Comparative Study of Phenolic Profile, Antioxidant Capacity, and Color-composition Relation of Roselle Cultivars with Contrasting Pigmentation

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: https://doi.org/10.1007/s11130-015-0522-5” 1 2 3 60 61 62 63 64 65 1 4 1 5 6 2 7 8 3 9 10 4 11 12 13 6 14 15 7 16 17 8 18 19 9 20 21 22 10 23 24 11 25 26 27 12 28 29 13 30 31 32 14 33 34 15 35 36 37 38 39 17 40 41 18 42 43 44 19 45 46 20 47 48 49 21 50 51 22 52 53 54 55 56 24 57 58 25 59 Comparative study of phenolic profile, antioxidant capacity, and color-composition relation of roselle cultivars with contrasting pigmentation Gustavo A. Camelo-Méndez a, M. José Jara-Palaciosb, M. Luisa Escudero-Gileteb, Belén Gordillob, Dolores Hernanzc, Octavio Paredes-Lópezd, Pablo E. Vanegas-Espinozaa, Alma A. Del Villar-Martíneza, Francisco J. Herediab a Instituto Politécnico Nacional, CEPROBI, Carretera Yautepec-Jojutla Km. 6, Calle CEPROBI 8, Col. San Isidro, Yautepec, Morelos, México C.P. 62731 b Food Colour & Quality Laboratory, Department Nutrition & Food Science, Universidad de Sevilla, Facultad de Farmacia, 41012 Sevilla, Spain c Department of Analytical Chemistry, Universidad de Sevilla, Facultad de Farmacia, 41012 Sevilla, Spain d Centro de Investigación y de Estudios Avanzados del IPN, Unidad Irapuato, Irapuato, Guanajuato, México Corresponding author at: e-mail: [email protected] 5 16 23 1 2 3 60 61 62 63 64 65 2 4 26 5 6 7 27 8 9 28 10 11 12 13 14 30 15 16 31 17 18 19 32 20 21 33 22 23 24 34 25 26 35 27 28 29 30 31 37 32 33 34 35 36 39 37 38 40 39 40 41 41 42 43 42 44 45 46 43 47 48 44 49 50 51 52 53 54 46 55 56 47 57 58 59 48 Abstract Roselle is a plant that accumulates anthocyanins significantly, hence its importance as food coloring and as a source of antioxidant compounds for human health. This study was aimed to determine phenolic composition and antioxidant capacity of methanolic extracts, and beverages obtained from roselle cultivars native to México (Negra, Sudan, Rosa and Blanca) with different degrees of pigmentation, and to establish the color-composition relationship. Chromatographic methods were used to determine phenolic compounds: flavanols, flavonols, benzoic, hibiscus and phenolic acids and two main anthocyanins (cyanidin 3-sambubioside and delphinidin 3-sambubioside), and antioxidant capacity was evaluated by ABTS and FRAP assays. Tristimulus colorimetry showed to be a useful technique for determination of color-composition relationship, leading to equations that allowed predicting anthocyanin content of roselle (R>0.84). Also a stepwise linear discriminant analysis (SLDA) was developed in order to classify roselle cultivars. Obtained mathematical model could be an important tool that can be used for colorimetric characterization of functional compounds used in food processing Keywords: Antioxidant capacity, color-composition, Hibiscus sabdariffa, multivariate statistical analysis, phenolic compounds, tristimulus colorimetry. Abbreviations: DIA: digital image analysis, Dp3Sa: delphinidin-3-sambubioside, Cy3Sa: cyanidin-3-sambubioside, LDA: linear discriminant analysis, SLDA: stepwise linear discriminant analysis, TA: total anthocyanins. Introduction Roselle (Hibiscus sabdariffa L.) is a plant belongs to family Malvaceae, their chalices are recognized as an important source of phenolic compounds [1, 2, 3]. Mexico is one the 29 36 38 45 1 2 3 61 62 63 64 65 3 4 49 5 6 7 50 8 9 51 10 11 12 13 14 53 15 16 54 17 18 19 55 20 21 56 22 23 24 57 25 26 58 27 28 29 30 31 60 32 33 34 35 36 62 37 38 63 39 40 41 64 42 43 65 44 45 46 66 47 48 67 49 50 51 52 53 69 54 55 70 56 57 58 71 59 60 72 main roselle producing countries, where it is widespread consumed in beverage. This beverage is obtained by thermal infusion of