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Optimization of the extraction of avanols and anthocyanins from the fruit pulp of Euterpe edulis using the response surface methodology

Graciele Da Silva Campelo Borges ⁎, Francilene Gracieli Kunradi Vieira, Cristiane Copetti,Luciano Valdemiro Gonzaga, Roseane Fett

Department of Food Science and Technology, Federal University of Santa Catarina, Florianópolis, SC, Brazil

a b s t r a c ta r t i c l e i n f o

Article history:Received 1 October 2010

Accepted 14 December 2010

Keywords:

ExtractionPolyphenols

Response surface methodology

Antioxidant activity

Euterpe edulis

The response surface methodology (RSM) was employed to optimize the ultrasound extraction of avanolsand anthocyanins from the pulp of jussara (Euterpe edulis), using a second-order polynomial equation todescribe the experimental data for total avanol (TF), total phenolic (TP), and total monomeric anthocyanin

(TMA) contents, as well as the total antioxidant activity (TAA). A central composite design with two-variables

(extraction time and solid to liquid ratio) was then applied. The optimized conditions that maximized theyields of avanol-enriched extract were a solvent methanol/0.1 M HCl, solid to liquid ratio of between 1:50and 1:100 and extraction time of 15 min. For anthocyanin-enriched extracts the respective optimal

parameters were a solvent methanol/1.5 M HCl, solid to liquid ratio of between 1:30 and 1:50 and extractiontime of 24 h. The results showed good ts with the proposed model for both the avanol-enriched extract

(R 2 =0.94) and for the anthocyanin-enriched extract (R 2 =0.99).© 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The extraction of bioactive compounds from permeable solid plant

materials using solvents constitutes an important step in themanufacture of phytochemical-rich products. The application of thislow-cost technology to obtain molecules to be used as food additivesor nutraceutical products is an appropriate strategy for the exploita-

tion of some non-timber products found in rain forests such as thefruits of the palm (Pompeu, Silva, & Rogez, 2009).

Euterpe edulis M. is a palm plant widely distributed in the AtlanticForest in Brazil, from the states of RioGrande do Norte to Rio Grande do

Sul (Henderson, 2000). Jussara, the fruit of the palm E. edulis, is roundand has a purple pulp covering a hardseed.Despiteits wide distributionin Brazil, jussara is much less commonly eaten than the other well-known palm fruits, for instance, açaí (Euterpe oleracea) (De Brito et al.,

2007). Currently, thefruits of these palmsare used forthe processing of juice, which is prepared by macerating the fruits in lukewarm water,triturating them to crush the softened pulp, adding water and lteringthe juice (Pompeu et al., 2009). Jussara fruits and pulp presented

two major anthocyanins identied as cyanidin 3-glucoside andcyanidin 3-rutinoside (Harbone, Saito, & Detoni, 1994). In a recentstudy De Brito et al. (2007) conrmed these results, and identied

minor compounds such as cyanidin 3-sambubioside, pelargonidin

3-glucoside, cyanidin 3-rhamnoside and pelargonidin 3-rutinosideby means LC-DAD-ESI/MS.

Açaí (E. oleracea) is historically commonly consumed as a food inthe form of a viscous pulp which has been associated with nutritionaland medicinal benets (Runo, Alves, Fernandes & De Brito, 2010b;Talcott, Pacheco-Palencia, & Talcott, 2008). It presents high concen-

trations of bioactive compounds such as anthocyanins (mainlycyanidin-3-glucoside and cyanidin-3-rutinoside) and other avanols(Gallori, Bilia,Bergonzi, Barbosa, & Vincieri,2004;Pompeu et al., 2009;Rosso et al., 2008; Runo, Pérez-Jiménez, et al., 2010; Schauss et al.,

2006). These components have been associated with the highantioxidant activity of this fruit (Pompeu et al., 2009; Rosso et al.,2008; Runo,Alves, Fernandes,& de Brito,2010a; Schauss et al., 2006).

A recent study has demonstrated the inuence of several factors

on the extraction of phenolic compounds from the fruits of E. oleraceausing response surface methodology (RSM) (Pompeu et al., 2009).However, to the best of our knowledge, there are no reports inliterature on the extraction of avanols and anthocyanins from the

pulp of E. edulis.Several studies on the optimized conditions for the extraction

of phenolic compounds from different sources using RSM have

been published (Hayouni, Abedrabba, Bouix, & Hamdi, 2007;Huang, Xue, Niu, Zhen, & Wang, 2009; Pinelo, Rubilar, Sineiro, &Nunez, 2005; Pompeu et al., 2009; Yang & Zhai, 2010; Zou, Xie,Fan, Gu, & Han, 2010). The RSM is a collection of statistical and

mathematical techniques useful for developing, improving andoptimizing processes, in which a response of interest is inuenced

Food Research International 44 (2011) 708–715

⁎ Corresponding author. Universidade Federal de Santa Catarina, Centro de Ciências

Agrárias, Departamento de Ciência e Tecnologia deAlimentos, Rodovia Admar Gonzaga,

1346,Itacorubi,CEP: 88034-001,Florianópolis, SC, Brazil.Tel.: +5548 33315375; fax:+55

48 33319943.

E-mail address: [email protected] (G.D.S.C. Borges).

0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.

doi:10.1016/j.foodres.2010.12.025

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by independent variables, and it generates a mathematical modelthat describes the chemical process (Montgomery, 2004). Knowledgeof the behavior of the factors inuencing the process conditions isnecessary to enhance the optimization extraction ef cient for any

bioactive compound (Pompeu et al., 2009). Previous ndings havereported the inuence of many independent variables, such as solventcomposition, pH, temperature, extraction time, and solid to liquid

ratio, on the yields of bioactive compounds which can be extracted

from diverse natural products (Bujić

-Kojić

, Planinic, Srécko,Jakabek, &Seruga, 2009; Pinelo et al., 2005; Santos, Veggi, & Meireles, 2010). Thepositive or negative role of each factor in the mass transfer of the

process is not always clear; the chemical characteristics of the solventand the diverse structures and compositions of the natural productsmean that each material–solvent system has a different behavior,which cannotbe predicted (Cacace & Mazza, 2002; Pinelo et al., 2005).

