Food Chemistry 132 (2012) Contents lists available at SciVerse ScienceDirect. Food Chemistry

Food Chemistry 132 (2012) 1908 1914 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Colour properties, stability, and free radical scavenging activity of jambolan (Syzygium cumini) fruit anthocyanins in a beverage model system: Natural and copigmented anthocyanins Puspita Sari a,, Christofora Hanny Wijaya b, Dondin Sajuthi c, Unang Supratman d a Department of Agricultural Products Technology, Faculty of Agricultural Technology, Jember University, Kampus Tegalboto Jl. Kalimantan I, Jember, East Java 68121, Indonesia b Department of Food Science and Technology, Faculty of Agricultural Technology, Bogor Agricultural University, Kampus IPB Darmaga, Bogor 16680, Indonesia c Faculty of Veterinary Medicine, Bogor Agricultural University, Kampus IPB Darmaga, Bogor 16680, Indonesia d Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Jl. Raya Bandung-Sumedang Km. 21, Jatinangor 45363, Indonesia article info abstract Article history: Received 13 May 2011 Received in revised form 29 November 2011 Accepted 8 December 2011 Available online 19 December 2011 Keywords: Syzygium cumini Anthocyanins Intermolecular copigmentation Colour enhancement and stability Bathochromic shift Hyperchromic effect Antioxidant activity The colour and stability properties of jambolan anthocyanins, both natural and copigmented forms, were investigated in beverage model as well as their radical scavenging ability. Natural anthocyanins of jambolan revealed low colour intensity due to glycosylation structure of the anthocyanins as diglucoside. The intermolecular copigmentation of anthocyanins with sinapic acid, caffeic acid, ferulic acid, and rosemary polyphenolic extract could enhance the colour intensity, which was observed through spectrometric parameters, such as hyperchromic effect (DA vis-max ) and bathochromic shift (Dk vis-max ). In addition of sinapic acid, caffeic acid, and rosemary polyphenolics also increased the stability of the anthocyanin colour during exposure to white fluorescent light and storage at refrigeration and room temperatures, whereas on high thermal treatments, this phenomenon was not observed. Furthermore, beverage model coloured with natural or copigmented anthocyanins revealed DPPH radical-scavenging activity. The AEAC values indicated that the beverage models with copigmented anthocyanins had higher scavenging activity than the beverage with natural anthocyanins. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Abbreviations: Acy, anthocyanin (natural); Acy + Caf, anthocyanin + caffeic acid; Acy + Fer, anthocyanin + ferulic acid; Acy + RP, anthocyanin + rosemary polyphenolic; Acy + Sin, anthocyanin + sinapic acid; AE, ascorbic acid equivalent; AEAC, ascorbic acid equivalent antioxidant capacity; CL, colour loss; CyE, cyanidin-3- glucoside equivalent; DPPH, 2,2-diphenyl-1-picrylhydrazyl; GAE, gallic acid equivalent; PC, polymeric colour; UV, ultraviolet; w/v, weight per volume. Corresponding author. Tel./fax: +62 331 321784. E-mail address: poespitha_s@yahoo.com (P. Sari). Anthocyanins are the best-known natural red colourants used in food due to their bright and attractive colours, non-toxicity, and water solubility, which allows their incorporation into aqueous food systems. However, they have stability problems. The colour and stability of anthocyanins are affected by several factors such as the chemical structure, concentration, ph, temperature, light, presence of copigments, metal ions, enzymes, oxygen, ascorbic acid, sugars and their degradation products, and sulphur dioxide (Cavalcanti, Santos, & Meireles, 2011; Markakis, 1982; Shipp & Abdel-Aal, 2010). Commonly, the colour of anthocyanins can be enhanced and stabilised through copigmentation reactions. Anthocyanin copigmentation gives bluer, brighter, stronger, and more stable colours than those of natural anthocyanin. Copigmentation can occur through several interactions, i.e. intramolecular interactions, which an organic acid, an aromatic acyl group, or a flavonoid (or some combination thereof) is covalently linked to an anthocyanin chromophore, or through loose intermolecular interactions, which colourless flavonoids or other phenolic compounds interact through weak hydrophobic forces with anthocyanins. Self-association and metal complexation are also possible means through which copigmentation occurs (Castaňeda-Ovando, Pacheco-Hernández, Páez-Hernández, Rodríguez, & Galán-Vidal, 2009; Eiro & Heinonen, 2002). The copigments can be flavonoids, organic acids, metals, or other anthocyanins (Castaňeda-Ovando et al., 2009). Beside their food colouring roles, anthocyanins are also important as antioxidant, which plays an important role in the prevention of many degenerative diseases (Castaňeda-Ovando et al., 2009; Shipp & Abdel-Aal, 2010). Incorporating anthocyanins as food colourants is not only valuable for improving overall appearance but also very beneficial to health (Shipp & Abdel-Aal, 2010). In recent years, the interest in anthocyanin pigments has dramatically increased because of their possible benefits for health. 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.12.025