dehydrated chalices [3]. It has been used in traditional medicine for treatment of some diseases because of its antioxidant properties [1, 2]. Roselle chalices are used worldwide for drinks production, as additives in cosmetics and pharmaceuticals [4]. Currently consumers demand healthy products, hence the importance of studies about extraction and purification of bioactive compounds such as phenolics from natural sources in order to identify phytochemicals for processing of food supplements or nutraceuticals, functional food ingredients and food additives [3]. There are few studies on roselle cultivars having as characteristic different pigmentation degree. Christian and Jackson [1] studied roselle grown in Jamaica, reporting that dark roselle chalices presented the highest anthocyanins content and antioxidant activity, followed by red chalices, while in the white ones, these compounds were not detected; these results were similar to reported by other authors [2,5]. Mexico is one the main roselle producing countries, recently breeding programs have generated new cultivars with different pigmentation levels, volatile compounds of these plants have been evaluated [5]. The aim of this study was to determine phenolic profile and antioxidant capacity of both methanolic extracts and beverages, obtained from roselle cultivars with contrasting pigmentation (Negra, Sudan, Rosa and Blanca) and, to establish the color-composition relationship. Materials and methods Plant material Roselle chalices were obtained from breeding programs and named according to pigmentation: three pigmented cultivars (Negra, Sudan and Rosa) and, one unpigmented cultivar (Blanca); chalices characteristics and storage conditions of raw material were previously described [5]. 52 59 61 68 1 2 3 61 62 63 64 65 4 4 73 5 6 7 74 8 9 75 10 11 12 13 14 77 15 16 78 17 18 19 79 20 21 80 22 23 24 81 25 26 82 27 28 29 30 31 84 32 33 34 35 36 86 37 38 87 39 40 41 88 42 43 89 44 45 46 90 47 48 91 49 50 51 52 53 93 54 55 94 56 57 58 95 59 60 96 Extraction procedure For methanolic extraction, samples (5 g) were homogenized in 25 mL of solvent (75% methanol, acidified with HCl 5% [1 N]), kept under shaking for 1 h and further centrifuged at 906 g for 5 min; supernatant was collected and the pellet was submitted to the same process three more times, supernatants were mixed. On the other hand, aqueous extraction was prepared in order to simulate the traditional beverage. For this purpose, 5 g of sample were macerated in 100 ml of water, according the method described by Sayago-Ayerdi et al. [6]. Extracts were carried out in triplicate. Analysis of phenolics compounds Determination was carried out in an Agilent 1260 chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with a diode-array detector (200-700 nm), and a C18 Poroshell 2.7 Micron (4x6x50 mm) [7]. The solvents were 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) at the following gradient: 0 5 min, 5% B linear; 5 20 min 50% B linear; 20 25 min, washing and reequilibration of the column. The flow rate was 1.5 mL/min, injection volume of 10 and the temperature of the column was set at 25°C. The wavelength of detection was 280 nm (flavanols and benzoic acids), 320 nm (phenolic acids), and 370 nm (flavonols). Anthocyanins determination was carried out following the methodology adapted from Ivanova et al. [8]. These compounds were separated using a Zorbax °C. A gradient of the mobile phase A (trifluoroacetic acid, 0.1%), and acetonitrile as solvent B, were used. The elution profile was as follows: 0-3.25 min 90% A - 10% B; 3.25- 15.62 min 90% A - 10% B; 15.62-20.83 min 85% A - 15% B; 20.83-26.04 min 85% A - 15% B; 26.04-46.88 min 82% A - 18% B; 46.88-52.08 min 70% A - 30% B; 52.08-60 min 65% A - 35% B; 60-65 