Betalain Profile, Phenolic Content, and Color ... - ACS Publications

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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-ESIToF-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

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(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 icusindica [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

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 advantageous 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 © 2014 American Chemical Society

Received: Revised: Accepted: Published: 8491

May 23, 2014 July 23, 2014 July 30, 2014 July 30, 2014 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 L* YS YW YP RS RW RP RYS RYW RYP

97.7 88.7 84.8 95.7 88.7 80.8 94.9 89.3 88.9

± ± ± ± ± ± ± ± ±

C*ab 0.7 0.9 7.8 2.8 1.4 1.7 3.5 0.2 0.9

4.4 57.1 54.5 6.2 54.9 61.1 7.7 62.5 66.5

± ± ± ± ± ± ± ± ±

hab 0.6 3.2 0.4 2.1 2.1 0.8 2.9 3.7 0.1

92.5 84.7 85.2 90.9 83.4 81.8 88.7 83.7 83.1

± ± ± ± ± ± ± ± ±

TP 1.8 0.1 1.1 4.1 0.1 0.3 2.6 0.1 0.4

3.8 13.3 8.1 4.1 6.6 11.8 4.0 8.4 12.9

± ± ± ± ± ± ± ± ±

0.5 1.1 0.5 0.4 1.5 1.3 0.1 1.0 1.4

Y

R

RY

S/W S/P W/P S/W S/P W/P S/W S/P W/P

by variety

L*

C*ab

hab

TP

* *

* *

* *

* * *

* * * * *

* * * * *

* * * * * * * * *

*

L* S

W

P

Y/R Y/RY R/RY Y/R Y/RY R/RY Y/R Y/RY R/RY

*

C*ab

* * * *

hab

TP

* *

* *

* * *

* *

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. length glass cells and distilled water as reference. The CIELAB parameters (L*, a*, b*, C*ab, and hab) were determined by using the original software CromaLab27 following the Commission Internationale 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 spectrophotometrically determined total polyphenol values. HPLC-DAD-ESI-ToF-MS Analysis of Betalains. HPLC separation, 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-

its practical application as a natural colorant by deepening the study of differential colorimetry and betalain profile.

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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 SigmaAldrich (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 icusindica [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 8492

dx.doi.org/10.1021/jf502465g | J. Agric. Food Chem. 2014, 62, 8491−8499


Article

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.

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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

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

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.

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

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

Figure 3. HPLC chromatogram corresponding to the fraction of betaxanthins and betacyanins of the whole fruit of the red-yellow Opuntia f icusindica 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 ficusindica by HPLC-DAD-ESI-ToF-MS peak 1 2 3 4 5 6 7 8 9 10 11 12

compound betaxanthins histidine-betaxanthin (muscaarin) glutamine-betaxanthin (vulgaxanthin I) aminobutyric acid-betaxanthin proline-betaxanthin (indicaxanthin) valine-betaxanthin valine-betaxanthin isomer isoleucine-betaxanthin leucine-betaxanthin (vulgaxanthin IV) phenylalanine-betaxanthin betacyanins betanin isobetanin gomphrenin

molecular formula

tr (min)

UV−vis maxima (nm)

C15H16N4O6 C14H17N3O7 C13H16N2O6 C14H16N2O6 C14H18N2O6 C14H18N2O6 C15H20N2O6 C15H20N2O6 C18H18N2O6

5.9 10.1 19.2 22.7 29.0 30.0 34.4 34.7 35.0

476 472 462 479 472 471 472 471 468

C24H26N2O13 C24H26N2O13 C24H26N2O13

26.5 28.1 29.8

534 534 535

8495

m/z [M + H]+

MS ion

297.1106 309.1102 311.1275 311.1295 325.1408 325.1369

253.1227 263.1018 137.0628 137.0654 219.1094 209.0844

551.1554 551.1485 551.1987

389.1302 389.1243 389.1223



Article

Table 3. Mean Areas and Standard Deviations of Betalain Compounds Belonging to Different Chemical Families (Betacyanins and Betaxanthins)a YW

a

RW

RYW

155.9 46.1 6.5 208.5

± ± ± ±

24.7 15.2 1.1 38.9

182.4 38.4 8.8 229.6

± ± ± ±

13.2 5.7 0.6 13.5

226.3 55.4 11.0 292.6

± ± ± ±

11.5 10.6 0.7 21.6

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

152.0 76.6 86.2 6550.6 34.1 30.7 58.5 64.8 32.6 7086.2

± ± ± ± ± ± ± ± ± ±

44.8 5.1 4.8 336.4 3.7 0.5 3.8 4.4 3.0 397.4

90.0 30.5 51.3 7224.0 15.4 12.4 36.5 44.2 26.3 7530.5

± ± ± ± ± ± ± ± ± ±

15.8 2.7 1.2 617.1 0.2 1.0 2.1 2.4 0.2 614.6

155.6 48.7 100.2 7656.1 43.9 33.0 72.9 69.7 51.1 8231.3

± ± ± ± ± ± ± ± ± ±

42.4 3.1 3.8 277.3 3.3 9.6 1.0 2.1 9.5 259.7

total betalains

7294.7 ± 427.6

betanin isobetanin gomphrenin I sum of betacyanins

7760.1 ± 619.5

8523.9 ± 248.8

YP

RP

25.3 10.8 5.4 37.9

± ± ± ±

1.1 2.4 1.2 1.8

55.0 38.3 61.3 6180.3 16.5 14.4 53.3 44.9 13.5 6477.3

± ± ± ± ± ± ± ± ± ±

1.4 9.8 10.1 279.0 0.5 4.0 6.0 9.2 1.3 279.4

6515.2 ± 280.2

RYP

169.1 82.5 8.8 260.4

± ± ± ±

6.3 12.6 0.7 17.9

216.2 45.3 11.8 273.3

± ± ± ±

39.8 3.1 1.3 39.2

50.6 25.4 84.9 8525.3 25.9 21.3 54.5 46.6 26.9 8861.3

± ± ± ± ± ± ± ± ± ±

7.1 10.6 12.4 297.9 2.7 3.4 5.0 5.6 4.0 336.6

21.3 11.5 68.6 8987.1 22.0 24.8 65.7 56.5 38.4 9295.9

± ± ± ± ± ± ± ± ± ±

1.5 0.8 12.8 502.5 3.8 1.0 8.1 8.7 7.6 524.5

9121.7 ± 319.3

9569.2 ± 528.8

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 compounds are summarized in Table 2, which shows the retention times, UV−vis data, and mass spectra of each betalain identified

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, welldescribed 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 identification 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 wavelengths have been monitored to carry out the semiquantification 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 8496



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

W/P

W/P

W/P

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

*

*

*

*

Y/R

R/RY

Y/R

Y/RY

R/RY

*

*

*

* *

* *

* * * *

* * * *

* *

* *

* * * *

* *

*

* *

* * * * * *

* * * * *

P

Y/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.

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.

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 highly correlated (p < 0.05), with Pearson correlation coefficients >0.7000 in the majority of individual betalains. Moreover, although Y was the variety with most differences on individual betaxanthins (p < 0.05), muscaarin, aminobutyric acid, and valine moiety compounds were the betalains with significance in all three prickly pears.

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Corresponding Author

*(F.J.H.) Phone: +34 954556495. Fax: +34 954556110. E-mail: [email protected]. 8497



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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.

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ACKNOWLEDGMENTS

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REFERENCES

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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