Food Chemistry 133 (2012) 256–263

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Food Chemistry

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Chemical characterization and evaluation of antioxidant properties of Açaífruits (Euterpe oleraceae Mart.) during ripening

André Gordon a, Ana Paula Gil Cruz b, Lourdes Maria Corrêa Cabral c, Sidinéa Cordeiro de Freitas c,Cristina Maria Araujo Dib Taxi d, Carmen Marino Donangelo b, Rafaella de Andrade Mattietto e,Mirko Friedrich a, Virgínia Martins da Matta c, Friedhelm Marx a,⇑a University of Bonn, Department of Nutrition and Food Sciences, Endenicher Allee 11-13, 53115 Bonn, Germanyb Federal University of Rio de Janeiro, Institute of Chemistry, Avenida Athos da Silveira Ramos 149 – Bloco A, 21941-909 Rio de Janeiro, Brazilc Embrapa Food Technology, Av. das Américas, POB 29.501, 23020-470 Rio de Janeiro, Brazild Institute of Agricultural Science, Federal Rural University of Amazon, Avenida Presidente Tancredo Neves, 2501, 66077-901 Belém, Brazile Embrapa Eastern Amazon, Trav. Dr. Enéas Pinheiro, POB 48, 66095-100 Belém, Brazil

a r t i c l e i n f o

Article history:Received 6 April 2011Received in revised form 7 November 2011Accepted 23 November 2011Available online 12 January 2012

Keywords:Açaí (Euterpe oleracea Mart.)AnthocyaninsRipeningFlavonesTOSCAntioxidant capacityLC–MSTEAC

0308-8146/$ - see front matter � 2012 Published bydoi:10.1016/j.foodchem.2011.11.150

⇑ Corresponding author. Tel.: +49 228 733713; fax:E-mail address: f.marx@uni-bonn.de (F. Marx).

a b s t r a c t

Consumption of açaí fruits has been linked to positive health effects due to its phenolic content and nutri-tive value. The objective of this study was to characterize açaí fruits chemically and to determine the anti-oxidant capacity at three different maturity stages. With the exception of fat, amounts of macronutrients,minerals and titratable acids decreased during the ripening process. The same trend was observed formost of the phenolic constituents identified by HPLC–ESI-MS/MS. A consistent decline was shown forflavones and hydroxycinnamic acids. The concentration of the anthocyanins increased in the course ofripening. In accordance with the total amount of the identified phenolic compounds, the antioxidantcapacity, measured by TEAC and TOSC, also decreased. However, the contribution of the main phenoliccompounds to the overall antioxidant capacity evaluated by TOSC was estimated to be low.

� 2012 Published by Elsevier Ltd.

1. Introduction

Açaí (Euterpe oleracea Mart.) is a tropical palm tree that occursnaturally in the Amazon region. Its spherical grape-sized fruitsare green when young and ripen usually to a dark purple (Strud-wick & Sobel, 1988). An important reloading point for açaí is thecity of Belém in Pará State, Brazil, where fruits can be collectedthroughout the year. However, a major harvesting period existsduring the ‘‘dry-months’’ from August to December. Usually, thefruits are used to prepare a liquid with creamy texture by macer-ating the pericarp and mixing it with different amounts of water,yielding commercially available açaí pulp (Lichtenthäler et al.,2005). In the production region, açaí is integrated in the daily die-tary habits of the native people and is normally used in main mealsfor lunch or dinner. In modern Brazilian society, it has gained inter-est as a nutritionally valuable wellness product (Strudwick & Sobel,1988). Meanwhile, açaí is favoured as an ingredient in fruit bever-

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ages beyond the Brazilian borders and is exported mainly to theUSA or to Europe (Sabbe, Verbeke, Deliza, Matta, & Van Damme,2009).

Interest in açaí has arisen especially due to its high in vitro anti-oxidant activity, explained by the considerably high content ofphenolic compounds, e.g., different anthocyanins, flavones, andphenolic acids (Lichtenthäler et al., 2005; Pacheco-Palencia,Duncan, & Talcott, 2009). Phenolic constituents are generallyassociated with health-promoting properties and the preventionof several degenerative diseases (Xia, Deng, Guo, & Li, 2010).Because of legislators and consumers’ growing concern over theuse of artificial food additives, there is a growing demand foradditives from natural resources (Giusti & Wrolstad, 2003). Forinstance, anthocyanins can be used as food colourants (Pazmino-Duran, Giusti, Wrolstad, & Gloria, 2001). Moreover, plant extractscontaining a broad range of polyphenols may act as antioxidantsor antimicrobial agents (El-Hela & Abdullah, 2010).

Generally, the ripening process of fruits is indicated by intensivemetabolism of primary and secondary plant compounds. Thisstudy gives information on quantitative changes of macronutri-

A. Gordon et al. / Food Chemistry 133 (2012) 256–263 257

ents, individual polyphenols and the antioxidant activity in açaífruits during ripening. In addition, the influence of major individualphenolic compounds on the overall antioxidant capacity wasestimated.

