Food Chemistry 117 (2009) 8893

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Food Chemistry
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Colour, pH stability and antioxidant activity of anthocyanin rutinosides isolated from tamarillo fruit (Solanum betaceum Cav.)
Nelson H. Hurtado a, Alicia L. Morales a, M. Lourdes Gonzlez-Miret b, M. Luisa Escudero-Gilete b, Francisco J. Heredia b,*
a b

Department of Chemistry, Universidad Nacional de Colombia, A.A. 14490 Bogot, Colombia Lab. Food Colour and Quality, Department of Nutrition and Food Science, Facultad de Farmacia, Universidad de Sevilla, 41012-Sevilla, Spain

a r t i c l e

i n f o

a b s t r a c t
Changes in colour and stability of anthocyanins have been evaluated over pH range 2.08.7. The study was made on crude extract (XAD-7 Amberlite-retained fraction) as well as on the following pure pigments isolated from tamarillo fruit (Solanum betaceum Cav.): delphinidin 3-O-(600 -O-a-rhamnopyranosyl-b-glucopyranosyl)-30 -O-b-glucopyranoside, delphinidin 3-O-(600 -O-a-rhamnopyranosyl)-b-glucopyranoside, cyanidin 3-O-(600 -O-a-rhamnopyranosyl)-b-glucopyranoside and pelargonidin 3-O-(600 -O-arhamnopyranosyl)-b-glucopyranoside. The relationships between the colour and the hydroxylation degree of the B ring and the pH have been studied for the rst time on rutinosides. The peel extract showed much more colour stability than the jelly extract at all the pH values studied. The replacement of the 30 -OH with a glycosyl group increased the stability of the colour to pH changes, although this sub0 stitution yields a less colourful (higher L* and lower C ab ) compound (Dp 3-rut-3 -glc), having both hypsochromic and hypochromic shifts relative to the non-glycosylated molecule (Dp 3-rut). Moreover, the inuence of the hydroxylation degree of the B ring on the quality and stability of colour, as well as on the antioxidant activity, was determined. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 14 November 2008 Received in revised form 20 March 2009 Accepted 23 March 2009

Keywords: Anthocyanins Solanum betaceum Tamarillo fruit Tree tomato Colour Stability Antioxidant

1. Introduction Most foodstuffs are exposed to some kind of processing before being consumed, which can cause loss of some quality properties, such as colour, aroma or taste; hence producers face the need of replacing these characteristics. Additives (e.g., colourings and avourings) are used to recover or to emphasize original features, to ensure uniformity, and to guarantee quality. Pigments are chemical components absorbing radiation in the visible region of the electromagnetic spectrum. The colour is due to a specic molecular group (chromophore) which absorbs energy and, as consequence, the excitation of an electron of external orbitals with major energy occurs; the non-absorbed energy is reected and refracted and detected by the eyes, where impulses are generated and sent to the brain, and then, interpreted as colour (Delgado-Vargas, Jimnez, & Paredes-Lopez, 2000). Based on the chromophore chemical structure, pigments can be classied as chromophore with conjugated systems (carotenoids, anthocyanins, betalains, caramel and synthetic pigments) or porphyrins with coordinated metals (myoglobin, chlorophyll, and their derivatives).

* Corresponding author. Tel.: +34 95455 6761; fax: +34 95455 7017. E-mail address: heredia@us.es (F.J. Heredia). 0308-8146/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.03.081

In the conjugated systems, anthocyanins are specically important because they are responsible for some red colours in the nature, as monomeric, oligomeric and polymeric anthocyanins. The use of natural extracts of these pigments as food additives has some limitations, due to colour variation caused by pH changes, light exposure and oxygen (Bridle & Timberlake, 1997; Markakis, 1982). In general, anthocyanins show their highest colour intensity in the avylium ion form (Harborne & Williams, 1995). It has been demonstrated that anthocyanin stability is inuenced by substituents in their structures, sugars and acyl groups (Giusti & Wrolstad, 2003). In recent years, research on anthocyanin chemical structure has increased (Bjory, Fossen, & Andersen, 2007; Byamukama, Kiremire, Andersen, & Steigen, 2005; Cabrita, Frystein, & Andersen, 2000; Fossen & Andersen, 2003; Mateus et al., 2003, 2006; Tian, Giusti, Stoner, & Schwartz, 2006; Wang, Race, & Shrikhande, 2003); however, it is important to study in depth the relationships between colour and chemical composition, which may help to understand the basic principles that inuence the anthocyanins colour. In Colombia, the tamarillo or tree tomato (Solanum betaceum Cav.) is a promising product for export, due to its colour; the red variety has been the most accepted internationally. In this work the stability of tamarillo fruit extracts and isolated individual