min 90% A -10% B. The flow rate was 0.8 mL/min, and the 76 83 85 92 1 2 3 61 62 63 64 65 5 12 29 34 51 4 97 5 6 7 98 8 9 99 10 11 100 13 14 101 15 16 102 17 18 19 103 20 21 104 22 23 24 105 25 26 106 27 28 107 30 31 108 32 33 109 35 36 110 37 38 111 39 40 41 112 42 43 113 44 45 46 114 47 48 115 49 50 116 52 53 117 54 55 118 56 57 58 119 59 60 120 The identification of phenolic compounds was achieved by comparison of the retention time with those of the available pure standards, our data library, and /or date in the literature. The quantification of phenolic compounds was done by external calibration from the areas of the chromatographic peaks recorded at 280, 320, 370 and 525 nm. Results were expressed as mg phenolic compound/100g of dry matter. Antioxidant capacity Antioxidant capacity was evaluated by FRAP and ABTS assays according to Benzie and Strain [9] and Re et al. [10], respectively. Different dilutions of each extract were assayed and the results were obtained by interpolating the absorbance on a calibration curve obtained with Trolox (30- Three independent experiments in triplicate were performed for each assayed extracts and results were expressed as Trolox-equivalent antioxidant capacity (TEAC; mmols of Trolox with the same antioxidant capacity as 100 g of dry matter). Color analysis Digital image analysis of color was developed using a DigiEye®, the conditions of image capture and lighting were previously reported [11-13]. In order to obtain CIELAB coordinates from RGB color space, the software DigiFood® [15] and the average of 18 measurements, were used. Statistical analysis The statistical analysis was done using the Statgraphics Plus version 5.1® (Manugistics, Inc., Rockville, MA, USA) software. Univariate analysis of variance (LSD test) was applied to establish: if phenolic composition differed significantly between roselle cultivars. Moreover, pattern recognition (PR) techniques, like stepwise linear discriminant 1 2 3 61 62 63 64 65 6 12 29 34 51 4 121 5 6 7 122 8 9 123 10 11 124 13 14 125 15 16 126 17 18 19 127 20 21 128 22 23 24 129 25 26 130 27 28 131 30 31 132 32 33 133 35 36 134 37 38 135 39 40 41 136 42 43 137 44 45 46 138 47 48 139 49 50 140 52 53 141 54 55 142 56 57 58 143 59 60 144 analysis (SLDA), was performed in order to classify roselle cultivars according to phenolic compounds, antioxidant capacity and color parameters. Results and discussion Phenolic compounds in roselle cultivars Chromatographic method allowed the separation of up to 24 compounds: two hibiscus acids, six phenolic acids, six flavanols, eight flavonols and two anthocyanins for all cultivars, except for Blanca cultivar, where anthocyanins were not found (Online Resource 1). Phenolic compounds of pigmented roselle extract have been widely studied [3, 14-16]; however, the phenolic compounds content in native Mexican cultivars with different levels of pigmentation have not been reported. Results indicate that Negra cultivar showed the highest content of hibiscus acid, phenolic acids and flavonols comparing to Sudan, Rosa and Blanca cultivars (p < 0.05). Sudan cultivar showed the highest anthocyanins content (3.02 ± 0.19 g/100g) followed by Negra (2.45 ± 0.43 g/100g) and Rosa (0.50 ± 0.06 g/100g) cultivars. Blanca cultivar showed higher flavanols content (p < 0.05) than the other analyzed cultivars. Dp-3-Sa and Cy-3-Sa were identified as the only anthocyanins in the cultivars analyzed. Dp- 3-Sa content was up to three times more than Cy-3-Sa in colored chalices; these pigments were also reported in red calyces of roselle from Mexico [16] and Senegal [16, 17]. The differences observed between our samples and previously reported in the number and concentration of the identified compounds may be due to the chemical complexity