Ital. J. Food Sci., vol. 30, 2018 - 614 PAPER JUICES OF PRICKLY PEAR FRUITS (OPUNTIA SPP.) AS FUNCTIONAL FOODS G. ZENTENO-RAMÍREZ1, B.I. JUÁREZ-FLORES1, J.R. AGUIRRE-RIVERA*1, M. MONREAL-MONTES1, J. MÉRIDA GARCÍA2, M. PÉREZ SERRATOSA2, M.Á. VARO SANTOS2, M.D. ORTIZ PÉREZ3 and J.A. RENDÓN-HUERTA4 1Instituto de Investigación de Zonas Desérticas, Universidad Autónoma de San Luis Potosí, Altair 200, Colonia Del Llano, 78377 San Luis Potosí, Mexico 2Departamento de Química Agrícola y Edafología, Universidad de Córdoba, España 3Facultad de Medicina, Universidad Autónoma de San Luis Potosí, Avenida Venustiano Carranza 2045, 78210. San Luis Potosí, Mexico 4Coordinación Académica Región Altiplano Oeste, Universidad Autónoma de San Luis Potosí, Avenida Insurgentes esquina Himno Nacional S/N, 78600, Salinas de Hidalgo, San Luis Potosí, Mexico *Corresponding author: Tel.: +52 4448422359; Fax: +52 444842435 E-mail address: iizd@uaslp.mx ABSTRACT The prickly pear (Opuntia spp.) usually is consumed as fresh fruit. In this study of prickly pear juice in vitro we characterized and quantified secondary metabolites including antioxidant capacity of ten Opuntia spp. variants. Gallic acid was abundant in most variants. Catechin and epicatechin isomers, and procyanidins B1 and B2 were present in most variants. Ascorbic acid content was higher than 84 mg. Betacyanins stand out in red- colored juices, betaxanthins in the yellow ones; this caused lack of relationship between antioxidant capacity and total phenolic content. The soluble fiber content, sugars, betalains and ascorbic acid position this juice as a functional food. Keywords: ABTS, antioxidant, betalains, FRAP, juice, prickly pears Ital. J. Food Sci., vol. 30, 2018 - 615 1. INTRODUCTION Normal metabolic processes produce free radicals, which cause oxidative damage. The human body possesses endogenous antioxidant mechanisms using substances that significantly delay or prevent oxidation. These substances include the cellular enzymes superoxide dismutase, glutathione peroxidase and catalase (WANG and QUINN, 2000). There are also non-enzymatic defense substances against oxidation stress, including vitamin E, an effective antioxidant of polyunsaturated membrane lipids, and vitamin C which, as a reducing agent or electron donor, reacts rapidly with the HO- and the superoxide anion and also prevents the oxidation of membrane lipids (WANG and QUINN, 2000). When endogenous antioxidant mechanisms are insufficient to offset the imbalance resulting from oxidative stress, physiological and biochemical changes take place such as protein glycosylation, lipid peroxidation, and glucose auto-oxidation (OPERA, 2004). Diseases associated with oxidative stress include Type-2 diabetes mellitus (DM2), hypertension, renal and hepatic impairment, cancer, and neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's, Parkinson's and Huntington (JELLINGER, 2003; D’AMICO et al., 2013). The intake of natural or synthetic antioxidants can reinforce the antioxidant capacity of the organism (HIDALGO et al., 2006). Recent research has shown that certain compounds present in plants, such as terpenes, flavonoids, betalains and anthocyanins, possess antioxidant properties that are more powerful than those of vitamins (HARASYM and OLEDZKI, 2014). Global trends in food and nutrition indicate a growing interest in the consumption of fruits and vegetables, given their nutritional value and benefits for the functions of the human body. These trends in eating patterns have led to a new area of research and development in nutrition related to the so-called "functional foods", defined as any food, either natural or processed, which in addition to its nutritional components contains substances that boost a person’s health, physical ability and mental state (KONIGSBERG- FAINSTEIN, 2008). Functional compounds include exogenous antioxidants, which safely interact with free radicals and disrupt their chain reaction before they damage vital molecules (OROIAN and ESCRICHE, 2015). The prickly pear, the fruit of cacti of the genus Opuntia is widely available throughout Mexico’s South Highland. More than 189 species of wild prickly pear cacti are known, 83 of which are Mexican; of these, 29 are distributed