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anthocyanin rutinosides (Fig. 1) to pH changes has been evaluated by means of colorimetric studies, to obtain more precise information about the change of colour in both crude extracts and individual anthocyanins. 2. Materials and methods 2.1. Chemicals and supplies acid) The 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and Amberlite XAD-7 were obtained from Rohm and Haas, Darmstadt, Germany. HPLC-grade acetonitrile, ACS-grade n-butanol, methanol, tert-butyl methyl ether (TBME), formic acid, hydrochloric acid and potassium persulfate were purchased from Merck, Bogot, Colombia. CD3OD, CF3COOD, and CF3COOH (TFA) were obtained from SigmaAldrich (St. Louis, MO). 2.2. Plant material Tamarillo fruits (5.09 kg) were collected in Puente Nacional (Santander, Colombia). A voucher specimen was coded as Col 343584 at the Instituto de Ciencias Naturales at Universidad Nacional de Colombia. 2.3. Isolation of crude anthocyanins extract Fresh ripe fruits were washed and peeled; the seeds and the surrounding jelly were manually separated from the esh. The jelly (250 g), ltered through glass wool, was applied onto an Amberlite XAD-7 column (800 mm long, 40 mm i.d.). The column was washed with 1.25 l of water, and elution of anthocyanins was carried out with 300 ml of a mixture of methanol:acetic acid (19:1, v/

v). The eluate was concentrated under reduced pressure at 35 C and the aqueous solution was lyophilised (crude jelly extract). This procedure was repeated four times to obtain 4.35 g of the crude jelly extract. The peelings (2.51 kg) were cut into small pieces (2 cm2) and extracted with 2 l of methanol:acetic acid (19:1, v/v) for 12 h (maceration). After ltration the organic solvent was evaporated at 35 C using a rotary evaporator and the remaining aqueous phase was applied onto a XAD-7 column (800 mm long, 40 mm i.d.). The pigments were eluted as indicated before (Degenhard, Knapp, & Winterhalter, 2000), to give an enriched anthocyanins extract of 0.935 g (crude peelings extract). Then, the XAD-7 isolates of jelly and peelings were fractionated by multilayer coil countercurrent chromatography (MLCCC). 2.3.1. Countercurrent chromatography (CCC) A multilayer coil countercurrent chromatograph (P.C. Inc., Potomac, MD) with tubular column of PTFE (400 ml total volume) was used. Solvent system consisted of n-butanol:TBME:acetonitrile:water (2:2:1:5) v/v/v/v, acidied with 0.1% TFA (Degenhard et al., 2000). The organic phase was used as the stationary phase; therefore elution mode was head to tail. Crude anthocyanins extract was dissolved in 5 ml of a mixture of stationary phase and mobile phase (1:1 v/v), and introduced through the injection port. The mobile phase was pumped at 1 ml min1, while centrifugation was carried out at 800 rpm. Four-millilitre fractions were collected. The sample loads in MLCCC were high (0.6 g), so fractionation of up to several hundred milligrams of sample was achieved in a single MLCCC run. To check the purity, each fraction was analysed by HPLC, and further purication was carried out using preparative HPLC. 2.3.2. High performance liquid chromatography The analytical HPLC results were obtained with an Agilent 1100 HPLC system (Agilent, Santa Clara, CA) tted with a photodiode array detector and a Zorbax-SB C18 column (4.6 mm 250 mm; 5 lm lm thickness). Two solvents were used for elution: A = acetonitrile:formic acid:water (3:10:87, v/v/v) and B = acetonitrile:formic acid:water (50:10:40, v/v/v). The elution prole consisted of a gradient from 6% to 20% B at 010 min, 20% to 40% B at 1020 min, 40 to 50% B at 2030 min, 50% to 6% B at 30 35 min. Aliquots of 100 ll (0.1 mg ml1) were injected and the ow rate was 0.8 ml min1. Prior to injection, all samples were ltered through a 0.45 lm Millipore membrane lter. Preparative HPLC was performed using a Luna C18 column (10 mm 250 mm: 5 lm lm thickness) and a 6000LP UV detector. An isocratic elution prole was applied (95% A, 5% B) using acetonitrile, formic acid and water (solvent A: 3:10:87, v/v/v; solvent B: 50:10:40, v/v/v). The ow rate was 4 ml min1 for 20 min and aliquots of 40 ll (250 mg ml1) were injected. 2.3.3. Spectroscopy UVVis absorption spectra of anthocyanins were recorded online during HPLC analysis, and the spectral measurements were made over the wavelength range 300680 nm in steps of 2 nm. ESIMS analyses were performed on a Shimadzu QP-8000 mass spectrometer (Shimadzu, Japan). The electrospray voltage applied was 4.5 kV, nebuliser gas ow of 4.5 l min1, probe voltage 4.5 kV, curved desolvation line (CDL) voltage 130 V, CDL temperature of 230 C, deector voltage at 45 and 60 V and acquisition from m/z 50 to m/z 800 in positive ionisation mode. A solution of 1 mg ml1 of each puried pigment was dissolved in a 1:1 mixture of solvent A (water:formic acid 9:1) and solvent B (acetonitrile:formic acid 9:1). The anthocyanin solutions were injected directly into the system at a ow rate of l00 ll min1. Low-resolution fast atom bombardment MS of the pigments was performed on an