2. Materials and methods

2.1. Chemicals

Ultrahigh quality (UHQ) water was prepared with a Direct-Q 3system (Millipore, Billerica, USA). Protocatechuic acid (P97%)and p-hydroxybenzoic acid (P99%) were purchased from Merck(Darmstadt, Germany) and caffeic acid (purum) from SERVA Fei-nbiochemica (Heidelberg, Germany). Gallic acid (P97.5%), vanillicacid (P97%), syringic acid (P95%), chlorogenic acid (5-O-(3,4-dihydroxycinnamoyl)-L-quinic acid) (P97%), taxifolin (P85%),diethylenetriaminepentaacetic acid (DTPA) (P99 %), a-keto-c-methylbutyric acid (KMBA) (P97%), 2,20-azobis(2-methylpropio-namidine) dichloride (ABAP) (P97%), 3-morpholinosydnonimineN-ethylcarbamide (SIN-1), 2,2-azinobis-(3-ehtylbenzothiazoline-6-sulphonic acid)-diammonium salt (ABTS), Folin–Ciocalteu re-agent, and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylicacid (Trolox�) were obtained from Sigma–Aldrich Chemie GmbH(Steinheim, Germany). Cyanidin 3-O-glucoside (P96%), cyanidin3-O-rutinoside (P96%), peonidin 3-O-glucoside (P95%), luteolin(P99%), isovitexin (P99%), chrysoeriol (P99%), orientin (P99%)and homoorientin (P99%) were purchased from Extrasynthèse(Genay, France). Luteolin 7-O-glucoside and chrysoeriol 7-O-gluco-side standards were a gift from Professor Dr. Galensa (University ofBonn). They were self-isolated and had a purity grade of P84% andP70%, respectively.

2.2. Raw material, sampling and preparation of the pulp

Açaí fruits of three different maturity stages (unripe – greenfruits, intermediate – reddish-brown fruits, ripe – deep purplefruits) were collected at the experimental station of the FederalRural University of Amazon in Castanhal, PA, Brazil. Fruits of eachmaturity stage were processed separately, following the specifica-tions that are usually applied by the industry. After reception,the fruits were selected, washed, weighed, and sanitized inchlorinated water. The subsequent maceration step consisted ofsteeping the fruits in hot water (50 �C) for 30 min to facilitatethe separation of the exo- and mesocarp from the seeds. Thisseparation was carried out in an açaí specific extractor by addingwater in a proportion of 0.6 l/kg fruit. The obtained pulp wasstored at �30 �C.

2.3. Determination of ash, minerals, lipids, protein, carbohydrates andtitratable acids

Minerals were quantified by using flame atomic absorptionspectrometry according to official AOAC methods (2005).Proximal composition of açaí pulp samples at three maturitystages was determined according to official AOAC methods(1998). Ash content was measured by a gravimetric assay. Lipidswere determined by acid hydrolysis and gravimetric measure ofthe final ether solution. Protein content was measured followingKjeldahl method. Total protein was calculated by multiplicationof the obtained nitrogen content with the fruit-specific conver-sion factor of 6.25. Carbohydrates were calculated as centesimaldifference of the previous determinations. All results are referredto dry matter (DM) basis. Titratable acids were determined bya titration method using an automatic titrator with sodium

hydroxide. Results are expressed in malic acid equivalent/100 gof DM.

2.4. Identification and quantification of phenolic compounds by HPLC–ESI-MS/MS

2.4.1. Extraction of phenolic compoundsAçaí pulp was lyophilized and defatted by Soxhlet extraction

with petroleum ether. The extraction of phenolic compoundswas performed using a modified sample preparation accordingto a method described in Pacheco-Palencia et al. (2009). Fortwo times, sample of each maturity stage (500 mg) was dissolvedin 10 ml of acetone–water–formic acid (70% + 29% + 1%; v/v/v),sonicated for 10 min, then centrifuged for 10 min with10,000 rpm at 10 �C. The residue was extracted once more with10 ml acetone–water–formic acid. Afterwards the supernatantswere combined. To get rid of the organic solvent, the extractwas vapourized using a rotary evaporator (Rotavapor R-210,Büchi, Essen, Germany) at 30 �C and the aqueous supernatantwas shaken with 10 ml ethyl acetate. The received ethyl acetatefraction was vapourized to dryness. The residue was solubilizedin 1 ml methanol–water–formic acid (50% + 49% + 1%; v/v/v) andfiltered through a 1.0/0.45 lm syringe filter (Chromafil GF/PET-45/25, Macherey–Nagel, Düren, Germany) prior to application toHPLC–ESI-MS/MS.