P. Sari et al. / Food Chemistry 132 (2012) 1908 1914 1909 Jambolan (Syzygium cumini) fruit, a tropical fruit found in Indonesia, is rich in anthocyanin pigments especially in its peel. The content of total monomeric anthocyanin in the peel of ripe fruit was 731 mg/100 g of fresh weight (Sari, Wijaya, Sajuthi, & Supratman, 2009). Its anthocyanin content was found to be higher than grapes, 6 600 mg/100 g of fresh weight and red cabbage, 25 mg/ 100 g of fresh weight (Giusti & Wrolstad, 2001). Jambolan anthocyanins had been identified as 3,5-diglucosides derivatives of delphinidin, petunidin, malvidin, cyanidin, and peonidin (Brito et al., 2007; Faria, Marques, & Mercadante, 2011; Sari et al., 2009) and determined their antioxidant properties (Banerjee, Dasgupta, & De, 2005; Veigas, Narayan, Laxman, & Neelwarne, 2007). Anthocyanins obtained from jambolan peel have potential as a novel source of natural colourants for food system. However, the colour and stability of jambolan anthocyanins for food uses had not been fully characterised. The objective of this present study was to evaluate the colour and stability properties of jambolan anthocyanins, both natural and copigmented forms. Pigment stability was tested toward the influence of heating temperature, exposure of white fluorescent light, and storage conditions. It was also of interest to investigate the radical scavenging activity of these anthocyanins incorporated in a beverage model system. 2. Materials and methods 2.1. Chemicals and reagents Folin Ciocalteau reagent, organic solvents, acids, and salts used were pro analysis purchased from Merck (Darmstadt, Jerman). Caffeic acid, sinapic acid, ferulic acid, and DPPH (2,2-diphenyl-1-picrylhydrazyl) were obtained from Sigma Aldrich (St. Louis, MO). 2.2. Sample preparation Fully ripened jambolan fruits were obtained from a traditional market in Jember district, Indonesia. Rosemary (Rosmarinus officinalis) leaves were obtained from Aljazaire. Jambolan fruits were selected for their highly coloured peel (deep purple), washed, manually peeled, and steam-blanched at approximately 80 C for 4 min. Meanwhile, the rosemary leaves were dried at room temperature and crushed to a 60 mesh powder. Jambolan peels and rosemary dried powder were separately packed in polyethylene plastic and stored at 20 C. 2.3. Anthocyanin extraction Jambolan peels were thawed at room temperature and homogenised in a high-speed hand blender (Braun, Spain). The pigments were extracted with ethanol 98% (material to solvent ratio of 1:2, w/v) under stirring for 1 h, followed by centrifugation at 3552g for 10 min. Then, the supernatant was collected and the residue was re-extracted (three times). The supernatants were vacuum-filtered then concentrated in a rotavapor at 40 C under reduced pressure to obtain aqueous extract by addition of distilled water. The monomeric anthocyanin content of aqueous extract was 4.37 mg CyE/mL, determined by the ph-differential methods (Giusti & Wrolstad, 2001). The total soluble solids and ph values were 11.00 ( Brix%) and 3.74, respectively. 2.4. Extraction of water soluble-rosemary polyphenolic Rosemary polyphenolics were extracted by stirring for 1 h with ethanol 98% (material to solvent ratio of 1:2, w/v) and centrifuged at 3552g for 10 min. The supernatant was collected and the residue was re-extracted (three times). The supernatants were vacuum-filtered and afterwards extracting solvent was removed using rotavapor at 40 C under reduced pressure to dryness. Water solublerosemary polyphenolics were dissolved with distilled water. The rosemary polyphenolic extract contained the total phenolic of 0.53 mg GAE/mg, as determined by Folin Ciocalteau method (Waterhouse, 2002). 2.5. Colour characterisation of natural anthocyanin Colour characterisation of natural anthocyanins was determined on different ph values from 1 to 8. Pigment solutions were prepared at ph 1 8 using potassium chloride buffer solution (0.025 M) for ph values 1 4 and sodium acetate buffer solution (0.4 M) for ph values 5 8. The mixtures were allowed to stand for 1 h at room temperature before spectral measurement at wavelength of 350 700 nm. 2.6. Intermolecular copigmentation treatment Intermolecular copigmentation reaction was conducted according to methods of Eiro & Heinonen, (2002) and Yawadio and Morita (2007) with some modification, using sinapic acid, caffeic acid, ferulic acid, and rosemary polyphenolic extract as copigment. The anthocyanin extract was dissolved in citrate buffer (0.1 M, citric acid sodium citrate) at ph 3.0 and the copigment at the concentrations of each 