R1
3' 2'

OH
4' 5'

HO
6

1' 2 3
4

B
6'

A
5 10

R2

O HO H
1'' 2''

OH

3''

OH
5'' 6'' 6''' 5''' 4''

HO

O O
1'''

HO

H3C

4'''

HO

3'''

H
2'''

HO

Fig. 1. Basic structure of tamarillo anthocyanins (Solanum betaceum Cav). Dp 3-rut30 -glc = delphinidin 3-O-(600 -O-a-rhamnopyranosyl-b-glucopyranosyl)-30 -O-b-glucopyranoside (R1 = O-glc, R2 = OH), Dp 3-rut = delphinidin-3-O-(600 -O-a-rhamnopyrCy 3-rut = cyanidin-3-O-(600 -O-aanosyl)-b-glucopyranoside (R1 = R2 = OH), rhamnopyranosyl)-b-glucopyranoside (R1 = OH, R2 = H), Pe 3-rut = pelargonidin-3O-(600 -O-a-rhamnopyranosyl)-b-glucopyranoside (R1 = R2 = H). glc = glucopyranoside.

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AutoSpec-Q (Waters Corporation, Milford, MA) in a glycerol-NaI matrix, using positive detection and acquisition from m/z 50 to m/z 900. Argon was used as collision gas. The complete structures of isolated anthocyanins were established by 1H- and 13C NMR analysis. Full assignments were performed with TOCSY, COSY, HSQC and HMBC experiments. Isolated anthocyanins dissolved in a mixture of CD3OD:CF3OOD (19:1, v/v) were measured using a Bruker AMX-500. Thus, the identities of anthocyanins were determined to be: delphinidin 3-O-(600 -O-a-rhamnopyranosyl-b-glucopyranosyl)-30 -O-bglucopyranoside (Dp 3-rut-30 -glu), delphinidin 3-O-(600 -O-arhamnopyranosyl)-b-glucopyranoside (Dp 3-rut), cyaniding 3-O(600 -O-a-rhamnopyranosyl)-b-glucopyranoside (Cy 3-rut) and pelargonidin 3-O-(600 -O-a-rhamnopyranosyl)-b-glucopyranoside (Pe 3-rut). The chemical structures of these anthocyanins are shown in Fig. 1. 2.4. Quantication of total phenols and determination of anthocyanic indices in the crude extracts Total phenols (TP) were estimated using the FolinCiocalteu method (Singleton & Rossi, 1965). Sample aliquots of 0.5 ml were added to 0.5 ml of water, 5 ml of FolinCiocalteau reagent (0.2 N), and 4 ml of a saturated solution of sodium carbonate (75 g/l), and mixed thoroughly. The absorbance was measured at 765 nm with an HP8452 spectrophotometer (Hewlett Packard, Palo Alto, CA) after incubation for 2 h at room temperature. Quantication was made based on a standard curve, generated with 2.6, 5.2, 7.9, 13.1 and 26.2 mg of gallic acid. TP values were expressed in % w/w (100 mg gallic acid/mg crude extract). The anthocyanic indices represent approximate measurements of the phenolic constituents and they can be used in comparative evaluations. The anthocyanin extracts include both monomeric anthocyanins and polymeric pigments. When a solution containing anthocyanins is treated with an excess of SO2 an immediate decolouration of the solution occurs, so the residual colour existing after such treatment is due to polymeric pigment forms. Aqueous solutions (2 mg ml1, pH 5.2) of crude extracts were prepared for the chemical indices determination. The index of polymeric pigments (IPP) was measured at 520 nm and total anthocyanic colour (AC) was measured in 1 M HCl at 520 nm. Polymeric colour (PC) was assumed to be equal to 5 IPP/3, and the colour of the monomeric anthocyanins (MC) in 1 M HCl was obtained by difference (MC = AC (5 IPP/3)). The concentration of total monomeric anthocyanins (AT) in 1 M HCl was expressed as delphinidin 3-glucoside chloride (molecular mass 500.5) using the molar absorptivity value (e) of 23,700 l mol1 cm1 (Heredia, Francia-Aricha, Rivas-Gonzalo, Vicario, & Santos-Buelga, 1998) at 520 nm. Hence, AT (mg/l) = 21.1 MC (Somers & Evans, 1977). 2.5. Determination of the total antioxidant capacity The total antioxidant capacity was determined by the TEAC method (Re et al., 1999), which is based on the capacity of antioxidants to capture the radical 2,20 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+). It was performed using the HP8452 spectrophotometer in kinetic mode. ABTS+ radical cation was produced by reacting 7 mM 2,20 -azino-bis-(3-ethylbenzothiazoline-6sulfonic acid) diamonium salt and 2.45 mM potassium persulfate, after incubation at room temperature in the dark for 16 h. The ABTS+ solution was diluted with ethanol to an absorbance of 0.70 0.1 at 734 nm. The ltered sample was diluted with ethanol, so as to give 2080% inhibition of the blank absorbance with 20 ll of sample. ABTS+ solution (1 ml; absorbance of 0.70 0.1) was read at 734 nm and 20 ll of the sample were added and mixed thoroughly. Trolox standards of nal concentration 015 lM in