in the north-central region of Mexico, in an area of approximately 300 000 km2 that stretches across part of the states of Aguascalientes, Guanajuato, Hidalgo, Jalisco, Queretaro, San Luis Potosí, Zacatecas, and around México City. In this area, a number of variants with different degrees of humanization can be found, from the wild O. streptacantha and the cultivated O. hyptiacantha, O. megacantha and O. albicarpa, to O. ficus-indica, the species considered as the one with the highest degree of domestication (REYES-AGÜERO et al., 2005). Prickly pears are consumed mainly as fresh fruit and display marked differences in size, shape, color and flavor, as well as in seed quantity, size and hardness; prickly pear is also processed to produce jelly, jam and paste. Chemical compounds found in prickly pear include polyphenols and betalains (FIGUEROA-CARES et al., 2010; YEDDES et al., 2013). These antioxidant metabolites either prevent or control the excessive production of highly unstable free radicals and reactive molecules that have the ability to disrupt the functions of various biomolecules, i.e., oxidative stress (RODRÍGUEZ et al., 2001; SOOBRATTEE et al., 2005). Evaluations of the prickly pear fruit indicate its potential to be considered as a functional food due to its content of ascorbic acid, phenols, carotenoids and betalains at levels that Ital. J. Food Sci., vol. 30, 2018 - 616 exceed those in plums, nectarines or peaches (FERNÁNDEZ-LÓPEZ et al., 2010). These phytochemicals may contribute to mitigation of the effects of prolonged hyperglycemia and reinforcement of the antioxidant system in normal glycemic patients. Antioxidants have been shown to increase the sensitivity of insulin receptors or may moderate the rise in blood glucose concentration after the ingestion of carbohydrates by inhibiting the action of digestive enzymes and glucose transporters SGLT-1 (BRYANS et al., 2007). In addition, these phytochemicals have been associated with anti-inflammatory, antioxidant, immunomodulatory and apoptotic properties (KAULMANN and BOHN, 2016). Phytochemicals, which locally reduce oxidative stress, are widely studied as cancer- protective agents (MOORE et al., 2016). Indeed, animal assays indicate that supplementation with green and black tea (rich in polyphenolic compounds) led to a decrease in postprandial blood glucose levels in Sprague-Dawley rats (ZEYUAN et al., 1998). Furthermore, in vivo studies showed a drop in glycosylated hemoglobin (FUKINO et al., 2008) and increased insulin activity after consumption of tea extracts (RICHARDA and DOLANSKY, 2002). Based on the above, the objective of this study was to supplement existing assessments of prickly pear juice as a functional food by identifying and quantifying antioxidant compounds in the juice of fruits of Opuntia and to investigate their antioxidant capacity in vitro. 2. MATERIALS AND METHODS 2.1. Selection of variants and sample preparation Ten prickly pear variants, six of them cultivated, were evaluated as ripe fruits: Rojo Pelón (Opuntia ficus-indica), Blanca (O. albicarpa), Amarilla Monteza, Pico Chulo, Torreoja and Sangre de Toro (O. megacantha), and four wild variants: Cardona (O. streptacantha), Charola (O. streptacantha ssp. aguirrana), Tapona and Tapón Rojo (O. robusta). Fruits were collected in the municipality of Villa de Arriaga, state of San Luis Potosí, México. Opuntia variants were selected based on: (a) degree of humanization, (b) abundance and economic potential in the state of San Luis Potosí, and (c) fruit color. The skin of prickly pears was removed, then the juice was extracted from the pulp with a stainless-steel blender (International LI-12-106), and seeds were separated with an 8 mesh filter; the juice was stored in sterile containers at -20°C until use. 2.2. Total phenolic compounds Total phenolic compounds in prickly pear juice was quantified using the Folin-Ciocalteu method modified by YEDDES et al. (2013), and expressed as gallic acid equivalents (mg GAE g-1). To extract phenols, cool absolute ethanol was added to 0.15 g of lyophilized juice stored at -50°C (Frezer dryers IIshin, Corea), the mixture was sonicated for 10 min and then maintained under constant stirring for 2 h at 4°C. The solution