Evaluation of bioactive compounds potential and antioxidant activity in some Brazilian exotic fruit residues

Accepted Manuscript Evaluation of bioactive compounds potential and antioxidant activity in some Brazilian exotic fruit residues

Romy Gleyse Chagas Barros, Julianna Karla Santana Andrade, Marina Denadai, Maria Lucia Nunes, Narendra Narain PII: DOI: Reference:

S0963-9969(17)30667-1 doi:10.1016/j.foodres.2017.09.082 FRIN 7031

To appear in:

Food Research International

Received date: Revised date: Accepted date:

7 July 2017 5 September 2017 26 September 2017

Please cite this article as: Romy Gleyse Chagas Barros, Julianna Karla Santana Andrade, Marina Denadai, Maria Lucia Nunes, Narendra Narain , Evaluation of bioactive compounds potential and antioxidant activity in some Brazilian exotic fruit residues. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Frin(2017), doi:10.1016/j.foodres.2017.09.082

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Evaluation of bioactive compounds potential and antioxidant activity in some Brazilian exotic fruit residues.

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Romy Gleyse Chagas Barros1, Julianna Karla Santana Andrade1, Marina Denadai1,

Laboratory of Flavor & Chromatographic Analysis, PROCTA, Federal University of

Sergipe, 49100-000 – São Cristóvão – SE, Brazil

Department of Food Technology, Federal University of Ceara, 60020-180 – Fortaleza-

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Maria Lucia Nunes2, Narendra Narain1*

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CE, Brazil

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* Corresponding author: Narendra Narain E-mail: [email protected]

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Phone: +55-79-3194 6514

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Abstract The agroindustrial residues have been recognized as important sources of some prominent chemical compounds and hence a viable strategy of obtaining bioactive compounds could be applied to them. The present study was aimed to investigate the

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presence of bioactive compounds and the antioxidant activity of some Brazilian exotic

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fruits (achachairu, araçá-boi, bacaba) residues. The antioxidant capacity of fruit residues was evaluated by ORAC, FRAP and ABTS assays. The contents of total phenolic

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compounds, flavonoids, chlorophylls and carotenoids were determined. The

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identification and quantification of the phenolic compounds were performed by using the UHPLC-QqQ-MS/MS system. The compounds cinnamic acid, p-coumaric acid,

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epicatechin and quercetin were identified and quantified in all fruits residues. The residue with the highest antioxidant capacity was bacaba for ORAC (15285.51 ± 20.38

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μmol TE/100g) and FRAP (16916.37 ± 10.01 μmol TE/100g) assays, as well as total

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phenolic compounds in its methanolic extract (1537.45 ± 73.35 mg GAE/100g). Keywords: Achachairu, Araça-boi, Bacaba, Fruit residues, Antioxidant capacity,

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Phenolic compounds.

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1. Introduction

Brazil occupies third position in the production of fruits in the world. There is a large industry which is involved with the processing of the fruit juices whereby a large volume of residues is generated which can be better exploited for the production of

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highly valorized substances (Forster-Carneiro et al., 2013). These by-products from

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different fruit processing industries are traditionally discarded as waste and these are

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currently being recognized as important source of valuable chemicals. In fruit processing, peel and seeds are the two main by-products and their extracts contain a

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considerable amount of bioactive compounds (Goot et al., 2016; Bataglion et al., 2015). Bioactive compounds occur in small amounts in food and are considered as

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non-nutritional ingredients, but vital for the maintenance of human health (Patil et al., 2009). In addition, polyphenols have been found to be the main constituents of fruits

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and in their residues (Bataglion et al., 2015). Polyphenols are compounds that have

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more than 9000 substances identified. These can be divided into some groups according to their chemical structure: flavonoids (isoflavonoids, anthocyanidins, flavanols,

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flavonols, flavanones, and flavones) and nonflavonoids (hydroxycinnamic and hydroxybenzoic acids, stilbenoids, lignoids, and coumarines) (Tsao, 2010). In general,

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these compounds exhibit anti-inflammatory and antioxidant effects (Kang et al., 2011), besides characterizing a reduced risk of diseases such as certain forms of cancer, inflammation, cataracts, macular and cardiovascular degeneration and degenerative diseases (Sergent et al., 2010; Snyder et al., 2011). The bioavailability and function of phenolic compounds present in foods of plant origin in the human body is dependent of many varying factors such as bioaccessibility, food matrix effect, transporters, molecular structures and metabolizing 3

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enzymes (Rein et al., 2013). In addition, a refined knowledge about these residues and its chemical characterization would contribute to its use as a source of bioactive compounds (Abdennacer et al., 2015). The achachairu (Garcinia humilis), araça-boi (Eugenia stipitata) and bacaba

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(Oenocarpus bacaba) are edible exotic fruits cultivated in the north and northeast region of Brazil. These fruits are used for the production of beverages and industrialized

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products such as pulp, ice creams etc. which are widely appreciated by the local

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population (Shanley & Medina, 2005). Most of the studies on these fruits have been undertaken on their pulp and practically no or very little work has been reported on their

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residues regarding the presence of bioactive compounds, mainly in relation to the

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identification and quantification of phenolic and flavonoid compounds. Genovese et al. (2008) determined the bioactive compounds and antioxidant

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activity of the araçá-boi pulp and characterized the fruit as an important source of

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phenolics and flavonoids. Gonçalves et al. (2010) investigated the antioxidant and antidiabetic capacity of araçá-boi pulp and mainly identified the presence of compounds viz. quercetin and kaempferol, highlighting its potential as inhibitor of enzymes of

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carbohydrate metabolism. Cui et al. (2010) studied the mangosteen (Garcinia

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mangostana), fruit of the same genus as achachairu (G. humilis), object of this study, and identified the presence of several phytochemical compounds in peel, rind and aril parts of the fruit, indicating that phenolic acids are mainly located in the pericarp of the fruit and hydroxybenzoic acid derivatives are the major phenolic acids. Finco et al. (2012) evaluated the antioxidant activity of the bacaba pulp and tentatively identified 14 compounds suggesting that bacaba is a promising source of phenolic compounds. Thus it could be concluded from the existing publications that no work has been reported on