Different solvents have been used for the extraction of poly-

phenols from plant material. The extraction method must allowextraction of the principal compounds of interest, and it must avoidtheir chemical modication (Hayouni et al., 2007). The polarity of thesolvent plays an important role in the selective extraction. Water and

aqueous mixtures of ethanol, methanol, and acetone are commonlyused in plant extraction (Hayouni et al., 2007; Pompeu et al., 2009;Sun & Ho, 2005).

In recent studies on extracting the valuable compounds from plantmaterials, ultrasound assisted technology has been used (Santos et al.,

2010; Zou et al., 2010) and has been proven to be a desirable methodof extraction with many advantages such as increasing extractionyield reducing solvent usage, economizing power consumption andshortening extraction duration. The increase in extraction ef cient by

ultrasound is attributed to the propagation of ultrasound pressurewaves and resulting cavitation phenomena (Vilkhu, Mawson, Simons,& Bates, 2008; Zou et al., 2010).

In this paper, the extraction of anthocyanin and avanols from

jussara pulp was optimized by applying the RSM, employing a two-variable, three-level central composite design, evaluating the inu-ence of factors, extraction time and solid to liquid ratio in the processof extraction. Finally, the optimized conditions were validated. The

RSM study was preceded by the selection of the best solvent forobtaining avanol-enriched extracts and, by maceration process,anthocyanin-enriched extracts of jussara pulp.

2. Materials and methods

2.1. Plant materials

Samples of frozen pasteurized jussara pulp (E. edulis) were kindlysupplied by Alicon Industry, Santa Catarina State, Brazil. The jussara

pulp samples were produced from fruits harvested during the 2008season. The general properties of the pulp were maintained duringtransportation to the laboratory by keeping them in isothermal boxes

containing ice. In the laboratory the samples were stored at − 18 °Cuntil analysis.

2.2. Chemicals

Folin–Ciocalteu's phenol reagent, 2,2′-azino-bis (3-ethylbenzothia-zoline-6-sulfonic acid) diammonium salt (ABTS), 4-dimethylaminocin-namaldehyde (DMACA) and 6-hydroxy-2,5,7,8-tetramethylchromane-

2-carboxyl acid (Trolox) were purchased from Sigma Chemical Co. (St.Louis, MO, USA). Gallic acid and potassium peroxydisulfate wereobtained from Vetec (Rio de Janeiro, RJ, Brazil). Acetone, methanol,ethanol and hydrochloric acid were purchased from Sigma-Aldrich

Chemical, S.A. (Madrid, Spain). All other chemicals were of analytical

grade.

2.3. Selection of the solvent for extraction of total avanols (TF)

The rst set of tests focused on selecting an appropriate solvent toextract the TF from jussara pulp. Three independent extractions were

carried out using 10 g of jussara pulp and 100 mL (1:10) of fourteendifferent solvents: water/0.01 M HCl;water/0.1 M HCl (Del Pozo-Insfran,Talcott, & Brenes, 2004); methanol (Revilla, Ryan, & Martin-Ortega,

1998); methanol/water (50:50 v/v) (Pérez-Jiménez & Sauro-Calixto,

2006); methanol/0.01 M HCl; methanol/0.1 M HCl; methanol/1 M HCl(Revilla et al., 1998); ethanol; ethanol/water (70:30v/v); ethanol/water(50:50 v/v); ethanol/0.01 M HCl (Sun & Ho, 2005); acetone; acetone/water (80:20 v/v) and acetone/water (80:20 v/v) 0.01 M HCl (Ou,

Hampsch-Woodill, & Prior, 2001; Pérez-Jiménez & Sauro-Calixto, 2006;Yilmaz & Toledo, 2006). The extractions were conducted in amber glassbottles (250 mL) using a Unique 1400A ultrasonic bath (Unique, SãoPaulo, Brazil) with a thermostat (25 °C) for 15 min. The extracts were

centrifuged at 2000× g for 10 min using a Fanem centrifuge model 280R (Fanem, São Paulo, Brazil) and the TF content of the supernatantrecovered was immediately determined.

2.4. Selection of solvent for extraction of total monomeric anthocyanins

(TMA)

Another set of tests focused on selecting an appropriate solvent toextract the TMA from jussara pulp. Three independent extractions were

carried out using 10 g of jussara pulp and 100 mL (1:10) of thirteendifferent solvents: water/0.1 M citric acid; water/0.01 M citric acid;water/0.1 M HCl; water/0.01 M HCl (Kahkonen, Hopia, & Heinomen,2001); methanol; methanol/water (50:50 v/v); methanol/0.01 M HCl

(Revilla et al., 1998); methanol/1.5 M HCl; ethanol; ethanol/water(70:30 v/v) (Gallori et al., 2004); ethanol/water (50:50 v/v); ethanol/0.01 M HCl; and ethanol/1.5 M HCl (Fan, Yonbin, Gu, & Chen, 2008;Rosso et al., 2008). In the extraction of the anthocyanins using a

maceration process, the pulp samples were extracted in the dark bystirring with 50 mL in amber glass bottles with the temperaturecontrolled at −5 °C for24 h.The samples were ltered using a Buchnerfunnel. The extracts werethen centrifugedat 2000× g for10 minusing a

Fanem centrifuge model 280R (Fanem, São Paulo, Brazil) and the TMAcontent of the supernatant recovered was immediately determined.

2.5. Experimental design

The optimization of the phenolic content of avanol-enriched andanthocyanin-enriched extracts of jussara pulp was carried out using

an experimental plan based on a 32 full factorial central compositedesign. The real and coded variables were the extraction time (min, T)and solid to liquid ratio (g mL −1, Slr) (Tables 2 and 4).In the avanol-enriched extracts the parameters measured were the total phenolic

content, total avanols content and total antioxidant activity. For theanthocyanin-enriched extracts the dependent variables determinedwere the total phenolic content, total monomeric anthocyanins

content and total antioxidant activity. The experimental resultswere analyzed according to a mathematical model (Eq. (1)). For anyresponse (dependent variables) Y is usually unknown, however, it canbe estimated accurately through a second-order polynomial expres-sion that accounts for variations caused by linear and quadratic order

effects as well as by interactions (Montgomery, 2004).