was filtered through Whatman Grade 42 filter paper. Extracts were brought to 15 mL with ethanol and stored protected from light at -20°C. Total phenols were measured in triplicate; to this end, 437.5 µL of 1N Folin-Ciocalteu reagent (Sigma) were added to 35 µL of the ethanol extract and were left to react at room temperature for 3 min. Afterwards, 2187.5 µL of a 20% Na2CO3 solution were added and the volume was brought to 3500 µL. The mixture was left to stand at room temperature in the dark for 2 h for the development of color. Absorbance was read at 760 nm in a spectrophotometer (Agilent Technologies, Germany), using blank samples made of distilled water and the reagents used. The amount of phenolic Ital. J. Food Sci., vol. 30, 2018 - 617 compounds was estimated by comparing the absorbance values of samples with those of the gallic acid standards. 2.3. Phenolic acids and flavan-3-ols Phenolic compounds were extracted with the method used by RODARTE et al. (2007). Two grams of lyophilized prickly pear juice were mixed with 3 mL of acidified methanol (0.1% hydrochloric acid), and sonicated in a water bath for 10 min; then, the supernatant was collected and the previous procedure was repeated five times with the precipitate to obtain six extractions. Supernatants were collected and centrifuged for 10 min at 5000 rpm; the centrifuged fraction was concentrated under vacuum on a rotary evaporator at 30°C (Heidolph, Alemania) followed by reconstitution with 2 mL of methanol. All samples were filtered through 0.45 µm nylon filters. Phenolic acids were identified and quantified in a liquid chromatograph (Spectra-Physics UV6000LP), with a LiChrospher® 100 RP-18 column (250 mm x 4.6 mm, 5 μm particle size). Formic acid (10% in water) (A) and acetonitrile/water/formic acid (45:45:10) (B) were used as mobile phase, with a flow rate of 1 mL/min. The identification was made by comparing the retention times and UV-Vis spectra obtained using a diode array detection system (Thermo Scientific, USA), with reference standards. Hydroxybenzoic acids were quantified at 280 nm; hydroxycinnamic acid esters, at 315 nm. Flavan-3-ols were identified and quantified in a liquid chromatograph (Thermo Spectra Physic Series P100, USA), coupled to a fluorescence detector (Perkin Elmer Series 200ª, USA), with a LiChrospher® 100 RP-18 column (250 mm x 4.6 mm and 5 μm particle size). Acetonitrile (A) and acetic acid (B) were used as mobile phase, with a flow rate of 1.4 mL/min. Flavanols were identified using the wavelengths λexc = 280 nm and λem = 320 nm. In this study, procyanidins were quantified as catechins. 2.4. Identification and quantification of betalains Betalains were measured using the method of CASTELLANOS-SANTIAGO and YAHIA (2008). To do this, 100 mg of lyophilized prickly pear juice were weighed and 10 mL of 80% methanol acidified with 0.5% HCl were added; this mixture was sonicated for 15 min and filtered through a 0.45 µm nylon filter (Agilent Technologies, Alemania). An electronic scan was run between 400 and 700 nm in an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Alemania), which identified the absorption peaks of betalains at 547 nm and 490 nm. Absorbance units were converted to concentration units. 2.5. Ascorbic acid quantification Ascorbic acid was quantified using the method of SDIRI et al. (2012). To this end, 2 mL of 4.5% meta-phosphoric acid were added to 0.1 g of lyophilized juice; this mixture was sonicated in a water bath for 2 min, then centrifuged for 10 min at 5000 rpm, and finally filtered through 0.45 µm nylon filters. Ascorbic acid was quantified in a HPLC chromatograph (Thermo Spectra Physic Series P100), coupled to a UV detector (Thermo Finnigan Spectra System UV2000), with a LiChrospher® 100 RP-18 column (250 mm x 4.6 mm and 5 μm particle size), and KH2PO4 (0.2 M at pH=2.3-2.4) was used as mobile phase, with a flow rate of 1.0 mL/min for 15 min at λ = 243 nm and an injection volume of 20 µL. This compound was estimated using the following calibration equation y = 76165x - 161251, r2= 0.9993. Ital. J. Food Sci., vol. 30, 2018 - 618 2.6. FRAP (ferric reducing antioxidant power) method The FRAP assay assessed the capacity of juice samples to reduce the ferric ion (Fe+3) in a complex