of the color intensity of W (Table 1). Therefore, it
is more suitable to use the pulp of R variety to confer a more
powerful red color.
With regard to the “variety” factor, P was considered the
most influential part of the fruit to discriminate among varieties,
above all regarding chroma and hue. Thus, if pulp extract was
considered to be used as natural colorant, the Y variety would
confer the significantly highest yellow tonalities.34 On the
contrary, if the whole fruit extract was taken into account, the
RY variety should be employed, due to its noticeably higher
chromatic intensity. It was also remarkable that the colorimetric
characteristics were not significantly affected by “variety” when
S extracts were taken into account.
From an industrial point of view, it is easier and economically
advantageous to avoid the time-consuming manipulation that
some technological procedures require, such as the separation
of the seeds from the pulp. Therefore, the whole Y fruit and the
pulp of R varieties are optimal for use as yellow and red
8494
Article
colorants, respectively, whereas the whole RY fruit could be
useful as an additive to confer vivid colors.
Differential Colorimetry. In an attempt to evaluate the
colorimetric differences among “varieties” and “parts of the
fruit”, the mean color difference (ΔE*ab) among samples was
calculated (Figure 2). Taking into account that ΔE*ab of up to
5 CIELAB units indicates “big color differences” appreciable to
the human eye,35,36 it was confirmed that visually perceptible
color differences were observed among the different parts of the
fruit of each variety (Figure 2a). On the one hand, the
significant variations among S/P and S/W for all varieties
previously commented were also appreciable to human eyes.
This fact is predictable because they greatly differ from the
phenolic profile and, consequently, from the color.34 When the
role of each color attribute with respect to Δ2E*ab was
calculated (%Δ2L, %Δ2C, %Δ2H), results evidenced that these
differences were mainly quantitative, with major values of
quadratic variations of chroma (%Δ2C > 90) and practically
negligible values of lightness and hue. In addition, also
noticeable and quantitative color differences were seen for W
and P in R variety, lightness being mainly responsible (%Δ2L >
65). The negligible quadratic percentages of hue demonstrated
that this parameter did not contribute to the establishment of
significant differences of color.
On the other hand, when a comparison among varieties was
carried out (Figure 2b), it was observed that several significant
differences in the pulps were mainly due to chroma (%Δ2C ∼
65 and 87 when Y/R and Y/RY, respectively, were compared).
However, it was lightness that was mainly responsible for the
discrepancy between R/RY varieties (%Δ2L = 68). In the case
of the whole fruit, only quantitative variations contributed to
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Journal of Agricultural and Food Chemistry Article

Figure 3. HPLC chromatogram corresponding to the fraction of betaxanthins and betacyanins of the whole fruit of the red-yellow Opuntia f icus-
indica variety as representative sample: DAD chromatogram at (a) 482 nm and (b) 535 nm and visible spectrum (c, f), EIC chromatogram in
positive mode (d, g), and MS positive mode (e, h) of the major compounds of each kind of betalains, indicaxanthin and betanin, respectively.

Table 2. Molecular Formula, Retention Times, UV−Vis Data, and Mass Spectral Data of Betalains Identified in Opuntia ficus-
indica by HPLC-DAD-ESI-ToF-MS
peak compound molecular formula tr (min) UV−vis maxima (nm) m/z [M + H]+ MS ion
betaxanthins
1 histidine-betaxanthin (muscaarin) C15H16N4O6 5.9 476
2 glutamine-betaxanthin (vulgaxanthin I) C14H17N3O7 10.1 472
3 aminobutyric acid-betaxanthin C13H16N2O6 19.2 462 297.1106 253.1227
4 proline-betaxanthin (indicaxanthin) C14H16N2O6 22.7 479 309.1102 263.1018
5 valine-betaxanthin C14H18N2O6 29.0 472 311.1275 137.0628
6 valine-betaxanthin isomer C14H18N2O6 30.0 471 311.1295 137.0654
7 isoleucine-betaxanthin C15H20N2O6 34.4 472 325.1408 219.1094
8 leucine-betaxanthin (vulgaxanthin IV) C15H20N2O6 34.7 471 325.1369 209.0844
9 phenylalanine-betaxanthin C18H18N2O6 35.0 468
betacyanins
10 betanin C24H26N2O13 26.5 534 551.1554 389.1302
11 isobetanin C24H26N2O13 28.1 534 551.1485 389.1243
12 gomphrenin C24H26N2O13 29.8 535 551.1987 389.1223