Y = β 0 + β 1 X 1 + β 2 X 2 + β 12 X 1 X 2 ð1Þ

where Y represents the dependent variable (estimated response); β0,β1, β2, and β12 represent the equation coef cients; X1 and X2represent the independent variables studied, extraction time and

solid to liquid ratio, respectively. Analysis of variance was performedfor each response variable using the full models where p-values

(partitioned into linear and interaction factors) indicated whether the

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terms were signicant or not. To verify the adequacy of the models,experimental data were compared to the values predicted byregression models.

2.6. Total avanol content

The total avanol (TF) content of the jussara avanol-enrichedextracts was estimated using a modied p-dimethylaminocinnamal-dehyde (DMACA) method (Arnous, Markis, & Kefalas, 2002). To each

jussara extract (1 mL), 5 mL of DMACA solution (0.1% in 1 N HCl in

MeOH) was added. Followingthis, the absorbance at 640 nm was readusing a Hewlett-Packard spectrophotometer model HP 8452A(Cheadle Heath, Stockport Cheshire, UK). The results were expressedas milligrams of catechin equivalents (CAE) per 100 g−1 fresh matter

(FM).

2.7. Total monomeric anthocyanin content

The total monomeric anthocyanin (TMA) content of the jussaraextracts was measured using a spectrophotometric pH differential

protocol (Giusti & Wrolstad, 2001). The absorbance of the mixture

was measured at 510 and 700 nm using a Hewlett-Packard spectro-photometer model HP 8452A (Cheadle Heath, Stockport Cheshire,UK). The TMA content (mg 100 g−1 of fresh matter) was calculated asfollows: TMA= A× MW×1000/ (×C) where A is absorbance =

(A515–A700)pH1.0–(A515–A700)pH4.5; MW is the molecular weightwhich for cyanidin 3-glucoside= 490; ε is the molar absorptivitywhich for cyanidin 3-glucoside= 26,900; andC is the concentration of the buffer in mg per milliliter. Anthocyanin content was expressed as

mg cyanidin 3-glucoside equivalents (cy-3-glu) 100 g−1 FM.

2.8. Total phenolic content

The total phenolic (TP) contents of the jussara extracts were

measured using a modied colorimetric Folin–Ciocalteu method(Budini, Tonelli, & Girotti, 1980). Briey, a volume of 2.5 mL of deionized water and 100 μ L of a known dilution of the extract were

added to a 10 mL volumetric ask. Folin–Ciocalteu reagent (0.5 mL)was added to the solution. Aliquots (1.5 mL) of 20% sodium carbonatesolution were placed into the volumetric asks, and the mixture wasmade up to 10 mL with deionized water. The color was left to

developed over 120 min, and the absorbance was read at 765 nmusing a Hewlett-Packard spectrophotometer model HP 8452A(Cheadle Heath, Stockport Cheshire, UK). The results were expressedas mg gallic acid equivalent (GAE) per 100 g−1 fresh matter (FM).

2.9. Total antioxidant activity (TAA)

The TAA of the extracts was determined using the ABTS methodevaluated according to the decolorization of the ABTS radical cation(ABTS+) expressed as percentage inhibition (Re et al., 1999). ABTSwas dissolved in water to give a concentration of 7 mM. The ABTS

radical cation (ABTS+) was produced by reacting the ABTS stocksolution with 2.45 mM potassium persulfate andallowingthe mixture(1:1) to stand in the dark at room temperature for 16 h before use.After addition of 1.0 mL of diluted ABTS+ solution (A734 nm =0.700±

0.020) to 0.01 mL of sample the absorbance reading was taken after7 min of reaction using a Hewlett-Packard spectrophotometer modelHP 8452A (Cheadle Heath, Stockport Cheshire, UK). The percentageinhibition was calculated as: (1−absorbance after 7 min of reaction/

initial absorbance without sample)×100. The antioxidant activity of the samples was expressed as μ Mol Trolox equivalent antioxidant

capacity per 100 g−1

of fresh matter (μ Mol TEAC 100 g−1

FM).

2.10. Statistical analysis

Data for the extracts were reported as mean ± standard deviation(S.D.) of three replicates. The results were compared by one-way

analysis of variance (ANOVA) and Tukey's test was carried out toidentify signicant differences between the mean values. Correlationsbetweenparameters were examined using the Pearson correlation.All

analyses were performed using the software STATISTICA 7.0 (Statsoft

Inc., Tulsa, OK, USA) and differences at the 5% probability level( pb0.05) were considered statistically signicant.

3. Results and discussion

3.1. Selection of the solvent for extraction of total avanols (TF)

Preliminary trials were performed in order to determine therequiredsolvent for the extraction of TF from jussara pulp. The results show thatthe extraction of TF was dependent on the solvent used (Table 1), with

statistically signicant differences ( pb0.05) between the values for thesamples. The TF yield in the methanol extract was higher than those inthe acetone, ethanol and water extracts, which is consistent withpreviously reported results for other berries (Oliveira-Brett, Novak,

Janeiro, & Seruga, 2008; Sun & Ho, 2005). The methanol/0.1 M HCl wasthe most ef cient solvent presenting 94.13 mg CAE 100 g−1 FM,followed by methanol/1 M HCl and methanol/0.01 M HCl with 67.26

and 59.50 mg CAE 100 g−1 FM, respectively. The acidied methanol(HCl 0.01 M, 0.1 M or 1 M) provided an increase in the TF extractionef cient when compared with methanol and methanol/water(50:50 v/v) applied to the same sample. The polar solvents have

greater ability to extract phenolic compounds compared withapolar solvents (Hayouni et al., 2007). As mentioned above, ourresults clearly showed that a higher content of avanols wasobtained with an increase in the polarity of the solvent used. On

the other hand, the solubility of polyphenolic compounds and theirdiffusion to the solvent are dependent on their chemical structure,which may vary from simple to highly polymerized. Studies withaçaí (E. oleracea) demonstrate the presence of avanols, ava-

nones, proanthocyanins, hydroxycinnamic acid and hydroxyben-zoic acid in methanol extracts (Del Pozo-Insfran et al., 2004;Schauss et al., 2006; Talcott et al., 2008). The monomers of catechinand epicatechin have been found in the fruits in the polymer formcalled tannins or proanthocyanins (Marete, Jacquier, & O'Riordan,

Table 1

Total avanols (TF) and total monomeric anthocyanins (TMA) in E. edulis pulp extracts

obtained from different solvent extractions.

