Changes during Maturation in the Bioactive Compounds and

Changes during Maturation in the Bioactive Compounds and Antioxidant Activity of Opuntia stricta (Haw.) Fruits R.L. Dantas, S.M. Silva, D.M. Brito Primo and A.S.B. Sousa Laboratory of Biologia e Tecnologia Pós-Colheita Departamento de Ciências Fundamentais e Sociais Universidade Federal da Paraíba-UFPB Areia, Paraíba Brazil

E.S. Brito and E.M.S. Macedo Laboratory of Fisiologia e Tecnologia Pós-Colheita Embrapa Agroindústria Tropical Fortaleza, Ceará Brazil

Keywords: Cactaceae, Opuntia genus, betacyanins, betaxanthins, quality, free radicals Abstract Increasingly evidences have pointed the benefits of fruit consumption on human health due to the presence of compounds capable of acting minimizing the incidence of diseases. Although in the Northeastern Brazil the Opuntia stricta species is mostly used as living fences, it produces small fruits that are of red purplish color, and source of betalains and ascorbic acid, compounds in general correlated to high antioxidant activity. This study evaluated changes in betalain content and antioxidant activity of fruits of O. stricta during maturation. Fruits were harvested from plants grown in area of family farming and graded in four maturity stages based on skin color. Evaluations were performed using the whole fruit. The content of betacyanin in the fruit increased from 6.9 to 51.6 mg/100 g during ripening. The pulp of fruit presented a intense red purplish color. The content of total extractable polyphenols (TEP) has also increased from 32.1 to 74.02 GAE mg/100 g during maturation, although no significant difference in TEP was observed between the first two maturity stages, in which some greenish traces was still present in the color. Antioxidant activity, measured by the radical ABTS•+, presented values that increased from 3.5 to 12.3 μM Trolox/g during ripening, showing similar profile to the TEP. The changes observed for the antioxidant activity of O. stricta fruits was correlated with the content of betacyanin (r=0.85**) and phenolics (r=0.99**), which can indicate that these compounds may act by capturing free radicals, minimizing oxidative stress. Therefore, these results suggest that fruits of O. stricta are a significant source of antioxidant compounds such as phenolics and betalains.

INTRODUCTION

Plants of the Cactaceae family are very important crops in the Brazilian Northeast semiarid landscapes, since they are well adjusted to adverse soil and climatic conditions, present important role on soil conservation, and are widely used for animal feeding and field management (Silva et al., 2009). Among the Cactaceae occurring in the Paraíba state semiarid lands the Opuntia stricta (Haw.) is considered an invasive species (Andrade Lima, 1989), which produces fruits that exhibit strong red-purple coloration when ripe (Castellar et al., 2012). In Northeastern Brazil, although this species is popularly used in the areas of occurrence for medicinal purposes, its use is restricted mainly to living fences in family farms, being used over periods of prolonged drought for feeding (Lucena et al., 2012), with little or no use for fruits of strong red-purple in the semiarid (Silva et al., 2009). Fruits and vegetables contain a wide range of phytochemicals, including carotenoids, flavonoids and other phenolic compounds, glucosinolates, dietary fibers, phytosterols and monoterpenes, among others, that may have a positive effect on human health (Yahia and Mondragon-Jacobo, 2011). The nutritional and health benefits of cactus fruit are associated with their antioxidant properties related to ascorbic acid, phenolic Proc. VIIIth IC on Cactus Pear and Cochineal Eds.: Inglese et al. Acta Hort. 1067, ISHS 2015

