colour, ph stability and antioxidant activity of anthocyanin rutinosides

8/13/2019 Colour, pH Stability and Antioxidant Activity of Anthocyanin Rutinosides

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Colour, pH stability and antioxidant activity of anthocyanin rutinosidesisolated 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 Department of Chemistry, Universidad Nacional de Colombia, A.A. 14490 Bogot, Colombiab 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

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

Keywords:AnthocyaninsSolanum betaceumTamarillo fruitTree tomatoColourStabilityAntioxidant

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 studywas made on crude extract (XAD-7 Amberlite-retained fraction) as well as on the following purepigments isolated from tamarillo fruit ( Solanum betaceum Cav.): delphinidin 3- O-(6 00-O-a -rhamnopyrano-syl-b-glucopyranosyl)-3 0-O-b-glucopyranoside, delphinidin 3- O-(6 00-O-a -rhamnopyranosyl)- b-glucopy-ranoside, cyanidin 3- O-(6 00-O-a -rhamnopyranosyl)- b-glucopyranoside and pelargonidin 3- O-(6 00-O-a -rhamnopyranosyl)- b-glucopyranoside. The relationships between the colour and the hydroxylationdegree of the B ring and the pH have been studied for the rst time on rutinosides. The peel extractshowed much more colour stability than the jelly extract at all the pH values studied. The replacementof the 3 0-OH with a glycosyl group increased the stability of the colour to pH changes, although this sub-stitution yields a less colourful (higher L* and lower C ab ) compound (Dp 3-rut-3

0-glc), having both hyp-sochromic and hypochromic shifts relative to the non-glycosylated molecule (Dp 3-rut). Moreover, theinuence of the hydroxylation degree of the B ring on the quality and stability of colour, as well as onthe antioxidant activity, was determined.

2009 Elsevier Ltd. All rights reserved.

1. Introduction

Most foodstuffs are exposed to some kind of processing beforebeing 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 a-vourings) are used to recover or to emphasize original features,to ensure uniformity, and to guarantee quality.

Pigments are chemical components absorbing radiation in thevisible region of the electromagnetic spectrum. The colour is dueto a specic molecular group (chromophore) which absorbs energyand, as consequence, the excitation of an electron of external orbi-tals with major energy occurs; the non-absorbed energy is re-ected and refracted and detected by the eyes, where impulsesare generated andsent to the brain, and then, interpreted as colour(Delgado-Vargas, Jimnez, & Paredes-Lopez, 2000 ). Based on thechromophore chemical structure, pigments can be classied aschromophore withconjugated systems (carotenoids, anthocyanins,betalains, caramel and synthetic pigments) or porphyrins withcoordinatedmetals (myoglobin, chlorophyll, and their derivatives).

In the conjugated systems, anthocyanins are specically impor-tant because they are responsible for some red colours in the nat-ure, as monomeric, oligomeric and polymeric anthocyanins. Theuse of natural extracts of these pigments as food additives hassome 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 inthe avylium ion form ( Harborne & Williams, 1995 ). It has beendemonstrated that anthocyanin stability is inuenced by substitu-ents in their structures, sugars and acyl groups ( Giusti & Wrolstad,2003). In recent years, research on anthocyanin chemical structurehas increased ( Bjory, Fossen, & Andersen, 2007; Byamukama,Kiremire, Andersen, & Steigen, 2005; Cabrita, Fr ystein, & Ander-sen, 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 relationshipsbetween colour and chemical composition, which may help tounderstand the basic principles that inuence the anthocyaninscolour.

In Colombia, the tamarillo or tree tomato ( Solanum betaceumCav.) is a promising product for export, due to its colour; the redvariety has been the most accepted internationally. In this workthe stability of tamarillo fruit extracts and isolated individual

0308-8146/$ - see front matter 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodchem.2009.03.081

* Corresponding author. Tel.: +34 95455 6761; fax: +34 95455 7017.E-mail address: [email protected] (F.J. Heredia).

Food Chemistry 117 (2009) 8893

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anthocyanin rutinosides ( Fig. 1) to pH changes has been evaluatedby means of colorimetric studies, to obtain more precise informa-tion about the change of colour in both crude extracts and individ-ual anthocyanins.