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 mg/ml. The beverage solution of natural anthocyanin (without copigment addition) was adjusted to give a final absorbance value of 0.6 units at 516 nm (k vis-max ); solutions with copigment addition had higher absorbance values. Subsequently, the solutions of beverage were agitated and equilibrated for 1 h at room temperature. The absorption spectra of beverage model solutions were monitored by using a UV visible spectrophotometer (Genesys 10, Thermo Electron Co., Marietto, OH, USA), scanning the visible wavelength range from 400 to 700 nm. A hyperchromic effect (DAvis-max) was detected as an increase in the absorbance value at k vismax and a bathochromic shift (Dk vis-max ) as a shift of the wavelength (nm) of k vis-max. 2.7. Pigment stability studies Beverage model systems were prepared using the jambolan anthocyanin extract, potassium sorbate as preservative, and citrate buffer (0.1 M, citric acid sodium citrate) at ph 3.0, adjusted to give an absorbance reading of 0.6 units at 516 nm. The copigment was added to contain final concentration in the beverage model of 1 mg/ml. The control sample was a beverage solution without copigment addition (natural anthocyanin). The solutions were agitated, partitioned into 20 ml transparent glass bottles, and allowed to stand for 1 h at room temperature to equilibrate. After reaching equilibrium, the absorbance at k vis-max of solutions were monitored on a UV visible spectrophotometer, and characterised as zero time. The colour stability of anthocyanins, both natural and copigmented forms, was analysed in which the effects of heating temperature, fluorescent light, and storage condition were taken into account. The influence of heating temperature on colour stability was done with test samples inside transparent glass bottles immersed in a water bath at 80 and 98 C for 0, 30, 60, 90, and 120 min. Light effect on colour stability of pigment was performed with test samples inside transparent glass bottles exposed to white fluorescent light of 23 W (Philips lamp) in the box dimension 58 72 60 cm (4000 lux) for 0, 2, 4, 6, 8, and 10 days at 32 C. Control samples for temperature and light assays were test samples inside transparent glass bottles covered with aluminium foil and stored at room temperature (28 C) and 32 C, respectively.

1910 P. Sari et al. / Food Chemistry 132 (2012) 1908 1914 The storage stability of test samples was investigated under storage conditions at refrigeration (7 C) and room temperatures (28 C) during 4 weeks in the dark. The colour loss was calculated from the values of percent colour retention at initial and final treatments. Polymeric colour was determined using the bisulphite bleaching method (Giusti & Wrolstad, 2001). The beverage solutions were bleached with sodium metabisulphite, using water as a control. The polymeric colour was measured at initial and final of treatments, expressed as DPC. L, a, b colour values of the beverage solutions were measured at initial and final of treatments using a Minolta Chroma CR-210 Colourimeter (Gonnet, 1998). Chroma (C =[(a ) 2 +(b ) 2 ] 1/ 2 ) and hue angle (h = arctan (b /a )) were calculated from a and b. Total colour difference (DE) was calculated through the equation of DE =[(DL ) 2 +(DC ) 2 +(DH ) 2 ] 1/2. The reaction rate constants (k) and half-life time (t 1/2 ) values were calculated according to Kirca and Cemeroglu (2003) following a first order kinetics. 2.8. Analysis of total phenolics and monomeric anthocyanins The Folin Ciocalteu method was used to determine phenolic content of beverage solutions, as described by Waterhouse (2002) with some modification. Total phenolics were quantified based on standard curves of gallic acid. The monomeric anthocyanin content of beverage solutions was determined using the phdifferential method (Giusti & Wrolstad, 2001). Absorbance was measured at 520 and 700 nm. Anthocyanin was calculated as cyanidin-3-glucoside using a molar extinction coefficient of 26900 and a molecular weight of 449.2. 2.9. DPPH radical-scavenging activity The free radical scavenging activity of beverage model was determined according to the DPPH method (Yamaguchi, Takamura, Matoba, & Terao, 1998) with some modifications. A solution of beverage (0.05 ml) was added to the test tube containing mixture of 0.95 ml ethanol and 3 ml of 100 lm DPPH solution in ethanol (freshly prepared). After incubation for 15 min at room temperature, the absorbance was immediately measured at 517 nm using a UV visible spectrophotometer. Results were expressed in term of ascorbic acid equivalent antioxidant capacity, AEAC. 3. Results and discussion 3.1. Colour property of natural anthocyanins The colour property of natural anthocyanins, described as absorption spectra, was evaluated in the ph range of 1 8 (Fig. 1). The results indicated that the absorbance values at maximum absorption wavelength