Solvent TF

(mg CAT 100 g−1

fresh weight)

TMA

(mg cy-3-glu 100 g−1

fresh weight)

Acetone 5.76± 1.13a –

Acetone/water (80:20 v/v) 8.78± 0.28cd –

Acetone/water (80:20 v/v) 0.01 M HCl 9.39±0.28

d–

Water/acid citric 0.1 M – 114.45±1.14e

Water/acid citric 0.01 M – 91.07±1.08d

Water/HCl 0.01 M 7.28± 1.03b 143.98±2.08g

Water/HCl 0.1 M 4.64± 0.78a 165.94±1.25i

Ethanol 5.50± 0.06a 21.60±1.35b

Ethanol/HCl 0.01 M 9.65 ±0.22d 136.12±1.62f

Ethanol/water (7 0:30 v/v) 44.37± 0.11e 197.56±1.72 j

Ethanol/water (50: 50 v/v) 7.35± 0.77bc 32.52±1.17c

Ethanol/HCl 1.5 M – 231.54±1.05m

Methanol 8.74± 0.85cd 17.74±1.56a

Methanol/water (50:50 v/v) 8.1± 0.11bcd 147.31±1.36h

Methanol/HCl 0.01 M 59.50 ±0.74f 205.90±1.58l

Methanol/HCl 0.1 M 94.13 ±0.90h –Methanol/HCl 1 M 67.26± 1.04g –Methanol/HCl 1.5 M – 254.57±1.84n

Values (mean± S.D., n =3) in thesame column followedby differentsuperscript letters

are signicantly different (Tukey's test, pb

0.05).

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2009; Pietta, 2000). The extraction of these components has beenreported in the literature using water/0.01 M HCl to break themolecules liberating residual sugars and organic acids; otherpolyphenolics with avanols were removed with methanol/

0.01 M HCl (Del Pozo-Insfran et al., 2004). Thus, as demonstratedin our study, methanol/0.1 M HCl is the most ef cient solvent forthe extraction of avanols such as catechin. The presence of an acid

in the solvent for the extraction of avanols enhances the

degradation of the plant material increasing the extraction capacityas well as the rate of extraction of bioactive compounds from thematrix. However, the acidity of the extraction solvent also affects

the chemical composition of extracts. Thus, according to the resultsof this study, and those previously reported in the literature, highconcentrations of mineral acids (1–2 M) cause hydrolysis of

avonoid glycosides, while some labile phenolic compoundspresent in the same material can be degraded under such harsh

conditions (Oliveira-Brett et al., 2008).

3.2. Selection of solvent for extraction of total monomeric anthocyanins

(TMA)

The selection of an appropriate solvent for the extraction of the

TMA from jussara pulp was the second stepin the preliminary studies.The different solvents clearly inuenced the TMA content of theextracts, with statistically signicant differences ( pb0.05) betweenthem (Table 1). The extraction of TMA with the solvents ethanol andmethanol was more ef cient than with water and mixed solvents.

Ethanol/1.5 M HCl, ethanol/1.5 M HCl and methanol/0.01 M HCl werethe most ef cient solvents, resulting in 254.57, 231.54 and 205.90 mgcya 3-glu 100 g−1, respectively (Table 1). The results of this studydemonstrate that acidied alcoholic solvents are effective in extract-

ing anthocyanins, which is in agreement with previous reports foranthocyanin extraction from jussara and açaí samples (Pompeu et al.,2009).

3.3. Response surface analysis

3.3.1. Flavanol-enriched extract The optimization of the extraction time and solid to liquid ratio in

terms of avanol compounds content in the avanol-enriched extract

(methanol/0.1 M HCl) of jussara pulp was carried out using anexperimental plan based on a 32 full factorial central composite design.According to the parameters in Table 2 and Fig. 1(A), the highest valuesof TF in the extract were reached when extraction was carriedout under

the conditions of experiments 3 (109.68 mg CAT 100 g−1 FW),6 (117.89 mg CAT 100 g−1 FW) and 9 (124.63 mg CAT 100 g−1 FW).These experiments have different extraction times (15, 30 and 45 min,

respectively) but the same solid to liquid ratio (1:100). The responsefunction models resulting from the statistical analysis of these valuesshowed their dependence on the two variables assayed for methanolextractions, whereas the variable extraction time was not signicant.

The calculated model is also able to explain 94% of the results for the TFcontent (Table 3). The statistical analysis also showed that the linearterms for solid-to-liquid ratio (F model =179.67; p =0.000001) and the

interaction extraction time and solid-to-liquid ratio (F model =8.60;

p= 0.0136) arehighly signi

cant( pb

0.05) intermsof TF content. Theseresults suggest that changes in the solid-to-liquid ratio and theinteractions between two variables had a signicant effect on the TF

content, although the effect of interactionwas less signicant, as shownby its smaller F value. The interaction indicated that the extraction timeand solid-to-liquid ratio enhance or reduce the extraction capability of the solvent. To obtain a simplied model for interpretation, all non-signicant terms ( pN0.05) and the linear term for extraction time

( p=0.87) were eliminated. The simplied model (Eq. (1)) for thevariables was obtained as:

Flavanols mg CAE 100 g 1

= 0 :37 Slr ð Þ + 0:0096 T ð Þ⋅ Slr ð Þ + 69:73 ð1Þ

where (Slr) is the solid-to-liquid ratio (g mL −1) and (T) is the

extraction time (min).The response surface for TP (Table 2 and Fig. 1(B)) indicates thatthe highest contents could also be achieved under the conditions of

experiments 3 (657.90 mg GAE 100 g− 1 FW), 6 (606.42 mgGAE 100 g−1 FW) and 9 (684.00 mg GAE 100 g−1 FW).