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the residues of achachairu, araçá-boi and bacaba fruits in order to tap their potential for bioactive compounds. Recent studies on bioactive compounds in fruit extracts have demonstrated that the Ultra High Performance Liquid Chromatography coupled to tandem Mass

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Spectometry (UHPLC-QqQ-MS/MS) is a powerful tool in the identification and characterization of organic compounds (Souza et al., 2016; Garzón et al., 2017). Thus,

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the aim of this work was to determine the presence of bioactive (carotenoids,

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chlorophylls, phenols and flavonoids) compounds, and to identify and quantify the phenolic and flavonoid compounds by the UHPLC-QqQ-MS/MS system in residues of

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Brazilian exotic tropical fruits viz. achachairu, araçá-boi, bacaba as well as to evaluate

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2.1. Samples

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2. Materials and Methods

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ther antioxidant capacity.

The residues of araçá-boi and bacaba were obtained from a juice processing

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industry in Manaus, Amazonas, while the residues of achachairu were collected from a local ice cream shop in Aracaju, Sergipe which processes the pulp of the fruit. The

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residues were transported in plastic containers maintained at −18 °C to the Laboratory of Flavor and Chromatographic Analysis, Federal University of Sergipe, São Cristóvão, Sergipe, Brazil. The residues of the achachairu, araçá-boi and bacaba were composed of the remains of seeds, peel and to a very small extent of pulp left after their processing for the production of juices or pasteurized pulp. Immediately after its arrival, these residues materials were grinded until they reach a particle size about 2x2 mm.

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The approximate yield of each residue corresponds to 70% of the fruit for the achachairu, 27% for the araçá-boi and 56% for the bacaba fruit. The residues were stored in different polyethylene bags at -18°C until analysis. All analyses were done in triplicate for each sample.

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2.2. Chemicals Sodium hydroxide, calcium carbonate, hydrochloric acid, sodium acetate,

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acetic acid, sodium carbonate, acetone, potassium persulfate were supplied from Synth

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(São Paulo, Brazil). Ethanol, methanol and ferric chloride were purchased from Neon (São Paulo, Brazil) and phenolphthalein from Dinâmica (São Paulo, Brazil). Folin-

acid

(ABTS),

2,4,6-tripyridyl-s-triazine

tetramethylchroman-2-carboxylic

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sulfonic

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Ciocalteu phenol reagent, aluminum chloride, 2,20-Azinobis-3-ethylbenzothiazoline-6-

acid

(TPTZ),

6-hidroxy-2,5,7,8-

(TROLOX),

2,2'-azobis(2-

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methylpropionamidine) dihydrochloride (APPH), flurescein, formic acid (HPLC grade),

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acetonitrile (HPLC grade), and the phenolic standards such as caffeic acid, cinnamic acid, chlorogenic acid, feluric acid, gallic acid, p-coumaric acid, vanillic acid, (+)catechin, (-)epicatechin, eriodictyol, ethyl gallate, naringenin, quercetin, rutin and

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vanillin were obtained from Sigma Aldrich (St. Louis, MO, USA).

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2.3. Physico-chemical composition of fruit residues 2.3.1. Moisture

The moisture content was determined on an infrared moisture meter (GEHAKA model IV 2500) by direct reading.

2.3.2. Titratable acidity

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The determination was made by titrating with the standardized alkali solution, according to the methodology cited by AOAC (2000, method no. 942.15). Five grams of the sample were weighed, which was transferred to a 125 mL Erlenmeyer flask with the aid of 50 mL of water. Two to four drops of phenolphthalein solution was added and

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subsequently the mixture was titrated with 0.1 M sodium hydroxide solution until pink.

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Titratable total acidity was expressed as a percentage of citric acid.

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2.3.3. Soluble solids (°Brix)

The soluble solids content was determinated according to the protocol

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determined by AOAC (2000, method no. 932.12). An aliquot of each sample was placed on the refractometer prism (The Electron Machine Corporation, model DSA E-Scan),

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duly calibrated with distilled water, under which direct reading was obtained.

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2.3.4. pH

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The determination was performed by direct reading in pHmeter (HANNA, model HI 2210), according to the method proposed by the AOAC (2000, method no.

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945.10). Ten grams of the sample was weighed into a becker and it was diluted by addition of 100 mL of water. The contents were stirred until the particles were

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uniformly suspended. With the apparatus previously calibrated with the buffer solutions of pHs 4 and 7, the pH of respective samples was read.

2.4. Extraction of residues The fruit residues were extracted with different solvents (water and methanol) in order to evaluate the best potential for the extraction of bioactive compounds. The methanolic extraction was followed by the methodology described by Rehman (2006),

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with alterations. Two grams of the residues of each fruit was weighed and diluted in 15 mL of methanol. The mixture was then maintained in shaker (SOLAB, Brazil, SL 222) at room temperature (29 ± 2°C) for 24 hours. The aqueous extraction was performed by autoclaving 2 g of the fruit residues with 15 mL of distilled water at 121°C for 15

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minutes. All extracts, obtained as above, were centrifuged (Eppendorf Centrifuge, 5810

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R) at 24°C at 12000 rpm for 15 minutes and the supernatants collected in 100 mL amber

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colored bottles which were stored in refrigerator (8°C) until the time of use.

2.5.1. Chlorophyll and carotenoids

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2.5. Bioactive compounds

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For the analysis of chlorophyll and carotenoids, the method of Lichtenthaler (1987) was used. For extraction, 2 g of the samples was weighed, macerated, added to

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0.2 g of calcium carbonate and 7 mL of 80% acetone. The extract was filtered through a

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25 mL volumetric flask and the volume filled with the solvent itself. The absorbance was measured in a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA;

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SpectraMax M2) at wavelengths of 470 nm for total carotenoids, 647 nm and 663 nm for chlorophyll a and b, respectively.