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Table 3. Mean Areas and Standard Deviations of Betalain Compounds Belonging to Different Chemical Families (Betacyanins
and Betaxanthins)a
YW RW RYW YP RP RYP
betanin 155.9 ± 24.7 182.4 ± 13.2 226.3 ± 11.5 25.3 ± 1.1 169.1 ± 6.3 216.2 ± 39.8
isobetanin 46.1 ± 15.2 38.4 ± 5.7 55.4 ± 10.6 10.8 ± 2.4 82.5 ± 12.6 45.3 ± 3.1
gomphrenin I 6.5 ± 1.1 8.8 ± 0.6 11.0 ± 0.7 5.4 ± 1.2 8.8 ± 0.7 11.8 ± 1.3
sum of betacyanins 208.5 ± 38.9 229.6 ± 13.5 292.6 ± 21.6 37.9 ± 1.8 260.4 ± 17.9 273.3 ± 39.2

histidine-betaxanthin (muscaarin) 152.0 ± 44.8 90.0 ± 15.8 155.6 ± 42.4 55.0 ± 1.4 50.6 ± 7.1 21.3 ± 1.5
glutamine-betaxanthin (vulgaxanthin) 76.6 ± 5.1 30.5 ± 2.7 48.7 ± 3.1 38.3 ± 9.8 25.4 ± 10.6 11.5 ± 0.8
aminobutyric acid-betaxanthin 86.2 ± 4.8 51.3 ± 1.2 100.2 ± 3.8 61.3 ± 10.1 84.9 ± 12.4 68.6 ± 12.8
proline-betaxanthin (indicaxanthin) 6550.6 ± 336.4 7224.0 ± 617.1 7656.1 ± 277.3 6180.3 ± 279.0 8525.3 ± 297.9 8987.1 ± 502.5
valine-betaxanthin 34.1 ± 3.7 15.4 ± 0.2 43.9 ± 3.3 16.5 ± 0.5 25.9 ± 2.7 22.0 ± 3.8
valine-betaxanthin isomer 30.7 ± 0.5 12.4 ± 1.0 33.0 ± 9.6 14.4 ± 4.0 21.3 ± 3.4 24.8 ± 1.0
isoleucine-betaxanthin 58.5 ± 3.8 36.5 ± 2.1 72.9 ± 1.0 53.3 ± 6.0 54.5 ± 5.0 65.7 ± 8.1
leucine-betaxanthin (vulgaxanthin) 64.8 ± 4.4 44.2 ± 2.4 69.7 ± 2.1 44.9 ± 9.2 46.6 ± 5.6 56.5 ± 8.7
phenylalanine-betaxanthin 32.6 ± 3.0 26.3 ± 0.2 51.1 ± 9.5 13.5 ± 1.3 26.9 ± 4.0 38.4 ± 7.6
sum of betaxanthins 7086.2 ± 397.4 7530.5 ± 614.6 8231.3 ± 259.7 6477.3 ± 279.4 8861.3 ± 336.6 9295.9 ± 524.5

total betalains 7294.7 ± 427.6 7760.1 ± 619.5 8523.9 ± 248.8 6515.2 ± 280.2 9121.7 ± 319.3 9569.2 ± 528.8
a
S, seeds; P, pulp; W, whole fruit; R, red variety; Y, yellow variety; RY, red-yellow variety.