decreased with increasing ph up to ph 6. The high absorbance values were observed only at ph 1 2 and the colour of anthocyanins was strong red. At ph 3, the colour of solution was fading and the decreasing of absorbance was found sharply at ph 4 6. With increasing ph, flavylium cation (red) concentration decreases as hydration to the colourless carbinol or pseudobase occurs, which then equilibrated to the open chalcone form (Mazza & Brouillard, 1987). Furthermore, the higher absorptions were found between 570 until 600 nm at ph 7 8, due to the presence of unstable blue quinonoidal structures. The bathochromic shifts were also observed at ph > 4. Jambolan anthocyanins showed lower colour intensity when compared to anthocyanins of red sweet potato, purple corn, purple carrot, red grape (Cevallos-Casals & Cisneros-Zevallos, 2004) and red cabbage (data not shown). It is due to glycosylation structure Absorbance 1.2 1 0.8 0.6 0.4 0.2 0 350 400 450 500 550 600 650 700 Wavelength (nm) of jambolan anthocyanins as 3,5-diglucoside. Mazza and Brouillard (1990) reported that malvidin 3,5-diglucoside (malvin) and cyanidin 3,5-diglucoside (cyanin) cannot confer much colour to a solution whose ph ranges from 3 to neutrality because of the high value of the equilibrium constant of the hydration reaction (K h ). The anthocyanin monoglucosides are known to possess K h constants about 10 times smaller than the corresponding diglucosides. 3.2. Intermolecular copigmentation effect on colour enhancement of anthocyanins Considering the low colour intensity of jambolan anthocyanins, it is necessary to improve the colour of these pigments through the term of intermolecular copigmentation using cinnamic acids (sinapic acid, caffeic acid, ferulic acid) and water soluble-rosemary polyphenolic extract as copigment. These copigments were commonly used to give enhancing and stabilising effect on anthocyanin colour through intermolecular copigmentation reaction (Brenes, Pozo-Insfran, & Talcott, 2005; Eiro & Heinonen, 2002; Gris, Ferreira, Falcao, & Bordignon-Luiz, 2007; Markovic, Petranovic, & Baranac, 2000; Yawadio & Morita, 2007). Intermolecular copigmentation reaction of jambolan anthocyanins with these copigments promoted an increase in the maximum absorption wavelength (bathochromic shift, Dk vis-max ) and absorbance (hyperchromic effect, DA vis-max ) in the beverage model at ph 3, as can be seen in Fig. 2. An increase in the concentration of copigment also produced in both an increment of absorbance at k vis-max and a bathochromic shift of k vis-max. In this study, a bathochromic shifts (Dk vis-max ) were observed ranging from 1.16% to 1.94% (Fig. 2A). The wavelength of anthocyanins in beverage model without addition of copigment (natural anthocyanin) was 516 nm and after copigmentation reaction, the wavelength increased to 522 526 nm. An increase of absorbance (DA vis-max ) was observed on 19.63 117.33% on copigment addition of 0.5 4 mg/ml (Fig. 2B). The observed hyperchromic effect and bathochromic shift are due to the increase in the electrons p p system (chromophore) resulting from the formation of intermolecular association between anthocyanin and copigment, through the chemical phenomena known as charge-transfer complex formation or p p interaction. This interaction results in an overlapping arrangement of both anthocyanin and copigment (Castaňeda-Ovando et al., 2009). In this study, the Dk vis-max and DA vis-max values of beverage model with natural anthocyanin were assumed of 0%, both hyper- 1 2 3 4, 5, 6 Fig. 1. Absorption spectra pattern of jambolan anthocyanins (natural) at ph 1 8. 8 7

P. Sari et al. / Food Chemistry 132 (2012) 1908 1914 1911 Δ λvis-max (%)/Bathochromic shift Δ Absorbance (%)/Hyperchromic effect 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 120 100 80 60 40 20 0 A Acy+Sin Acy+Caf Acy+Fer Acy+RP B Acy+Sin Acy+Caf Acy+Fer Acy+RP Fig. 2. Intermolecular copigmentation effect of jambolan anthocyanins with sinapic acid, caffeic acid, ferulic acid, and rosemary polyphenolic extract, observed as bathochromic shift (A) and hyperchromic effect (B). Bars from left to right for each copigments represent copigment concentration 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 mg/ ml, with Acy + Sin = anthocyanin + sinapic acid, Acy + Caf = anthocyanin + caffeic acid, Acy + Fer = anthocyanin + ferulic acid, Acy + RP = anthocyanin + rosemary polyphenolic. chromic effect and bathochromic shift were not observed. The highest hyperchromic effect (DA vis-max ) and bathochromic shift (Dk vis-max ) were observed in the beverage model with copigmented anthocyanin using rosemary polyphenolic extract. It is likely to occur because of high solubility of rosemary polyphenolic extract in water. On the other hand, sinapic acid, caffeic acid, and ferulic acid have lower water solubility, giving the lower hyperchromic effect and bathochromic shift, as observed in this study. 