Chemical characterisation of anthocyanins in tamarillo (Solanum betaceum Cav.) and Andes berry (Rubus glaucus Benth.) fruits

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 Chemistry on 15 June 2012, available at: https://doi.org/10.1016/j.foodchem.2011.12.026 .” 1 Chemical characterisation of anthocyanins in tamarillo (Solanum betaceum Cav.) and Andes berry (Rubus glaucus Benth) fruits CORALIA OSORIO, NELSON HURTADO, CORINNA DAWID, THOMAS HOFMANN, FRANCISCO JOSÉ HEREDIA-MIRA,§ AND ALICIA LUCIA MORALES Departamento de Química, Universidad Nacional de Colombia, AA 14490, Bogotá, Colombia; Chair of Food Chemistry and Molecular Sensory Science, Technische Universität München, Lise-Meitner-Strasse 34, D-85354 Freising-Weihenstephan, Germany; and Laboratory of Food Colour & Quality, University of Seville, 41012, Sevilla, Spain Title running header: Anthocyanins from Solanum betaceum and Rubus glaucus * Author to whom correspondence should be addressed: C. Osorio. Telephone: +57-1- 3165000, ext. 14472. Fax: +57-1-3165220. E-mail: [email protected] Departamento de Química, Universidad Nacional de Colombia. Technische Universität München § University of Seville 2 1 ABSTRACT 2 3 The anthocyanin composition of tamarillo (Solanum betaceum Cav., red variety) 4 and Andes berry (Rubus glaucus Benth) was determined by HPLC-PDA and HPLC- 5 ESIMS. From the anthocyanin-rich extracts (AREs), pure compounds (1-7) were obtained 6 by MLCCC (multilayer countercurrent chromatography) and further preparative HPLC, and 7 their unequivocal structures were obtained by 1D and 2D NMR analyses. The new 8 anthocyanin delphinidin 3-O- -L-rhamnopyranosyl- - -D-glucopyranoside- -O- - 9 D-glucopyranoside, as well as the known cyanidin-3-O-rutinoside, pelargonidin-3-O- 10 rutinoside, and delphinidin-3-O-rutinoside were identified as constituents of tamarillo fruit. 11 Although the anthocyanin composition of Andes berry had been reported before in the 12 literature, the unequivocal structure elucidation of the major compound, cyanidin-3-O- -L- 13 rhamnopyranosyl-(1 6)-O- -D-xylopyranosyl-(1 2)- -D-glucopyranoside, was achieved 14 for the first time. 15 16 Keywords: Anthocyanins, Solanum betaceum, Tamarillo, Rubus glaucus Benth, Andes 17 berry. 3 18 1. Introduction 19 20 Anthocyanins are the largest group of water-soluble pigments in the plant kingdom. 21 They are responsible for most red and blue colours in fruits and vegetables, and recently, 22 have been used in the food industry as pigments, because of their bright attractive colours, 23 high water solubility, and associated health benefits (Castañeda-Ovando et al., 2009). It is 24 important to note that the studies on the sensory and biofunctional properties of tropical 25 fruits have increased due to their novelty (Sousa De Brito et al. 2007; Mahattanatawee et 26 al., 2006). These fruits and their derived-products have conquered new markets in 27 subtropical regions because consumer interests have turned to the natural foods. 28 Solanum betaceum Cav. syn Cyphomandra betacea Sendt. (Solanaceae) is a shrub 29 native to the Andes, specifically in Peru, Ecuador, and Colombia, that belongs to the 30 Solanaceae family. In Colombia, the most common varieties are the traditional yellow- 31 fleshed, and the red one (yellow fleshed with red peel and purple jelly) commonly named as 32 tamarillo (Vasco, Avila, Ruales, Svanberg & Kamal-Eldin¸ 2009). The red-fleshed fruit is 33 the most commercialised variety in Colombia and one of the highly-marketed Colombian 34 tropical fruits in Europe. The fruit is ovoid in shape, a length of 6-8 cm, and a diameter of 35 4-5 cm; when ripe, its peel is dark red, and exhibits a slightly bitter, sour, and astringent 36 taste with a delicate and characteristic aroma. It is generally consumed fresh, or blended 37 together with water and sugar to make juices and desserts. Early studies on phenolics 38 constituents of tamarillo