Pigment Composition and Antioxidant Capacity of Betacyanins and Betaxanthins Fractions of Opuntia Dillenii (Ker Gawl) Haw Cactus Fruit

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in Food Research International on November 2017, available at: https://doi.org/10.1016/j.foodres.2017.09.007” 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 10 17 32 38 41 Pigment composition and antioxidant capacity of betacyanins and betaxanthins 1 2 fractions of Opuntia dillenii (Ker Gawl) Haw cactus fruit 3 4 5 6 7 Carolina Betancourta, María Jesús Cejudo-Bastanteb*, Francisco J. Herediab, Nelson 8 9 Hurtadoa 11 12 a Departamento de Química, Grupo de Investigación en Productos de Importancia 13 14 Biológica (GIPIB), Universidad de Nariño, San Juan de Pasto, Nariño, Colombia 15 16 b Food Colour and Quality Lab., Área de Nutrición y Bromatología, Facultad de 18 19 Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain 20 21 22 23 24 25 26 27 28 Carolina Betancourt: [email protected] 29 María Jesús Cejudo-Bastante: [email protected] 30 Francisco J. Heredia: h[email protected] 31 Nelson Hurtado: [email protected] 33 34 35 36 37 * Corresponding author: 39 María Jesús Cejudo-Bastante 40 Food Color & Quality Lab., Área de Nutrición y Bromatología. Facultad de Farmacia. 42 Universidad de Sevilla. 41012-Sevilla, Spain 43 Tel.: +34 954557017 44 e-mail: [email protected] 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 5 10 22 27 32 Abstract 1 2 A purification and fractionation of Opuntia dillenii (Ker-Gawl) Haw extracts followed 3 4 by betalainic and phenolic characterization by HPLC-DAD-ESI-MS were carried out. 6 7 Several betalains and polyphenols have been identified, the majority of them not 8 9 previously described in Opuntia dillenii: 17-decarboxybetanin and 17- 11 12 decarboxyisobetanin, -O-sinapoyl-O- -O-sinapoyl-O- 13 14 -O-apiosyl-4-O- -O-E-sinapoyl- -apiosyl- 15 16 17 phyllocactin as betacyanins, tryptophan-betaxanthin and tyrosine-betaxanthin 18 19 (portulacaxanthin II) as betaxanthins and isoramnethin-3-glucuronide and quercetin-3- 20 21 O-glucoside as polyphenolics. Moreover, the antioxidant activity of extracts of Opuntia 23 24 dillenii (Ker-Gawl) Haw fruit followed the ensuing decreasing order (crude extract (CE) 25 26 > purified extract (PE) > red fraction (RF) > yellow fraction (YF)), being in 28 29 concordance with the total reducing power (TRP). These evidences suggest that 30 31 Opuntia dillenii is a rich source of bioactive compounds, with a high amount of total 33 34 betacyanins (16.63 mg betanin/100 g fresh fruit) and betaxanthins (7.55 mg 35 36 indicaxanthin/100 g fresh fruit). 37 38 39 Keywords: Opuntia dillenii; betacyanins; betaxanthins; polyphenols; antioxidant 40 41 activity; HPLC-DAD-ESI-MS. 42 43 44 45 59 60 61 62 63 64 65 4 5 10 22 27 32 44 49 1. Introduction 1 2 Opuntia ss. is the most abundant genus in the Cactaceae family, growing in semi-desert 3 4 climate with water-scarce conditions and an optimal low pluviometry for its growth 6 7 (150-200 mm median annual precipitation) (Cejudo-Bastante, Hurtado, & Heredia, 8 9 2015). Their yellow flowers and red fruits are characteristic from Opuntia genus, 11 12 together with thick, flat and stiff stems. Among this genus, Opuntia ficus-indica is the 13 14 most widespread and investigated fruit (Cejudo-Bastante, Chaalal, Louaileche, Parrado, 15 16 17 & Heredia, 2014; Stintzing, Schieber, & Carle, 2002), but there are further many 18 19 species of Opuntia which deserve special attention for their potential beneficial effects. 