3.3. Colour stability of natural and copigmented anthocyanins The colour stability of jambolan anthocyanins, both natural and copigmented forms, was studied in the beverage model system at ph 3 in which the copigment concentration of 1 mg/ml was used in stability study. The chromaticity parameters (L, C, and h )of beverage are presented in Table 1. The copigmentation process decreased the lightness (L ) value and increased the chroma (C ), indicating that the colour of copigmented anthocyanin in the beverage model is more intense and saturated than the colour of natural anthocyanin. The colour of natural anthocyanin in beverage Table 1 Chromaticity of jambolan anthocyanins, both natural and copigmented forms, in beverage model system (citrate buffer, ph 3) with copigment addition of 1 mg/ml. Anthocyanin Lightness (L ) Chroma (C ) Hue angle (h ) Natural anthocyanin 62.44 24.89 0.63 Anthocyanin + sinapic acid 55.81 35.63 354 Anthocyanin + caffeic acid 57.00 34.40 355 Anthocyanin + ferulic acid 54.41 37.57 352 Anthocyanin + rosemary polyphenolic 49.07 43.37 345 model was red (h = 0.63) and the colour of copigmented anthocyanin was purplish-red, ranging for h values of 345 355, indicating the bluing effect on copigmentation reaction of jambolan anthocyanins. Gonnet (1998) and Francis (1989) explained that the bluing effect was consistently reported on copigmentation reaction as the consequence of the bathochromic shift affecting the pigment visible absorption band. Among the sample tests, copigmented anthocyanin with rosemary polyphenolic gave the most intense colour, saturation, and purplish-red colour. The influence of heating process of 80 and 98 C on colour stability of natural and copigmented anthocyanins was reported as values of DPC (polymeric colour difference), DE (total colour difference), CL (colour loss), and t 1/2 (half-life time), presented in Table 2. Heating accelerated in degradation of anthocyanins, resulting the decrease in colour intensity and formation of polymeric colour. The anthocyanins, both natural and copigmented forms, were more susceptible to degradation at the heating process of 98 C than 80 C. Further, at the two heating temperature of 80 and 98 C, the natural anthocyanin in beverage model revealed higher stability than the copigmented anthocyanin, as confirmed by DPC, DE, CL values of the natural anthocyanins were smaller and t 1/2 values higher than the copigmented anthocyanin. It can be explained that the intermolecular copigmentation interaction possesses weak hydrophobic forces, so the heat can cause degradation of copigmentation complexes. The derivative products of thermally copigmented anthocyanin degradation had lower stability than natural anthocyanin. In addition, Mazza and Brouillard (1990) explained that the interaction between the pigment and copigment is exothermic and the temperature increase causes dissociation of the copigmentation complexes, giving colourless compounds, thus resulting in a loss of colour. Light is another factor that affects the colour stability of anthocyanins. The effects of white fluorescent light on the colour stability of anthocyanin, both natural and copigmented forms, are presented in Table 2, as confirmed by DPC, DE, CL, and t 1/2 values. The result revealed that exposure of the anthocyanins to fluorescent light accelerated their degradation. During light exposure, the copigmented anthocyanins with sinapic acid, caffeic acid, and rosemary polyphenolic extract were degraded slower than the copigmented anthocyanin with ferulic acid and natural anthocyanin. As shown in Table 2, the copigmented anthocyanin with sinapic acid, caffeic acid, and rosemary polyphenolic extract had the values of DPC, DE, CL smaller and the values of t 1/2 higher than the copigmented anthocyanin with ferulic acid and natural anthocyanin. It revealed that copigmentation of anthocyanin contributed to an increase in the colour stability, suggesting a protective effect of intermolecular copigmentation. This result is in accordance with the study reported by Gris et al. (2007), that the colour stability of Carbenet Sauvignon grape anthocyanins under fluorescent light was increased with the presence of anthocyanin caffeic acid complex. Colour stability of jambolan anthocyanins, both natural and copigmented forms, in the beverage model was studied during storage at refrigeration and room temperature of which result is presented in Table 2. The addition of sinapic acid, caffeic acid, and rosemary polyphenolic extract increased the colour stability of anthocyanin in both storage refrigeration and room temperature, promoting an increase in the t 1/2 values and decrease in the DPC, DE, CL values of these copigmented forms. The increased stability effect was not observed in the beverage model with copigmented anthocyanin using ferulic acid. The different storage temperature affected the colour stability of anthocyanins, in which the increase in storage temperature, from refrigeration (7 C) to room temperature (28 C) greatly accelerated the degradation of both natural and copigmented anthocyanins. There was greater increase in t 1/2 values and decrease in DPC, DE, CL values in the bev-