2. Materials and methods

2.1. Chemicals and supplies

The 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchro-man-2-carboxylic acid (Trolox) and Amberlite XAD-7 were ob-tained from Rohm and Haas, Darmstadt, Germany. HPLC-gradeacetonitrile, ACS-grade n-butanol, methanol, tert -butyl methylether (TBME), formic acid, hydrochloric acidand potassiumpersul-fate were purchased from Merck, Bogot, Colombia. CD 3OD,CF3COOD, and CF3COOH (TFA) were obtained from SigmaAldrich(St. Louis, MO).

2.2. Plant material

Tamarillo fruits (5.09kg) were collected in Puente Nacional(Santander, Colombia). A voucher specimen was coded as Col343584 at the Instituto de Ciencias Naturales at Universidad Nac-ional de Colombia.

2.3. Isolation of crude anthocyanins extract

Fresh ripe fruits were washed and peeled; the seeds and thesurrounding jelly were manually separatedfrom theesh. The jelly(250 g), ltered through glass wool, was applied onto an AmberliteXAD-7 column (800 mm long, 40 mm i.d.). The column waswashed with 1.25 l of water, and elution of anthocyanins was car-ried out with 300 ml of a mixture of methanol:acetic acid (19:1, v/

v). The eluate was concentrated under reduced pressure at 35 Cand the aqueous solution was lyophilised (crude jelly extract). Thisprocedurewas repeatedfour times to obtain 4.35 g of thecrude jel-ly extract.

The peelings (2.51 kg) were cut into small pieces (2 cm 2) andextractedwith 2 l of methanol:acetic acid (19:1, v/v) for 12 h (mac-eration). After ltration the organic solvent was evaporated at35 C using a rotary evaporator and the remaining aqueous phasewas 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 jellyand peelings were fractionated by multilayer coil countercurrentchromatography (MLCCC).

2.3.1. Countercurrent chromatography (CCC)A multilayer coil countercurrent chromatograph (P.C. Inc., Poto-

mac, MD) with tubular column of PTFE (400 ml total volume) wasused. Solvent system consisted of n-butanol:TBME:acetoni-trile:water (2:2:1:5) v/v/v/v, acidied with 0.1% TFA ( Degenhardet al., 2000 ). The organic phase was used as the stationary phase;therefore elution mode was head to tail. Crude anthocyanins ex-tract was dissolved in 5 ml of a mixture of stationary phase andmobile phase (1:1 v/v), and introduced through the injection port.The mobile phase was pumped at 1 ml min 1, while centrifugationwas carried outat 800 rpm. Four-millilitre fractions were collected.The sample loads in MLCCC were high (0.6 g), so fractionation of upto several hundred milligrams of sample was achieved in a singleMLCCC run. To check the purity, each fraction was analysed byHPLC, and further purication was carried out using preparativeHPLC.

2.3.2. High performance liquid chromatographyThe analytical HPLC results were obtained with an Agilent 1100

HPLC system (Agilent, Santa Clara, CA) tted with a photodiode ar-ray detector and a Zorbax-SB C 18 column (4.6mm 250mm;5 l m lm thickness). Two solvents were used for elution: A = ace-tonitrile:formic acid:water (3:10:87, v/v/v) and B = acetoni-trile:formic acid:water (50:10:40, v/v/v). The elution proleconsisted 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 3035 min. Aliquots of 100 l l (0.1 mg ml 1) were injected and theow rate was 0.8 ml min 1. Prior to injection, all samples were l-tered through a 0.45 l m Millipore membrane lter.

Preparative HPLC was performed using a Luna C 18 column(10 mm 250 mm: 5 l m lm thickness) and a 6000LP UV detec-tor. An isocraticelution prole was applied(95% A, 5%B) using ace-tonitrile, formic acid and water (solvent A: 3:10:87, v/v/v; solventB: 50:10:40, v/v/v). The ow rate was 4 ml min 1 for 20 min andaliquots of 40 l l (250 mg ml 1) were injected.