20 21 That is the case of Opuntia dillenii (Ker-Gawl) Haw. 23 24 The origin, distribution and taxonomy of Opuntia dillenii have been previously 25 26 described by Böhm (2008) and Sharma (2015). It is considered native from India and 28 29 America, but it could be also grown in the Mediterranean area, Asia or Australia. In 30 31 Colombia, Opuntia dillenii is wildly found in the arid zones of Santander, Nariño and 33 34 Guajira departments. Their fruits are characterized by the red colour and contain 35 36 cladode spines, an average length and width around 4.0 and 3.2 cm, respectively, an 37 38 39 acidic taste and a great number of seeds (Medina, Rodríguez, & Romero, 2007). This 40 41 feedstock has been studied from different points of view: the analysis of 42 43 polysaccharides (Li, Yuan, Zhou, Zeng, & Lu, 2016), the physicochemical 45 46 characterization (total fibre, proteins, fat, minerals and ascorbic acid, among others) 47 48 (Medina, Rodríguez, & Romero, 2007; Méndez, Flores, Martín, Rodríguez Rodríguez, 50 51 & Díaz Romero, 2015), and the analysis in terms of health amelioration (such as the 52 53 protective effect of the pulp, skin and seeds against the low-density lipoprotein 54 55 56 peroxidation) (Chang, Hsieh, & Yen, 2008). Concretely, our research groups have 57 58 contributed to increase the knowledge of Opuntia dillenii by deepening in the stability 59 60 61 62 63 64 65 5 5 10 22 27 32 44 49 of the colorimetric characteristics, betalains and antioxidant activity under different 1 2 conditions of pH and temperature (Cejudo-Bastante, Hurtado, & Heredia, 2015; 3 4 Montes-Lora, Hurtado, Mosquera, Heredia, & Cejudo-Bastante, 2016), being pioneers 6 7 in the study of betalains in this raw material. 8 9 Betalains are water-soluble compounds present in a restricted number of families of the 11 12 plant order of Caryophyllales and of the genus Amanita of the Basidiomycetes. Their 13 14 basic structure consists of a moiety of betalamic acid, and, depending on the residue, 15 16 17 betalains could be classified as red/purple betacyanins and yellow-orange betaxanthins 18 19 (when cyclo-Dopa and hydroxycinnamic acid derivatives or sugars, and amines or 20 21 amino acids residues are linked to betalamic acid, respectively) (Herbach, Stintzing, & 23 24 Carle, 2006). Betacyanins do not only vary by the degree and type of glycosylation in 25 26 C5 and C6 positions of cyclo-Dopa, but also by the acylation of sugars with organic 28 29 acids such as ferulic, cinnamic or malonic acids (Herbach, Stintzing, & Carle, 2006). 30 31 They are present in a large extent number of plants, vegetables, flowers and fruits such 33 34 as Opuntia. Although individual betalains have been well-described in other species of 35 36 Opuntia, to the best of our knowledge, any accurately characterization study on 37 38 39 individual betalains have not been already reported in Opuntia dillenii. 40 41 In terms of biological activity, betalain-rich products have been described as important 42 43 sources of free radical-scavenging activity. Although red beet and red pitahaya fruits 45 46 have been assigned as two important sources of antioxidant activity 47 48 & Wiczkowski, 2016; Vaillant, Perez, Davila, Dornier, & Reynes, 2005), other raw 50 51 materials, such as ulluco tuber (Campos, Noratto, Chirinos, Arbizu, Roca, & Cisneros- 52 53 Zevallos, 2006; Cejudo-Bastante, Hurtado, Mosquera, & Heredia, 2014) or amaranthus 54 55 56 flowers (Li, Deng, Liu, Zhu, Draves, Marcone, et al., 2015), showed remarkable 57 58 antioxidant ability related to betalains. With regard to Opuntia genus, several studies 59 60 61 62 63 64 65 6 5 10 22 27 32 44 49 have been conducted about determining the antioxidant capacity, such as Jiménez- 1 2 Aguilar, López-Martínez, Hernández-Brenes, Gutiérrez-Uribe, & Welti-Chanes (2015) 3 4 