Anthocyanins were extracted using a modified method explic-itly described in Wu, Gu, Prior, and McKay (2004). Briefly, freeze-dried sample (250 mg) of each maturity stage was extracted induplicate with 4 ml of methanol–UHQ water–acetic acid(MeOH–H2O–HAc) (50% + 49.5% + 0.5%; v/v/v). After vortexing,sonication and centrifugation, the supernatant was removed andthe sample once more extracted but with 2.5 ml of MeOH–H2O–HAc. Both sample solutions were combined and filteredthrough a 1.0/0.45 lm syringe filter (Chromafil GF/PET-45/25,Macherey–Nagel, Düren, Germany) prior to application toHPLC–ESI-MS/MS.

2.4.2. Analysis of phenolic compoundsQuantification of the phenolic compounds was performed

following a method described in Gordon, Schadow, Quijano, andMarx (2011). HPLC instruments consisted of a pump system anda UV-detector of the HP 1050 series (Hewlett Packard, Waldbronn,Germany), a degasser Degasys Populair DP3010 (Uniflows, Tokyo,Japan) and an analytical column Aqua 3 lm C18, 150 mm, 2 mmi.d., with a guard column Security Guard, C18, 4 mm, 2 mm i.d.(both Phenomenex, Aschaffenburg, Germany). The solvents wereUHQ water with 1% (v/v) formic acid (mobile phase A) and 1%(v/v) formic acid in acetonitrile (mobile phase B). The HPLC gradi-ent, using a flow rate of 0.2 ml/min started at 5% B and rose to 35%B after 55 min, 100% B after 65 min and re-equilibrated for 15 minat 5% B. Another gradient was used to obtain a better separation forquantification of the luteolin derivatives, orientin and homoorien-tin. Starting at 5% B, the gradient rose to 17.5% B after 50 min witha subsequent washing and re-equilibration procedure. Twentymicrolitres of each sample extract were injected for analysis. Thecoupled API 2000 HPLC–ESI-MS/MS system was controlledwith Analyst 1.5 Software (both Applied Biosystems, Darmstadt,Germany). Mass spectra for the determination of anthocyaninswere obtained by using positive ionization, whereas all otherphenolic compounds were detected in negative ionization mode.

Identification of phenolic compounds was performed by com-paring fragmentation patterns in multiple reaction mode andretention times with those of authentic standard substances. Stan-dards were also used to create calibration curves for quantification.Results were recalculated to the non-defatted material and ex-pressed in mg/100 g DM.

Table 2Minerals at three different maturity stages of açaí referred to the dry matter (DM)content.

Parameter (mg/100 g DM) Unripe Intermediate Ripe

Sodium n.d. 51.3 ± 7.2 6.8 ± 0.7Magnesium 397 ± 0.4 287 ± 0.8 172 ± 0.3Phosphorus 262 ± 0.5 232 ± 1.8 186 ± 1.5Potassium 4271 ± 21.7 2314 ± 11.4 930 ± 9.9Calcium 962 ± 2.3 846 ± 1.7 423 ± 1.2Manganese 30.9 ± 0.1 17.7 ± 0.1 13.3 ± 0.1Iron 23.9 ± 0.9 12.8 ± 0.0 7.8 ± 0.2Zinc n.d. 1.2 ± 0.2 2.1 ± 0.0

258 A. Gordon et al. / Food Chemistry 133 (2012) 256–263

2.5. Antioxidant activity

2.5.1. Total oxidant scavenging capacity (TOSC) assayAnalysis of the TOSC assay was performed as described in Licht-

enthäler, Marx, and Kind (2003). Briefly, the measurement of theantioxidant capacity is based on an ethylene-yielding reaction ofKMBA with either generated peroxyl radicals or peroxynitrite.Antioxidative compounds present in the sample inhibit the ethyl-ene formation. Ethylene is measured gas chromatographically(GC-17A, Shimadzu, Tokyo, Japan) over a time course of 1 h. Quan-tification of generated ethylene results in a kinetic curve and thearea under the curve (AUC) is calculated. Only those data with avariance (standard deviation/arithmetic mean) of the AUC after re-peat determination below 5% are further processed. Mean data of asample are compared to those of an uninhibited reaction withwater, which gives rise to the TOSC values.

Authentic standard compounds for TOSC analysis were solubi-lized in DMSO. Lyophilized samples (1 g) of each maturity stagewere suspended in UHQ water to obtain a total weight of 10 g(w/w). The suspension was sonicated for 10 min and centrifugedfor 7 min with 10,000 rpm at 10 �C. The supernatant of the waterextract was stored until further application at �30 �C. Results indi-cate the sample concentration (in mg/100 ml) which is needed toobtain a radical inhibition of 50%.

2.5.2. Trolox equivalent antioxidant capacity (TEAC)The antioxidant activity with ABTS�+ radicals was determined

after an extraction of the pulp in a methanol/acetone solution asdescribed by Rufino et al. (2007). Trolox was used as referencecompound. Results were expressed in lmol trolox equivalent/100 g DM.

2.5.3. Folin–Ciocalteu total phenolsTotal phenolic compounds were determined by the Folin–

Ciocalteu assay, based on Georgé, Brat, Alter, and Amiot (2005).Results were expressed in mg gallic acid equivalent/100 g DM.