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compounds, and a mixture of yellow betaxanthin and red betacyanin pigments (SumayaMartínez et al., 2011). In general, the fruit of some species of Opuntia contain two betalain pigments, the purple-red betanin and the yellow indicaxanthin, both with radicalscavenging and reducing properties (Stintzing et al., 2005). In this direction, Opuntia stricta is considered an invasive plant in some countries (Andrade Lima, 1989), however phytochemical studies have been conducted primarily evaluating betalains whose content in the ripe fruits is the highest (80 mg/100 g), and betanin and isobetin the main constituents (Castellar et al., 2012). In ripe fruits of O. stricta, the total betalain content is approximately 5-fold higher than those of O. ficus-indica of red-purple coloration (14 to 19 mg/100 g fresh mass), O. undulate (20 mg/100g fresh mass), O. lasiacantha (19 to 28 mg/100 g fresh mass) and even higher than some commercial varieties of sugar beets (40 to 60 mg/100 g fresh mass) (Fernández-López et al., 2002; Castellar et al., 2003; Sánchez et al., 2006). In addition, in fruits of different varieties of Opuntia ficus-indica the content of betalains was highly correlated with the antioxidant activity assessed by the reduction of ABTS•+ radical (TEAC) and ORAC (Stintzing et al., 2005; Sumaya-Martínez et al., 2011). In this sense, Castellar et al. (2012) showed that in fruits of O. stricta the total phenolic and betacyanins contents increased considerably from 4 to 9 months after anthesis. In red pulp pitaya Ding et al. (2009) verified that betanin and isobetanina are the main betalains (betacyanins) both in the flesh and in the skin, representing, respectively, an increase in the total content of betacyanins of 5- to 36-fold from 20 to 35 days after anthesis. Cardador-Martinez et al. (2011) studied the changes in by-products (skin and seed) in fruits of different varieties of O. ficus-indica at two maturity stages (green and ripe), showing the influence of maturation for the content of total phenolics, tannins and flavonoids from the skin, while this was not observed for the seeds. In general, due to the non-climacteric respiratory pattern, fruits of cacti do not present abrupt changes in the attributes associated with flavor, as soluble solids and titratable acidity, such as those associated with appearance as color, size, and shape (ElGharras et al., 2006; Duru and Turker, 2005). However, other features are relevant in characterizing the quality of these fruits especially during maturation, when changes in the content of nutritionally important compounds, in addition to taste and appearance, undergo irreversible alterations. Because the cactus fruit are significant sources of compounds with functional properties, such as phenolic compounds and betalains, detailed studies of unexploited species are of fundamental importance in view of the need of natural sources of compounds that can be used for medicinal and higher quality food purposes. However, so far there are few studies that address the changes resulting from maturation in fruits of Opuntia stricta, especially in plants occurring in the semiarid region of Northeastern Brazil. Thus, this study aims to evaluate the changes during maturation in the total extractable polyphenolic compounds and betalains in fruits of Opuntia stricta. MATERIAL AND METHODS Fruits of Opuntia stricta were harvested early in the morning from living fences found in an agricultural area of family farm located at the semiarid region of the Paraíba State, Northeastern Brazil. Fruits were harvested in four maturity stages based on the skin color: I – At the beginning of the red-purple coloration; II – Predominant purple-red coloration; III – Totally purple-red coloration; and IV – Dark red-purple coloration. The previous preparation of samples for evaluations consisted of the manual removal of the thin skin that surrounds the fruit, followed by the separation the seeds from the pulp using a 0.6 mm mesh sieve, which resulted in a pulp of pasty consistency. The quantification of the betalains was performed spectrophotometrically using the equations of Nilson (1970). For each maturity stage, the extracts were prepared using water for extraction. Thus, subsamples of the pulp of the fruits were weighed and diluted in distilled water followed by centrifugation at 12,000 rpm during 25 min at 4°C. For 160