An increase in the solid-to-liquid ratio increases the phenoliccompound extraction, however the variable extraction time has a

signicant inuence on the model ( p=0.0033); in contrast to thebehavior observed for the TF content (Fig. 1(A)). Thus, the extractiontime and solid to liquid ratio variables were highly signicant(F model =515.37; pb0.00001), however, the interaction between

these two variables was not signicant (F model =4.72, p=0.0525).In addition, the statistical analysis carried out with the modelobtained demonstrated that the model explains 97% of the results(Table 4). The variable extraction time showed no signicant linear

effect on the TP content, suggesting a progressive release of solutefrom the solid matrix to the solvent for the extraction timesconsidered.

For TAA, Table 2 and Fig. 1(C) demonstrated that a higher solid toliquid ratio contributes to anincrease in the TAAas shownby the resultsof experiments 3, 6 and 9, which reveal differences in the TAA values( pb0.05). The highest extraction yield is achieved when the ultrasound

exposure time is in the range of 45–90 min (Huang et al., 2009; Vilkhuet al., 2008). As a consequence, an increase in the extracted solids wasobserved at higher solid-to-liquid ratio values. The interaction has a

Table 2

Extraction conditions of theexperimental design 32 setting in theoriginaland coded and uncoded formthe independentvariables (Ta, Slrb) and experimentalresults of total phenolic(TP) content, total avanols (TF) content, and total antioxidant activity (TAA).

Expt Ta Slrb TPc TFd TAAe

Exp. Pred. Exp. Pred. Exp. Pred.

1 −1 (15) −1 (1:10) 353.31 348.29 62.61 74.88 408.28 483.14

2 −1 (15) 0 (1:50) 497.78 468.69 104.50 95.48 670.47 783.14

3 −1 (15) 1 (1:100) 657.90 619.19 109.68 121.23 1435.53 1158.14

4 0 (30) −1 (1:10) 346.69 320.09 80.86 76.32 508.88 483.14

5 0 (30) 0 (1:50) 488.10 440.49 97.53 102.68 798.93 783.14

6 0 (30) 1 (1:100) 606.42 590.99 117.89 135.63 1567.99 1158.14

7 1 (45) −1 (1:10) 292.56 291.89 50.78 77.76 678.04 483.14

8 1 (45) 0 (1:50) 432.95 412.29 122.87 109.88 810.08 783.149 1 (45) 1 (1:100) 684.00 562.79 124.63 150.03 1696.55 1158.14

a T: extraction time (min).b Slr: solid to liquid ratio (g mL −1).c mg GAE 100 g−1 fresh weight.d mg CAT 100 g−1 fresh weight.e

μ mol TEAC 100 g−1

fresh weight.


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positive effect, in fact, the higher the solid-to-liquid ratio, the higher the

total amount of solids obtained, regardless of the time used. This isconsistent with masstransfer principles, which is greater when a highersolid-to-liquid ratio is used(Pastor-Cavada,Juan,Pastor, Alaiz, & Vioque,2009; Pinelo et al., 2005; Vilkhu et al., 2008).

3.3.2. Anthocyanin-enriched extractsFor the extraction of TMA methanol/1.5 M HCl was used, which

showed the best ef cient in the extraction of these compounds in thepreliminary tests (Table 1). Experiment 9 led to the highest content of

TMA with 456.78 mg cy-3-glu 100 g−1. As can be observed in Table 4and Fig. 2(A) these experiments differ in terms of extraction time

(1 to 24 h) and solid to liquid ratio (1:30 at 1:50). The statistical

analysis also demonstrated that the linear terms for solid to liquid

ratio (F model =5620.79; p=0.000001), extraction time (F model =22.04;

p= 0.00065) andthe interactionextraction timeand solid to liquidratio(F model =23.06; p =0.000556) for the anthocyanin-enriched extractswerehighly signicant( pb0.05). The results suggest that the changes in

the extraction mass–solvent ratio had a more signicant effect(F model =5620.79; p=0.000001) than the other variables in thisstudy. The simplied model (Eq. (2)) for the variables was obtained as:

Anthocyanins mg cy 3 glu 100 g 1

= 9:46 Slr ð Þ+ 151:12 T ð Þ + 9:67 Slr ð Þ T ð Þ

ð2Þ

where (Slr) is the solid to liquid ratio (g mL −1) and (T) is the

extraction time (h).

A

160

140

120

100

80

60

40

20

1 0 0

1 2 0

8 0

6 0

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2

0

0 1 0

1 5

2 0

2 5

3 0

3 5

5 0

4 0

4 5

700

600

500

400

300

B

t i m e

( m i n u

t e s )

1 0

1 5

2 0

2 5

3 0

3 5

5 0

4 0

4 5

t i m

e ( m i

n u t e s

)

1 0

1 5

2 0

2 5

3 0

3 5

5 0

4 0

4 5

t i m e ( m i

n u t e s

)

R a t i o ( g . m

l )

1 0 0

1 2 0

8 0

6 0

4 0

2 0

0

R a t i o ( g . m l )

1 0 0

1 2 0

8 0

6 0

4 0

2 0

0

R a t i o ( g . m l )

200

300

400

500

600

700

800

200

600

1000

1400

1800

f l av an ol em g.1 0 0 g-1

ph en ol i c s m g.1 0 0 g-1

µm ol T r ol ox .m

g.1 0 0 g-1 1800

1600

1400

1200

1000

800

600

400

C

60

80

100

120

140

Fig. 1. Response surface plot for avanol-enriched extract for the effect of the extraction time (min), solid to liquid ratio (g mL −1) on the extraction of total avanols (TF) (A), total

phenolics (TP) (B), and total antioxidant activity (C) (TAA) from E. edulis pulp.