and/or the method of extraction and analysis [2], since the content of compounds is influenced by both internal (genotype) as external (environment, cultivation and storage) factors [17,18]. Online Resource 2 shows the average normalized area of extraction efficiency of chemical compounds in roselle beverages, anthocyanins showed the lowest 1 2 3 61 62 63 64 65 7 12 29 34 51 4 145 5 6 7 146 8 9 147 10 11 148 13 14 149 15 16 150 17 18 19 151 20 21 152 22 23 24 153 25 26 154 27 28 155 30 31 156 32 33 157 35 36 158 37 38 159 39 40 41 160 42 43 161 44 45 46 162 47 48 163 49 50 164 52 53 165 54 55 166 56 57 58 167 59 60 168 extraction efficiency, near of 4%, while phenolic acids presented an extraction efficiency in a range of 30 to 60%. Proportion of phenolics and flavonols in roselle beverages of Negra, Sudan, Rosa and Blanca cultivars was analyzed (Online Resource 2).Chemical data about roselle beverages showed high phenolic compounds content represented as hibiscus and phenolic acids, flavanols, flavonols and anthocyanins in pigmented cultivars, the significant amounts of phenolics can be an important issue to develop of functional foods and, benefits to human health. Antioxidant capacity of roselle cultivars Negra cultivar showed higher values (p < 0.05) for FRAP assay than Sudan and Rosa cultivars; on the other hand, Negra and Sudan cultivars had the highest values for ABTS assay (Online Resource 1). Methanolic extracts include a closer determination of a total antioxidant activity present in roselle calyx, while aqueous extracts refer to the activity present in beverage. Beverages showed higher antioxidant properties compared to those found in calix, values were up to 50% and 60% for ABTS and FRAP assays, respectively (Online Resource 2). It is noteworthy that is not easy the comparison of antioxidant activity data between obtained in this study and other previously published, due that the content of these kind of compounds could be influenced by extraction conditions and, samples origin (geographic location) [18]. Antioxidant capacity of roselle extracts could be associated to efficiency of chlorogenic acid derivatives and anthocyanins as reducing agents [14, 15]. Correlation analysis showed a positive and significant relationship (p < 0.05, r > 0.9) between antioxidant capacity and chemical compounds, these results are similar to those reported by Mohd-Esa et al [2]. 1 2 3 59 60 61 62 63 64 65 8 12 29 34 51 4 169 5 6 7 170 8 9 171 10 11 172 13 14 173 15 16 174 17 18 19 175 20 21 176 22 23 24 177 25 26 178 27 28 179 30 31 180 32 33 181 35 36 182 37 38 183 39 40 41 184 42 43 185 44 45 46 186 47 48 187 49 50 188 52 53 189 54 55 190 56 57 58 191 Color Analysis The color data were plotted in a CIELAB diagram (Figure 1), pigmented cultivars overlap each other due to anthocyanins presence, color data representation converged to redness. Rosa cultivar showed higher value of chroma (p < 0.05) than the other cultivars. The color intensity of the pigmented samples was similar, however, anthocyanins content showed significant differences (p < 0.05), the co-pigmentation is suggested, as it naturally occurs in anthocyanins containing plants. Christian and Jackson [1] reported color values similar to found in this study using a Lab Scan XE 1669 Hunter colorimeter. We propose DIA as a useful, non-invasive and fast tool for color determination in roselle cultivars with different pigmentation degrees, moreover, minimal sample processing is required and it may be applied to other food and pharmaceutical products. Color-composition relationships At first, using simple correlations (Person coefficient), the relationship between color parameters of roselle cultivars and their anthocyanin content, was analyzed. The p value for anthocyanin