each subsample, the supernatant was reserved and the residue was re-extracted twice more. The resulting supernatants were received in the same vial and the volume adjusted to 30 ml with distilled water, which was analyzed immediately. The absorbance of the extracts was measured at 476, 538, and 600 nm and the content of betalains (mg/100 g) were estimated, respectively, by the following equations: x = 1.095(A538 – A600) and y= –0.258 × А538 + A476 – 0.742 × A600. Finally, the betacyanin contents were obtained by [BTC= (x × R × 100)/1120] and betaxanthin [BTX = (y × R × 100)/750], where R is the dilution factor, and % 1120 and % 750 the extinction coefficients for betanin and vulgaxantina-I, respectively. The preparation of the extracts for evaluation of the total extractable polyphenols and antioxidant activity was conducted according to Rufino et al. (2010). The samples were weighted and added 4 ml of 50% methanol and the vials shaken for 1 min, followed by 1 h rest. The extract was centrifuged at 4°C and 15,000 rpm during 15 min. The supernatant was reserved and to the residue was added 4 ml of 70% acetone, and then subjected to the same procedure. The two supernatants were put together and the final volume of the vial adjusted to 10 ml with distilled water. The extracts were kept at -20°C until analyzed. The total extractable polyphenol contents were determined using a spectrophotometer by the Folin-Ciocalteu method, with modifications (Rufino et al., 2010). Briefly, based on prior testing, aliquots of the extract were taken, 300 µl (maturity stages I and II) and 150 µl (maturity stages III and IV), which were diluted to 1000 µl with distilled water. The oxidation was performed by adding 1 ml of Folin-Ciocalteu’s reagent in distilled water (1:3), followed by neutralization with 2.0 ml of 20% sodium carbonate and addition of 2.0 ml distilled water. The reading was performed in a spectrophotometer at 700 nm, after being kept in the dark for 30 min at room temperature. The estimated content of phenolic compounds was performed using a standard curve of gallic acid (R = 0.99) and the results expressed in mg of gallic acid per 100 g fresh weight. The antioxidant activity of the extracts of the pulp of the fruits of Opuntia stricta, harvested in four maturity stages, was evaluated by the capture of the ABTS•+ radical. The radical’s preparation consisted of mixing 5 ml of the ABTS•+ solution at 7 mM with 88 µl of the solution of potassium persulfate at 140 mM, letting to rest at room temperature for 16 h in absence of light. Before the essay, the radical was diluted with ethanol until the absorbance of 0.700 ± 0.05, measured at 734 nm. From the extracts, it was prepared three dilutions of 50, 150, and 300 mg/ml. To an aliquot of 3.0 ml of ABTS•+ radical (absorbance of 0.700) was added 30 µl of each dilution and the absorbance was read after 6 min at 734 nm. The Trolox was used as standard, with a standard curve ranging from 100 to 2000 µM (R=0.996). The results were expressed as µM of Trolox/g fresh weight (Rufino et al., 2010). All evaluations were performed considering six replications, in triplicate. Data were subjected to analysis of variance and the means presented with their standard errors and compared by the Tukey test at 5% of probability. RESULTS AND DISCUSSION The content of betalains in fruits of O. stricta increased as maturation progressed (Fig. 1). Fruits at the beginning of maturation (ripening stage I) presented betacyanins content of 6.9 mg/100 g, increasing to 51.6 mg/100 g (maturation stage-IV), approximately 7.5-fold higher, when fruit presented skin with dark red-purple coloration. Furthermore, the internal color of these fruits initially developed in the endocarp, where some seeds are found, then the flesh coloration develops in similar pattern (Castellar et al., 2012). This explains the large difference in the content of betacyanins throughout maturation. It was also observed a clear increase in the levels of betaxantinas with advancing of maturity, for which an average range of 1.04-7.74 mg/100 g was obtained. However, these contents were about 7-fold lower as compared with those for betacyanins, indicating 161