fruit from New Zealand, which were performed by paper 39 chromatography and TLC (thin layer chromatography), reported the presence of 3- 40 rutinosides and 3-glucosides of pelargonidin, cyanidin and delphinidin (Wrolstad & 4 41 Heatherbell, 1974); whereas pelargonidin 3-glucosyl-glucose, peonidin 3-glucosyl-glucose, 42 and malvidin 3-glucosyl-glucose were identified in tamarillo fruits from Brazil by UV-Vis 43 spectrophotometry and TLC (Bobbio, Bobbio & Rodriguez-Amaya, 1983). Recently, 44 delphinidin 3-rutinoside, cyanidin 3-rutinoside, and pelargonidin 3-glucoside-5-rhamnoside 45 (tentatively) were identified by LC/MS from fruits of Brazil (Vera de Rosso & Mercadante, 46 2007). In fruits from Ecuador, the presence of the anthocyanins, delphinidin glucosyl 47 rutinoside, delphinidin rutinoside, cyanidin rutinoside, and pelargonidin rutinoside; the 48 hydroxycinnamic acids, dicaffeoylquinic acid, caffeoylquinic acid, caffeoyl glucose, and 49 feruloyl glucose; as well as, the esterified carotenoids, lutein and -cryptoxanthin were 50 reported (Mertz et al, 2009; Mertz, Brat, Caris-Veyrat & Gunata, 2010). As can be seen, 51 numerous studies on tamarillo anthocyanins have been performed, but little attention has 52 been paid to the isolation and unequivocal identification by spectroscopic methods. 53 Rubus glaucus Benth (Rosaceae), commonly k 54 (Figure 1) is a berry native to South America which is found between 2600 and 55 3100 meters above the sea level and highly consumed in Colombia (annual production 56 surpassing 10000 ton.). The fruit consists of numerous small drupes on a receptacle about 57 1- 2.5 cm long, which are dark-red or purple. It is characterised by an intense aroma and 58 sweet-sour taste; however, this fruit is highly perishable and susceptible during postharvest 59 handling. This fact has motivated the development of processed products with higher shelf- 60 life time as strategy to overcome those problems. The phenolic composition of this fruit and 61 other related ones have been studied by different research groups (Mertz, Cheynier, Günata 62 & Brat, 2007; Vasco, Riihinen, Ruales & Kamal-Eldin, 2009; Cuevas-Rodríguez et al., 5 63 2010). Thus, high amounts of ellagitannins and proanthocyanidins have been found, as well 64 as hydroxycinnamic acid derivatives, flavonols (mainly quercetin glycosides), and 65 anthocyanins. The main anthocyanins were cyanidin-3-O-glucoside (67% of total 66 anthocyanin content), and cyanidin-3-O-rutinoside (31% of total anthocyanins). Minor 67 anthocyanins were tentatively reported as cyanidin-3-O-malonyl glucoside and a 68 pelargonidin derivative. Garzón, Riedl and Schwartz (2009) reported the anthocyanin 69 content of Andes berry as 45 mg/100 g FW and cyanidin 3-sambubioside, cyanidin 3- 70 glucoside, cyanidin 3-xylorutinoside, cyanidin 3-rutinoside, pelargonidin 3-glucoside, and 71 pelargonidin 3-rutinoside as major constituents. All of the above reported data were 72 obtained only by HPLC-MS analyses, so the unequivocal anthocyanin composition of 73 Rubus glaucus Benth is still unknown because the lack of NMR studies on the purified 74 compounds. 75 Tamarillo and Andes berry fruits are a promising source of natural colorants (Osorio 76 et al., 2007), and they have exhibited in vitro antioxidant activity (Vasco, Ruales & Kamal- 77 Eldin, 2008; Mertz et al, 2009; Hurtado, Morales, González-Miret, Escudero-Gilete & 78 Heredia, 2009) that gives them an interesting added-value. Thus, as part of our ongoing 79 studies on the pigment composition of Colombian fruits (Osorio et al., 2010; Barrios et al., 80 2010; Jaramillo, Dawid, Hofmann, Fujimoto & Osorio, 2011), the anthocyanin composition 81 of the above-mentioned fruits was investigated, by purification and subsequent 82 spectroscopical analyses of these compounds. 