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The regression model is able to explain 99% of the results foranthocyanins extraction (Table 3).

This may be explained by the fact that glycosylated anthocyaninsin açaí are localized in the most external region of the fruits ( Pompeuet al., 2009) and thus less energy is required for their extraction.Extraction procedures have generally involved the use of acidic

solvents, which denature the membranes of the tissue cells andsimultaneously dissolve pigments. The acid tends to stabilize theanthocyanins, butit may also changethe nativeform of the pigment inthe tissue by breaking associations with metals, co-pigments, or other

factors (Castañeda-Ovandro, Pacheco-Hernández, Páez-Hernández,Rodriguez, & Andrés, 2009; Revilla et al., 1998).

The response surface for jussara total phenolic (TP) content as afunction of extraction time and solid to liquid ratio is shown in Table 4

and Fig. 2(B), and demonstrates that experiments 6 (756.98 mgGAE 100 g−1 FW), 8 (766.45 mg GAE 100 g−1 FW) and 9 (766.45 mgGAE 100 g−1 FW) have different extraction times (12 and 24 h) andthe same values for the solid to liquid ratio (1:30 and 1:50). The

statistical analysis demonstrated that the linear terms for extractiontime ( p =0.3600) and the interaction extraction time and solid toliquid ratio ( p= 0.2082) are not signicant variables for modelstatistics ( pN0.05). However, the variable solid to liquid ratio had a

signicant effect (F model =274.15; p =0.000001) with the concentra-tion of phenolic compounds increasing with increasing solid to liquidratio for the same time. Similar results were observed in a study byPompeu et al. (2009), which showed that the extraction of the

phenolic compounds from açaí was dependent on the mass-to-solvent ratio. The calculated model is also able to explain 97% of theresults in the case of phenolics extraction (Table 3).

For TAA, Table 4 and Fig. 2(C) demonstrated that a higher time

contributes to an increase in the TAA.

3.4. Validation of the optimized conditions

The optimized conditions obtained by RSM were used to validatethe predictive model of TF extraction in avanol-enriched extracts

and TMA in anthocyanin-enriched extracts of jussara pulp. The results(Table 4) showed that the experimental optimized conditionsselected for avanol-enriched extracts were: solid to liquid ratio of between 1:50 and 1:100 and extraction time of 15 min. These

conditions may also be applied for TP and TAA extractions. Theoptimized conditions for anthocyanin-enriched extracts were: solid toliquid ratio of between 1:30 and 1:50 and extraction time of 24 h.Again, the same optimized conditions can be applied to TP and TAAextractions.

3.5. Correlation analysis

For the avanol-enriched extract the correlations between TF andTAA, TF and TP, and TP and TAA were statistically signicant ( pb0.05),with R 2 values of 0.64, 0.61 and 0.62, respectively. For the

anthocyanin-enriched extract the correlations between TMA andTAA, TMA and TP, and TP and TAA also were statistically signicant( pb0.05), with R 2 values of 0.99, 0.87 and 0.98, respectively. Severalstudies have suggested that the anthocyanin content and its

corresponding antioxidant activity, contribute to the protective effectof fruits and vegetables against degenerative and chronic diseases(Castañeda-Ovandro et al., 2009; Gao & Mazza, 1996; Yilmaz &Toledo, 2006).

4. Conclusions

The experimental design approach was successfully applied in theoptimization of the conditions for the extraction of avanols andanthocyanins from jussara (E. edulis) pulp. The variables investigatedin this study, extraction time and solid to liquid ratio, can inuence

the extraction performance. The antioxidant capacity was moderatelycorrelated with avanol content, but highly correlated with anthocy-anin content. Further work is required to investigate the contributionof individual anthocyanins, avanols and other compounds to the

Table 3

Analysis of variance (ANOVA) and regression coef cients of the second order

polynomial equation for total phenolic (TP), total avanols (TF), total monomeric

anthocyanins (TMA) and total antioxidant activity (TAA) in the avanol-enriched

extract and anthocyanin-enriched extract from E. edulis pulp.

Regression

coef cients

Extract

(avanol-enriched)

Extract

(anthocyanin-enriched)

TPa TFb TAAc TPa TMAb TAAc

Intercept β0 346.39⁎ 69.73⁎ 408.13⁎ 532.58⁎ 178.79⁎ 615.42⁎

Linear β1 −1.88 −0.50 −0.68 −11.38 151.12⁎ −11.12

β2 3.01⁎ 0.37⁎⁎ 7.50⁎⁎ 197.41⁎ 9.46⁎ 12.89⁎

Interaction β12 0.02 0.009⁎⁎ 0.093 15.94 9.66⁎ −0.03

r2 0.97 0.94 0.62 0.98 0.99 0.99

a mg GAE 100 g−1 fresh weight.b mg CAT 100 g−1 fresh weight.c μ mol TEAC 100 g−1 fresh weight.⁎ p≤0.01.

⁎⁎ p≤0.05.

Table 4

Extraction conditions of theexperimental design 32 setting in theoriginaland coded and uncoded formthe independentvariables (Ta, Slrb) and experimentalresults of total phenolic(TP) content, total monomeric anthocyanins (TMA) content, and total antioxidant activity (TAA).

Expt Ta Slrb TPc TMAd TAAe

Exp. Pred. Exp. Pred. Exp. Pred.

1 −1 (1) −1(1:10) 364.43 483.14 30.86 25.54 709.65 744.32

2 −1 (1) 0 (1:30) 567.90 633.14 60.89 57.70 994.56 1002.12

3 −1 (1) 1 (1:50) 721.00 783.14 313.77 303.40 1873.83 1259.92

4 0 (12) −1 (1:10) 525.44 483.14 166.84 140.24 1339.25 744.32

5 0 (12) 0 (1:30) 584.86 633.14 203.56 193.63 1213.89 1002.12

6 0 (12) 1 (1:50) 756.98 783.14 307.89 247.04 1435.99 1259.92

7 1 (24) −1 (1:10) 303.63 483.14 300.56 265.33 678.05 744.32

8 1 (24) 0 (1:30) 766.45 633.14 352.04 341.93 1764.40 1002.129 1 (24) 1 (1:50) 806.37 783.14 456.78 418.52 1459.00 1259.92

a T: extraction time (min).b Slr: solid to liquid ratio (g mL −1).c mg GAE 100 g−1 fresh weight.d mg cy-3-glu 100 g−1 fresh weight.e

μ mol TEAC 100 g−1

fresh weight.