2.3.3. SpectroscopyUVVis absorption spectra of anthocyanins were recorded on-

line during HPLC analysis, and the spectral measurements weremade over the wavelength range 300680 nm in steps of 2 nm.ESIMS analyses were performed on a Shimadzu QP-8000 massspectrometer (Shimadzu, Japan). The electrospray voltage appliedwas 4.5 kV, nebuliser gas ow of 4.5 l min 1, probe voltage4.5 kV, curved desolvation line (CDL) voltage 130 V, CDL tempera-ture of 230 C, deector voltage at 45 and 60 V and acquisitionfrom m/z 50 to m/z 800 in positive ionisation mode. A solution of 1mgml 1 of each puried pigment was dissolved in a 1:1 mixtureof solvent A (water:formic acid9:1) and solvent B (acetonitrile:for-mic acid 9:1). The anthocyanin solutions were injected directly

into the system at a ow rate of l00 l l min1

. Low-resolution fastatom bombardment MS of the pigments was performed on an

O

O

O

O

H

HO

OH

HO

O

HO

HO

HOH

H 3 C

R 1

OH

R 2

+HO

OH

2

3

45

6

7

8

9

10

1'

2'

3'

4'

5'

6'

1'' 2''

3''

4''5''

6''

1'''

2'''

3'''4'''

5'''6'''

A C

B

Fig. 1. Basic structure of tamarillo anthocyanins ( Solanum betaceum Cav). Dp 3-rut-30-glc = delphinidin 3- O-(6 00-O-a -rhamnopyranosyl- b-glucopyranosyl)-3 0-O-b-gluco-pyranoside (R 1 = O-glc, R 2 = OH), Dp 3-rut = delphinidin-3- O-(6 00-O-a -rhamnopyr-anosyl)- b -glucopyranoside (R 1 = R 2 = OH), Cy 3-rut = cyanidin-3- O-(6 00-O-a -

rhamnopyranosyl)- b-glucopyranoside (R 1 = OH, R 2 = H), Pe 3-rut = pelargonidin-3-O-(600-O-a -rhamnopyranosyl)- b-glucopyranoside (R 1 = R 2 = H). glc= glucopyranoside.

N.H. Hurtado et al./ Food Chemistry 117 (2009) 8893 89


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AutoSpec-Q (Waters Corporation, Milford, MA) in a glycerol-NaImatrix, using positive detection and acquisition from m/z 50 tom/z 900. Argon was used as collision gas. The complete structuresof isolated anthocyanins were established by 1H- and 13C NMR analysis. Full assignments were performed with TOCSY, COSY,HSQC and HMBC experiments. Isolated anthocyanins dissolved ina mixture of CD 3OD:CF3OOD (19:1, v/v) were measured using aBruker AMX-500.

Thus, the identities of anthocyanins were determinedto be:del-phinidin3- O-(6 00-O-a -rhamnopyranosyl- b-glucopyranosyl)-3 0-O-b-glucopyranoside (Dp 3-rut-3 0-glu), delphinidin 3- O-(6 00-O-a -rhamnopyranosyl)- b -glucopyranoside (Dp 3-rut), cyaniding 3- O-(600-O-a -rhamnopyranosyl)- b -glucopyranoside (Cy 3-rut) andpelargonidin 3- O-(6 00-O-a -rhamnopyranosyl)- b-glucopyranoside(Pe 3-rut). The chemical structures of these anthocyanins areshown 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 FolinCiocalteumethod ( Singleton & Rossi, 1965 ). Sample aliquots of 0.5 ml wereadded to 0.5 ml of water, 5 ml of FolinCiocalteau reagent(0.2N), and 4 ml of a saturated solution of sodium carbonate(75 g/l), and mixed thoroughly. The absorbance was measured at765 nm with an HP8452 spectrophotometer (Hewlett Packard,Palo Alto, CA) after incubation for 2 h at room temperature. Quan-tication 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 expressedin % w/w (100 mg gallic acid/mg crude extract).

The anthocyanic indices represent approximate measurementsof the phenolic constituents and they can be used in comparativeevaluations. The anthocyanin extracts include both monomericanthocyanins and polymeric pigments. When a solution containinganthocyanins is treated with an excess of SO 2 an immediate decol-ouration of the solution occurs, so the residual colour existing aftersuch treatment is due to polymeric pigment forms.