content in methanolic extracts and, color parameters (a*, b* and C*ab), showed significant relationship (p < 0.05), but the same parameters in aqueous extracts were not significant. To achieve accurate evaluation of the correlation between color and pigments, multiple linear regressions were calculated. Color parameters were accounted as dependent variables while anthocyanin were considered independent variables, to determinate total and individual anthocyanin content, simple models were proposed (p < 0.01) (Table 1). Moreover, models to evaluate the extraction efficiency were designed; however, those models did not show good correlation coefficients (data not showed). It has been reported that the use of high number of variables allows a better predictive model [13]. The 1 2 3 58 59 60 61 62 63 64 65 15 48 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 294 23 24 25 295 26 27 28 296 29 30 31 297 32 33 34 298 35 36 37 38 299 39 40 41 300 42 43 44 301 45 46 47 302 49 50 51 303 52 53 54 304 55 56 57 305 Figure 2. Representation of roselle beverages in the space defined by the first and second discriminant functions obtained from chemical and color data. 1 2 3 4 5 6 58 59 60 61 62 63 64 65 14 4 306 5 Table 1. Color composition relation between anthocyanins content and CIELAB (L*, a* and 6 7 307 8 b*) and polar coordinates (C* and h ab) for roselle cultivars (p<0.01). 9 10 11 12 13 14 15 16 17 18 19 Total 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Beverage 36 37 38 39 40 41 42 43 44 Model R 0.86 0.91 0.90 0.92 0.88 0.84 0.92 0.92 0.90 0.87 45 308 46 47 309 48 49 50 310 51 52 53 54 311 55 56 57 312 Dp3Sa: delphinidin-3-sambubioside and Cy3Sa: cyanidin-3-sambubioside; TA: total anthocyanins 0.92 ab 0.90 1 2 3 58 59 60 61 62 63 64 65 15 ab 8 18 20 7 314 9 315 10 Table 2. Linear discriminant analysis (LDA) and stepwise linear discriminant analysis (SLDA) to classify Negra, Sudan, Rosa and Blanca roselle cultivars. 11 12 Data LDA SLDA 13 14 % of classification % of classification Retained variables 15 16 Chromatographic a 100 100 Ha, Pa, Fa, An 17 Color b 100 100 L * , C * , hab 19 Antioxidant c 75 75 ABTS, FRAP 21 Total d 100 100 L * ,C * b , h ab , An, FRAP 22 a 23 316 24 a Group of compounds: Hibiscus acids, phenolic acids, flavanols, flavonols and 25 317 26 anthocyanins. b Polar coordinates (L*, C* and hab). c FRAP and ABTS assays.d 27 318 28 29 319 30 31 320 32 33 34 35 321 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Chromatographic, color and antioxidant data. Abbreviations: Ha: hibiscus acids, Pa: phenolic acids, Fa: flavanols, An: anthocyanins. ab 1 2 3 4 5 6 313 58 59 60 61 62 63 64 65 16 Manuscript title: Comparative study of phenolic profile, antioxidant capacity, and color-composition relation of roselle cultivars with contrasting pigmentation Manuscript title: Comparative study of phenolic profile, antioxidant capacity, and color-composition relation of roselle cultivars with contrasting pigmentation Manuscript title: Comparative study of phenolic profile, antioxidant capacity, and colorcomposition relation of roselle cultivars with contrasting pigmentation Manuscript title: Comparative study of phenolic profile, antioxidant capacity, and colorcomposition relation of roselle cultivars with contrasting pigmentation Manuscript title: Comparative study of phenolic profile, antioxidant capacity, and colorcomposition relation of roselle cultivars with contrasting pigmentation Manuscript title: Comparative study of phenolic profile, antioxidant capacity, and color-composition relation of roselle cultivars with contrasting pigmentation Manuscript title: Comparative study of phenolic profile, antioxidant capacity, and colorcomposition relation of roselle cultivars with contrasting pigmentation