ethanol were prepared and assayed under the same conditions. Trolox equivalent antioxidant capacity of sample was calculated based on the inhibition exerted by standard Trolox solution at 6 min. 2.6. Colorimetric study The evaluation of the colour was based on the spectrophotometric measurement of the transmission spectrum in the visible region (380770 nm) using an HP8452. The colour parameters were obtained through weighted ordinates method (Dk = 2 nm) from transmission spectra, by using the CromaLab software (Heredia, lvarez, Gonzlez-Miret, & Ramrez, 2004), which takes into consideration the International + Commission on Illumination recommendations (CIE, 2004). D65 standard illuminant, corresponding to the natural daylight, and 10 standard observer were considered in the calculations. Reference blank measurements were made with the cuvette lled with distilled water. CIE 1976 ( L* a* b*) (CIELAB) uniform colour space was taken into account for the colorimetric analysis. Within the CIELAB uniform space a psychometric index of lightness, L* (ranging from 0, black, to 100, white), and two colour coordinates, a* (which takes positive values for reddish colours and negative values for greenish ones) and b* (positive for yellowish colours and negative for the bluish ones), are dened. From these coordinates, other colour parameters are dened: the hue angle (hab) is the qualitative attribute of colour, and the chroma (C ab ) is the quantitative attribute of colour intensity. 2.6.1. The pH effect The CIELAB parameters (L*, a*, b*, C ab , hab) were determined in 5 105 M solutions of each anthocyanin at different pH values, ranged from 2.0 to 8.7. Modications in pH were made by addition of small volumes of NaOH (1 M or 10 M). Crude extracts were diluted in water until 0.8 absorbance units at k = 520 nm were obtained in order to study the inuence of the pH on the colour; the concentration of the diluted jelly extract was 2.1 mg/ml, and that of the diluted peelings extract was 35 mg/ml. The colour differences DE ab were calculated between the initial pH value (pH = 2.0) and after each increase of pH, considering the Euclidean distance between the two colour points: *2 *2 * 2 1/2. Data consisted of the average DE ab = ((DL ) + (Da ) + (Db ) ) of two experimental values. 3. Results and discussion Once both jelly and peelings crude extracts were obtained, different chemical characteristics were determined (anthocyanic indices). The XAD-7 jelly extract showed higher content of total phenols (TP) and total anthocyanins (TA), while the peelings extract showed higher index of polymeric pigments (IPP). The higher content of TP and TA in the crude jelly extract is in accordance with the higher antioxidant capacity of this extract (Table 1). 3.1. Inuence of the pH on the colour of the anthocyanin crude extracts The colour variation in the aqueous solutions of anthocyanin crude extracts was studied within the pH range 2.06.2, that is to say under and above the most common pH values in foods. As revealed in Table 1 both jelly and peelings extracts showed reddish hues at the lowest pH value (peelings: 32.1; jelly: 35.0). As the pH increased to 6.2 the colour of the peelings extract gradually changed towards orange hues (until hab = 64.9) and the jelly extract towards red-purple hues (until hab = 0.4). The colour differences DE ab regarding the pH = 2.0 sample (the most colourful sample) are summarised in Table 2. These differences were higher

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Table 1 Total Phenolic (TP), Polymeric Pigment Index (PPI), Total Anthocyanins (TA) and Antioxidant Activity (TEAC) of crude extract and pure anthocyanins isolated from tamarillo fruit (Solanum betaceum Cav.). Sample Jelly Peelings Dp 3-rut-30 -glc Dp 3-rut Cy 3-rut Pe 3-rut Ascorbic acid
a

kmax 500 530 514 518 512 500

e
15974 25874 27268 36660

TEAC (pH 5.2) 1.90 0.125b 1.09 0.076b 2.20 0.026a 4.91 0.146a 1.80 0.098a 1.48 0.045a 1.09 0.093a