that fruits of Opuntia stricta could be used as a source of natural coloring. Stintzing et al. (2005) evaluating fruit of clones of the O. ficus-indica reported for the Purple cultivar the content of 28.8 mg/100 g; for the Red cultivar, 6.5 mg/100 g and for O. robusta (clone 1240), the content of 58.7 mg/100 g. In another study with the clone 1279 of the O. ficus-indica presenting increasing percentages of red coloration (0, 50, and 100%) in the skin, Felker et al. (2008) reported an increase in the betacyanin content as a function of the maturation of the fruit. These authors demonstrated that, for the total betalains in the pulp of these fruits, the contribution of betacyanin ranged from 70.875.7% and from 24.3-29.3% for betaxantinas. However, they also showed that in both type of clones, the richest in batacyanins as those in betaxanthins, the development of the characteristic pigmentation of different tissues (endocarp, mesocarp, and epicarp) appeared to be regulated by different mechanisms. Furthermore, they stated that the complete coloring of the skin was achieved only after the internal tissues become fully pigmented possibly in response to the increase of sugars content. In turn, the content of total extractable phenolics in the pulp had a significant increased during maturation, especially for fruits of the maturity stages III and IV. Partially pigmented fruits (maturity stages I and II) presented a mean value of 33.9 mg GAE/100 g, while those heavily pigmented fruits (maturity stage IV) had the value of 74.02 mg GAE/100 g (Fig. 2A). For fruit of Opuntia megacantha, varieties orange and yellow, it was reported an increased in the content of phenolic compounds of 28 and 42%, respectively, over a period of 8 weeks (Cayupán et al., 2011). In this context, it has been shown that, in Cactaceae fruits, the maturation results in a significant increase in these compounds. However, the varietal difference can provide disparity among the contents and not always fruits with higher contents exhibit the strongest coloration (Yahia et al., 2011). Fruits of O. stricta showed values for antioxidant activity ranging from 3.49 to 12.33 mM Trolox/g fresh weight (Fig. 2B), observing that the extracts of fully pigmented fruits exhibited higher Trolox equivalent antioxidant capacity, which was paralleled to the increase in the phenolic compounds observed herein with the intensification of the redpurple coloration. Cayupán et al. (2011) also observed an increase in the antirradical activity as maturation advanced in fruits of two varieties of O. megacantha, which was attributed, in part, to the content of ascorbic acid, but also due to the presence of phenolic compounds and betalains, as also reported by Yahia and Mondragon-Jacobo (2011). The changes observed in this study on the antioxidant activity during the maturation of fruit of O. stricta were significantly correlated with the content of betacyanins (R = 0.85**), betaxanthins (R = 0.89**), and phenols (R = 0.99**), indicating that these compounds are probably responsible for capturing of the ABTS•+ radical. A positive and very significant correlation of the antioxidant activity of the extracts of Opuntia spp. with the bioactive compounds present in the pulp was also reported by Stintzing et al. (2005), which highlighted the marked contribution of phenolic and betalains. Therefore, these results indicate that fruits of Opuntia stricta are a significant source of antioxidant compounds such as phenolics and betalains. The functional potential in fruits of O. stricta is high and increases as maturation progresses. In addition, as the natural red pigments from plants are of growing interest as substitutes for synthetic red dyes in the food and pharmaceutical industry (Castellar et al., 2003), based on its higher content of betalainic pigments, the fruit of O. stricta may be regarded as an alternative for the family farming agricultural economy in the semiarid region of Northeastern Brazil. However, more studies are needed to evaluate the bioavailability and toxicity of the compounds present in these fruits, especially those completely pigmented. ACKNOWLEDGEMENTS Thanks to the Banco do Nordeste do Brasil and CNPq for financial support.