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The concentrations of total chlorophyll (a and b) and total carotenoids contents were determinated according to equations 1.1, 1.2 and 1.3, respectively. Chlorophyll a (Ca) = [12.25 A663.2 – 2.79 A646.8]

(Equation 1.1)

Chlorophyll b (Cb) = [21.50 A646.8 – 5.10 A663.2]

(Equation 1.2)

Total Carotenoids = [100 A470 – (1.82Ca – 104.96 Cb)/ 198]

(Equation 1.3)

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2.5.2. Total phenolic compounds Total phenolic compounds content was determined according to the methodology suggested by Shetty et al. (1995) and adapted by Shori et al. (2014). One mililiter aliquot of extract solution was mixed with 1 mL of 95% ethanol solution, 5 mL

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of distilled water and 0.5 mL of 1 N Folin-Ciocalteu phenol reagent. Subsequently, 1 mL of 5% sodium carbonate solution (Na2CO3) was added followed by

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homogenization. The mixtures were allowed to rest in a darkroom for 60 minutes and

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then it was again homogenized. The absorbance was measured at 725 nm using a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA; SpectraMax M2). The

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total phenolic compounds content was expressed in milligrams equivalent of gallic acid

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per 100 grams (mg GAE/100g) of sample. The calibration curve was prepared by analyzing gallic acid as standard in the concentration varying from 0.00078 to 0.1

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mg/mL.

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2.5.3. Total flavonoids content

The determination of total flavonoids content was done according to the

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methodology proposed by Meda et al. (2005) with some modifications. A 0.5 mL aliquot of extract solution was mixed with 0.5 mL of 20 mg/mL aluminum chloride

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(AlCl3) solution. The samples were stored in a darkroom for 30 minutes before reading in a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA; SpectraMax M2) at 427 nm. Total flavonoids content was determined using a standard quercetin curve, prepared by its analysis at different concentrations ranging from 0.00078 to 0.025 mg/mL. The results were expressed in terms of milligrams of quercetin per 100 g of residue.

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2.6. Antioxidant Activity 2.6.1. ABTS assay The method was developed according to Moo-Huchin et al. (2014). The ABTS cation was generated by the interaction of 19.2 mg of ABTS dissolved in 5 mL of

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distilled water and 88 μL of 0.0378 g/mL of potassium persulfate. The solution was incubated in a darkroom for a period of 16 hours. The active ABTS radical was diluted

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in ethanol to an absorbance of 0.7 ± 0.02 at 734 nm. Later, 30 μL of sample or standard

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was added to 2970 μL of diluted ABTS solution. The absorbance value was recorded after 6 minutes of mixing. A calibration curve was prepared using ascorbic acid

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(vitamin C) at different concentrations ranging from 25 to 350 ppm and antioxidant

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capacity results were expressed as vitamin C equivalent (VCE) mg/100g of fresh

2.6.2. FRAP assay

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weight.

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The FRAP assay was performed according to the method of Thaipong et al. (2006). Stock solutions included 300 mM of acetate buffer (3.1 g C2H3NaO.3H2O and

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16 mL C2H4O2), pH 3.6, 10 mM TPTZ in 40 mM HCl and 20 mM FeCl3.6H2O. The working solution was prepared by mixing 25 mL of the acetate buffer, 2.5 mL of the

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TPTZ solution and 2.5 mL of FeCl3·6H2O and then heated to 37 °C before use. The 150 μL of each extract was allowed to react with 2850 μL of FRAP and the solution was incubated in the dark for 30 minutes. The absorbance reading was measured at 593 nm. The calibration curve was prepared using solutions of 20 to 800 mM Trolox. The results were expressed in µmol TE/100g of fresh weight.

2.6.3. ORAC assay

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The ORAC procedure was performed using the methodology proposed by Thaipong et al. (2006) with some modifications. For the analysis, 1.5 mL of flurescein working solution (25 µL of flurescein with 50 mL of phosphate buffer) was added to 0.75 mL of the sample and vortexed. The solution was then incubated in a water bath at

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37 °C for 15 minutes. Finally, 0.75 mL of the vortex-agitated APPH solution was added and absorbance was read under fluorescence conditions: excitation at 485 nm and

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emission at 520 nm. The calibration curve was prepared by using eight data points

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covering the range from 0 to 50 mM trolox. The results were expressed as μmol

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TE/100g of fresh weight.

2.7. Identification and quantification of phenolic and flavonoid compounds by

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UHPLC-QqQ-MS/MS

2.7.1. Chromatographic conditions

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The flavonoid and phenolic acid standards were analyzed using a UHPLC

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system - model 1290 Infinity coupled to a 6490 Triple Quadruple mass spectrometer equipped with an electrospray ionization (Agilent Technologies, Palo Alto, USA) in

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positive mode for all compounds, in Multiple Reaction Monitoring (MRM) mode of acquisition. The chromatographic separation was performed on an Ascentis Express F5

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(150 x 2.1 mm, 2.7 μm particle; Sigma Aldrich) column. The mobile phase consisted of: Solvent A (deionized water with 0.1% formic acid) and Solvent B (acetonitrile with 0.1% formic acid). The flow rate was 0.2 mL/min, at a temperature of 40°C and an injection volume of 2 μL. The gradient profile was: 0-1 min, 15% B; 1-7 min, 25% B; 79 min, 25% B; 9-13 min, 30% B; 13-16, 30% B; 16-21 min, 40% B; 21-23, 40% B; 2325, 45% B; 25-28, 50% B; 28-33, 60% B; 33-37, 75% B; 37-38, 15% B.

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2.7.2. Parameters of the mass spectrometer UHPLC-QqQ-MS/MS is a powerful tool in screening and determination of phenolic compounds in complex matrices. The analysis was conducted in the system with triple quadrupole mass analyzer (Agilent 6490 Triple Quad UPHLC-MS/MS)

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having electrospray ionization in positive mode. The mass spectrometer conditions were: gas temperature 200 °C, gas flow rate 12 L/min; Nebulizer 20 psi; Sheat gas

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temperature 400 °C; Sheat gas flow 11 L/min; capillary voltage 3500 V; nozzle 500 V;

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Dwell time 9.8 and acceleration cell voltage 5 V. The LC-MS/MS data was acquired

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using Mass Hunter software (Agilent, version B.07).