2.6. Data analysis

To prove significant differences between maturity stages, statis-tical analysis of data was accomplished by one-way analysis of var-iance, followed by the Tukey test at 95% of probability, usingXLSTAT 7.5 software. Results were significantly different betweenmaturity stages unless otherwise indicated.

3. Results and discussion

3.1. Nutrients and titratable acidity

Variations among the samples of different ripeness are as ex-pected because maturation can generally be defined as a sequenceof changes in colour, flavour, and texture of fruits and vegetables(Chitarra & Chitarra, 2005). As shown in Table 1, the ash contentdecreases during the ripening process. This development is concor-

Table 1Nutrients and titratable acids at three maturity stages of açaí referred to the drymatter (DM) content of the fruit.

Parameter (g/100 g DM) Unripe Inter-mediate Ripe

Ash 14 ± 0 8 ± 0 4 ± 0Lipids 7 ± 0 31 ± 0 48 ± 4Total protein 21 ± 0 13 ± 0 12 ± 0Carbohydrates 58 ± 14 48.0 ± 2.6 36 ± 4Titratable aciditya 2.8 ± 0.0 1.5 ± 0.0 1.2 ± 0.0

a Expressed in malic acid equivalents.

dant with the results of the mineral composition (Table 2). Withexception of sodium and zinc, all other values of the determinedminerals decline with increasing maturity. Amounts of total pro-teins, carbohydrates and titratable acids indicate the same regres-sive trend. Values of total carbohydrates are approximately in thesame range as those reported previously for açaí pulp obtainedfrom fruits harvested in February (31.6 g/100 g DM). However, val-ues are lower than those of pulp from fruits harvested in July(48.0 g/100 g DM) (Sanabria & Sangronis, 2007). In contrast tothe aforementioned parameters, the lipid content increased duringripening, generating (partly) the expected alterations in flavourand texture. Our findings represent an average content of total lip-ids of 48% in the DM of ripe fruits, similar to the value of 42% foundby Sanabria and Sangronis (2007).

3.2. Identification and quantification of phenolic compounds

3.2.1. AnthocyaninsMonomeric anthocyanins were mass spectrometrically identi-

fied by comparing fragmentation patterns and retention timeswith those of authentic standards. According to Fig. 1, cyanidin3-O-glucoside (peak 18) and cyanidin 3-O-rutinoside (peak 19)were assigned to the main anthocyanins in ripe açaí. Cyanidin 3-O-sambubioside (peak 17), pelargonidin 3-O-glucoside (peak 20),and peonidin 3-O-glucoside (peak 21) were found in traces. Inaddition to these anthocyanins, peonidin 3-O-rutinoside (peak22) was tentatively identified. Peak 22 shows a molecular ion[M+H]+ at m/z 609 and a product ion at m/z 301. The mass differ-ence of m/z 308 suggests the loss of a hexosyl-deoxyhexoside. Thisassignment also agrees with regularities in the elution order ofanthocyanins. Generally, the 3-O-rutinoside of an anthocyanidinis retained after the 3-O-glucoside moiety (Wu & Prior, 2005a).

Fig. 1. HPLC chromatogram of ripe açaí recorded at 520 nm.

A. Gordon et al. / Food Chemistry 133 (2012) 256–263 259

Additionally, a sugar moiety bound to a certain anthocyanidin willlikely be linked to another anthocyanidin distributed in the samefruit (Wu & Prior, 2005b). As the presence of cyanidin 3-O-rutino-side was already established, the linkage of a rutinoside to peoni-din is certain.

The overall findings concerning anthocyanins in açaí are inaccordance with other published papers. Lichtenthäler et al.(2005) found cyanidin 3-O-glucoside and cyanidin 3-O-rutinosideas the major anthocyanin components. Peonidin 3-O-rutinosidewas found as a minor component in their açaí sample. Schausset al. (2006) also found mainly cyanidin 3-O-glucoside and cyani-din 3-O-rutinoside. Additionally, cyanidin 3-O-sambubioside,peonidin 3-O-glucoside, and peonidin 3-O-rutinoside were foundas minor anthocyanins. Del Pozo-Insfran, Brenes, and Talcott(2004) found pelargonidin 3-O-glucoside as a major anthocyanincomponent in açaí, beside cyanidin 3-O-glucoside. Further, pelarg-onidin 3-O-glucoside was present in fruits of Euterpe precatoria(Pacheco-Palencia et al., 2009).

Quantification in the intermediate maturity stage of açaí couldonly be performed for cyanidin 3-O-glucoside and 3-O-rutinoside(Table 3). Peonidin 3-O-glucoside, peonidin 3-O-rutinoside, andpelargonidin 3-O-glucoside were also identified but just achievedthe limit of detection defined by a peak/noise-ratio of 3:1. Cyanidin3-O-sambubioside was not detected in the intermediate maturitystage. No monomeric anthocyanins were present in unripe açaí.Thus, the anthocyanin concentration rises in açaí during maturity,as well as in many other plant species, probably since the anthocy-anin biosynthesis proceeds faster than does fruit expansion(Bureau, Renard, Reich, Ginies, & Audergon, 2009).