TP 25.11 0.9 13.69 0.8

PPI 0.20 0.034 1.16 0.107

TA 20.03 0.85 0.20 0.03

e: molar absorption (0.1% HCl in ethanol). TEAC: Trolox equivalent antioxidant capacity.
mmol of Trolox/mmol compound. mmol Trolox/g. Data given are the average of six measurement expressed in terms of mean S.D. TP: total phenolics (% p/p), PPI: polymeric pigment index (absorbance units), TA: total anthocyanins (mg delphinidin 3-glucoside/l).
b

for the jelly extract across the pH range, which is evidence of the lower colour stability (higher sensitivity to pH changes) of this extract. The higher stability of the peelings extract may be due not only to the higher polymeric anthocyanins content, which are components more stable to pH changes (Somers & Evans, 1977), but also to the possible presence of some compounds stabilising the colour in the extract, i.e., phenolic acids, avones, avonols, avanones, avanols and organic acids (Eiro & Heinonen, 2002; Markovic, Petranovic, & Baranac, 2000; Rein & Heinonen, 2004). The crude extracts were shown to be relatively more stable under low acid conditions (pH 2.03.4); however, even in the crude peelings extract (more stable) the colour differences were up to 3 CIELAB units (Table 2) indicating that they can be visually discriminated (Martnez, Melgosa, Prez, Hita, & Negueruela, 2001). 3.2. UVvis spectroscopy of individual anthocyanins For this study, the following anthocyanins isolated from the fruit were studied: Dp 3-rut-30 -glc, Dp 3-rut, Cy 3-rut and Pe 3-

rut. The maximum absorption in the visible spectrum of the aqueous solution (5 105 M; pH 2.0) and the molar absorption (e) of each anthocyanin are shown in Table 1. Comparing the three rutinosides, of which the structural differences are the number of hydroxyl groups in the B ring, it is observed that Pe 3-rut, having one hydroxyl group, shows a kmax at 500 nm. However, as the number of hydroxyl groups in the ortho position increases (Cy 3-rut and Dp 3-rut) the maximum absorption shifts towards higher wavelengths and the absorption intensity decreases. Analysing the spectral characteristics of the delphinidin derivatives, it can be observed that the replacement of the 30 -hydroxyl of the Dp 3-rut with a glucose moiety causes both hypsochromic and hypochromic shifts in the visible spectrum (Table 1). The spectral measurements were important to study the effect of hydroxylation and glycosylation in the B ring of the aglycone on the visible spectrum; however, with only this data we do not have enough information to explain the chromatic characteristics of the anthocyanins. It is well known that the properties of anthocyanins, including the colour expression, are inuenced by the chemical structure and pH (Heredia et al., 1998).

Table 2 CIELAB colour parameters (L*, a*, b*, C ab , hab) and colour differences (DE ) of crude extracts (jelly and peelings) and pure anthocyanins isolated from tamarillo fruit (Solanum betaceum Cav.). pH 2.03.4 Jelly a i a f bi bf L i L f C ab;i C ab;f hab,i hab,f DEab 62.1 55.4 43.5 22.5 45.5 49.0 75.8 59.8 35.0 22.1 22.3 pH 2.03.4 Dp 3-rut-30 -glc a i a f bi bf L i L f C ab;i C ab;f hab,i hab,f DEab 28.1 6.5 4.8 0.6 79.7 87.5 32.0 7.8 28.6 33.3 23.3 Dp 3-rut 59.1 24.6 15.2 1.1 70.8 87.2 61.0 24.7 14.4 2.5 41.5 Peelings 51.3 52.4 32.2 42.5 51.2 46.3 60.6 67.4 32.1 39.0 11.5 pH 3.46.2 Jelly 55.4 28.7 22.5 0.2 49.0 66.1 59.8 28.7 22.1 0.4 38.8 pH 3.4 6.2 Pe 3-rut 44.4 34.3 62.7 34.8 78.3 80.1 76.9 48.9 54.7 45.4 29.7 Dp 3-rut-30 -glc 6.5 1.0 4.3 0.4 87.5 89.0 7.8 1.0 33.3 21.0 7.4 Dp 3-rut 24.6 0.3 1.1 0.4 87.2 98.5 24.7 1.9 2.5 23.9 27.4 Peelings 52.4 24.1 42.5 51.6 46.3 40.5 67.4 56.9 39.0 64.9 30.3

Cy 3-rut 52.7 28.9 26.8 5.8 74.2 85.0 59.1 29.5 26.1 11.4 33.5

Cy 3-rut 28.9 5.4 5.8 2.6 85.0 85.4 29.5 29.4 11.4 22.5 24.9

Pe 3-rut 34.3 6.0 34.8 2.1 80.1 97.6 48.9 6.3 45.4 19.0 46.7

i = initial (lowest pH value of the range); f = nal (highest pH value of the range).