2.7.3. Determination of phenolic compounds in residues

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In this work, 20 phenolic analytical standards were monitored simultaneously. The presence of these was confirmed according to MS/MS2 transitions (quantification

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transition/confirmation transition) of each target compound and the corresponding

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retention time. The UHPLC-MS/MS parameters, retention time and linearity limits for phenolic compounds are presented in Table 1.

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Determinations were carried out based on the peak areas of target compounds and these compared with the corresponding standards. Analysis of standard compounds at 8

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different levels over a concentration range of 20-1000 ng/mL was performed in the system in triplicate. Calibration curves were constructed for each standard by plotting the peak area versus the nominal concentration, producing values of correlation coefficients (r2) greater than 0.990. The lower limit of quantification (LOQ) was considered the concentration at initial level of calibration curve (20 ng/mL) with the accepted criteria that the precision and the accuracy for three samples had variability of less than 20%. The UHPLC-MS/MS analysis of phenolic and flavonoid compounds in 12

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the fruit residues was followed by the analysis of standard reagents and consequent preparation of calibration curves. The lower limits of detection (LOD) were calculated, considering a signal-to-noise (S/N) ratio of >3. Samples which had analytes concentration above the detection limit of the method, but below the LOQ, were

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denominated as traces (TR), since the measured minimum concentration of the

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substance could be reported with 99% confidence.

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2.8. Statistical analyses

The statistical analysis was performed using SAS software (SAS Institute, Cary,

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NC) Version 9.1.3. Significant differences between the mean values of different

the probability of 5% (p≤0.05).

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3. Results and Discussion

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characteristics were determined by applying Tukey´s test for multiple comparisons at

3.1. Physico-chemical composition of fruit residues

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Table 2 presents the contents of moisture, total soluble solids, pH and titratable acidity of fruit residues. Regarding the moisture, it can be verified that among all fruit

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residues, the araçá-boi contained the highest moisture content (79.05%), followed by achachairu (60.24%) and bacaba (37.67%). These data on fruit residues differ from those reported on their fruit pulp by Canuto et al. (2010), Carvalho et al. (2013) and Domingues et al. (2014) who presented values of 90.1 ± 0.5% for araçá-boi pulp, 87.86 ± 0.00% for achachairu pulp and 88.6 ± 0.14% for bacaba pulp, respectively. The content of total soluble solids presented values close to those described by Canuto et al. (2010) for araçá-boi pulp (4.5 ± 1.4) and bacaba pulp (2.0 ± 0.7), while

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Pimentel (2011) reported (16.4 ± 0.54) for achachairu pulp. As for pH, all residues were close to those reported by different authors, being for achachairu pulp (3.78 ± 0.27) (Pimentel, 2011), for bacaba pulp (5.3 ± 0.1) by Canuto et al. (2010) and for araçá-boi pulp (2.51 ± 0.0) by Gomes et al. (2010).

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In this work, the titratable acidity parameter showed levels higher than those

3.2.

Total contents of bioactive compounds

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araçá-boi pulp (1.8 ± 0.1) and bacaba pulp (0.1 ± 0.0).

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reported by Pimentel (2011) for achachairu pulp (1.37 ± 0.41), Canuto et al. (2010) for

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The fruit residues were submitted to the analysis of total phenolic and flavonoid compounds from extractions carried out with different solvents (distilled water and

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methyl alcohol), in order to determine the best extractive solvent of the mentioned compounds. Table 3 presents the data on total phenolic and flavonoid contents of the

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residues extracts obtained with different solvents.

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It can be observed from the Table 3 that the residue of bacaba contained the highest value of extracted phenolic compounds in the methanolic extract. Araçá-boi did

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not have any significant difference (p≤0.05) in their aqueous and methanolic extractions. Regarding flavonoid content, aqueous extracts of achachairu and bacaba

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showed higher extractive potential than their methanolic extracts, while araçá-boi presented similar data in both extractions. For Naczk & Shahidi (2006), the extraction of phenolic compounds or specific class in foods needs further investigation, since factors such as sample preparation, polarity of the solvent used, the technique employed and also the temperature can interfere in the extraction and contents of these compounds.

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With respect to the content of phenolic compounds it can be observed that the results obtained for methanolic extracts of bacaba are close to those reported by Finco et al. (2012) for the pulp of the same fruit of 1759.27 ± 1.01 mg/100g and higher than those presented by Carvalho et al. (2016) for bacaba-de-leque pulp (589.00 ± 11.03

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mg/100g). The aqueous extracts of achachairu residue obtained higher values than those described by Rufino et al. (2010) for bacuri pulp (23.8 ± 0.7 mg/100g) and lower than

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those reported by Koolen et al. (2013) for buriti pulp of 378.07 ± 3.12 mg/100g. Both

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extractions of araçá-boi residue presented a lower value when compared with araçá pulp (111.00 ± 3.64 mg/100g) (Contreras-Calderón et al., 2011).

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Regarding total flavonoid contents, when compared to the values reported by

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Rufino et al. (2010), it was verified that both extracts of bacaba residue presented higher levels than acerola pulp (9.6 ± 1.4 mg/100g) and lower than acai pulp (91.3 ± 20.6

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mg/100g). The extracts of achachairu and araçá-boi residues showed lower amounts

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compared to fruits pulps such as bacuri (16.9 ± 1.7 mg/100g), mango (15.0 ± 1.1 mg/100g) and umbu (6.9 ± 1.7 mg/100g). The total carotenoid content found in the fruit residues demonstrated that the

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araçá-boi presented the highest amount (33.39 ± 0.00 μg/g) of this compound followed

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by bacaba and achachairu (Table 4). On comparing the data of our study with results reported by Denardin et al. (2015), it was observed that the araçá-boi residue had a higher carotenoid content than that presented by the authors for the araçá pulp (6.27 ± 0.06 μg/g), as well as for guava pulp (22.98 ± 0.00 μg/g) (Nora et al., 2014). Bacaba residue showed a value (15.47 ± 0.01 μg/g) close to that described by Santos et al. (2015) for bacaba oil of 13.53 ± 0.97 μg/g. The achachairu residue contained much higher value (9.32 ± 0.03 μg/g) of

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carotenoids when compared to that of the araticum do mato pulp (0.89 ± 0.16 μg/g) according to Pereira et al. (2013).