Beside the general increase of the anthocyanin concentration, itwas also observed that the relative amounts of the individualanthocyanins were different at each ripening stage. The ratio ofcyanidin 3-O-glucoside to cyanidin 3-O-rutinoside was 1:1 at theintermediate ripening stage. In ripe fruits, by contrast, the ratio in-creased in favour of cyanidin 3-O-rutinoside to approximately 4:1.

Table 3Content of phenolic compounds at three maturity stages of açaí in mg/100 g dry matter.

Peak Compound [M�H]�/product ion m/

1 Gallic acid 169/1252 Protocatechuic acid 154/1093 p-Hydroxybenzoic acid 137/934 Vanillic acid 167/1525 Chlorogenic acid 353/1916 Caffeic acid 179/1357 Syringic acid 197/1828 Orientin 447/3279 Homoorientin 447/327– p-Coumaric acid 163/11910 Luteolin 7-O-glucosidea 447/28511 Vitexin 431/31112 Isovitexin 431/31113 Chrysoeriol 7-O-glucoside 461/25514 Taxifolin 303/28515 Luteolin 285/13316 Chrysoeriol 299/285

[M+H]+/product ion m/z

17 Cyanidin 3-O-sambubioside 581/28718 Cyanidin 3-O-glucoside 449/28719 Cyanidin 3-O-rutinoside 595/28720 Pelargonidin 3-O-glucoside 433/27121 Peonidin 3-O-glucoside 463/30122 Peonidin 3-O-rutinoside 609/301

Sum of the identified phenolic compounds

n.d.: Not detected.a Estimated.b Not significant.

Siriwoharn, Wrolstad, Finn, and Pereira (2004) reported changes inanthocyanin formation in two blackberry cultivars. Cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside were determined as majoranthocyanin compounds. But, unlike in açaí, the content of cyani-din 3-O-glucoside increased steadily whereas amounts of cyanidin3-O-rutinoside decreased from unripe to ripe fruits. Fernández-López, Almela, Muñoz, Hidalgo, and Carreño (1998) observed a de-cline of cyanidin 3-O-glucoside concentration in ripening grapes.They noted that, in the biosynthesis of anthocyanidins in Vitisssp., cyanidin typically occurs as a precursor molecule of peonidinderivatives. Conclusively, according to these results, there are nogeneral regularities deducible for the formation of individualanthocyanins during ripening in fruits. Only the increase of totalanthocyanins, in general, is apparent.

3.2.2. Phenolic acidsAccording to Table 3 and Fig. 2, in total eight phenolic acids and

phenolic acid derivatives were identified in açaí: gallic acid (peak1), protocatechuic acid (peak 2), p-hydroxybenzoic acid (peak 3),vanillic acid (peak 4), p-coumaric acid (peak not present inFig. 2), caffeic acid (peak 6), syringic acid (peak 7), and chlorogenicacid (peak 5). With the exception of p-coumaric acid, all com-pounds were present at each of the three different ripening stages.During the ripening process, the individual phenolic acids werefound in different amounts. The highest concentrations of individ-ual phenolic acids were generally found in unripe açaí. Vanillicacid, p-hydroxybenzoic acid and syringic acid were quantitativelythe most dominating phenolic acids. Gallic acid, protocatechuicacid, caffeic acid and chlorogenic acid were found in loweramounts. p-Coumaric acid was only present in traces. Amountsof hydroxycinnamic acids decreased during ripening. Chlorogenicacid and caffeic acid vanished nearly completely. Similar findingswere reported by Dragovic-Uzelac, Levaj, Mrkic, Bursac, and Boras(2007) and Gruz, Ayaz, Torun, and Strnad (2011). Interestingly, Xu,Ye, Liu, Ma, and Chen (2008) found the biggest quantitative loss

z Maturity stage

Unripe Intermediate Ripe

0.01 ± 0.00 0.04 ± 0.00 0.02 ± 0.000.75 ± 0.00 0.63 ± 0.06 0.65 ± 0.026.48 ± 0.27 2.56 ± 0.18 1.90 ± 0.0725.9 ± 0.54 12.3 ± 0.73 6.97 ± 0.301.64 ± 0.20 0.06 ± 0.02 0.02 ± 0.000.56 ± 0.03 0.06 ± 0.01 0.02 ± 0.004.95 ± 0.38 0.46 ± 0.08 1.10 ± 0.12109 ± 5.01 19.0 ± 0.7 11.2 ± 0.8467.1 ± 1.85 14.0 ± 0.32 3.06 ± 0.26Tracesb n.d. n.d.60.04b