with 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), to the ferrous ion (Fe+2), in accordance with FIRUZI et al. (2005). The FRAP reagent was prepared daily by mixing 10 mL of 300 mM sodium acetate buffer solution (pH 3.6) with 1 mL of 20 mM ferric chloride hexahydrate and 1 mL of 10 mM TPTZ dissolved in 40 mM hydrochloric acid. Twenty five microliters of prickly pear juice diluted 1/20 with methanol were added to 96-well flat bottom microplates in triplicate, followed by the addition of 175 µL FRAP solution. A control treatment was prepared with 200 µL methanol; another control was prepared by mixing 25 µL methanol with 175 µL FRAP; for a third control, 25 µL ferric sulphate and 175 µL sodium acetate buffer were added; finally, 25 µL of juice sample were added to 175 µL sodium acetate buffer solution. Readings were recorded with a Multiskan Ascent reader (Thermo Electron Corporation 100-240 VAC Type: 354) at 595 nm. The first reading was taken at time 0, and the plate was incubated at 37°C immediately afterwards. The second reading was taken after 60 min. The calibration curve was constructed with ferric sulphate heptahydrate (7.194 mM) dissolved in methanol at concentrations of 108 µM to 864 µM. The resulting calibration equation was y = 0.0011x - 0.069, r2= 0.9971. The FRAP value for the curve was calculated according to the following equation: 𝐹𝑅𝐴𝑃 𝑀 = ∆𝑎!𝐹𝐼 ∆𝑎!𝐹𝑒!! 𝑥10!! Where ∆𝑎!𝐹𝐼 = change in absorbance of the analyte after the time interval. ∆𝑎!𝐹𝑒!!= change in absorbance of iron sulfate at the same concentration and after the time interval. The results of each sample are expressed as µM FeSO4 eq. 2.7. Estimate of the trolox equivalent antioxidant capacity (TEAC) with the chemical mediator ABTS•⁺ This estimate was made in accordance with the methodology of NENADIS et al. (2004). The TEAC of samples was based on 2,2'-azino-bis(3-ethylenebenzothiazoline-6-sulfonic acid) (ABTS), which produces the radical ABTS•⁺ and is compared with an antioxidant (trolox). For each evaluation, the ABTS•⁺ solution was prepared by mixing 5 ml of 7 mM ABTS and 88 µL of 140 mM potassium persulphate; the mixture was stored in the dark covered with aluminum foil and left to stand for 12 h at room temperature to produce the radical; afterwards, 500 µL of the solution were mixed with 25 mL of ethanol and its absorbance was read in an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Alemania) to confirm that it was between 0.7 and 1 at 734 nm. Samples and controls were measured in triplicate (20 µL sample plus 230 µL ABTS•⁺) and placed in a 96-well flat bottom plate; after adding the radical ion, the plate was covered with aluminum foil and after 6 min the reading was recorded at 734 nm in a Multiskan Ascent Reader (Thermo Electron Corporation 100-240 VAC Tip: 354). The percent inhibition of the standard was obtained with the following equation: Ital. J. Food Sci., vol. 30, 2018 - 619 % inhibition = !"# !"#$%"&!!"# !"#$%& !"# 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 ∗ 100 The absorbance of the sample was subtracted from the absorbance of the control to obtain the true absorbance. The calibration curve was constructed with 50 µM, 100 µM, 200 µM, 300 µM, 400 µM and 500 µM trolox standards, adding 20 µL of the standard solution and 230 µL ABTS•⁺; the resulting regression equation was y = 0.2263x + 7.5033; r2= 0.9979. The results were expressed as µg·mol trolox equivalent (TE) per gram of juice (dry weight). 2.8. Experimental design and statistical analysis The experiment was performed according to a completely randomized experimental design. Treatments were the juices from the 10 prickly pear variants, which were tested for content of phenolic compounds, betalains and ascorbic acid, as well as their antioxidant capacity through FRAP and ABTS. Three replicates were used for each of these measurements. The data were subjected to an analysis of variance and Tukey’s multiple comparison test. A Pearson correlation was carried out between FRAP and ABTS variables (SAS, version 8.0; SAS Institute, Cary, North Carolina). 