1912 P. Sari et al. / Food Chemistry 132 (2012) 1908 1914 Table 2 Colour changes, rate constant (k) and half-life time (t 1/2 ) of jambolan anthocyanins, both natural and copigmented forms, in model beverage (citrate buffer, ph 3) during heating, exposure to fluorescent light, and storage condition. Anthocyanin Parameters of colour change Kinetic parameters DPC a DE b CL (%) c k d t 1/2 e Heating at 80 C f Natural anthocyanin 7.61 7.13 32.30 0.0032 3.68 h Anthocyanin + sinapic acid 9.12 9.76 33.45 0.0033 3.55 h Anthocyanin + caffeic acid 11.27 9.62 34.74 0.0035 3.35 h Anthocyanin + ferulic acid 8.39 9.71 36.29 0.0036 3.21 h Anthocyanin + rosemary polyphenolic 8.69 10.00 33.32 0.0032 3.61 h Heating at 98 C f Natural anthocyanin 20.75 16.85 62.23 0.0079 1.46 h Anthocyanin + sinapic acid 33.87 26.77 66.61 0.0090 1.28 h Anthocyanin + caffeic acid 36.60 28.45 68.23 0.0094 1.23 h Anthocyanin + ferulic acid 24.81 26.13 69.17 0.0097 1.20 h Anthocyanin + rosemary polyphenolic 25.20 28.65 64.51 0.0085 1.37 h Exposure to white fluorescent light g Natural anthocyanin 54.48 43.07 78.60 0.1678 4.13 d Anthocyanin + sinapic acid 24.61 22.53 57.43 0.0869 7.98 d Anthocyanin + caffeic acid 34.66 26.13 61.43 0.0946 7.33 d Anthocyanin + ferulic acid 51.51 43.00 81.12 0.1701 4.07 d Anthocyanin + rosemary polyphenolic 30.23 29.67 54.79 0.0804 8.63 d Storage at refrigeration temperature h Natural anthocyanin 3.37 3.89 13.15 0.0324 21.59 w Anthocyanin + sinapic acid 1.31 1.86 6.61 0.0075 40.19 w Anthocyanin + caffeic acid 1.70 3.05 8.50 0.0215 32.35 w Anthocyanin + ferulic acid 3.10 3.79 12.42 0.0333 20.88 w Anthocyanin + rosemary polyphenolic 1.64 3.16 10.89 0.0265 26.22 w Storage at room temperature h Natural anthocyanin 17.54 21.59 52.50 0.1894 3.66 w Anthocyanin + sinapic acid 12.45 14.61 43.32 0.1426 4.86 w Anthocyanin + caffeic acid 15.58 17.36 50.27 0.1799 3.91 w Anthocyanin + ferulic acid 21.00 23.99 61.63 0.2437 2.84 w Anthocyanin + rosemary polyphenolic 14.46 20.47 49.14 0.1669 4.17 w Colour changes calculated from colour parameter values before and after treatments (heat, light, and storage). Time unit: h = hours, d = days, w = weeks. a Polymeric colour. b Chromaticity colour (CIELAB system). c Colour loss. d Rate constant. e Half-life time. f Exposed to heat for 2 h. g Exposed to white fluorescent light for 10 d. h Storage for 4 w. erages stored at refrigeration temperature. The increasing temperature causes dissociation of the copigmentation complexes due to the weak bond formation, subsequently resulting colourless compounds and loss of colour, as explained by Mazza and Brouillard (1990). These results are in agreement with those found by Gris et al. (2007), who verified that caffeic acid addition conferred greater colour stability to Carbenet Sauvignon grape anthocyanin solution under the storage condition at 4 ± 1 and 29 ± 3 C; storage at 4 ± 1 C gave higher colour stability than that of 29 ± 3 C. Some investigation suggested that the copigmentation of anthocyanins with other compounds (copigments) is the main mechanism of colour stabilisation. The increase colour stability occurs because of the anthocyanin copigment complexes, formed through charge transfer or electrons p p interaction, giving protection against water nucleophilic attack in the position 2 of the flavylium cation, as described by Mazza and Brouillard (1987) and Castaňeda-Ovando et al. (2009). Therefore, formation of the colourless carbinol (hemiketal), open chalcone forms, and subsequent colour loss is at least partially prevented. 3.4. Phenolics content and radical scavenging activity of beverage model The total phenolics and monomeric anthocyanins content of beverage model coloured with natural or copigmented anthocyanins of jambolan are presented in Fig. 3A. Determination of monomeric anthocyanin revealed that there was no variation in anthocyanin contents in beverage model, ranging from 63.27 to 65.73 lg CyE/mL. It revealed that copigmentation reaction did not increase the monomeric anthocyanin content. The content of total phenolic varied greatly among the beverages ranging from 65.32 to 578.99 lg GAE/mL. The highest and the lowest values were found in the beverage models with copigmented anthocyanin using rosemary polyphenolic and natural anthocyanin, respectively. It was also observed that the addition of the copigments into beverage contributed an increase in the total phenolic content as compared to the beverage without copigment addition. The beverage models only added with copigment (as control sample) revealed the total phenolic content, ranging from 154.79 to 524.30 lg GAE/mL.