in Mexican commercial varieties of cactus pear, Gandía-Herrero, Escribano, & García- 6 7 Carmona (2016), Osorio-Esquivel, Alicia Ortiz, Álvarez, Dorantes-Álvarez, & Giusti 8 9 (2011) and Tamer E. Moussa-Ayoub, Jaeger, Knorr, El-Samahy, Rohn, & Kroh (2011) 11 12 in Opuntia ficus-indica, Opuntia joconostle and Opuntia macrorhiza fruits, 13 14 respectively. However, up to the present, scarce studies about the antioxidant capacity 15 16 17 of Opuntia dillenii have been developed. Among them, Kumar, Ganesh, Peng, & Jang 18 19 (2014) demonstrated that Indian Opuntia dillenii flowers showed effective antioxidant 20 21 properties against DPPH free radicals. Another example is Montes-Lora, Hurtado, 23 24 Mosquera, Heredia, & Cejudo-Bastante (2016), who studied the influence of 25 26 technological treatments on the antioxidant activity of Opuntia dillenii extracts, and 28 29 Ghazi, Ramdani, Tahri, Rmili, Elmsellem, El Mahi, et al. (2015) studying the 30 31 antioxidant activity of seeds oils and fruit juice of Opuntia dillenii from Morocco. 33 34 However, the antioxidant activity of Opuntia dillenii extracts after purification and 35 36 fractionation has not been previously carried out. 37 38 39 Overall, this research study sought to develop a great chemical characterization of 40 41 Opuntia dillenii extracts, in the light of any detailed study concerning individual 42 43 betalains and phenolic compounds of this fruit have been performed up to now. To 45 46 bring something to fruition, a purification and fractionation based on the colour of the 47 48 extracts was carried out, followed by the identification of each compound by HPLC- 50 51 DAD-ESI-MS. The study was complemented with the determination of the antioxidant 52 53 capacity against ABTS + radical of each fraction, in order to value the under-utilized 54 55 56 Opuntia dillenii as source of bioactive compounds. 57 58 59 60 61 62 63 64 65 7 5 10 22 27 32 44 49 2. Material and methods 1 2 2.1. Chemical and solvents 3 4 All solvents (Folin-Ciocalteau reagent and HPLC-grade methanol, acetonitrile and 6 7 water) and standards (gallic acid and Trolox) were supplied by Sigma-Aldrich (St. 8 9 Louis, MO, USA). 11 12 2.2. Vegetal material 13 14 Prickly pear samples (Opuntia dillenii) were collected in the village of Chachagüí 15 16 17 (Nariño, Colombia), which is located at approximately 25 Km north of the city of San 18 19 Juan de Pasto. It is situated at 1° 21 latitude and 77° 16 W longitude at an altitude of 20 21 1982 m above the mean sea level. 23 24 A specimen of the plant reposes in the herbarium of the Universidad de Nariño (code 25 26 No 13691). A representative set of samples of up to a weight of about 2 kg was 28 29 harvested, collecting mature fruits according to visual characteristics and similar size. A 30 31 simple random sampling model of ten plants was underwent. After homogenization, 33 34 only 1450 g of fruits was considered, with an average weight of each fruit around 11 g. 35 36 They were carefully washed and dried, and prickles were manually removed. Fruits 37 38 39 were kept under refrigeration at 4 ºC until their analysis. 40 41 2.3. Preparation of extracts 42 43 The crude extract (CE) was obtained according to the method described by Fernández- 45 46 López, Giménez, Angosto, & Moreno (2012). Fruits were cut into small pieces (1 cm2) 47 48 and extracted with 1.5 L of methanol:water (60:40) for 24 h at 10 ºC (chemical 50 51 maceration). The procedure was developed several times until the complete 52 53 discoloration of the plant material. After vacuum filtration (Whatman filter No 1), the 54 55 56 organic solvent was evaporated at 35 ºC using a rotary evaporator (Heidolph, 57 58 Schwabach, Germany) and the re-dissolved extract with distilled water (relation 1 57 58 59 60 61 62 63 64 65 8 5 10 22 27 32 44 49 g/mL) was lyophilized (Labconco, MO, USA). Lyophilized samples were stored at 4 ºC 1 2 until their analysis. 