3. RESULTS AND DISCUSSION The potential of these prickly pear juices as functional foods is high due to outstanding content of soluble fiber and the adequate content and proportion of glucose to fructose (ZENTENO-RAMÍREZ et al., 2015). In order to determine whether consumption of prickly pear juices can help prevent or cure diseases associated with the excess of free radicals it is essential to identify and quantify the compounds with antioxidant capacity contained in prickly pear juice. Phenols in fruits, flowers and vegetables have attracted the attention due to their antioxidant potential. It has been shown that various parts of Opuntia (pulp, fruit skin, seeds and cladodes) are rich in polyphenols (GALATI et al., 2003; VALENTE et al., 2010; TOUNSI-SAIDANI et al., 2011). In addition, various studies have demonstrated their antioxidant effects (DOK-GO et al., 2003; TESORIERE et al., 2004; SIRIWARDHANA and JEON, 2004; OSORIO-ESQUIVEL et al., 2011). 3.1. Total phenol content Due to their ubiquitous presence in plant foods, phenolic compounds are normally included in the daily human diet. The daily intake ranges between 25 mg and 1 g, depending on the amount of fruits, vegetables, pulses, tea and spices consumed (HAGERMAN et al., 1998). Raw extracts of phenol-rich plant products are attracting interest in the food industry, since these slow down the oxidative degradation of lipids, and hence improve the quality and nutritional value of food; their antioxidant power protects against heart disease and cancer, in addition to other chronic degenerative diseases (KÄKHÖNEN et al., 1999). The protection against LDL oxidation is not due to a single compound, but results from the effect of several phenolic compounds (RICCHELLE et al., 2001). The total content of polyphenols was estimated in the ethanol extracts of lyophilized juice samples (Table 1). The statistical differences between prickly pear variants seem to be unrelated to fruit color and degree of humanization, and it should be noted that the four Ital. J. Food Sci., vol. 30, 2018 - 620 O. megacantha variants evaluated showed the highest total content of phenolic compounds. Among the variants evaluated by MABROUKI et al. (2015), the highest concentration was observed in the pulp of O. streptacantha, followed by O. ficus-indica, with 104.6 GAE per 100 g of juice. In general, it has been pointed out that the concentration of phenolic compounds in prickly pears range from 54 mg/100 g to 104 mg/100 g fresh weight (KATABI el al., 2013; FIGUEROA-CARES, et al., 2010). Thus, the concentration of phenolic compounds in prickly pear juice is similar or higher than in pineapple, tomato, banana, mango and cucumber (1.7, 2.0, 2.3, 2.6, and 3.8, all in mg/g dry weight, respectively) (MUÑÓZ-JÁUREGUI and RAMOS-ESCUDERO, 2007). 3.2. Quantification of phenols by high performance liquid chromatography (HPLC) Table 1 shows the concentration of phenolic acids in the studied prickly pear variants, which show significant differences (P < 0.0001). Gallic acid was recorded in all variants except Tapona, and was the main phenolic compound in most of them, with varying concentrations between 32.6 µg/g and 81.2 µg/g. Syringic acid was absent only in Torreoja and Cardona, and ellagic acid in Blanca, Sangre de Toro and Tapón Rojo. Protocatechic acid was recorded only in Pico Chulo (41.6 µg/g). Pico Chulo showed the four phenolic acids and recorded the highest total phenolic acid concentration (176 µg/g). By contrast, Blanca showed the lowest total phenolic acid content (79.4 µg/g). Table 1. Average concentration (µg/g) of total phenols and phenolic acids in lyophilized juices of 10 prickly pear variants. Variant* Total phenols Gallic acid Syringic acid Ellagic acid Total phenolics acids Rojo Pelón 1.92±0.11de 32.6±0.6g 29.2±0.9d 25.0±0.9e 86.9±2.3e Blanca 1.93±0.25de 53.7±0.6e 25.6±0.4e n.d. 79.4±0.8e Amarilla Monteza 3.81±0.75bc 74.8±3.6bc 13.6±0.3h 33.5±0.1d 122.0±3.8b Pico Chulo 2.81±0.39bcd 63.6±0.6d 20.0±2.7f 50.5±2.3b 176±7.9a Torreoja 3.90±0.06ab 49.7±1.8e n.d. 41.9±1.9c 91.6±3.6d Sangre de Toro 5.21±0.83a 42.4±0.5 f 66.5±0.1a n.d. 109±1.2c Cardona 1.67±0.36de 81.2±0.7a n.d. 26.7±0.4e 108.0±1.0c Charola 1.69±0.48de 78.3±1.0ab 16.9±0.3 g 73.2±1.5a 168±2.2a Tapona 2.52±0.42cde n.d. 45.3±1.1b 68.3±4.1a 114.0±5.2bc Tapón Rojo 1.45±0.14e 71.5±0.1c 38.4±0.3c n.d. 110.0±1.2c P value ˂0.0001 <0.0001 <0.0001 <0.0001 <0.0001 *Variants are sorted from highest to lowest degree of humanization. n = 3. Treatments with different letters in the same column are statistically different (<0.05). n.d.