3.3.

Identification and quantification of phenolic compounds in residues The concentrations of bioactive compounds present in the extracts of different

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fruit residues are presented in Table 5. The chromatograms and main structures

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identified and quantified of these compounds can be observed in Figure 1. Twenty

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compounds were identified in the residues, but only sixteen were within the limits of quantification. When comparing the aqueous and methanolic extractions, it was possible

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to conclude that for most phenolic acids (caffeic acid, chlorogenic acid, feluric acid, gallic acid, p-coumaric acid, protocatechinic acid and vanillic acid), the aqueous

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extraction was more efficient than the methanolic extraction.

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For methanolic extractions, it was observed that the compound naringenin was

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identified and quantified only in the residues of achachairu and bacaba extracted with the organic solvent. In addition, other compounds also obtained more expressive values in methanolic extracts e. g. the ethyl gallate which was identified in the residue of araçá-

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boi, eriodictyol in the residue of achachairu and bacaba, rutin in the residue of bacaba

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and quercetin in the residues of achachairu, araçá-boi and bacaba. The presence of these bioactive compounds is reported for the first time in the fruit residues. The catechin and epicatechin were the majority compounds detected and

quantified in the bacaba residue and these presented no significant difference (p≤0.05) between the aqueous and methanolic extracts, but cinnamic acid contained higher content (0.27 ± 0.06 μg/g) in methanolic extract than the aqueous extract (0.19 ± 0.02

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μg/g) of the same residue. Artepellin C compound was identified in methanolic extract of this residue, but was lower than the limits of quantification. Regarding the quantification of the compounds in the bacaba residue, it was evident that it has significant amounts of catechin (135.15 ± 1.11 μg/g in the aqueous

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extract) and epicatechin (123.86 ± 7.16 μg/g in the aqueous extract). These data when related to other fruit pulps with higher antioxidant capacity such as açai (Euterpe

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oleracea), blueberrys (Vaccinium spp.) and grapes (Vitis spp.) showed higher values,

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since the açai pulp presented the respective compounds above the limit of detection, but below the limit of quantification (Garzón et al., 2017), blueberrys have catechin values

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varying between 1.80 ± 0.09 μg/g and 3.09 ± 0.01 μg/g (Wang et al., 2017) and

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different grape varieties have catechin ranged between 29.1 ± 1.9 μg/g and 117.6 ± 0.7 μg/g and epicatechin in a range of 14.5 ± 0.9 and 21.1 ± 0.8 μg/g (Silva & Queiroz,

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2016). When compared to different varieties of tea, the bacaba residue presented values

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of catechin close to those found by Gárcia-Villalba et al. (2017) varying from 6.61 ± 0.73 to 177 ± 7.09 μg/g and to raw cocoa beans that avaraged 180.81 ± 4.0 µg/g (Zyzelewicz et al., 2016).

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Catechin and epicatechin are studied in detail, concerning their antioxidant

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capacities as polyphenolic compounds, which is well-established by various in vitro and in vivo methodologies. Catechins affect the molecular mechanisms involved in angiogenesis, degradation of the extracelular matrixes, regulation of cell deaths and reduction of oxidative stress, resulting in a series of benefits to health due to several actions such as antioxidative, antihypertensive, anti-inflammatory, antiproliferative, antithrombogenic and anti-hyperlipidemic (Zanwar et al., 2014).

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Finco et al. (2010) when conducting a study on the antioxidant activity and composition of bacaba phenolics in the fruit pulp identified the presence of fourteen different compounds, including quercetin, rutin, luteolin and rhamnetin. However, indepth studies on the phenolic compounds present in the pulp and mainly on bacaba

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residue are still scarce and insufficient. Other compounds found in açai such as rutin (400.00 ± 100.00 μg/100g) and protocatechuic acid (200.00 ± 0.100 μg/100g) were

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present in lower concentrations than the bacaba residue, according to Garzón et al.

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(2017).

For araçá-boi the compounds of greater value were cinnamic acid (44.61 ±

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0.76 μg/g in the aqueous extract) followed by gallic acid (30.18 ± 5.47 μg/g in the

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aqueous extract). Vanillin and cinnamic acid showed better results in aqueous extracts, and there was significant difference (p≤0.05) between their concentrations. Vanillic

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acid, kaempferol and naringenin were detected but were lower than the limit of

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quantification. Denardin et al. (2015) analyzed the antioxidant capacity and bioactive compounds of araçá fruit pulps which contained major phenolic compounds such as gallic acid, quercetin, apigenin and isoquercetin.

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For the achachairu residue, the epicatechin and ferulic acid compounds were

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the most predominant. Compounds cinnamic acid, feluric acid, p-coumaric acid and epicatechin were in higher concentrations in aqueous extracts, having significant difference (p≤0.05) compared to methanolic extracts. Vanillic acid, daidzein, pinocembrin and vanillin were lower than the quantification limits. The genus Garcinia, to which achachairu pertains has a number of biflavonoids identified, such as naringenin, apigenin, eriodictyol, luteonin and quercetin (Carrillo-Hormaza et al., 2016). As for quantification, achachairu presented superior results than pitomba (Talisia

18

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esculenta) fruit in relation to ferulic acid (1.8 ± 0.1 μg/g) and epicatechin (2.9 ± 0.1 μg/g). Meanwhile, the p-coumaric acid (3.4 ± 0.3 μg/g) and quercetin concentrations (0.6 ± 0.0 μg/g) of the pitomba had higher amounts when compared to their values in achachairu residue (Souza et al., 2016).