3 4 2.4. Precipitation of hydrocolloids and proteins 6 7 In order to remove hydrocolloids and proteins which could interfere in the 8 9 determination of betalains, the methods described by Stintzing, Schieber, & Carle 11 12 (2002) was followed. 20 mL of ethanol at 96% were added to 10 g of CE previously 13 14 dissolved in 10 mL of water. The solution was stirred and then was reposed for 20 min. 15 16 17 The mucilages were separated from the aqueous phase by vacuum filtration (Whatman 18 19 No 1 filters), and the organic solvent was evaporated at reduce pressure using a rotary 20 21 evaporator (Heidolph, Schwabach, Germany). The purified extract (PE) was then 23 24 lyophilized and kept at 4 ºC until its further analysis. 25 26 2.5. Fractionation on C18 column 28 29 Column chromatography was carried out in order to separate different fractions of 30 31 pigments from the PE by means a differential fractionation based on the hydrophobicity. 33 34 A C18 column (28 x 2 cm) (Sigma-Aldrich, USA) was previously conditioned by 35 36 passing, separately, 3 volumes of 100 % methanol and then rinsed with 3 volumes of 37 38 39 deionized water acidified with formic acid (pH 3), according to the method proposed by 40 41 Stintzing, Schieber, & Carle (2002). 0.3 g of PE dissolved in 1 mL of deionized water 42 43 was then added to the column. For fractionation, two fractions were distinguished based 45 46 on their colour. The less hydrophobic compounds (yellow fraction, YF) were eluted 47 48 from the column by passing 2 volumes of 100% methanol. The red fraction (RF) were 50 51 eluted with two volumes of a mixture of methanol:acidified water (pH 3) (95:5, v/v). 52 53 Both fractions were concentrated at reduce pressure and then lyophilized. 54 55 56 59 60 61 62 63 64 65 15 5 10 22 27 32 44 49 betanin/100 g fresh fruit and 7.55 ± 0.04 mg indicaxanthin/100 g fresh fruit, 1 2 respectively). To draw a comparison with other Opuntia species, Opuntia ficus-indica 3 4 and Opuntia undulata displayed greater content of total betaxanthins and similar order 6 7 of magnitude of betacyanins (Fernández-López, Almela, Obón, & Castellar, 2010), 8 9 whereas Sepúlveda (2003) exhibited that betacyanins content of Chilean Opuntia ficus- 11 12 indica cacti ranged between 16.6 and 62.4 mg of betanin/100 g of fresh fruit. With 13 14 regard to Opuntia dillenii, only Cejudo-Bastante, Hurtado, & Heredia (2015) previously 15 16 17 provided a concentration of total betacyanins and betaxanthins (around 7 and 6 mg 18 19 betanin and indicaxanthin/100 g of dry fruit, respectively). 20 21 3.2. Total reducing power (TRP) and Antioxidant Activity 23 24 Table 2 summarizes the values of total reducing power and antioxidant activity of the 25 26 different fractions of Opuntia dillenii. The values of antioxidant activity of ascorbic acid 28 29 used as positive control was in agreement with other authors (Re, Pellegrini, 30 31 Proteggente, Pannala, Yang, & Rice-Evans, 1999) (1.08 mmol Trolox/mmol ascorbic 33 34 acid). 35 36 The results evidenced a diminution of the antioxidant activity along the purification 37 38 39 process (CE > PE > RF > YF), with significant differences (p < 0.05) between samples. 