= not detected Flavonoids are the dominant class of phenols in food, accounting for approximately two thirds of the phenols consumed in the human diet (LOTITO and FREI, 2006). Table 2 shows the concentrations of the flavan-3-ol derivatives found in the juice of all prickly pear variants studied, with significant differences (P < 0.0001) between them. These four derivatives were recorded in the juice of all variants; however, catechin was not found in Blanca, being the derivative found at the lowest concentration in all variants except Charola, where epicatechin attained the lowest concentration. The derivative registered at the highest concentration in these prickly pear juices was either epicatechin or procyanidin Ital. J. Food Sci., vol. 30, 2018 - 621 B2, according to the variant. The highest epicatechin concentrations were found in Tapona juice, and the lowest in Cardona, with 90.8 µg/g and 17.2 µg/g, respectively. With regard to the total content of flavan-3-ol derivatives, the Tapona juice showed the highest concentration (223±6.09 µg/g), and also the highest levels of each individual derivative; in contrast, the Cardona juice showed the lowest concentration of these derivatives (73.7 µg/g). Table 2. Average concentration (µg/g) of flavan-3-oles in lyophilized juices of 10 prickly pear variants. Species Variant* Catechin Epicatechin Procyanidin B1 Procyanidin B2 Total flavan- 3-oles O. ficus-indica Rojo Pelón 13.30±0.63de 19.26±1.68f 16.71±0.13f 28.36±0.38e 77.6±1.30f O. albicarpa Blanca n.d. 60.94±1.26b 32.50±1.49d 38.76±0.43c 132±3.18c O. megacantha Amarilla Monteza 14.23±0.69 cd 37.4±0.16c 21.03±0.74ef 33.93±0.55d 107±4.82d Pico Chulo 14.87±0.6cd 24.58±0.02e 23.59±1.25e 29.51±1.19de 92.5±1.86e Torreoja 19.61±0.94b 32.10±0.39d 25.40±0.87e 20.37±0.40f 97.5±1.80de Sangre de Toro 19.61±1.60 b 61.93±0.77b 40.67±1.40c 51.62±1.31a 174±1.88b O. streptacantha Cardona 10.44±0.18e 17.15±0.45f 22.41±0.51e 23.68±1.08f 73.7±2.21f O. streptacantha ssp. aguirrana Charola 27.25±0.95 a 17.97±0.98f 47.06±2.19b 44.45±0.28b 137±4.40c O. robusta Tapona 27.89±0.97a 90.81±2.18a 59.47±0.90a 45.20±2.05b 223±6.09a Tapón Rojo 17.56±1.14bc 19.16±0.30f 24.67±1.56e 23.63±1.81f 85.1±4.21ef P value ˂0.0001 ˂0.0001 ˂0.0001 ˂0.0001 ˂0.0001 *Variants are sorted from highest to lowest degree of humanization. n = 3. Treatments with different letters in the same column are statistically different (<0.05). n.d.= not detected 3.3. Concentration and identification of betalains According to STINTZING et al. (2005), prickly pear color is due to betalains, since these authors recorded indicaxanthin and betaxanthins (84 mg/kg and 100 mg/kg, respectively) in yellow-orange prickly pears, while red prickly pears contained betacyanins at concentrations of 400 mg/kg, as the chemicals responsible for this color. As shown in Table 3, the juice of red-colored prickly pears have a higher betacyanin content, while betaxanthins predominate in the yellow variants (Amarilla Monteza and Pico Chulo), a finding that is consistent with other studies (STINTZING et al., 2005; CHÁVEZ et al., 2009; YAHIA and MONDRAGÓN, 2011). The Tapona juice showed the highest content of betacyanins and betaxanthins, but its purple-red color derives from the prevalence of betacyanins. The intermediate values of both compounds in the juice of the red O. megacantha variants is worth noting, as well as the minimum content of them in Blanca juice; this finding coincides with the results of CASTELLANOS-SANTIAGO and YAHIA (2008) for the same species. Ital. J. Food Sci., vol. 30, 2018 - 622 Table 3. Average content of betaxanthins and betacyanins (mg/g dry weight) in lyophilized juices of 10 prickly pear variants. Species Variant* Betaxanthins Betacyanins Total betalalains O. ficus-indica Rojo Pelón 0.148±0.005f 0.149±0.010g 0.298g O. albicarpa Blanca 0.018±0.003 g 0.021±0.004h 0.044h O. megacantha Amarilla Monteza 0.120±0.007 f 0.011±0.001h 0.130h Pico Chulo 0.085±0.017 f 0.019±0.004h 0.105f Torreoja 0.313±0.029 e 0.358±0.030f 0.671ef Sangre de Toro 0.810±0.007 c 1.580±0.030c 2.390c O. streptacantha Cardona 0.423±0.030d 0.800±0.008d 1.213d O. streptacantha ssp. aguirrana Charola 0.290±0.007e 0.660±0.020e 0.945e O. robusta Tapona 1.450±0.017a 2.610±0.030a 4.074a Tapón Rojo 1.230±0.04 b 2.380±0.060b 3.610b P value ˂0.0001 ˂0.0001 ˂0.0001 *Variants are sorted from highest to lowest degree of humanization. n = 3. Treatments with different letters in the same column are statistically different (<0.05). 