the color differences among RY/Y and R/RY (%Δ2C > 95).
The contribution of quadratic variations of lightness was
negligible. No appreciable color differences among varieties
were found in seeds.
Polyphenolic Content. The values of phenolic compounds
of the different parts of cactus pears are shown in Table 1. The
ANOVA test applied to the whole set of data revealed that no
significant differences were assigned either by “part of the fruit”
or by “variety” (data not shown). However, as was performed
with CIELAB parameters, the Tukey test was applied to
elucidate possible significant variations by pairs. It is highlighted
that the “part of the fruit” factor showed significance for all
three varieties (Table 1). In the case of the Y variety, as
expected, the phenolic content of W was the most
predominant, followed by P and S. Khatabi et al.34 also
reported an abundant amount of polyphenols in the whole fruit
and pulp of Moroccan prickly pears. Furthermore, according to
the “variety” factor, Y variety (both P and S) had the
significantly lowest phenolic content in comparison with the
rest of the varieties. Similar results were also obtained by
Khatabi et al.34 when comparing pulps of yellow and red
varieties of prickly pears. Furthermore, seeds were attributed
minor values of total polyphenols, but, even so, prickly pear
seeds could be considered as an important natural source of
polyphenols with direct application in the food or health
industry.18,19 No variance in the polyphenolic content among
varieties was found, contrarily to that observed by Morales et
al.22 in seeds of diverse species of Opuntia.
Betalain Identification. In this research, several types of
betalains have been identified by HPLC coupled with
electrospray mass spectrometry. The identities of betalains
were achieved on the basis of the retention times, spectra, and
m/z values. Figure 3 shows the chromatographic pattern and
peak assignment of the two families of betalains (betacyanins
and betaxanthin), monitored at 535 and 482 nm, respectively.
As well, the EIC chromatogram and MS positive mode of the
major compounds belonged to each kind of betalains (betanin
and indicaxanthin, respectively) were included. These com-
pounds are summarized in Table 2, which shows the retention
times, UV−vis data, and mass spectra of each betalain identified
8496
of O. f icus-indica by HPLC-DAD-ESI-ToF-MS. Concretely, the
whole fruit of the red-yellow variety has been selected as
representative because of its higher values of areas. Individual
betalains were identified in both pulp and whole fruit. However,
seeds lacked betalains.
A great number of betaxanthins have been identified in this
study. Among them, the presence of indicaxanthin, well-
described in cactus pears, has been confirmed (peak 4,
molecular ion at 309.1102 m/z units, and a daughter ion at
m/z 263.1018) (Table 2; Figure 3). Moreover, the identi-
fication of several additional betaxanthins is highlighted, the
structures of which involved amino acids such as aminobutyric
acid (peak 3, m/z 297.1106 and 253.1227), valine (peak 5, m/z
311.1275 and 137.0628), isovaline (peak 6, m/z 311.1195 and
137.0654), isoleucine (peak 7, m/z 325.1408 and 219.1094),
and leucine (peak 8, m/z 325.1369 and 209.0844) (Table 2).
Other betalains have been tentatively identified on the basis of
UV−vis spectra and the retention times of the chromatographic
method used:14 muscaarin (peak 1, 476 nm), vulgaxanthin
(peak 2, 472 nm), and phenylalanine (peak 9, 468 nm). In
addition, three betacyanin compounds have been correctly
assigned: the well-known betanin (peak 10, m/z 551.1554), its
isomer isobetanin (peak 11, m/z 551.1485), and a minor one,
poorly described, called gomphrenin (peak 12, m/z 551.1987).
The different attachment of the glucose moiety (C-6 and C-5)
caused the different elution times between gomphrenin and
betanin, respectively.14 All betacyanins produced a fragment
near m/z 389.1243 units corresponding to the protonate
aglycones ([betanidin + H]+ or [isobetanidin + H]+) (loss of a
fragment of m/z 162 units corresponding to the glucose
molecule).
Semiquantification of Betalain. Two different wave-
lengths have been monitored to carry out the semi-
quantification of betalains, 535 nm for betacyanins and 482
nm for betaxanthins. It is noteworthy that, although both
betacyanins and betaxanthins were present in all cactus pear
varieties, the mean areas of betaxanthins were, by far,
considerably higher in comparison with that of betacyanins
(Table 3). Among them, indicaxanthin was the major
betaxanthin compound in O. f icus-indica, with an average
dx.doi.org/10.1021/jf502465g | J. Agric. Food Chem. 2014, 62, 8491−8499
Journal of Agricultural and Food Chemistry Article

Table 4. Influence of the Tested Factors (Part of the Fruit and Variety) on Betalain Compounds As Assessed by Univariate
Statistical Analysisa
by part of fruit by variety
Y R RY W P
W/P W/P W/P Y/R Y/RY R/RY Y/R Y/RY R/RY
betanin * * * * *
isobetanin * * * * *
gomphrenin I * * * * * *
sum of betacyanins * * * * *

histidine-betaxanthin (muscaarin) * * * * *
hlutamine-betaxanthin (vulgaxanthin) * * * * * *
aminobutyric acid-betaxanthin * * * * * *
proline-betaxanthin (indicaxanthin) * * * * *
valine-betaxanthin * * * * * * *
valine-betaxanthin isomer * * * * *
isoleucine-betaxanthin * * * *
leucine-betaxanthin (vulgaxanthin) * * *
phenylalanine-betaxanthin * * * * * *
sum of betaxanthins * * * * * *

total betalains * * * * * *
a
Asterisks denote significant differences by pairs according to Tukey test (p < 0.05). S, seeds; P, pulp; W, whole fruit; R, red variety; Y, yellow
variety; RY, red-yellow variety.