40 41 The fact that PE had lesser antioxidant activity than CE might be from the elution of 42 43 some uncoloured compounds (such as organic acids), responsible for supplying 45 46 antioxidant capacity. These results were in accordance with the values of total reducing 47 48 power (TRP) (Table 2), as the crude extract (CE) evidenced the highest antioxidant 50 51 activity and TRP. In fact, univariate lineal correlation was applied to these data and a 52 53 very strong correlation (R2 = 0.9684) was observed between the TRP and the 54 55 56 antioxidant activity measured by TEAC, which indicate that Opuntia dillenii represent a 57 58 good source of natural antioxidants with high added value. Similar relations were 59 60 61 62 63 64 65 16 5 10 22 27 32 44 49 observed by Moussa-Ayoub et al. (2016) in Opuntia dillenii juices when compared 1 2 antioxidant activity, measured by electron paramagnetic resonance spectrometry, and 3 4 total phenolic content. 6 7 It is also remarkable that RF manifested significantly (p < 0.05) higher values of 8 9 antioxidant activity and TRP compared to YF. Similar results were found by Butera, 11 12 Tesoriere, Di Gaudio, Bongiorno, Allegra, Pintaudi, et al. (2002), who demonstrated 13 14 that the radical-scavenging capacity of purified betanin and indicaxanthin from Opuntia 15 16 17 ficus-indica were highly effective, but betanin was more active than indicaxanthin. 18 19 4. Conclusions 20 21 It is demonstrated that Opuntia dillenii is a rich source of betalain compounds. Apart 23 24 from the well-known betanin, isobetanin and indicaxanthin previously described in 25 26 Opuntia dillenii, a large extent of betalains and polyphenols have been identified for the 28 29 first time in this fruit: 17-decarboxybetanin and 17-decarboxyisobetanin, -O-sinapoyl- 30 31 O- -O-sinapoyl-O- -O-apiosyl-4-O-phyllocactin and 33 34 -O-E-sinapoyl- -apiosyl-phyllocactin as betacyanins; tryptophan-betaxanthin and 35 36 portulacaxanthin II as betaxanthins; and isorhamnetin-3-glucuronide and quercetin-3-O- 37 38 39 glucoside as polyphenols. This fact evidenced that the purification and subsequent 40 41 fractionation of the extract (scarcely studied in betalain-rich products), make possible to 42 43 identify a greater number of betalains and phenolic compounds. Moreover, it is also 45 46 proved that Opuntia dillenii exhibited a potent antioxidant activity against ABTS + 47 48 radical, strongly related to the total reducing power. Therefore, the described properties 50 51 could make interesting the use of this low-exploited natural resource in terms of 52 53 nutrition and future industrial applicability. The commercialization and industrialization 54 55 56 of fruits with high functional value could generate competitive advantages and 57 58 economic opportunities for the producers. 59 60 61 62 63 64 65 17 5 10 22 27 32 44 49 Funding 1 2 This research did not receive any specific grant from funding agencies in the public, 3 4 commercial, or not-for-profit sectors. 6 7 Acknowledgments 8 9 We are indebted to Vicerrectoría de Investigaciones Postgrados y Relaciones 11 12 Internacionales VIPRI UDENAR (Universidad de Nariño, Colombia). Authors are 13 14 also grateful to Biology Service (SGI, Universidad de Sevilla) for technical assistance. 15 16 17 Conflict of interest. None. 18 19 References 20 21 Butera, D., Tesoriere, L., Di Gaudio, F., Bongiorno, A., Allegra, M., Pintaudi, A. M., 23 24 Kohen, R., & Livrea, M. A. (2002). Antioxidant activities of sicilian prickly pear 25 26 (Opuntia ficus indica) fruit extracts and reducing properties of its betalains: 28 29 Betanin and indicaxanthin. 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HPLC chromatogram corresponding to the yellow fraction (YF) of Opuntia 8 9 dillenii (480 nm). 11 12 Fig. 3. -O-sinapoyl-6-O-gomphrenin 13 14 -O-apiosyl-4-O-malonyl-betanin (compound 8). 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 22 63 64 65 Figure 1.