Aqueous solutions (2 mg ml 1, pH 5.2) of crude extracts wereprepared for the chemical indices determination. The index of polymeric pigments ( IPP ) was measured at 520 nm and total anth-ocyanic colour ( AC ) was measuredin 1 M HCl at 520 nm. Polymericcolour ( 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 dif-ference ( MC = AC (5 IPP /3)). The concentration of total mono-meric anthocyanins ( AT ) in 1 M HCl was expressed as delphinidin3-glucoside chloride (molecular mass 500.5) using the molarabsorptivity value ( e) of 23,700 l mol 1 cm 1 (Heredia, Francia-Ari-cha, 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 TEACmethod ( Re et al., 1999 ), which is based on the capacity of antiox-idants to capture the radical 2,2 0-azino-bis-(3-ethylbenzothiazo-line-6-sulfonic acid) (ABTS +). It was performed using the HP8452spectrophotometer in kinetic mode. ABTS + radical cation was pro-duced by reacting 7 mM 2,2 0-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diamonium salt and 2.45 mM potassium persulfate,after incubation at room temperature in the dark for 16 h. TheABTS+ 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 l lof sample. ABTS+ solution (1 ml; absorbance of 0.70 0.1) was

read at 734 nm and 20 l l of the sample were added and mixedthoroughly. Trolox standards of nal concentration 015 l M in

ethanol were prepared and assayed under the same conditions.Trolox equivalent antioxidant capacity of sample was calculatedbased on the inhibition exerted by standard Trolox solution at6 min.

2.6. Colorimetric study

The evaluation of the colour was based on the spectrophoto-metric measurement of the transmission spectrum in the visibleregion (380770nm) using an HP8452. The colour parameterswere obtained through weighted ordinates method ( D k = 2 nm)from transmission spectra, by using the CromaLab software(Heredia, lvarez, Gonzlez-Miret, & Ramrez, 2004 ), which takesinto consideration the International + Commission on Illuminationrecommendations ( CIE, 2004). D65 standard illuminant, corre-sponding to the natural daylight, and 10 standard observer wereconsidered in the calculations. Reference blank measurementswere made with the cuvette lled with distilled water. CIE 1976( L*a*b*) (CIELAB) uniform colour space was taken into accountfor the colorimetric analysis. Within the CIELAB uniform space apsychometric index of lightness, L* (ranging from 0, black, to 100,white), and two colour coordinates, a* (which takes positivevaluesfor 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 aredened: the hue angle ( hab) is the qualitative attribute of colour,and the chroma ( C ab ) is the quantitative attribute of colourintensity.

2.6.1. The pH effect The CIELAB parameters (L*, a*, b*, C ab , hab) were determined in

5 10 5 M solutions of each anthocyanin at different pH values,ranged from 2.0 to 8.7. Modications in pH were made by additionof small volumes of NaOH (1 M or 10 M). Crude extracts were di-luted in water until 0.8 absorbance units at k = 520 nm were ob-tained in order to study the inuence of the pH on the colour;the concentration of the diluted jelly extract was 2.1 mg/ml, andthat of the diluted peelings extract was 35 mg/ml.

The colour differences DE ab were calculated between the initialpH value (pH = 2.0) and after each increase of pH, considering theEuclidean distance between the two colour points:DE ab = ((D L*)2 + (D a*)2 + (D b*)2)1/2 . Data consisted of the averageof two experimental values.

3. Results and discussion

Once both jelly and peelings crude extracts were obtained, dif-ferent chemical characteristics were determined (anthocyanicindices). The XAD-7 jelly extract showed higher content of totalphenols (TP) and total anthocyanins (TA), while the peelings ex-

tract showed higher index of polymeric pigments ( IPP ). The highercontent of TPand TAin the crude jelly extract is in accordance withthe 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 anthocyanincrude extracts was studied within the pH range 2.06.2, that isto say under and above the most common pH values in foods. Asrevealed in Table 1 both jelly andpeelings extracts showed reddishhues at the lowest pH value (peelings: 32.1 ; jelly: 35.0 ). As thepH increased to 6.2 the colour of the peelings extract graduallychanged towards orange hues (until hab = 64.9 ) and the jelly ex-tract towards red-purple hues (until hab = 0.4 ). The colour differ-

ences DE ab regarding the pH = 2.0 sample (the most colourfulsample) are summarised in Table 2 . These differences were higher