3.4. Quantification of ascorbic acid by HPLC Ascorbic acid is one of the most effective and abundant antioxidants in fruits and vegetables (LOGANAKI and MANIAN 2010), participating in various biological functions that include the synthesis of collagen, hormones and neurotransmitters. The increase in the consumption of ascorbic acid is associated with a lower risk of chronic diseases such as cancer, cardiovascular disease and cataracts. This may be due to its ability to eliminate free radicals in biological systems. This study only measured ascorbic acid content (Table 4) without performing the reduction of dehydroascorbic acid (DHAA), necessary to obtain the total vitamin C content (SDIRI et al., 2012). Table 4. Average concentration of ascorbic acid (mg/g dry weight) in lyophilized juices of 10 prickly pear variants. Species Variant* Ascorbic acid O. ficus-indica Rojo Pelón 1.328±0.003a O. albicarpa Blanca 0.316±0.003d O. megacantha Amarilla Monteza 0.327±0.016d Pico Chulo 0.542±0.004 c Torreoja 0.327±0.004 d Sangre de Toro 0.652±0.041 b O. streptacanta Cardona 0.325±0.006d O. streptacanta ssp. aguirrana Charola 0.191±0.000e O. robusta Tapona 0.527±0.029c Tapón Rojo 0.691±0.006 b P value ˂0.0001 n=3. Treatments with different letters in the same column are statistically different (<0.05). *Variants are sorted from highest to lowest degree of humanization. Ital. J. Food Sci., vol. 30, 2018 - 623 Ascorbic acid concentration showed significant differences between the prickly pear juices evaluated (P < 0.0001). The highest ascorbic acid content was recorded in Rojo Pelón, followed by Sangre de Toro and Tapón Rojo; Charola was the variant with the lowest ascorbic acid concentration in juice. However, all concentrations measured were sufficient to meet easily the minimum daily intake (84 mg) of ascorbic acid in the human diet (SÁENZ et al., 2007). Among the variants evaluated by YAHIA and MONDRAGÓN (2011), the highest ascorbic acid concentration was recorded in the juice of the Camuesa prickly pear (O. robusta), followed by Cardona (O. streptacantha), with 4.0 mg/100 g and 2.1 mg/100 g fresh weight, respectively, while the lowest concentration was observed in the juice of Naranjona (O. megacantha), ranging between 1.2 mg/100 g and 1.4 mg/100 g fresh weight; according to these authors, DHAA showed a pattern similar to that of ascorbic acid. In general, it has been pointed out that the concentration of ascorbic acid in prickly pear (Opuntia spp.) ranges from 12 mg/100 g to 81 mg/100 g fresh weight (FEUGANG et al., 2006). Thus, the concentration of ascorbic acid in prickly pear juice is similar to or higher than in grapes, apple and pear (0.5 mg/g, 0.3 mg/g and 0.2 mg/g edible dry weight, respectively), but lower than in guava and kiwi fruit (9.4 mg/g and 4.9 mg/g edible dry weight, respectively) (LOTITO and FREI, 2006). To note, the variants with the highest and lowest degree of humanization showed the highest concentrations of this antioxidant, suggesting that this process is unrelated to the concentration of this antioxidant. 3.5. Antioxidant capacity of prickly pear juice in vitro The antioxidant capacity of prickly pear juice was estimated through ABTS and FRAP, since both are the assays most frequently used and they measure most antioxidants present. ABTS is typically used for mixtures or complex beverages, and measures mainly SET (single electron transfer) antioxidants, without excluding HAT (hydrogen atom transfer) antioxidants, in both water-soluble and fat-soluble media. In contrast, FRAP is applicable mostly to vegetables with SET and HAT antioxidants, mainly phenols and ascorbic acid (SURVESWARAN et al., 2007; GÜLÇIN, 2012). These methods were considered as mutually complementary and were contrasted through the correlation between their respective results. SURVESWARAN et al. (2007) point out that