percentage of area around 90%. Similar results were reported in
the pulps of different Mexican and Moroccan prickly pear
cultivars.14,34 Muscaarin took second place, with 2% of the total
mean area of betalains, followed by the aminobutyric acid
moiety betaxanthin (around 1%). With regard to betacyanins,
betanin was the dominate compound in O. f icus-indica, but
represents only 2% of the total area of betalains.12 The rest of
the identified betaxanthins and betacyanins were found in an
area percentage below 1%.
When ANOVA was applied to the set of data, and similarly
to that described for the color of extracts, the “part of the fruit”
was largely the most influential factor of significant variance in
virtually all betalain compounds (data not shown). In fact, all
individual betalains were highly (p < 0.05) correlated with
chromatic characteristics (positive for L* and hab, and negative
for C*ab). Concretely, C*ab and hab were the chromatic
parameters more correlated with betalains (Pearson correlation
coefficients: C*ab, 0.8500; hab, −0.7000; L*, −0.5500), so the
chromatic intensity and the tonality of the samples were
directly related with the content of betalains. The “variety”
factor, however, practically did not affect this kind of
compound. To test the diversity among pairs of samples,
statistical main differences (Tukey test) on the main areas of
betalain compounds were scrutinized (Table 4). As can be seen,
the major considerable differences on betacyanins among W
and P were ascribed to the Y variety, with remarkable areas in
the whole fruit. With regard to betaxanthins, pulps of R and RY
varieties had main areas in plenty in comparison with that
observed in the whole fruit. These results were in concordance
with the phenolic content previously commented. In fact,
individual betalains and phenolic content were positively and
With regard to the “variety” factor, when the whole fruit was
considered, RY significantly held the first position of total area
of individual betacyanins and betaxanthins, whereas Y and R
were at the tail end (p < 0.05) (Table 3). However, different
behaviors followed the pulp extracts, with significantly lower
values of Y variety and without variance among R and RY
varieties. That pattern was in concordance with the
aforementioned content of total polyphenols. In short, the Y
variety showed the significantly lowest areas of betalains, the
pulp of the RY variety being the one that adopted the greater
values.
In summary, it might be concluded that O. f icus-indica could
be an important choice for use as a natural colorant and source
of phenolic and betalain compounds. The choice of the best
part of cactus pears with that objective has been directed not
only toward the highest values of polyphenols and betalains but
also to the best color properties. Thus, the whole grain of
yellow cactus pear could be more suitable as yellow colorant,
whereas it could be more appropriate to use the pulp of the red
variety to obtain a more reddish colorant. Finally, also the pulp
of red-yellow prickly pear is optimal as a complement due to its
high colorant intensity. Moreover, regardless of the variety,
Opuntia seeds could be used as an important source of
polyphenols. In addition, the possibility of the presence of
essential amino acids in the structure of betaxanthins makes
possible the use of O. f icus-indica as an important essential
dietary colorant or as a diet fortification with essential amino
acids. This study could be a great step forward in the food,
cosmetics, and drug industries, among others.

■
highly correlated (p < 0.05), with Pearson correlation
coefficients >0.7000 in the majority of individual betalains. AUTHOR INFORMATION
Moreover, although Y was the variety with most differences on
individual betaxanthins (p < 0.05), muscaarin, aminobutyric Corresponding Author
acid, and valine moiety compounds were the betalains with *(F.J.H.) Phone: +34 954556495. Fax: +34 954556110. E-mail:
significance in all three prickly pears. heredia@us.es.
8497 dx.doi.org/10.1021/jf502465g | J. Agric. Food Chem. 2014, 62, 8491−8499
Journal of Agricultural and Food Chemistry
Funding
We are indebted to Consejeriá de Economia,́ Innovación y
Ciencia, Junta de Andalucı ́a, Spain (Projects P11-AGR-7843
and P11-RNM-7887), and Ministerio de Ciencia y Tecnologia,́
Spain (Projects Plan Nacional I+D CTM2011-29930) for
financial support. We are also grateful to the Algerian Ministry
of Higher Education and Scientific Research for the award of a
grant.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank the technical staff of Biology Service (SGI,
Universidad de Sevilla) and Instituto de Biomedicina de Sevilla
(IBiS, Universidad de Sevilla) for technical assistance. We also
thank Marı ́a Gil Vicente for her support in the analytical assays.
■
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