90 N.H. Hurtado et al./ Food Chemistry 117 (2009) 8893


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for the jelly extract across the pH range, which is evidence of thelower colour stability (higher sensitivity to pH changes) of this ex-tract. The higher stability of the peelings extract may be due notonly to thehigher polymeric anthocyaninscontent, whicharecom-ponents more stable to pH changes ( Somers & Evans, 1977 ), butalso to the possible presence of some compounds stabilising thecolour in the extract, i.e., phenolic acids, avones, avonols, ava-nones, avanols and organic acids ( Eiro & Heinonen, 2002; Marko-vic, Petranovic, & Baranac, 2000; Rein & Heinonen, 2004 ). Thecrude extracts were shown to be relatively more stable underlow acid conditions (pH 2.03.4); however, even in the crude peel-ings extract (more stable) the colour differences were up to 3 CIE-LAB units (Table 2 ) indicating that they can be visuallydiscriminated ( Martnez, Melgosa, Prez, Hita, & Negueruela,2001 ).

3.2. UVvis spectroscopy of individual anthocyanins

For this study, the following anthocyanins isolated from thefruit were studied: Dp 3-rut-3 0-glc, Dp 3-rut, Cy 3-rut and Pe 3-

rut. The maximum absorption in the visible spectrum of the aque-ous solution (5 10 5 M; pH 2.0) and the molar absorption ( e) of each anthocyanin are shown in Table 1 . Comparing the three ruti-nosides, of which the structural differences are the number of hy-droxyl groups in the B ring, it is observed that Pe 3-rut, having onehydroxyl group, shows a k max at 500 nm. However, as the numberof hydroxyl groups in the ortho position increases (Cy 3-rut andDp3-rut) the maximum absorption shifts towardshigher wavelengthsand the absorption intensity decreases. Analysing the spectralcharacteristics of the delphinidin derivatives, it can be observedthat the replacement of the 3 0-hydroxyl of the Dp 3-rut with a glu-cose moiety causes both hypsochromic and hypochromic shifts inthe visible spectrum ( Table 1 ).

The spectral measurements were important to study the effectof hydroxylation and glycosylation in the B ring of the aglyconeon the visible spectrum; however, with only this data we do nothave enough information to explain the chromatic characteristicsof the anthocyanins. It is well known that the properties of antho-cyanins, including the colour expression, are inuenced by thechemical structure and pH ( Heredia et al., 1998 ).

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 k max e TEAC (pH 5.2) TP PPI TA

Jelly 500 1.90 0.125 b 25.11 0.9 0.20 0.034 20.03 0.85Peelings 530 1.09 0.076 b 13.69 0.8 1.16 0.107 0.20 0.03Dp 3-rut-3 0-glc 514 15974 2.20 0.026 a Dp 3-rut 518 25874 4.91 0.146 a

Cy 3-rut 512 27268 1.80 0.098 a Pe 3-rut 500 36660 1.48 0.045 a Ascorbic acid 1.09 0.093 a

e: molar absorption (0.1% HCl in ethanol). TEAC: Trolox equivalent antioxidant capacity.a mmol of Trolox/mmol compound.b 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).

Table 2

CIELAB colour parameters ( L* ,a * ,b * , C ab , h ab ) and colour differences ( D E ) of crude extracts (jelly and peelings) and pure anthocyanins isolated from tamarillo fruit ( Solanumbetaceum Cav.).