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antioxidant capacity of extracts of jussara produced in the conditionsoptimized.

Acknowledgements

The authors are grateful to the National Council of Technologicaland Scientic Development (CNPq) fornancial support and to AliconIndustrial Ltda for their collaboration in the sample collection.

References

Arnous, A., Markis, D., & Kefalas, P. (2002). Correlation of pigment and avanol contentwith antioxidant properties in selected aged regional wines from Greece. Journal of Food Composition and Analysis, 15, 655−665.

Budini, R., Tonelli, D., & Girotti, S. (1980). Analysis of total phenols using the prussianblue method. Journal of Agricultural and Food Chemistry, 28, 1236−1238.

Bujić-Kojić, A., Planinic, M., Srécko, T., Jakabek, L., & Seruga, M. (2009). Inuence of solvent and temperature on extraction of phenolic compounds from grape seed,antioxidant activity and colour of extract. International Journal of Food Science &

Technology, 44, 2394−2401.

Cacace, J. E., & Mazza, G. (2002). Extraction of anthocyanins and other phenolics fromBlack currants with sulfured water. Journal of Agricultural and Food Chemistry, 50,5939−5946.

Castañeda-Ovandro, A., Pacheco-Hernández, M., Páez-Hernández, M., Rodriguez, J., &Andrés, C. (2009). Chemical studies of anthocyanins: A review. Food Chemistry, 113,859−871.

De Brito, E. S., De Araújo, M. C. P., Alves, R. E., Carkeet, C. C., Clevidence, B., & Novotny, J.(2007). Anthocyanins present in selected tropical fruits: Acerola, jambolão, jussarae guarabiju. Journal of Agricultural and Food Chemistry, 55, 9389−9394.

Del Pozo-Insfran, D., Talcott, S., & Brenes, C. H. (2004). Phytochemical composition andpigment stability of açaí (Euterpe oleracea Mart.). Journal of Agricultural and FoodChemistry, 52, 1539−1545.

Fan, G., Yonbin, H., Gu, Z., & Chen, D. (2008). Optimizing conditions for anthocyaninsextraction from purple sweet potato using response surface methodology (RSM).LWT Food Science and Technology, 41, 155−160.

Gallori,S., Bilia, A. R.,Bergonzi, M. C.,Barbosa, W. L. R.,& Vincieri, F. F. (2004).Polyphenolicconstituents of fruit pulp of Euterpe oleraceae Mart. (açai). Cromatographia, 59,739−743.

Gao, L., & Mazza, G. (1996). Extraction of anthocyanin pigments from purple sunowerhulls. Journal of Food Science, 61, 600−603.

Giusti, M. M., & Wrolstad, R. E. (2001). Anthocyanins: Characterization andmeasurement with uv–visible spectroscopy. In R. E. Wrolstald (Ed.), Current protocols in food analytical chemistry (PP) (pp. 1−13). New York: John Wiley and

Sons.

400

800

700

600

500

400

300

600

800

1000

1200

1400

1600

1800

2000

300

200

100

0

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200

600

1000

1400

1800

2200 µm ol T r ol ox .m g.1 0 0 g-1

C

1 0 0

1 2 0

-100

0

100

200

300

400

500

8 0

6 0

4 0

2 0

0 0

4

8

1 2

1 6

2 0

2 4

R a t i o ( g . m l )

1 0 0

1 2 0

8 0

6 0

4 0

2 0

0

R a t i o ( g . m l )

1 0 0

1 2 0

8 0

6 0

4 0

2 0

0

R a t i o ( g . m l )

200

300

400

500

600

700

800

900 an t h o c y an

i n s m g.1 0 0 g-1

ph

en ol i c s m g.1 0 0 g-1

T i m e ( h o

u r )

0

4

8

1 2

1 6

2 0

2 4

T i m e ( h o

u r )

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1 2

1 6

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T i m e ( h o

u r )

Fig. 2. Response surface plot for total anthocyanin-enriched extract for the effect of time (h),solid to liquid ratio (g mL −1) on the extraction of total monomeric anthocyanins (TMA)

(A), total phenolics (TP) (B) and total antioxidant activity (TAA) (C) from E. edulis pulp.


8/13/2019 1-s2.0-S0963996910004953-main

8/8

Harbone, J. B., Saito, N., & Detoni, C. H. (1994). Anthocyanins of Cephalis, Cynomorium,Euterpe, Lavatera and Pinanga. Biochemistry System Ecology, 22, 835−836.

Hayouni, E.A., Abedrabba, M.,Bouix, M.,& Hamdi,M. (2007). Theeffectsof solvents andextrraction method on the phenolic contents and biological activities in vitro of Tunisian Quercus coccifera L. and Juniperus phoenicea L. fruits extracts. Analytical,Nutritional and Clinicals Methods, 105, 1126−1134.

Henderson, A. (2000). The genus Euterpe in Brazil. In M. S. Reis, & A. Reis (Eds.), Euterpeedulis Martius – (Palmiteiro) biologia, conservação e manejo (pp. 1−22). Itajaí:Herbário Barbosa Rodrigues.

Huang, W., Xue, A., Niu, H., Zhen, J., & Wang, J. (2009). Optimised ultrasonic-assistedextraction of avonoids from Folium eucommiae and evaluation of antioxidant

activity in multi-test systems in vitro. Food Chemistry, 114, 1147−1154.Kahkonen, M. P., Hopia, A. I., & Heinomen, M. (2001). Berry phenolics and theirantioxidant activity. Journal of Agricultural and Food Chemistry, 49, 4076−4082.