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Literature Cited Andrade Lima, D. 1989. Plantas das Caatingas. Academia Brasileira de Ciências, Rio de Janeiro, 243p. Cardador-Martinez, A., Jimenez-Martinez, C. and Sandoval, G. 2011. Revalorization of cactus pear (Opuntia spp.) wastes as a source of antioxidants. Ciênc. Tecnol. Aliment. 31:782-788. Castellar, M.R., Solano, F. and Obón, J.M. 2012. Betacyanin and other antioxidants production during growth of Opuntia stricta (Haw.) fruits. Plant Foods Hum. Nutr. 67:337-343. Castellar, R., Obón, J.M., Alacid, M. and Fernández-López, J.A. 2003. Color properties and stability of betacyanins from Opuntia fruits. J. Agric. Food Chem. 51:2772-2776. Cayupán, Y.S.C., Ochoa, M.J. and Nazareno, M.A. 2011. Health-promoting substances and antioxidant properties of Opuntia sp. fruits. Changes in bioactive-compound contents during ripening process. Food Chem. 126:514-519. Ding, P., Chew, M.K., Abdul Aziz, S., Lai, O.M. and Abdullah, J.O. 2009. Red-fleshed pitaya (Hylocereus polyrhizus) fruit colour and betacyanin content depend on maturity. Int. Food Res. J. 16:233-242. Duru, B. and Turker, N. 2005. Changes in physical properties and chemical composition of cactus pear (Opuntia ficus-indica) during maturation. J. Prof. Assoc. Cactus Develop. 6:22-33. El Gharras, H., Hasib, A., Jaouad, A. and El Bouadili, A. 2006. Caracterización química y física de tres variedades de higos Chumbos amarillas de Marruecos (Opuntia ficusindica) en tres etapas de madurez. Cienc. Tecnol. Alimentaria. 5:93-99. Felker, P., Stintzing, F.C., Müssig, E., Leitenberger, M., Carle, R., Vogt, T. and Bunch, R. 2008. Colour inheritance in cactus pear (Opuntia ficus-indica) fruits. Ann. Appl. Biol. 152:307-318. Fernández-López, J.A., Almela, L., Obón, J.M. and Castellar, R. 2010. Determination of antioxidant constituents in cactus pear fruits. Plant Foods Hum. Nutr. 65:253-259. Nilson, T. 1970. Studies into the pigments in beetroot (Beta vulgaris L. ssp. vulgaris var. rubra L.). Lantbrukhogskolans Annaler. 36:179-219. Rufino, M.S.M., Alves, R.E., Brito, E.S., Pérez-Jiménez, J., Saura-Calixto, F. and Mancini-Filho, J. 2010. Bioactive compounds and antioxidant capacities of 18 nontraditional tropical fruits from Brazil. Food Chem. 121:996-1002. Sánchez, F.D., Santos, E.M., Filardo, S., Villagómez, R. and Scheinvar, L. 2006. Colorant extraction from a red prickly pear (Opuntia lasiacantha) for food application. Elec. J. Env. Agricult. Food Chem. 5:1330-1337. Silva, S.M., Brito Primo, D.M., Torres, L.B.V., Martins, L.P., Lima, A.B. and Silva, F.V.G. 2009. Features of postharvest physiology and quality of Cactaceae fruits from Brazilian Northeast. Acta Hort. 811:113-122. Silva, S.M., Lopes, M.F., Brito Primo, D.M. and Torres, L.B.V. 2009. Pulp color Changes during storage of cactus pear fruit coated with yam starch. Acta Hort. 811:173-178. Stintzing, F., Herbach, K.M., Mosshammer, M.R., Carle, R., Yi, W., Sellappan, S., Akoh, C.C., Bunch, R. and Felker, P. 2005. Color, betalain pattern, and antioxidant properties of cactus pear (Opuntia spp.) clones. J. Agric. Food Chem. 53:442-451. Sumaya-Martínez, M.T., Jaime, S.C., Santillán, E.M., Paredes, J.D.G., Cortés, R.C., Cansino, N.C., Vega, C.V., Cardenas, L.M. and García, E.A. 2011. Betalain, acid ascorbic, phenolic contents and antioxidant properties of purple, red, yellow and white cactus pears. Int. J. Mol. Sci. 12:6452-6468. Yahia, E.M. and Mondragon-Jacobo, C. 2011. Nutritional components and anti-oxidant capacity of ten cultivars and lines of cactus pear fruit (Opuntia spp.). Food Res. Int. 44: 2311-2318.

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Figures

Fig. 1. Betalains (betacyanins and betaxanthins) in different stages of maturity of the fruits of Opuntia stricta. Bars (± Standard Error) with different letters differ among them by the Tukey’s test at 5% of probability. n=6.

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Fig. 2. Total extractable polyphenol contents (A) and antioxidant activity by ABTS•+ radical method (B) in different stages of maturity of the fruits of Opuntia stricta. Bars (± Standard Error) with the same letter do not differ between them by the Tukey’s test at 5% of probability. n=6.

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