83 84 2. Materials and methods 85 6 86 2.1. General 87 88 The 1D and 2D NMR spectra were obtained at 303 K using a 400 MHz (DRX) and 89 a 500 MHz Avance III (Bruker, Rheinstetten, Germany) spectrometers in 90 CD3OD/CF3COOD (19:1, v/v, and 98:2 v/v for compound 7) with solvent peaks as 91 references. The 13C signal and the 1H signal of the CD3OD were used as secondary 92 references ( C 49.3 and H 3.35 ppm). For structural elucidation and NMR signal 93 assignment in 2D NMR experiments, such as COSY-, DEPT-, TOCSY-, ROESY, g- 94 HSQC-, and g-HMBC-spectroscopy were carried out using the pulse sequences taken from 95 the Bruker software library. Data processing was performed by using XWin-NMR software 96 (version 3.5; Bruker, Rheinstetten, Germany) as well as Mestre-C (Mestrelab Research, A 97 Coruña, Spain). HPLC analyses were performed on an instrument HP 1100 series (Hewlett 98 Packard, Palo Alto, CA, USA) equipped with photodiode array detector (PDA). For 99 preparative purposes, an HPLC apparatus consisting of a Merck-Hitachi L-6000A pump, a 100 Rheodyne injection valve with a 500 L loop, and a Merck-Hitachi UV-Vis L-4250 101 detector, was used. The CCC system was a multilayer coil countercurrent chromatograph 102 series 521 (P.C. Inc., Potomac, MD, USA). A UV-Vis HP 8452 spectrometer was used for 103 monitoring MLCCC fractions. ESI-MS analyses were performed on a Shimadzu QP-8000 104 (Shimadzu Corp. Kyoto, Japan). FAB-MS analyses of pure compounds were performed in 105 an AutoSpecQ spectrometer by using positive mode, argon as collision gas and glycerol- 106 sodium iodide as matrix. 107 7 116 118 119 HPLC grade acetonitrile, and ACS grade n-butanol, methanol, and tert- 120 butylmethylether (TBME) were purchased from Merck (Darmstadt, Germany). Water for 121 chromatographic separation was purified by means of a Milli-Q water advantage A 10 122 water system (Millipore, Molsheim, France). For LCMS analyses, acetonitrile, water, and 123 formic acid were purchased from Honeywell Burdick and JacksonTM (Muskegon, Michigan, 124 USA). The following compounds were obtained commercially from the sources given in 125 parentheses: hydrochloric acid (Merck, Darmstadt, Germany); CD3OD and CF3COOD 126 (Euriso-top, Saarbrücken, Germany); CF3CO2H (TFA) (Aldrich Chemical Company, 127 Milwaukee, WI, USA); sodium hydroxide, glucose, and rhamnose (Fluka, Steinheim, 128 129 Germany); and xylose (Lancaster, Eastgate, England). 130 2.4. Isolation of anthocyanin-rich extracts (AREs) 108 2.2. Plant material 109 110 Ripe tamarillo fruits (red variety, peel 100% red, pH 3.5) were collected in Puente 111 Nacional, Santander, Colombia. A voucher specimen was coded as COL 510177 at the 112 Instituto de Ciencias Naturales, Universidad Nacional de Colombia. Pulp, peelings, seeds, 113 and jelly were manually separated. Mora fruits were purchased from different local markets 114 in Chinchiná, Caldas, Colombia, and selected according to their colour ripeness qualities 115 (more than 75% dark-red to wine red), good consistency, and shape. 117 2.3. Chemicals and reagents 8 131 132 Tamarillo fruits (5 kg) were washed, and after removal of peel and seeds, the jelly 133 was manually separated from the flesh and diluted with water (1:1, w/w). Then, portions of 134 250 g were separately applied to an 80 x 4 cm Amberlite XAD-7 resin open column (Rohm 135 and Haas, Darmstadt, Germany). The column was rinsed with water, and the adsorbed 136 pigments were eluted with 1 L of methanol-acetic acid (19:1, v/v), according to the 137 procedure by Degenhardt, Knapp, and Winterhalter (2000). Methanol was removed under 138 vacuum at 35°C and the residue was freeze-dried. The final product was 4.0 g of 139 anthocyanin-rich extract (ARE). Whole Andes berry fruits (945 g) were processed as it was 140 141 before described to obtain 13.7 g of ARE. 142 143 2.5. Fractionation of anthocyanin-rich extracts 144 The ARE of tamarillo was separately fractionated by MLCCC (multilayer 145 countercurrent chromatography) in portions of 0.6 g. The solvent system was a mixture of 146 TBME/n-butanol/acetonitrile/water (2:2:1:5, v/v/v/v, acidified with 0.1% TFA, v/v). A 147 single coil (75 x 2.6 mm i.d. PTFE tubing, total volume approx. 