60.02b60.01b

24.7 ± 0.99 11.3 ± 0.18 3.41 ± 0.0429.0 ± 0.69 10.8 ± 0.94 2.66 ± 0.140.44 ± 0.03 0.08 ± 0.01 0.03 ± 0.000.98 ± 0.05 0.46 ± 0.01 0.20 ± 0.014.98 ± 0.15 1.32 ± 0.09 0.24 ± 0.075.27 ± 0.19 2.53 ± 0.11 0.68 ± 0.04

n.d. n.d. 0.02 ± 0.00b

n.d. 0.29 ± 0.05 4.94 ± 0.55n.d. 0.31 ± 0.04 17.9 ± 1.59n.d. Tracesb 0.06 ± 0.01b

n.d. Tracesb 0.08 ± 0.01b

n.d. Tracesb 0.29 ± 0.00b

282 ± 10.35 77.2 ± 3.53 55.4 ± 4.07

Fig. 2. HPLC chromatogram of açaí at intermediate maturity stage recorded at 280 nm.

260 A. Gordon et al. / Food Chemistry 133 (2012) 256–263

during ripening in two Citrus varieties for chlorogenic acid, whichis in agreement with results found in açaí. However, amounts ofcaffeic acid and syringic acid increased during ripening in Citrusfruits.

In contrast to the hydroxycinnamic acids, there was no cleartrend observable for the hydroxybenzoic acids in açaí (Table 3).Amounts of vanillic acid and p-hydroxybenzoic acid decreasedfrom immature fruits to ripe fruits. Declines of 90% and 16% wereobserved for syringic acid and protocatechuic acid, respectively,from unripe fruits to the intermediate ripening stage. Subse-quently, the concentrations increased with further ripeness. In re-gard to gallic acid, the highest amount was noted in theintermediate ripening stage. Beside gallic acid, the biggest lossesof the phenolic acids were observed between unripe and interme-diate ripe fruits. With the exception of syringic acid, a furtherquantitative decrease between the intermediate and ripe maturitystage was generally less distinctive. Different amounts of hydroxy-benzoic acids during the ripening process were also observed inmedlar fruits (Mespilus germanica L.). Contents of free proto-catechuic acid and syringic acid became lower whereas amountsof p-hydroxybenzoic acid increased (Gruz et al., 2011).

Concerning the findings of phenolic acids in ripe açaí, our re-sults are largely in common with previously published reports.Protocatechuic acid was identified by Lichtenthäler et al. (2005).Del Pozo-Insfran et al. (2004) identified and quantified ferulic acid,p-hydroxybenzoic acid, gallic acid, protocatechuic acid, vanillicacid and p-coumaric acid in fresh açaí pulp. Furthermore,Pacheco-Palencia et al. (2009) found protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, syringic acid and ferulic acidin E. oleracea and E. precatoria fruits. Contrary to these reports,ferulic acid and p-coumaric acid could not be found in our samples.On the other hand, chlorogenic acid and caffeic acid were detectedfor the first time in açaí fruits.

3.2.3. Flavones and flavanonolsIn total, eight flavones and one flavanonol were detected in açaí.

All compounds were present throughout the three different

maturity stages. According to Fig. 2 and Table 3, peaks 11 and 12were assigned to vitexin and isovitexin, respectively, whichshowed a deprotonated molecular ion [M�H]� at m/z 431 and afragment ion at m/z 311.

Four luteolin derivatives were found in peaks 8, 9, 10, and 15.Peaks 8 and 9 showed a base peak [M�H]� at m/z 447 and a prod-uct ion at m/z 327 which is typically found for orientin and homo-orientin due to the likely fragmentation of the attached C-glycoside(Pacheco-Palencia et al., 2009). Peak 10 had a base peak [M�H]� atm/z 447, as well, but produced a daughter ion at m/z 285, indicatingthe presence of luteolin 7-O-glucoside in consequence of retentiontime and fragmentation pattern corresponding to that of a stan-dard solution. Luteolin was found in peak 15, providing a deproto-nated molecular ion [M�H]� at m/z 285 and a product ion at m/z133. Chrysoeriol and a chrysoeriol glycoside were found in peaks16 and 13, respectively. Peak 16 exhibited a base peak [M�H]�

at m/z 299 and a fragment ion at m/z 285 after loss of the methylgroup, indicating the presence of the chrysoeriol. A deprotonatedmolecular ion [M�H]� at m/z 461, and a fragment ion at m/z 255found in peak 13, showed the presence of chrysoeriol 7-O-gluco-side. The flavanonol taxifolin was detected in peak 14, providinga deprotonated molecular ion [M�H]� at m/z 303 and a corre-sponding daughter ion at m/z 285.

The findings of flavones in açaí were partially consistent withthose of previous reports. Homoorientin, orientin and isovitexinwere already found by Schauss et al. (2006). Additionally, Kanget al. (2010) reported vitexin, luteolin, and chrysoeriol as polyphe-nolic constituents in açaí pulp. No reports are known about the 7-O-glucosides of luteolin and chrysoeriol as constituents of açaí. Theflavanonol taxifolin was identified for the first time. Previous re-ports were only based on the occurrence of taxifolin derivatives(Pacheco-Palencia et al., 2009; Schauss et al., 2006).