pH 2.03.4 pH 3.46.2

Jelly Peelings Jelly Peelings

a i 62.1 51.3 55.4 52.4a f 55.4 52.4 28.7 24.1bi 43.5 32.2 22.5 42.5b f 22.5 42.5 0.2 51.6Li 45.5 51.2 49.0 46.3L f 49.0 46.3 66.1 40.5C ab ; i 75.8 60.6 59.8 67.4C ab ; f 59.8 67.4 28.7 56.9hab,i 35.0 32.1 22.1 39.0hab,f 22.1 39.0 0.4 64.9D E ab 22.3 11.5 38.8 30.3

pH 2.03.4 pH 3.4 6.2

Dp 3-rut-3 0-glc Dp 3-rut Cy 3-rut Pe 3-rut Dp 3-rut-3 0-glc Dp 3-rut Cy 3-rut Pe 3-rut

a i 28.1 59.1 52.7 44.4 6.5 24.6 28.9 34.3a f 6.5 24.6 28.9 34.3 1.0 0.3 5.4 6.0bi 4.8 15.2 26.8 62.7 4.3 1.1 5.8 34.8b f 0.6 1.1 5.8 34.8 0.4 0.4 2.6 2.1Li 79.7 70.8 74.2 78.3 87.5 87.2 85.0 80.1L f 87.5 87.2 85.0 80.1 89.0 98.5 85.4 97.6C ab ; i 32.0 61.0 59.1 76.9 7.8 24.7 29.5 48.9C ab ; f 7.8 24.7 29.5 48.9 1.0 1.9 29.4 6.3hab,i 28.6 14.4 26.1 54.7 33.3 2.5 11.4 45.4hab,f 33.3 2.5 11.4 45.4 21.0 23.9 22.5 19.0D E ab 23.3 41.5 33.5 29.7 7.4 27.4 24.9 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 rutinosidessolutions 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 repre-sent variation of the hydroxylation grade of the B ring of manyanthocyanins isolated from fruits and vegetables ( Brouillard,1982 ). Aqueous solutions (5 10 5 M) of Dp 3-rut-3 0-glc, Dp 3-rut, Cy 3-rut and Pe 3-rut were studied at two pH range: (2.03.4) and (3.46.2). According to the CIELAB parameters ( L*,a*,b*,C ab , hab , DE ab ) their colour stabilities depend highly on the pHand the structure ( Fig. 2, Table 2). At the lowest pH value (pH2.0, where anthocyanins exit basically in avylium form), Dp 3-rut-3 0-glc, Dp 3-rut and Cy 3-rut showed reddish hues(hab = 28.6 , 14.4 and 26.1 , respectively), while Pe 3-rut showedorange ones ( hab = 54.7 ). At this pH, the number of hydroxylgroups of the B ring clearly inuenced the colour characteristic;the hue angle ( hab) and lightness ( L*) values were clearly higherfor Pe 3-rut (only one OH), followed by Dp 3-rut-3 0-glc (two OH),Cy 3-rut (two OH) and Dp 3-rut (three OH). Dp 3-rut-3 0-glc under-went small decreases of the hue angle as the pHincreased from 2.0to 6.2, while great decreases in hue were found for Dp 3-rut, Cy 3-rut andPe 3-rut. It is noticeable that anthocyanins containing agly-cones with only two or three hydroxyl groups on the B ring (Cy 3-rut and Dp 3-rut) showed smaller colour differences ( DE ab ) in themore 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 Dp3-rut (three OH) was more unstable (the highest DE ab ).

Comparing the DE ab values at both pH ranges, it was observedthat Dp 3-rut, Cy 3-rut and Pe 3-rut were clearly more unstablethan Dp 3-rut-3 0-glc. The loss of colour occurring due to the baseattack on the pigment structure could be related to the solvatinggrade of each molecule, in such a way that the greater the solvatinggrade was the higher the stability to pH changes. According to astudy 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 (lowestsolvating) at low pH (2.03.4). Nevertheless, at pH 3.46.2, whichinduces the formation of quinoidal bases ( Brouillard, 1982 ), differ-ent behaviours were observed. Anthocyanins having two or threehydroxyl 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 intramolecularinteractions between neighbouring OH, since it is reasonable tosuppose 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 relationsbetween the colour and the chemical composition, so that solvat-ing degree signicantly inuences the nal colour characteristics.