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3.3. Inuence of pH on the colour of the individual anthocyanins Fig. 2 represents the chromatic characteristics of the rutinosides solutions on the (a*b*)-diagram. This study reveals the impact of anthocyanins structures, such as hydroxylation and glycosidation, on colour and stability at various pH values. The pigments represent variation of the hydroxylation grade of the B ring of many anthocyanins isolated from fruits and vegetables (Brouillard, 1982). Aqueous solutions (5 105 M) of Dp 3-rut-30 -glc, Dp 3rut, Cy 3-rut and Pe 3-rut were studied at two pH range: (2.0 3.4) and (3.46.2). According to the CIELAB parameters ( L*, a*, b*, C ab , hab, DEab ) their colour stabilities depend highly on the pH and the structure (Fig. 2, Table 2). At the lowest pH value (pH 2.0, where anthocyanins exit basically in avylium form), Dp 3rut-30 -glc, Dp 3-rut and Cy 3-rut showed reddish hues (hab = 28.6, 14.4 and 26.1, respectively), while Pe 3-rut showed orange ones (hab = 54.7). At this pH, the number of hydroxyl groups of the B ring clearly inuenced the colour characteristic; the hue angle (hab) and lightness (L*) values were clearly higher for Pe 3-rut (only one OH), followed by Dp 3-rut-30 -glc (two OH), Cy 3-rut (two OH) and Dp 3-rut (three OH). Dp 3-rut-30 -glc underwent small decreases of the hue angle as the pH increased from 2.0 to 6.2, while great decreases in hue were found for Dp 3-rut, Cy 3rut and Pe 3-rut. It is noticeable that anthocyanins containing aglycones with only two or three hydroxyl groups on the B ring (Cy 3rut and Dp 3-rut) showed smaller colour differences (DE ab ) in the more alkaline region pH (3.46.2), contrary to Pe 3-rut (one OH) which showed greater colour differences (DE ab ) at these pH values (Table 2). On the other hand, at the most acid pH (2.03.4) the Dp 3-rut (three OH) was more unstable (the highest DE ab ). Comparing the DE ab values at both pH ranges, it was observed that Dp 3-rut, Cy 3-rut and Pe 3-rut were clearly more unstable than Dp 3-rut-30 -glc. The loss of colour occurring due to the base attack on the pigment structure could be related to the solvating grade of each molecule, in such a way that the greater the solvating grade was the higher the stability to pH changes. According to a study on avonoids (Rezende, Moll, Gonzlez, Beezer, & Mitchell, 1999), the internal hydrogen bonding (intramolecular interactions)

between neighbouring OH reduces the availability of these groups (by both steric and electronic factors) to interact with the solvent. This could explain the lowest colour stability of Dp 3-rut (lowest solvating) at low pH (2.03.4). Nevertheless, at pH 3.46.2, which induces the formation of quinoidal bases (Brouillard, 1982), different behaviours were observed. Anthocyanins having two or three hydroxyl groups on the B ring showed similar colour differences, DE ab being 27.4 and 24.9 CIELAB units for Dp 3-rut and Cy 3-rut, respectively. This can be also explained based on intramolecular interactions between neighbouring OH, since it is reasonable to suppose that two neighbouring OH groups allow the formation of hydrogen bonding, which would yield to the most stable system. According to these results, it is evident the existence of relations between the colour and the chemical composition, so that solvating degree signicantly inuences the nal colour characteristics. With the purpose of observing the effect of replacing the 30 -hydroxyl of Dp 3-rut with a glucosyl group (giving Dp 3-rut-30 -glc) on the colour characteristics and stability to pH changes, the chromatic characteristics of the dilutions of these two pigments are presented on the (a*b*)-plane (Fig. 3). The net red hue (hab values between 10 and 30) at very acid pH, gradually changed to yellow hues as the pH increased. It is clear that the glycosidic substitution at the 30 - position of the aglycone compared to non-substitution produced relative large decrease of hab, and less intense colour * (C ab : 32 and L : 79.7 CIELAB units), at pH = 2.0 (Table 2); however, Dp 3-rut-30 -glc is the most resistant to colour changes (lowest DE ab ) as described above. The major colour stability of this anthocyanin compared to the other pigments could be explained by the presence of three sugars in the molecule which protect the avylium ion from the base attack, due to the sandwich conguration of this type of compound (Giusti & Wrolstad, 2003), restricting the hydration possibilities. On the other hand, the antioxidant capacity of the four pure anthocyanins was determined: Dp 3-rut, Cy 3-rut, Pe 3-rut and Dp 3-rut-30 -glc (all of them glycosylated in the 3-position of the C ring). Dp 3-rut-30 -glc has an additional glycoxyl group in the 30 position of the B ring. The results showed that the isolated anthocyanins have higher capacity to capture free radicals in aqueous