According to Table 3, amounts of all flavones and taxifolin con-stantly decreased throughout ripening. C-glycosidic derivatives ofluteolin (homoorientin, orientin) and apigenin (isovitexin, vitexin)were found to be quantitatively dominant at all three maturitystages, followed by luteolin and chrysoeriol, taxifolin, and the

A. Gordon et al. / Food Chemistry 133 (2012) 256–263 261

7-O-glycosides of luteolin and chrysoeriol. Especially in unripeaçaí, orientin and homoorientin occurred in outstandingly highconcentrations of 109 and 67.1 mg/100 g DM, showing 3- to 4-foldamounts when compared to other polyphenolic ingredients. Also,in another report, homoorientin, orientin, and isovitexin were cal-culated as the highest concentrated non-anthocyanin polyphenolsin ripe açaí (Pacheco-Palencia et al., 2009).

To our knowledge, literature on changing amounts of non-anthocyanin flavonoid compounds in the course of fruit ripeningis scarce. No reports are available on flavones or flavanonols. Flav-anols and flavonol glycosides were determined at different matu-rity stages of apricots. The quantities of flavonoids variedbetween the ripening stages but it was generally not possible toestablish a correlation between flavonoid content and ripeningstages. However, regarding flavonols, the highest values weremostly found at the initial maturity stage of apricot cultivars(Dragovic-Uzelac et al., 2007). Amounts of ellagic acids and totalflavonols decreased during ripening in camu camu fruits whereasvalues of total flavanols and total flavanones did not change mark-edly (Chirinos, Galarza, Betallelez-Pallardel, Pedreschi, & Campos,2010).

3.3. Antioxidant activity (Folin–Ciocalteu, TEAC, TOSC)

As shown in Table 4, a change during ripening was observed forthe Folin–Ciocalteu assay. The highest value of Folin–Ciocalteu to-tal phenols was found in unripe açaí. Only one quarter of the con-centration was present at the intermediate ripening stage,followed by a slight increase in the ripe fruits.

Comparing the results of the Folin–Ciocalteu assay to those ofother fruits during ripening is difficult. Results are often presentedin wet matter by other authors, not taking into account likely alter-ations of the water content during the ripening process. A determi-nation of the total phenolic content in blackberries at threedifferent maturity stages showed, e.g., a continuous decrease fromunripe to ripe fruits (Tosun, Ustun, & Tekguler, 2008). By contrast,some raspberry cultivars behaved like açaí or showed even a re-versed trend by showing highest amounts in the ripe condition(Wang & Lin, 2000).

The antioxidant capacity, by both TEAC and TOSC, was the high-est in unripe açaí fruits and decreased continuously with increas-ing ripening as shown in Table 4. The TEAC antioxidant capacityshowed four times higher values in unripe fruits than at the inter-mediate ripening stage. A further decline of the TEAC value (ofaround 30%) was observed from intermediate to ripe fruits. TOSCresults indicate the sample concentration that is needed to obtaina radical inhibition of 50%. For peroxyl radicals (px) and peroxyni-trite (pn), the antioxidant capacity decreased from the unripe tothe ripe maturity stage. For both radicals, the decline was the mostdistinctive between the intermediate and ripe fruits.

The TEAC antioxidant capacity of the ripe açaí fruits is in accor-dance with values found for different commercial açaí pulps.

Table 4Antioxidant capacity of açaí fruits at three different maturity stages. Results are

Parameter

Folin–Ciocalteu total phenols (in mg GAEa/100 g DM)TEAC (in lmol trolox/100 g DM)TOSCb (mg DM/100 ml) px

pn

a Gallic acid equivalent.b TOSC values represent the concentration of the sample that is needed to obt

(pn).

Depending on the water content of the pulps, the TEAC rangesapproximately between 1.02 and 5.25 lmol trolox equivalents/100 g dry matter (Dos Santos et al., 2008). Regarding both radicals,TOSC values of ripe açaí were at least 1.5-fold higher thanthose found in pulps of different harvest periods described byLichtenthäler et al. (2005).

The trend of a decreasing antioxidant capacity during ripeningwas also observed in strawberries by Pineli et al. (2011) (on adry matter basis). In contrast to these results, durian fruits showedthe lowest antioxidant capacity in the immature ripening stage(Haruenkit et al., 2010). Corresponding to the total phenolic con-tent, there is also no generalization or a consistent trend derivablefor the antioxidant capacity at different ripening stages of fruits.

3.4. Contribution of the phenolic content to the antioxidant capacity

The sum of the individual phenolic compounds in unripe fruitsis four times higher when compared to the intermediate ripeningdegree (Table 3). Amounts decreased further during the ripeningprocess, by approximately 30%. Interestingly, the same percentagewas observed for the reduction of TEAC values.