With the purpose of observing the effect of replacing the 3 0-hy-droxyl of Dp3-rut with a glucosyl group (giving Dp3-rut-3 0-glc) onthe colour characteristics and stability to pH changes, the chro-matic characteristics of the dilutions of these two pigments arepresented on the ( a*b*)-plane ( Fig. 3). The net red hue ( hab valuesbetween 10 and 30 ) at very acid pH, gradually changed to yellowhues as the pH increased. It is clear that the glycosidic substitutionat the 3 0- position of the aglycone compared to non-substitutionproduced 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-3 0-glc is the most resistant to colour changes (lowestDE ab ) as described above. The major colour stability of this antho-cyanin compared to the other pigments could be explained by thepresence of three sugars in the molecule which protect the avyli-um ion from the base attack, due to the sandwich congurationof this type of compound ( Giusti & Wrolstad, 2003 ), restricting thehydration possibilities.

On the other hand, the antioxidant capacity of the four pureanthocyanins was determined: Dp 3-rut, Cy 3-rut, Pe 3-rut andDp 3-rut-3 0-glc (all of them glycosylated in the 3-position of theC ring). Dp 3-rut-3 0-glc has an additional glycoxyl group in the 3 0

position of the B ring. The results showed that the isolated antho-cyanins have higher capacity to capture free radicals in aqueous

-20 -10 0 10 20 30 40 50 60 70 80

a*

-20

-10

0

10

20

30

40

50

60

70

80

b *

pH 2.0

pH 2.0

pH 2.0

pH 2.9

pH 3.5

pH 3.4

pH 3.3pH 3.2

pH 3.1

pH 2.7pH 2.5

pH 4.7

pH 9.0pH 2.4

pH 2.6

pH 2.9pH 3.0

pH 3.2pH 3.5

pH 2.3pH 2.5

pH 2.7pH 2.9pH 3.1

pH 8.7

pH 8.9

pH 7.5

pH 6.8

pH 3.3pH 3.7

pH 8.7

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

b *

10

20

30

40

50

60

70 80 90

#37

-10

0

10

20

30

40

50

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pH 2.0

pH 4.1

pH 3.7pH 3.0

pH 5.5

pH 2.5pH 2.1

pH 2.0

pH 6.8

pH 7.5

pH 8.7

pH 3.7 pH 3.3pH 3.1

pH 2.9pH 2.7

pH 2.5pH 2.3

-10 0 10 20 30 40 50 60 70 80

a*

Fig. 3. (a*b*)-diagram. Colour changes of Dp 3-rut-30

-glc (N ) and Dp3-rut ( d ) atdifferent pH values.

92 N.H. Hurtado et al./ Food Chemistry 117 (2009) 8893


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solution (pH 5.2) than ascorbic acid ( Table 1 ). It was also observedthat the hydroxylation degree of the isolated rutinosides havegreat inuence on the antioxidant capacity.

As observed in Table 1 , Dp 3-rut was more efcient at capturingthe ABTS radical than Cy 3-rut, and this in turn was more efcientthan Pe 3-rut. Thus, the rutinosides having hydroxyl groups inortho position of the B ring, as in the case of Dp 3-rut, Cy 3-rutand Dp 3-rut-3 0-glc, have a greater efciency in capturing free rad-icals, which can be attributed to the fact that hydroxyl groups inthe ortho position confer high stability on the formed radical, thatis to say they stabilise the formation of the O-semiquinone radical(Rice-Evans, Miller, & Paganga, 1996 ). In comparison, Dp 3-rut-3 0-glu, having an additional glycosyl in the 3 0-position, was less ef-cient in capturing radicals in aqueous solution than the similarDp 3-rut. This indicates that a different glycosylation pattern canconsiderably 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 antioxi-dant activity of the crude extracts and isolated rutinosides pig-ments of Tamarillo fruit have been studied in aqueous solution.Through the application of tristimulus colorimetry, the relationsof chemical structure and antioxidant pigments colour, and theirrelevance in pH change have been shown.

A relationship between antioxidant activities in vitro and phe-nolic contents has been observed in crude extracts; however,whether this antioxidant potential has an effective role in vivo re-mains to be demonstrated. This study shows the potential value of these extracts as antioxidants and in the improvement of nutri-tional value of foods and their preservation. Furthermore, the pos-sible use of the peelings (usually waste material) for theproduction of anthocyanins or natural antioxidant extracts canprovide some economic benets and added value to this material.

Acknowledgements

The authors sincerely acknowledge to Colciencias (Colombia),Universidad de Nario (Colombia), and IPICS-Uppsala (UniversitySweden) for the nancial support.

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