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Compared to a fruit of the same genus, mangosteen (Garcinia mangostana), the residue of the achachairu contained higher values of ferulic acid (96.01 ± 7.72 μg/g)

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than that reported for the peel of mangosteen fruit (12.0 ± 1.0 μg/g). Moreover, the

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compounds eriodictyol and epicatechin were not detected in mangosteen, while the cinnamic acid content of mangosteen was higher (27.9 ± 2.3 μg/g) than its value (8.16 ±

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1.69 μg/g) found in this work in achachairu residue (Zadernowski et al., 2009). Thus in

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this work, all the fruit residues studied may be considered promising sources of bioactive compounds, which may be used for the development of nutraceuticals and

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3.4. Antioxidant Activity

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functional foods.

There are various methods to determine antioxidant capacity and these differ in relation to the principle and experimental conditions of the assay. A single assay does

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not accurately account for all of the groups of antioxidant compounds, particularly in a

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complex system, such as fruit matrices. Therefore, different antioxidant assays (ORAC, FRAP, ABTS) need to be performed in order to ensure a better comparison of data among different fruit residues. Table 6 presents the data on antioxidant capacity of the various fruit residues. The bacaba residue had a higher antioxidant capacity (12648.13 ± 8.76 μmol TE/100g for ORAC and 16916.37 ± 10.01 μmol TE/100g for FRAP) than its value (3330.00 ± 219.00 μmol TE/100g for ORAC assay) reported for bacaba-de-leque pulp by Carvalho

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et al. (2016) and the one presented by Finco et al. (2012) for bacaba pulp (13440.0 ± 0.20 µmol TE/100g in FRAP assay). However, it presented value close to that cited by Finco et al. (2012) for the pulp of the same fruit (10750.71 ± 1496.51 µmol TE/100g) for ORAC analyses, but inferior (63000 ± 0.0 µmol TE/100g) to those demonstrated for

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açai pulp (Schauss et al., 2010). The methanolic extracts of the achachairu (14391.12 ± 9.26 µmol TE/100g) and

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araçá-boi (6791.07 ± 10.20 µmol TE/100g) residues presented higher values than those

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presented by Floegel et al. (2011) in relation to ORAC methodology for banana (879.00 µmol TE/100g) and mango (1002.00 µmol TE/100g). Neri-Numa et al. (2013) evaluated

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the antioxidant activity of the araçá-boi according to ORAC method and obtained a

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lower value (371.98 μmol TE/100 g), while Garzón et al. (2012) reported an antioxidant activity in FRAP assay of 1240.00 μmol TE/100 g of araçá-boi peel which is similar to

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the value (1380.74 ± 7.71 μmol TE/100 g) found in this study.

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In antioxidant activity measured by FRAP, the hightest activity was found in bacaba residue, the values being 16916.37 ± 10.01 μmol TE/100g in methanolic extract

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and 6567.45 ± 4.25 μmol TE/100g in its aqueous extract, however lower values were found in the aqueous extract of araçá-boi (768.92 ± 1.52 μmol TE/100g) and achachairu

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(712.35 ± 6.61 μmol TE/100g). The Figures 2 and 3 show the correlation coefficients between the three different methods of evaluation of antioxidant activity of the samples, ORAC, FRAP, ABTS vs. total phenolics compounds (PC), in their methanolic and aqueous extracts, respectively. These important correlations show the individual contributions of phenolic compounds to the antioxidant activity.

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FRAP-ABTS, ABTS-PC, FRAP-PC, ORAC-ABTS, ORAC-PC, ORAC-FRAP had positive correlation values, which suggests that phenolic compounds and antioxidant capacity respond proportionally to the methods used. It is thus concluded that the high antioxidant capacities found in the residues are based on its high total phenolic contents,

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besides their concentrations in the fruit residues matrix.

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4. Conclusion

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This study elucidated the total contents of bioactive compounds present in

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tropical fruit residues of achachairu, araçá-boi and bacaba, as well as identified sixteen phenolic compounds, out of which cinnamic acid, p-coumaric acid, epicatechin and

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quercetin were present in all resudues. Among the fruit residues, achachairu contained the principal compounds of ferulic acid (96.01 ± 7.72 μg/g) and epicatechin (96.01 ±

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7.72 μg/g); araçá-boi contained the major compounds as cinnamic acid (44.61 ± 0.76

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μg/g) and gallic acid (30.18 ± 5.46 μg/g) and bacaba contained cathechin (135.15 ± 1.11 μg/g) and epicatechin (123.86 ± 7.15 μg/g). Aqueous extraction of fruit residues for

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bioactive compounds was much more efficient than the methanolic extraction. Therefore, it is concluded that all residues present great potential as sources of bioactive

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compounds, but the residue of bacaba is the most promising in relation to the presence of bioactive compounds and their antioxidant capacity. In addition, all these residues can be used as raw materials for the isolation and purification of these compounds, which are highly essential for the well-being of human system. Acknowledgements

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All authors gratefully acknowledge the financial support received from CNPq, Brazil vide research project ‘Instituto Nacional de Ciência e Tecnologia de Frutos Tropicais’ (Project 465335/2014-4) in developing this work. Authors (Romy Barros, Julianna Andrade, Marina Denadai) acknowledge and thank CAPES (Ministry of Education,

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ACCEPTED MANUSCRIPT Figure captions: Figure 1. SRM chromatograms of aqueous extracts of achachairu (1-3), araçá-boi (4-6) and bacaba (7-8) residues based on quantification transitions of main bioactive compounds. Figure 2. Correlation coefficients between the different methods of measuring antioxidant capacities ORAC, FRAP, ABTS and total phenolic compounds (PC) based on data of

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metanolic extracts of the residues of achachairu, araça-boi and bacaba. Figure 3. Correlation coefficients between the different methods of measuring antioxidant

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capacities ORAC, FRAP, ABTS and total phenolic compounds (PC) based on data of aqueous

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extracts of the residues of achachairu, araça-boi and bacaba.

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Table 1 - MS/MS parameters, retention time and linearity limits for phenolic compounds identified in various fruits residues.