It was observed that the reduction of the antioxidant capacityin the course of maturity, especially between unripe and mediumripe fruits, is less distinctive for TOSC than for TEAC. In order toelucidate which compounds are responsible for the antioxidantcapacity, authentic standard compounds, that occur in relevantamounts in açaí fruits, were determined by TOSC against px.Results of the determination are given in Table 5. It turns outthat the antioxidant capacity of these standard compounds de-pended on the extent of hydroxylation, position of glycosylation,and the kind of glycosylation: orientin and homoorientin differ,by the presence of one additional hydroxyl group in the 30-posi-tion (B-ring), from vitexin and isovitexin, consequently showing ahigher antioxidant capacity. Vanillic acid had the weakest antiox-idant capacity, presenting the fewest hydroxyl groups in compar-ison with all measured standard compounds. Due to the varyingposition of the glucose residue between homoorientin (6-C-glu-coside) and orientin (8-C-glucoside), the two compounds showeddifferent radical-scavenging behaviours. Orientin turned out to bea more efficient radical-scavenger than homoorientin. Vice versa,vitexin (8-C-glucoside of apigenin) exhibited weaker antioxidantproperties than isovitexin (6-C-glucoside). In comparison tothe flavones, anthocyanins tend to be more potent radical-scavengers. In agreement with Lichtenthäler et al. (2005), cyani-din 3-O-glucoside showed higher TOSC values than did cyanidin3-O-rutinoside. Our results are in accordance with those of Kanget al. (2010) to a certain extent. In this report, flavonoids isolatedfrom açaí were measured with ORAC. Luteolin also showed,e.g., higher ORAC values than did its corresponding C-glyco-sides. In contrast to our findings, vitexin exhibited a 2–10 timeshigher antioxidant capacity than did luteolin, orientin andhomoorientin.

referred to the dry matter (DM) content.

Maturity stage

Unripe Intermediate Ripe

12,317 ± 264 3039 ± 149 3437 ± 15417.0 ± 0.71 4.04 ± 0.05 2.78 ± 0.1012.1 15.0 24.046.4 60.3 87.2

ain an inhibition rate of 50% against peroxyl radical (px) and peroxynitrite

Table 5Concentration of standard compounds that is needed to obtain aninhibition rate of 50% against peroxyl radicals by TOSC.

Compound TOSC (in mg/l)

Luteolin 4.8Cyanidin 3-O-glucoside 5.65Orientin 6.52Cyanidin 3-O-rutinoside 6.72Homoorientin 7.11Chrysoeriol 7.49Isovitexin 7.87Vitexin 9.12Vanillic acid 10.8

262 A. Gordon et al. / Food Chemistry 133 (2012) 256–263

Despite the determination of the antioxidant capacity of indi-vidual phenolic compounds, it is difficult to estimate their contri-bution to the overall antioxidant capacity. The decrease of thesequantitatively dominating compounds from unripe and to mediumripe fruits is not in proportion to the decline of the TOSC antioxi-dant capacity, which is less distinctive. Regarding the standardcompounds, a greater reduction of the TOSC values would be ex-pected. Further, the TOSC antioxidant capacity decreased byapproximately 75% from intermediate ripe fruits to ripe fruitswhereas the total amount of identified phenolic compounds wasreduced by only 30%. The reduction of TOSC values was also notcompensated by increasing amounts of anthocyanins, which arepotent radical-scavengers. Thus, it is striking that the antioxidantcapacity of açaí fruits, against px, during ripening, is strongly influ-enced by unknown compounds. Most likely is a contribution ofproanthocyanidins (Lichtenthäler et al., 2005).

4. Conclusions

The chemical characterization of açaí fruits at three differentmaturity stages showed significant alterations in all determinedparameters. Concentrations of nutritionally relevant compounds,e.g., minerals, proteins and carbohydrates, as well as acidity, de-creased in the course of ripening. An increase was observed forthe fat content. Manifold alterations also occurred regarding thephenolic composition. The concentration of anthocyanins in-creased during ripening whereas amounts of hydroxycinnamicacids and other flavonoids steadily decreased. Hydroxybenzoicacids did not show a consistent trend. A very high phenolic contentwas found in unripe açaí, and the flavones orientin and homoorien-tin were quantitatively dominant. Values of the antioxidant capac-ity, determined by TOSC and TEAC, decreased consistently withincreasing maturity. All in all, changes of the determined parame-ters generate expected alterations in flavour and texture of açaífruits during ripening. Processing of açaí fruits seems to be useful,and not only in the ripe condition, as a health-promoting product.Extracts of unripe fruits may also be interesting for the industry, as,e.g., food additives, dietary supplements or ingredients in cosmet-ics due to their high content of bioactive compounds.

Acknowledgements

The study was funded by the European Union (PAVUC-FP6-INCO, Contract No. 0015279). The authors would also like to thankAndrea Memmesheimer for TOSC measurements of phenolic stan-dard compounds.

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