80 70 60 50 40
pH 2.0 pH 2.5 pH 2.7 pH 2.9 pH 3.1 pH 3.2 pH 3.3 pH 3.4

80 70 60 50 40
pH 2.0 pH 2.4 pH 2.6

90

80

70 60 50 40

b*

pH 8.7

pH 9.0

pH 3.5

b*

30 20 10
0
pH 8.9 pH 8.7 pH 7.5 pH 6.8 pH 4.7

30 pH 8.7

30 20 10

20

pH 2.9 pH 2.3 pH 3.0 pH 3.2 pH 2.5 pH 2.7 pH 3.5 pH 3.1 pH 2.9 pH 3.7 pH 3.3

pH 2.0

-10 -20 -20

pH 2.0 pH 2.0 10 pH 2.1 pH 2.3 pH 2.5 pH 2.5 pH 7.5 pH 3.0 pH 2.7 pH 3.7 pH 6.8 #37 pH 2.9 pH 5.5 0 pH 3.3 pH 3.1 pH 3.7 pH 4.1

-10

10

20

30

40

50

60

70

80

-10 -10

10

20

30

40

50

60

70

80

a*
Fig. 2. (a*b*)-diagram. Colour changes of the major anthocyanins studied at different pH values (j Pe 3-rut; d Cy 3-rut; N Dp 3-rut).

a*
Fig. 3. (a*b*)-diagram. Colour changes of Dp 3-rut-30 -glc (N) and Dp3-rut (d) at different pH values.

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solution (pH 5.2) than ascorbic acid (Table 1). It was also observed that the hydroxylation degree of the isolated rutinosides have great inuence on the antioxidant capacity. As observed in Table 1, Dp 3-rut was more efcient at capturing the ABTS radical than Cy 3-rut, and this in turn was more efcient than Pe 3-rut. Thus, the rutinosides having hydroxyl groups in ortho position of the B ring, as in the case of Dp 3-rut, Cy 3-rut and Dp 3-rut-30 -glc, have a greater efciency in capturing free radicals, which can be attributed to the fact that hydroxyl groups in the ortho position confer high stability on the formed radical, that is to say they stabilise the formation of the O-semiquinone radical (Rice-Evans, Miller, & Paganga, 1996). In comparison, Dp 3-rut-30 glu, having an additional glycosyl in the 30 -position, was less efcient in capturing radicals in aqueous solution than the similar Dp 3-rut. This indicates that a different glycosylation pattern can considerably modify the antioxidant activity of the anthocyanins, and the extent of this change also depends on the aglycone type. In summary, the characteristics of colour, stability and antioxidant activity of the crude extracts and isolated rutinosides pigments of Tamarillo fruit have been studied in aqueous solution. Through the application of tristimulus colorimetry, the relations of chemical structure and antioxidant pigments colour, and their relevance in pH change have been shown. A relationship between antioxidant activities in vitro and phenolic contents has been observed in crude extracts; however, whether this antioxidant potential has an effective role in vivo remains to be demonstrated. This study shows the potential value of these extracts as antioxidants and in the improvement of nutritional value of foods and their preservation. Furthermore, the possible use of the peelings (usually waste material) for the production of anthocyanins or natural antioxidant extracts can provide some economic benets and added value to this material. Acknowledgements The authors sincerely acknowledge to Colciencias (Colombia), Universidad de Nario (Colombia), and IPICS-Uppsala (University Sweden) for the nancial support. References
Bjory, ., Fossen, T., & Andersen, . M. (2007). Anthocyanins 3-galactosides from Cornus alba Sibirica with glucosidation of the B-ring. Phytochemistry, 68(5), 640645. Bridle, P., & Timberlake, C. F. (1997). Anthocyanins as natural food colours-selected aspects. Food Chemistry, 58(12), 103109. Brouillard, R. (1982). Chemical structure of anthocyanins. In P. Markakis (Ed.), Anthocyanins as food colors (pp. 138). London, UK: Academic Press. Byamukama, R., Kiremire, B. T., Andersen, . M., & Steigen, A. (2005). Anthocyanins from fruits of Rubus pinnatus and Rubus rigidus. Journal of Food Composition and Analysis, 18(6), 599605. Cabrita, L., Frystein, N. A., & Andersen, . M. (2000). Anthocyanins trisaccharides in blue berries of Vaccinium Padifolium. Food Chemistry, 69(1), 3336.