Compounds

Molecular Formula

Molecular mass

Collision

Precursor

(g/mol)

ion

MS/MS2

C9H8O4

1,801,574

181

135a; 163

25;15

(+)-Catechin

C15H14O6

2,902,681

291

139a;123

15

N A 15

Chlorogenic acid

C19H24O3

3,003,921

a

301

C16H18O9

3,543,087

355

Cinnamic acid

C9H8O2

1,481,586

149

p-coumaric acid

C9H8O3

164,158

Daidzein

C15H10O4

2,542,375

(-)-Epicatechin

C15H14O6

Eriodictyol

245 ; 189 163a; 145

D E

C S U

M

4.41 3.1

T P

Correlation coefficient

LOD

2

(r )

(ng/mL)

y=443.1-558.4x

0.9980

0.400

y=1460-2538x

0.9980

0.116

Calibration equation*

I R

(min)

(eV)

Caffeic acid

Artepelin C

Retention time

energy

y=

29.3

0.9950 12979+1680x+0.7351x2

0.017

15;25

3.2

y=6477x-250659

0.9930

0.149

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103a; 131

25;15

11.5

y=1838.2x-10296

0.9979

0.749

165

147a; 91

15;35

6.5

y=1832.9x-20780

0.9968

1.120

255

199a;137

25

11.78

y=164591+8489x-2.96x2

0.9970

0.229

2,902,681

291

139a; 123

25

3.6

y=1461.4x-67066

0.9953

2.390

C15H12O6

2,882,522

289

153a;145

30;25

13.1

y=2707-15399x

0.9904

1.610

Ethyl gallate

C9H10O5

1,981,727

199

153;127a

15

5.6

y=408.47x-67066

0.9995

0.103

Ferulic acid

C10H10O4

194,18

195

177a; 134

15;20

7.67

y=3390.6x-63793

0.9926

0.192

E C

C A

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Gallic acid

C7H6O5

1,701,195

171

109a;125

15

2.17

y=171-2135x

0.9950

16.500

Kaempferol

C15H10O6

2,862,363

287

153a; 121

35;25

17.8

y = 1193-6547x

0.9970

1.800

Naringenin

C15H12O5

2,722,528

273

153a; 119

35

16.4

y=4168.4x-53991

0.9941

0.040

Pinocembrin

C15H12O4

2,562,534

257

153a; 131

15;25

22.96

y=-68899+4477x

0.9980

0.040

C7H6O4

1,541,201

155

137; 65a

15;35

2.85

y=414.6-2985x

0.9970

0.270

Quercetin-3-glucoside

C21H20O12

464,376

465

303a; 85

15;35

7.24

y=2053x-27605

0.9962

0.926

Rutin

C27H30O16

6,105,175

611

303a; 465

35;15

6.5

y=317.49x+7847

0.9991

0.066

Vanillic acid

C8H8O4

1,681,467

169

93a; 65

35;15

4.6

y=3312.6x+17603

0.9943

0.221

Vanillin

C8H8O3

1,521,473

153

93a; 65

6.5

y=8707.5x-102922

0.9952

0.054

Protocatechinic acid

D E

M

15:35

I R

C S U

N A

T P

*Y is the value of the peak area. X is the concentration of the standard compound. a

T P E

Quantification transitions.

C C

A

33

ACCEPTED MANUSCRIPT Table 2 - Physico-chemical composition of fruit residues. Moisture

Soluble Solids

pH

Total titratable acidity

Fruit residue

(%)

(°Brix)

Achachairu

60.24b ± 0.20

17.76a ± 0.11

3.48b ± 0.01

9.99b ± 0.24

Araçá-boi

79.05a ± 0.24

5.06b ± 0.05

3.06c ± 0.01

43.31a ± 0.69

Bacaba

37.67c ± 0.38

1.96c ± 0.05

5.66a ± 0.01

8.53c ± 1.90

PT

(%)

RI

Results expressed as mean ± standard deviation (n=5);

AC

CE

PT E

D

MA

NU

SC

Means in each column followed by different superscript letters were significantly different (p ≤0.05).

34

ACCEPTED MANUSCRIPT Table 3 - Total content of phenolic and flavonoid compounds in the fruit residues extracted with different solvents. Total phenolic contents (mg/100g)

Total Flavonoids (mg/100g)

Fruit residues Methanolic extract

Aqueous extract

Methanolic extrac

Achachairu

33.51bA ± 0.91

20.24cB ± 0.30

2.15bA ± 0.44

1.01cB ± 0.05

Araçá-boi

42.81bA ± 2.23

41.59bA ± 6.26

2.52bA ± 0.25

2.52bA ± 0.30

398.97aB ± 67.98

1537.45aA ± 73.35

Bacaba

PT

Aqueous extract

8.69aB ± 0.33

RI

Results expressed as mean ± standard deviation (n=3);

10.25aA ± 3.48

SC

Means in each column followed by different superscript lowercase letters were significantly different (p ≤0.05) among the fruit residues.

NU

Means in each row of aqueous and methanolic extracs per fruit residue followed by different

AC

CE

PT E

D

MA

superscript capital letters were significantly different (p ≤0.05).

35

ACCEPTED MANUSCRIPT Table 4 - Total chlorophyll and carotenoid contents of fruit residues. Total content* Fruit residues

Chlorophyll B

Carotenoids

(μg/g)

(μg/g)

(μg β-carotene/g)

Achachairu

3.95a ± 0.07

5.77a ± 0.01

9.32c ± 0.03

Araçá-boi

0.67b ± 0.03

1.69b ± 0.02

33.39a ± 0.00

Bacaba

0.78b ± 0.02

1.53b ± 0.01

*

15.47b ± 0.01

RI

All data are the means ± standard deviation (n=5)

PT

Chlorophyll A

AC

CE

PT E

D

MA

NU

SC

Means in each column followed by different superscript letters were significantly different (p ≤0.05).