Marete, E. N., Jacquier, J. C., & O`Riordan, D. (2009). Effects of extraction temperature onthe phenolic and parthenolide contents, and colour of aqueous feverfew(Tanacetum parthenium) extracts. Food Chemistry, 117 , 226−231.

Montgomery, Douglas C. (2004). Design and analysis of experiments . Eds: John Wiley. 6edição.

Oliveira-Brett, A. M., Novak, I., Janeiro, P., & Seruga, M. (2008). Ultrasound extractedavonoids from four varieties of Portuguese red grape skins determined byreverse-phase high-performance liquid cromatography with electrochemicaldetection. Analytica Chimica Acta, 630, 107−115.

Ou, B. X., Hampsch-Woodill, M., & Prior, R. L. (2001). Development and validation of animproved oxygen radical absorbance capacity assay using uorescein as theuorescent probe. Journal of Agricultural and Food Chemistry, 49, 4619−4626.

Pastor-Cavada, E., Juan, R., Pastor, J., Alaiz, M., & Vioque, J. (2009). Antioxidant activity of seed polyphenols in fteen wild Lathyrus species from South Spain. LWT FoodScience and Technology, 42, 705−709.

Pérez-Jiménez, J., & Sauro-Calixto, F. (2006). Effect of solvent and certain food

constituents on different antioxidant capacity assays. Food Research International, 39, 791−800.

Pietta, P. G. (2000). Flavanoids as antioxidants. Journal of Natural Products, 63,1035−1042.

Pinelo, M., Rubilar, M., Sineiro, J., & Nunez, M. J. (2005). Effect of solvent, temperature,and solvent-to-solid ration the total phenolic content and antiradical activity of extracts from different components from grape pomace. Journal of Agriculture andFood Chemistry, 53, 2111−2117.

Pompeu, D. R., Silva, E. M., & Rogez, H. (2009). Optimization of the solvent extraction of phenolic antioxidants from fruits of Euterpe oleracea using response surfacemethodology. Bioresource Technology, 100, 6076−6082.

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Liu, M., & Rice-Evans, C. (1999).Antioxidant activity applying an improved ABTS radical cation descolorizationassay. Free Radical Biology & Medicine, 26, 1231−1237.

Revilla, E., Ryan, J., & Martin-Ortega, G. (1998). Comparison of several procedures usedfor the extraction of anthocyanins from red grapes. Journal of Agricultural and FoodChemistry, 46, 4592−4597.

Rosso, V. V., Hillebrand, S., Montilla, E. C., Bobbio, F. O., Winterhalter, P., & Mercadante,A. Z. (2008). Determination of anthocyanins from acerola (Malpighia emarginataDC.) and açaí (Euterpe oleraceae Mart .) by HPLC-PDA-MS/MS. Journal of FoodComposition and Analysis, 21, 291−299.

Runo, M. S. M., Alves, R. E., Fernandes, F. A. N., & de Brito, E. S. (2010a). Free radical

scavenging behavior of ten exotic tropical fruits extracts. Food Research International,doi:10.1016/j.foodres.2010.07.002.Runo, M. S. M., Pérez-Jiménez, J., Arranz, S., Alves, R. E., Brito, E. S., Oliveira, M. S. P.,

et al. (2010b). Açaí (Euterpe oleraceae) “BRS Pará”: a tropical fruit source of antioxidant dietary ber and high antioxidant capacity oil. Food ResearchInternational, doi:10.1016/j.foodres.2010.09.011.

Santos, D. T., Veggi, P. C., & Meireles, A. M. A. (2010). Extraction of antioxidantcompounds from jabuticaba (Myrciaria cauli ora) skins. Yield, composition andeconomical evaluation. Journal of Food Engineering , 101, 23−31.

Schauss, A. G., Wu, X., Prior, R. L., Ou, B., Huang, D., Owens, J., et al. (2006).Phytochemical and nutrient composition of the freeze-dried Amazonian PalmBerry, Euterpe oleraceae Mart. (Acaí). Journal of Agricultural and Food Chemistry, 54,8598−8603.

Sun,T., & Ho, C. (2005). Antioxidant activitiesof buckwheat extracts.FoodChemistry, 90,743−749.

Talcott, S. T., Pacheco-Palencia, L. A., & Talcott, S. M. (2008). Chemical composition,antioxidant properties, and thermal stability of phytochemical enriched oil fromAçaí (Euterpe oleracea Mart.). Journal Agriculture and Food Chemistry , 56, 4631−4636.

Vilkhu, K., Mawson, R., Simons, L., & Bates, D. (2008). Applications and opportunities for

ultrasound assisted extraction in the food industry—A review. Innovative FoodScience & Emerging Technologies, 9, 161−169.

Yang, Z., & Zhai, W. (2010). Optimization of microwave-assisted extraction of anthocyanins from purple corn ( Zea mays L.) cob and identication with HPLC-MS.Innovative Food Science & Emerging Technologies, 11, 470−476.

Yilmaz, Y., & Toledo, R. T. (2006). Oxygen radical absorbance capacities of grape/wineindustry byproducts and effect of solvent type on extraction of grape seedpolyphenols. Journal of Food Composition and Analysis , 19, 41−48.

Zou, Y., Xie, C., Fan, G., Gu, Z., & Han, Y. (2010). Optimization of ultrasound-assistedextraction of melanin from Auricularia auricular fruitbodies. Innovative FoodScience& Emerging Technologies, doi:10.1016/j.ifset.2010.07.002.

http://dx.doi.org/10.1016/j.foodres.2010.07.002http://dx.doi.org/10.1016/j.foodres.2010.07.002http://dx.doi.org/10.1016/j.foodres.2010.09.011http://dx.doi.org/10.1016/j.ifset.2010.07.002http://dx.doi.org/10.1016/j.ifset.2010.07.002http://dx.doi.org/10.1016/j.foodres.2010.09.011http://dx.doi.org/10.1016/j.foodres.2010.07.002

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