410 mL) was used and the 148 revolution speed was set to 800 rpm. The less dense layer was always used as the stationary 149 phase and flow rate of mobile phase was 1.0 mL/min. Eighty fractions (4 mL each one) 150 were collected and pooled based on their UV-Vis absorption as follows: F1 (1-8), F2 (9- 151 24), F3 (25-48), F4 (49-67), F5 (67-80), and F6 (stationary phase). In the same way, 4 g of 152 Andes berry ARE were separately fractionated in portions of 1 g, and ninety fractions of 153 5mL were obtained and pooled as mentioned: F1 (1-11), F2 (12-15), F3 (16-30), F4 (31- 15 287 spectrum of this compound (Table 2) showed characteristic signals of a p-disubstituted 288 benzene ring at H 6.80 (d, J = 8.3 Hz, 2H) and 7.37 (d, J = 9.5 Hz, 2H), as well as those of 289 an E-olefinic bond at H 6.28 (d, J = 15.7 Hz) and 7.59 (d, J = 15.7 Hz), thus confirming the 290 presence of a p-coumaroyl residue in this compound. The 1H NMR data of sugar moiety 291 were in agreement with the data of compound 1, evidencing the presence of a 3-rutinoside 292 moiety. Due to the tiny amount of this compound 13C and 2D-NMR spectra were not 293 acquired, so the attached position of p-coumaroyl residue was not able to be defined. Thus, 294 compound 2 was tentatively identified as pelargonidin-p-coumaroyl-rutinoside. 295 Based on 1D- and 2D-1H and 13C-NMR data (Table 3), the identities of the main 296 compounds were found to be delphinidin-3-O- -O- -rhamnopyranosyl)- - 297 glucopyranoside (3), cyanidin-3-O- -O- -rhamnopyranosyl)- -glucopyranoside (4), and 298 pelargonidin-3-O- -O- -rhamnopyranosyl)- -glucopyranoside (5). 1H-NMR data of 299 these compounds are not shown because the values for aglycon protons were in agreement 300 with those published previously by Andersen and Fossen (2001), and the data of sugar 301 moieties were similar to compound 1. These results partially agree with those obtained for 302 the tamarillo from Brazil (Vera de Rosso & Mercadante, 2007). They only used HPLC- 303 PDA-MS/MS for the analysis and reported delphinidin 3-rutinoside, cyanidin 3-rutinoside, 304 and pelargonidin-3-glucoside-5-rhamnoside as major tamarillo pigments. In the present 305 306 work, the structure of the anthocyanin 5 could be unequivocally established. 307 308 3.2. Identification of anthocyanins in Andes berry fruit 16 309 The contents of the major anthocyanins in Andes berry fruit are seen in Table 1. 310 The HPLC-PDA profile at 520 nm (Figure 2B) revealed the presence of two major 311 anthocyanins in Andes berry ARE, cyanidin-3-O- -O- -rhamnopyranosyl)- - 312 glucopyranoside (4) and compound 7. 313 The UV-Vis spectra of the compound 7 showed typical absorption maxima at 271 314 and 516 nm. LC-MS analyses evidenced a pseudomolecular ion in the ESI+ mode [M]+ at 315 m/z 727. The fragment ions at m/z 287 [M-132-146-162]+, 449 [M-132-146]+, 581 [M- 316 146]+, and 595 [M-132]+ further revealed the presence of one hexose, one methylpentose, 317 and one pentose. Moreover the fragment ion at m/z 287 is in agreement with cyanidin being 318 the aglycone. For structure elucidation and NMR signal assignment 2D NMR 319 measurements were performed. The cyanidin moiety was confirmed from the protons of the 320 two 3H ABX systems in the 1H-NMR spectrum. The first typical 3H ABX system could be 321 seen for H-C(6´) at 8.29 ppm (dd, 1H, J = 8.6 Hz, 2.4 Hz), H-C(2´) at 8.02 ppm (d, 1H, 322 J = 2.3 Hz) and H-C(5´) at 7.02 ppm (d, 1H, J = 8.8 Hz) and the second 3H ABX system 323 for H-C(4) at 8.87 (brs, 1H), H-C(8) at 6.89 (d,1H, J = 2.0 Hz) and H-C(6) at 6.66 (d, 324 1H, J = 1.9 Hz). Additionally, the 1H NMR spectrum of compound 7 displayed the three 325 anomeric protons of one hexosyl, one methylpentose, and one pentose moieties, resonated 