CIE 15:2004, Technical Report Colorimetry, third ed. Commission International de lEclairage, Central Bureau. Degenhard, A., Knapp, H., & Winterhalter, P. (2000). Separation and purication of anthocyanins by high-speed countercurrent chromatography and screening for antioxidant. Journal of Agricultural and Food Chemistry, 48(2), 338343. Delgado-Vargas, F., Jimnez, A. R., & Paredes-Lopez, O. (2000). Natural pigments: Carotenoids, anthocyanins, and betalains-characteristic, biosynthesis, processing, and stability. Critical Reviews in Food Science and Nutrition, 40(3), 173289. Eiro, M. J., & Heinonen, M. (2002). Anthocyanin color behavior and stability during storage: Effect on intermolecular copigmentation. Journal of Agricultural and Food Chemistry, 50(25), 74617466. Fossen, T., & Andersen, . M. (2003). Anthocyanins from red onion, Allium cepa, with novel aglycone. Phytochemistry, 62(8), 12171220. Giusti, M. M., & Wrolstad, R. (2003). Acylated anthocyanins from edible sources and their application in food systems. Biochemical Engineering Journal, 14(3), 217225. Harborne, J. B., & Williams, C. A. (1995). Anthocyanins and other avonoids. Natural Product Reports, 12(6), 639657. Heredia, F. J., Francia-Aricha, E., Rivas-Gonzalo, J. C., Vicario, I. M., & Santos-Buelga, C. (1998). Chromatic characterization of anthocyanins from red grapes. I. pH effect. Food Chemistry, 63(4), 491498. Heredia. F. J., lvarez, C., Gonzlez-Miret, M. L., & Ramrez, A. (2004). CromaLab, anlisis de color. Registro General de la Propiedad Intelectual SE-1052-04. Sevilla, Spain. Markakis, P. (1982). Stability of anthocyanins in food. In P. Markakis (Ed.), Anthocyanins as food colors (pp. 163180). London, UK: Academic Press Inc.. Markovic, J. M. D., Petranovic, N. A., & Baranac, J. M (2000). A spectrophotometric study of the copigmentation of malvin with caffeic and ferulic acids. Journal of Agricultural and Food Chemistry, 48(11), 55305536. Martnez, J. A., Melgosa, M., Prez, M. M., Hita, E., & Negueruela, A. I. (2001). Note: Visual and instrumental color evaluation in red wines. Food Science and Technology International, 7(5), 439444. Mateus, N., Carvalho, E., Carvalho, A. R. F., Melo, A., Gonzlez-Params, A. M., SantosBuelga, C., et al. (2003). Isolation and structural characterization of new acylated anthocyanins-Vinyl-avanol pigments occurring in aging red wines. Journal of Agricultural and Food Chemistry, 51(1), 277282. Mateus, N., Oliveira, J. P., Pissarra, J., Gonzlez-Params, A. M., Rivas-Gonzalo, J. C., Santos-Buelga, C., et al. (2006). A new vinylpyranoanthocyanin pigment occurring in aged red wine. Food Chemistry, 97(4), 689695. Re, R., Pellegrini, N., Proteggente, A., Pannala, M., Yang, C., & Rice, E. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26(910), 12311237. Rein, M. J., & Heinonen, M. (2004). Stability and enhancement of berry juice color. Journal of Agriculture and Food Chemistry, 52(10), 31063114. Rezende, M. C., Moll, A. U., Gonzlez, L. C., Beezer, A., & Mitchell, J. C. (1999). Solute solvent interactions from diffusion of avonoids in methanol. Journal of Solution Chemistry, 28(9), 11071112. Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structureantioxidant activity relationship of avonoids and phenolic acids. Free radical Biology and Medicine, 20(7), 933956. Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16(3), 144158. Somers, T. C., & Evans, M. E. (1977). Spectral evaluation of young red wines: Anthocyanins equilibria, total phenolics, free and molecular SO2, chemical age. Journal of the Science of Food and Agriculture, 28, 279282. Tian, Q., Giusti, M. M., Stoner, G. D., & Schwartz, S. J. (2006). Characterization of a new anthocyanin in black raspberries (Rubus accidentalis) by liquid chromatography electrospray ionization tandem mass spectrometry. Food Chemistry, 94(3), 465468. Wang, H., Race, E. J., & Shrikhande, A. J. (2003). Characterization of anthocyanins in grape juice by ion trap liquid chromatographymass spectrometry. Journal of Agricultural and Food Chemistry, 51(7), 18391844.