36

ACCEPTED MANUSCRIPT

Table 5. Quantification of bioactive compounds in aqueous and methanolic extracts of various fruit residues. Residues (μg/g) Compounds Achachairu (A)

Achachairu (M)

Araçá-boi (A)

Araçá-boi (M)

Artepelin C

ND

ND

TR

ND

Caffeic acid

ND

ND

0.46a ± 0.06*

0.28b ± 0.29*

(+)-Catechin

ND

ND

ND

Chlorogenic acid

ND

ND

ND

8.16a ± 1.69*

1.46b ± 0.22*

44.61a ± 0.76*

Cinnamic acid p-coumaric acid Daidzein

a

b

ND

TR

0.81a ± 0.00*

0.60b ± 0.02*

ND

135.15a ± 1.11*

132.27a ± 7.15*

ND

1.53a ± 0.06*

0.79b ± 0.06*

10.42b ± 1.09*

0.19b ± 0.02*

0.27a ± 0.05*

A M

I R

SC

1.55 ± 0.20*

0.95 ± 0.10*

1.57 ± 0.15*

0.47b ± 0.02*

2.38a ± 0.11*

0.56b ± 0.02*

TR

ND

PT

ND

ND

ND

ND

18.05a ± 5.35*

1.67b ± 0.29*

123.86a ± 7.16*

122.65a ± 4.89*

0.31a ± 0.09*

ND

ND

TR

0.28 ± 0.04*

ND

0.37b ± 0.02*

0.43a ± 0.03*

ND

ND

(-)-Epicatechin

96.01a ± 7.72*

Eriodictyol

0.16b ± 0.08*

Ethyl gallate

Bacaba (M)

U N

C A ND

D E

73.40b ± 15.12*

E C

a

T P

Bacaba (A)

Ferulic acid

96.01 ± 7.72*

TR

0.44 ± 0.02*

TR

0.81a ± 0.01*

0.76b ± 0.01*

Gallic acid

ND

ND

30.18a ± 5.47**

8.26b ± 0.17**

ND

ND

Kaempferol

ND

ND

TR

TR

ND

ND

Naringenin

TR

1.09 ± 0.31*

TR

TR

ND

0.28 ± 0.00*

37

ACCEPTED MANUSCRIPT

Pinocembrin

TR

TR

ND

ND

ND

ND

0.55 ± 0.04*

TR

ND

ND

16.07a ± 0.51*

2.78b ± 0.31*

0.34b ± 0.05*

0.52a ± 0.15*

2.47b ± 0.54**

5.79a ± 0.17**

Rutin

ND

ND

ND

ND

Vanillic acid

TR

ND

TR

Vanillin

TR

TR

0.09a ± 0.00*

Protocatechinic acid Quercetin-3-

T P

2.32b ± 0.04**

9.32a ± 0.53**

2.03b ± 0.09**

6.26a ± 0.04**

TR

1.38a ± 0.04*

0.88b ± 0.00*

0.04b ± 0.01*

1.24a ± 0.05*

1.14b ± 0.02*

I R

glucoside

U N

SC

A M

LQ – Limit of quantification; ND – Not detected; TR – Traces; A – Aqueous extract; M – Methanolic extract; Results expressed as mean ± standard deviation (n=3);

D E

*Compounds identified and quantified for the first time in the fruit residues;

T P E

**Compounds identified in fruit pulps in other studies;

Mean values of aqueous and methanolic extracts followed by different superscript letters were significantly different (p ≤0.05).

C C

A

38

ACCEPTED MANUSCRIPT

Table 6 - Antioxidant activity of aqueous and methanolic extracts of various fruit residues.

Fruit residues

ORAC

FRAP

(µmol TE/100g)

(µmol TE/100g)

Aqueous Extract

Methanolic Extract

Aqueous Extract

Achachairu

5485.70bB ± 12.60

14391.12aA ± 9.26

712.35bB ± 6.61

Araçá-boi

3302.47cB ± 3.11

6791.07cA ± 10.20

798.92bB ± 1.52

15285.51aA ± 20.38

12648.13bB ± 8.76

6567.45aB ± 4.25

Bacaba

Results expressed as mean ± standard deviation (n=3):

D E

TE – trolox equivalent; VCE – Vitamin C equivalent.

ABTS

T P

I R

Aqueous Extract

Methanolic Extract

1460.98bA ± 1.45

218.85cB ± 11.25

1427.45cA ± 53.20

1380.74bA ± 7.71

1553.39bA ± 32.67

1548.54bA ± 42.3

16916.37aA ± 10.01

3374.64aB ± 31.88

5588.23aA ± 62.4

Methanolic Extract

C S U

N A

(mg VCE/100g)

M

T P E

Means in each column followed by different superscript lowercase letters were significantly different (p ≤0.05) among the fruit residues. Means in each row of aqueous and methanolic extracs per fruit residue followed by different superscript capitalt letters were significantly different (p ≤0.05).

C C

A

39

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

Fig. 1

40

ACCEPTED MANUSCRIPT FRAP.ABTS

0.983107089

ABTS.PC

0.971081141

FRAP.PC

0.998163108

ORAC.ABTS

0.599413473

ORAC.PC

0.436784598

ORAC.FRAP

PT

0.487812273

RI

Correlation

AC

CE

PT E

D

MA

NU

SC

Fig. 2

41

ACCEPTED MANUSCRIPT FRAP.ABTS

0.912316458

ABTS.PC

0.916131353

FRAP.PC

0.999955677

ORAC.ABTS

0.821548319 0.981202436

ORAC.FRAP

0.982975876

PT

ORAC.PC

RI

Correlation

AC

CE

PT E

D

MA

NU

SC

Fig. 3

42

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Graphical abstract

43

ACCEPTED MANUSCRIPT Highlights

PT

RI

SC NU MA



D



PT E



CE



Agroindustrial residues of Brazilian exotic fruits achachairu, araçá-boi and bacaba analyzed; Bioactive compounds viz. carotenoids, chlorophylls, phenols and flavonoids determined in the residues; Sixteen phytochemical compounds were identified and quantified for the first time in the residues of achachairu, araçá-boi and bacaba; The antioxidant activity of the residues and its correlation with the presence of phenolic compounds was established; Among all fruits studied, bacaba residue was the most promising in bioactive compounds and antioxidant capacity.

AC



44