various herbs, fruits and vegetables show a direct relationship between antioxidant capacity and total phenolic content. In the prickly pear juices evaluated, this trend was not observed due to their contrasting differences in color, related to the presence of antioxidants such as ascorbic acid and betalains (Table 5). The data obtained with ABTS were normally distributed, but those with FRAP had to be log-transformed before being analyzed. The estimates of antioxidant capacity obtained with both methods (FRAP and ABTS) for total phenols, betalains and ascorbic acid in the juice of the 10 prickly pear variants were compared through a simple linear correlation analysis (Table 6). All correlation values for each comparison had the same sign, which evidences a consistent general trend in the estimates obtained with both methods. The results show that the estimates of the reduction ability of betalains with FRAP and ABTS were positively and significantly correlated (P < 0.0001). On the other hand, the estimates for ascorbic acid and phenolic compounds were not significantly correlated. The significant correlation of the total antioxidant capacity between both methods is explained by the abundance of betalains and because both methods produced similar estimates of the antioxidant capacity for all other compounds tested. Ital. J. Food Sci., vol. 30, 2018 - 624 Table 5. Antioxidant total capacity of juices of 10 prickly pear variants. Species Variant* ABTS •+ TEAC (µM/g dry weight) FRAP (µM eq. FeSO4/g dry weight) O. ficus-indica Rojo Pelón 43837±2601abc 49102±4280cd O. albicarpa Blanca 39570±8473 c 45202±4098cd O. megacantha Amarilla Monteza 37504±6726c 38490±2591d Pico Chulo 38307±6833 c 39157±2583d Torreoja 46264±8198 abc 42378±9272cd Sangre de Toro 51422±400 abc 79066±7562b O. streptacantha Cardona 45501±3565abc 60141±4645bc O. streptacantha spp. aguirrana Charola 40778±3741bc 57543±4843cd O. robusta Tapona 62117±10439a 112651±15066a Tapón Rojo 59968±12243 ab 118790±16262a P value ˂0.0016 ˂0.0001 *Variants are sorted from highest to lowest degree of humanization. n=3. Treatments with different letters in the same column are statistically different (<0.05). Table 6. Correlation (r) between estimates of antioxidant capacity generated by ABTS and FRAP methods in the juices of 10 prickly pear variants. Antioxidant ABTS FRAP Total 0.7845 *** 0.9436 *** Total phenolic compounds 0.0848 -0.11811 Phenolic acids -0.2033 -0.0943 Flavan-3 ols 0.3943 0.4781 Betalains 0.7828*** 0.9511*** Ascorbic acid 0.1829 0.1635 Significance *** P < 0.0001. 4. CONCLUSIONS The higher content of betalains in the red prickly pear variants Tapona, Tapón Rojo and Sangre de Toro, and of ascorbic acid in Rojo Pelón, Tapón Rojo and Sangre de Toro, resulted in their total antioxidant capacity being higher than in the other color variants, and explains the lack of a significant correlation between the estimates of the antioxidant capacity of phenols. Of the variants evaluated, Pico Chulo was the richest in phenolic acids, and Tapona in flavan-3-ols. Unlike other table fruits, prickly pear is an important source of phenols, in addition to having the most common antioxidant phytochemicals, such as betalains and ascorbic acid. Therefore, this study of prickly pear juice in vitro provides further support for recommending consumption of prickly pear juice as a functional food due to its antioxidant properties similar or superior to the juice of various marketed fruits. The study confirms the antioxidant capacity of the analyzed fruit, however before prickly pear fruit can be considered a potential functional food it is important to highlight the importance of running in vivo studies (animals and humans) in order to confirm Ital. J. Food Sci., vol. 30, 2018 - 625 bioavailability of the compounds analyzed in the study and to find whether consumption of prickly pear fruit actually induces positive effect in promoting a healthy status. ACKNOWLEDGEMENTS This study was supported by Fundación Produce de San Luis. Ing. Roberto Canovas Garfias, President of Sistema Producto Nopal San Luis Potosí, promoted and supported this project and provided all raw materials (prickly pears) required. 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