P. Sari et al. / Food Chemistry 132 (2012) 1908 1914 1913 Acy+RP RP Acy+Caf Caf Acy+Sin Sin Acy+Fer Fer Fig. 3B illustrates the free radical scavenging activities (ascorbic acid equivalent antioxidant capacity, AEAC) of beverage models assayed in the DPPH system. The DPPH radical scavenging activity values of beverages ranged from 47.54 to 354.58 lg AE/mL. The highest and lowest of activity values were also found in beverage model with copigmented anthocyanin using rosemary polyphenolic and natural anthocyanin, respectively. The addition of copigments into beverage increased the radical scavenging activity. However, there was no synergy effect on activities observed on anthocyanin copigment complexes. Result of this study is in agreement with the previous reports (Markovic, Ignjatovic, Markovic, & Baranac, 2003a, 2003b) that evaluated the antioxidant properties of the copigmentation complexes of malvidin 3,5- diglucoside (malvin) with caffeic, chlorogenic, ferulic, sinapic, and tannic acids through measuring of the oxidation potential. The result confirmed that the antioxidative properties of the malvin copigment with the above acids were greater than the antioxidative property of pure malvin (natural anthocyanin), as reported in our results. Besides enhancing and stabilising of anthocyanin colour, the intermolecular copigmentation of jambolan anthocyanins in the beverage model also increased the total phenolic content and radical scavenging activity. Therefore, the jambolan anthocyanins, natural and copigmented forms, could be utilised as natural food colourants with additional property of an antioxidant capability. 4. Conclusion 63.73 64.15 63.27 65.73 154.79 207.75 241.85 268.50 65.32 Acy 64.20 0 100 200 300 400 500 600 700 The low colour intensity of the diglucosides structure of jambolan anthocyanins could be enhanced through intermolecular copigmentation using copigments of sinapic acid, caffeic acid, ferulic acid, and rosemary polyphenolic extract. The complexes formed 317.65 300.52 total phenolic 524.30 578.99 monomeric anthocyanin Anthocyanin (µg CyE/mL) / total phenolic ( µg GAE/mL) content Acy+RP 354.58 RP 320.10 Acy+Caf 239.79 B Caf 205.72 Acy+Sin 143.87 Sin Acy+Fer 99.45 117.91 Fer 80.39 Acy 47.54 0 50 100 150 200 250 300 350 400 Antioxidant capacity AEAC (µg AE/mL) Fig. 3. Total phenolic, monomeric anthocyanins (A), and DPPH radical-scavenging activity (B) of beverage models coloured with natural or copigmented anthocyanins of jambolan, copigment concentration of 1 mg/ml. Acy = anthocyanin (natural), Acy + Sin = anthocyanin + sinapic acid, Acy + Caf = anthocyanin + caffeic acid, Acy + Fer = anthocyanin + ferulic acid, Acy + RP = anthocyanin + rosemary polyphenolic. A between anthocyanins and copigment resulted in an enhancement of absorbance at maximum wavelength and a shift to the higher k vis-max values, and gave purplish-red, stronger and more stable colour on jambolan anthocyanins. In addition, the stability of jambolan anthocyanins to light exposure and storage condition was increased through copigmentation of jambolan anthocyanins with sinapic acid, caffeic acid, and rosemary polyphenolic extract. The incorporating of jambolan anthocyanins, both natural or copigmented forms, in the beverage model provided two benefits as colourants and antioxidant, which are beneficial for health. Among copigments used in this study, addition of rosemary polyphenolic in beverage model gave the strongest colour enhancement and antioxidant activity and moderate stability. Accordingly, it can be selected as copigment for the jambolan anthocyanins when incorporated in the food system. Acknowledgements This research was supported by the Directorate General of Higher Education (DGHE), the Ministry of National Education, the Republic of Indonesia; the International Foundation for Science (IFS), Stockholm, Sweden; and the Organization for the Prohibition of Chemical Weapons (OPCW), The Hague, The Netherlands, through Grants to Puspita Sari. The authors are grateful to Prof. Dr. Suminar Setiati Achmadi of Bogor Agricultural University for assistance in correction of manuscript. References Banerjee, A., Dasgupta, N., & De, B. (2005). In vitro study of antioxidant activity of Syzygium cumini fruit. Food Chemistry, 90, 727 733. Brenes, C. H., Pozo-Insfran, D. D., & Talcott, S. T. (2005). Stability of copigmented anthocyanins and ascorbic acid in a grape juice model system. Journal of Agricultural and Food Chemistry, 53, 49 56. Brito, E. S., Araŭjo, M. C. P., Alves, R. E., Carkeet, C., Clevidence, B. A., & Novotny, J. A. (2007). Anthocyanins present in selected tropical fruits: Acerola, jambolão, jussara, and guajiru. Journal of Agricultural and Food Chemistry, 55, 9389 9394. Castaňeda-Ovando, A., Pacheco-Hernández, M. L., Páez-Hernández, M. E., Rodríguez, J. A., & Galán-Vidal, C. A. (2009). Chemical studies of anthocyanins: A review. Food Chemistry, 113, 859 871. Cavalcanti, R. N., Santos, D. T., & Meireles, M. A. A. (2011). Non-thermal stabilization mechanisms of anthocyanins in model and food systems An overview. Food Research International, 44, 499 509. Cevallos-Casals, B. A., & Cisneros-Zevallos, L. (2004). Stability of anthocyanin-based aqueous extracts of Andean purple corn and red-fleshed sweet potato compared to synthetic and natural colorants. Food Chemistry, 86, 69 77. Eiro, M. J., & Heinonen, M. (2002). Anthocyanin color behaviour and stability during storage: Effect of intermolecular copigmentation. Journal of Agricultural and Food Chemistry, 50, 7461 7466. Faria, A. F., Marques, M. C., & Mercadante, A. Z. (2011). Identification of bioactive compounds from jambolão (Syzygium cumini) and antioxidant capacity evaluation in different ph conditions. Food Chemistry, 126, 1571 1578. Francis, F. J. (1989). Food colorants: Anthocyanins. Critical Reviews in Food Science and Nutrition, 28, 273 314. Giusti, M. M., & Wrolstad, R. E. (2001). Characterization and measurement of anthocyanins by UV visible spectroscopy. In R. E. Wrolstad, T. E. Acree, E. A. Decker, M. H. Penner, D. S. Reid, S. J. Schwartz, C. F. Shoemaker, D. Smith, & P. Sporns (Eds.), Handbook of food analytical chemistry: Pigments, colorants, flavors, texture, and bioactive food components (pp. 19 31). New Jersey: John Wiley & Sons Inc. Gonnet, J. F. (1998). Colour effects of co-pigmentation of anthocyanins revisited-1, a colorimetric definition using the CIELAB scale. Food Chemistry, 63(3), 409 415. Gris, E. F., Ferreira, E. A., Falcao, L. D., & Bordignon-Luiz, M. T. (2007). Caffeic acid copigmentation of anthocyanins from Cabernet Sauvignon grape extracts in model systems. Food Chemistry, 100, 1289 1296. Kirca, A., & Cemeroglu, B. (2003). Degradation kinetics of anthocyanins in blood orange juice and concentrate. Food Chemistry, 81, 583 587. Markakis, P. (1982). Stability of anthocyanins in food. In P. Markakis (Ed.), Anthocyanins as food colors (pp. 163 180). New York: Academic Press Inc. Markovic, J. M. D., Ignjatovic, L. M., Markovic, D. A., & Baranac, J. M. (2003a). Antioxidant capabilities of some organic acids and their co-pigments with malvin Part I. Journal of Electroanalytical Chemistry, 553, 169 175. Markovic, J. M. D., Ignjatovic, L. M., Markovic, D. A., & Baranac, J. M. (2003b). Antioxidant capabilities of some organic acids and their co-pigments with malvin Part II. Journal of Electroanalytical Chemistry, 553, 177 182.

1914 P. Sari et al. / Food Chemistry 132 (2012) 1908 1914 Markovic, J. M. D., Petranovic, N. A., & Baranac, J. M. (2000). A spectrophotometric study of the copigmentation of malvin with caffeic and ferulic acid. Jounal of Agricultural and Food Chemistry, 48, 5530 5536. Mazza, G., & Brouillard, R. (1987). Recent developments in the stabilization of anthocyanins in food products. Food Chemistry, 25, 207 225. Mazza, G., & Brouillard, R. (1990). The mechanism of co-pigmentation of anthocyanins in aqueous solutions. Phytochemistry, 29(4), 1097 1102. Sari, P., Wijaya, C. H., Sajuthi, D., & Supratman, U. (2009). Identification of anthocyanins in jambolan (Syzygium cumini) fruit by high performance liquid chromatography diode array detection. Journal of Food Technology and Industry, 20(2), 102 108 (Indonesian). Shipp, J., & Abdel-Aal, E. M. (2010). Food applications and physiological effects of anthocyanins as functional food ingredients. The Open Food Science Journal, 4, 7 22. Veigas, J. M., Narayan, M. S., Laxman, P. M., & Neelwarne, B. (2007). Chemical nature, stability and bioefficacies of anthocyanins from fruit peel of Syzygium cumini Skeels. Food Chemistry, 105, 619 627. Waterhouse, A. L. (2002). Determination of total phenolics. In R. E. Wrolstad, T. E. Acree, E. A. Decker, M. H. Penner, D. S. Reid, S. J. Schwartz, C. F. Shoemaker, D. Smith, & P. Sporns (Eds.), Handbook of food analytical chemistry: Pigments, colorants, flavors, texture, and bioactive food components (pp. 463 470). New Jersey: John Wiley & Sons Inc. Yamaguchi, T., Takamura, H., Matoba, T., & Terao, J. (1998). HPLC method for evaluation of the free radical-scavenging activity of food by using 1,1-diphenyl- 2-picrylhydrazyl. Bioscience Biotechnology Biochemistry, 62, 1201 1204. Yawadio, R., & Morita, N. (2007). Color enhancing effect of carboxylic acids on anthocyanins. Food Chemistry, 105, 421 427.