326 at H 5.45, 4.63, and 4.76 ppm. The anomeric proton of the rhamnosyl moiety showed a 327 coupling constant of 1.6 Hz, which indicates an -configuration, while the glycosyl and 328 xylosyl moieties showed coupling constants of 7.5 and 7.6 Hz, thus indicating - 329 configurations. The identification of glycosidically bound carbohydrates was further 330 confirmed by acid hydrolysis followed by high performance ion chromatography of the 17 331 corresponding monosaccharides. The sugars of compound 7 were determined to be glucose, 332 rhamnose, and xylose by comparison with authentic compounds. The assignment of the 333 sugar moieties as well as the linkage of the sugar to the aglycone was performed by COSY- 334 , HMQC-, HMBC-, and TOCSY-experiments. On the basis of the heteronuclear data, the 335 structure of compound 7 was identified as cyanidin-3-O-(2´´-O- -D-xylopyranosyl-6´´-O- 336 -L-rhamnopyranosyl- -D-glucopyranoside) previously reported in fruit skin of Kadsura 337 japonica (Ishikura, 1971), red currant Ribes rubrum L. (Goiffon, Mouly & Gaydou, 1999), 338 berries of Viburnum opulus L. (Jordheim, Giske, & Andersen, 2007), and black raspberry 339 and raspberry extracts (Tulio et al., 2008), among others. However, to the best of our 340 knowledge this is the first time that is reported as a constituent of Rubus glaucus Benth. 341 With an analysis similar to that described above and based on ESI-MS and 1H and 342 13C NMR data, the identity of anthocyanins 5 and 6 was elucidated as pelargonidin-3-O- 343 -O- -rhamnopyranosyl)- -glucopyranoside, and cyanidin-3-O- -D-glucopyranoside, 344 respectively. The NMR data were in agreement with those before published (Andersen & 345 346 Fossen, 2001). 347 4. Conclusion 348 349 The novel delphinidin 3-O- -L-rhamnopyranosyl- -D-glucopyranoside- - 350 O- -D-glucopyranoside was isolated and identified for the first time in tamarillo ARE 351 (Solanum betaceum Cav.) as a minor constituent. In addition, the previously reported 3-O- 352 rutinosides of cyanidin, delphinidin, and pelargonidin were isolated and their identification - 18 353 was mainly based on 2D-NMR spectroscopy and MS. 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Identification of anthocyanins and distribution 459 of flavonoids in tamarillo fruit (Cyphomandra betaceae (Cav.) Sendt.). Journal of the 460 Science of Food Agriculture, 25(10), 1221-1228. 23 Figure Captions Fig. 1. a) Andes berry (Rubus glaucus Benth) and b) tamarillo (Solanum betaceum Cav., red variety) fruits. Fig. 2. HPLC analyses ( = 520 nm) of a) ARE of tamarillo (Solanum betaceum Cav.) fruit, and b) Andes berry (Rubus glaucus Benth.) fruit. Peak numbers correspond to the compound numbers in Table 1. Fig. 3. Chemical structure of tamarillo and mora anthocyanins. Glc = glucose, Xyl = xylose. 24 Figure 1. A B 31 Table 3 C 1 a 3 a 4 a 5 a 7 b Aglycone 2 162.9 163.8 163.2 163.4 164.3 3 144.5 145.8 144.3 144.2 145.4 4 136.9 135.7 136.5 137.6 135.5 5 157.4 158.0 156.9 157.1 159.1 6 103.4 103.2 103.4 103.5 103.5 7 170.3 170.3 168.4 170.1 170.4 8 95.2 93.8 95.4 95.1 95.2 9 155.8 156.3 156.2 156.5 157.6 10 112.7 113.0 111.8 112.0 113.1 119.5 120.0 120.1 119.8 121.3 113.0 112.8 118.4 135.7 118.6 146.6 147.7 146.2 117.5 147.6 156.4 144.8 154.6 165.7 156.0 145.2 147.7 117.7 117.5 117.4 114.4 112.8 128.6 135.7 128.9 3-O-glucoside 103.6 103.5 103.8 104.0 101.3 76.5 74.6 74.9 74.4 81.7 77.9 76.6 77.5 74.8 77.4 71.2 77.6 78.0 77.2 71.0 74.2 72.4 74.0 73.6 77.9 67.9 67.8 67.7 67.6 67.7 -O-rhamnosyl 102.4 102.1 102.2 102.1 102.2 71.9 69.7 72.6 72.5 71.9 72.3 73.9 70.5 73.9 72.5 74.7 71.3 71.2 69.7 73.9 69.8 67.7 67.7 71.3 69.8 17.9 17.8 17.9 17.9 17.9 -O-glucoside 104.5 72.3 74.9 71.2 74.0 62.8 2´´-O-xylofuranosyl 1´´´´ 105.6 13C NMR chemicals shifts for the anthocyanins 1, 3-5, 7 ( in ppm). 32 a CD3OD-CF3COOD (19:1, v/v), 125 MHz, b CD3OD- CF3COOD (98:2, v/v), 125 MHz. 2´´´´ 75.8 3´´´´ 78.1 4´´´´ 70.9 5´´´´ 67.3