Antioxidant phytochemicals of Byrsonima ligustrifolia throughout fruit developmental stages

Journal of Functional Foods 18 (2015) 400–410

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Antioxidant phytochemicals of Byrsonima ligustrifolia throughout fruit developmental stages Camila Ramos Pinto Sampaio a,b,*, Fabiane Hamerski c, Rosemary Hoffmann Ribani c a

Nutrition Department, Federal University of Paraná, 632 Lothário Meissner Av., 80210-170 Curitiba, PR, Brazil b Graduate Program in Food Engineer, Chemical Engineering Department, Federal University of Paraná, 100 Cel. Francisco H. Santos Av., 81531-980 Curitiba, PR, Brazil c Chemical Engineering Department, Federal University of Paraná, 100 Cel. Francisco H. Santos Av., 81531-980 Curitiba, PR, Brazil

A R T I C L E

I N F O

A B S T R A C T

Article history:

Byrsonima ligustrifolia is a small and coloured underutilized fruit with no scientific litera-

Received 7 June 2015

ture about its antioxidant capacity and content of individual phytochemicals. The present

Received in revised form 19 July

work evaluated the antioxidant capacity of B. ligustrifolia fruit throughout maturation process

2015

by Folin–Ciocalteu, ABTS, and DPPH methods, and quantified by HPLC-DAD, six polyphe-

Accepted 12 August 2015

nolic compounds: cyanidin-3-glucoside, pelargonidin-3-glucoside, gallic acid, p-hydroxybenzoic

Available online

acid, p-coumaric acid, and catechin. Besides that, total flavonoids (TF), total monomeric anthocyanins (TMA), and ascorbic acid (AA) were analysed. AA did not show correlation with

Keywords:

antioxidant activity. TF and phenolic acids decreased with maturation process and had strong

Byrsonima ligustrifolia

positive correlation with antioxidant capacity. TMA, cyanidin-3-glucoside and gallic acid were

Maturation

present in the fruit in levels that are difficult to find in other berries. These results expose

Antioxidant capacity

a potential approach for improving human health through consumption of B. ligustrifolia fruit.

Phenolic compounds

© 2015 Published by Elsevier Ltd.

Anthocyanins HPLC-DAD

1.

Introduction

In recent years, the increasing prevalence of chronic diseases, such as cancer, cardiovascular, and chronic inflammatory diseases, has resulted in increased interest in the consumption

of foods with high nutraceutical value. Fruits and vegetables are excellent sources of bioactive compounds, with their consumption associated with disease prevention. This protective effect has been attributed partially to the dietary antioxidants including ascorbic acid (vitamin C), tocopherols (vitamin E), carotenoids, and phenolic compounds.

* Corresponding author. Nutrition Department, Federal University of Paraná, 632 Lothário Meissner Av., 80210-170 Curitiba, PR, Brazil. Tel.: +55 41 3360 4003; fax: +55 41 33604133. E-mail address: [email protected] (C.R.P. Sampaio). Chemical compounds: Cyanidin-3-O-glucoside (PubChem CID: 441667); Gallic acid (PubChem CID: 370); p-Hydroxybenzoic acid (PubChem CID: 135); p-Coumaric acid (PubChem CID: 637542); Catechin (PubChem CID: 9064); Ascorbic acid (PubChem CID: 54670067). http://dx.doi.org/10.1016/j.jff.2015.08.004 1756-4646/© 2015 Published by Elsevier Ltd.

Journal of Functional Foods 18 (2015) 400–410

Small and coloured berry fruits are good dietary sources of antioxidant compounds. Phenolic compounds are the major group of phytochemicals in berry fruits including flavonoids, stilbenes, tannins, and phenolic acids. They are secondary metabolites from plants that are produced for protection against UV light, insects, viruses, and bacteria (Heleno, Martins, Queiroz, & Ferreira, 2015). The antioxidant activity of a compound depends mainly on its high activity against free radicals and the stability of the intermediate species formed (Ferreyra, Viña, Mugridge, & Chaves, 2007). The antioxidant capacity of fruits is directly related to the phytochemical profile that depends on factors such as stage of maturation, geographic location, and climatic conditions, amongst others. Fruits are usually eaten in the optimum ripened stage determined by sensory attributes. However, the immature fruits are usually rich in bioactive compounds. Therefore, if the aim is to use the fruit as a functional food or as an ingredient for the food industry, the knowledge of changes in fruit composition throughout the developmental process is essential to select the most suitable maturity stage (Maieves et al., 2015). Murici-vermelho (Portuguese) or red-Murici (Byrsonima ligustrifolia A. Juss.) is an underutilized berry fruit that, in Brazil, is geographically distributed in the southeastern region, and, in the south region, it can be found in the states of Santa Catarina and Paraná. It is an astringent fleshy fruit that can be consumed fresh or be pulped for use in juices and sweets with greatly appreciated colour and flavour. The fruit’s colour changes during the maturation process from green to red, vinaceous, and then purple when over-ripe (Fig. S1). There is no information regarding antioxidant capacity and content of individual phytochemical of B. ligustrifolia fruit, and there is no report about the development of these compounds during the maturation process and ripening of this fruit. Therefore, the aim of this work was to evaluate the antioxidant capacity of B. ligustrifolia fruit extracts, exploring the contribution of polyphenols and vitamin C to the antioxidant activity, as well as to study the polyphenolic profile of this fruit.

2.

Materials and methods

2.1.

Plant materials

Samples of B. ligustrifolia A. Juss. (popularly known as Muricivermelho) were obtained from Guaraqueçaba city (Paraná, Brazil), geographic coordinates 25°17′50″S 48°19′8″W, in March of 2014. Fruits with different ripening stages were collected at the same time from the same trees. The species’ official identification was conducted by Municipal Botanical Museum (MBM) of Curitiba city (Paraná, Brazil) and one specimen (exsiccate) was deposited in the Herbarium of this Museum, registered under number 388371. The fruits were selected, washed, sorted into five ripening stages (RS) according to external colour (Fig. S1), weighed, whole freeze-dried (L101-Liotop, São Carlos, São Paulo, Brazil) to ensure the retention of antioxidant compounds, stored at −20 °C and analysed after 50 days.

2.2.

401

Extraction procedure and sample preparation

Freeze-dried pulp was ground until a fine and visually homogenous powder (50 mesh) was obtained. The extraction procedure was conducted using pulp powder (0.5 g) with 15 mL of methanol and acetone (80:20, v/v), acidified with 2% of acetic acid and added butylated hydroxyanisole (BHA) (2 g/L). The solvent combination was determined in a previous study, not yet published, that aimed to evaluate the best conditions amongst three solvents (water, methanol and acetone) which allowed the maximum extraction of phenolic compounds. Therefore, the mixture was sonicated in an ultrasonic bath (UNIQUE model USC-1880A and 37 kHz) (Indaiatuba, São Paulo, Brazil) at room temperature for 40 min. After that, the extract was centrifuged (3256 g for 15 min), the supernatant was collected, and the sediment was subjected to an additional extraction using the same procedure. Both supernatants were mixed, the solvent was totally evaporated in a rotary evaporator (350 mmHg, 30 °C) (IKA, Campinas, São Paulo, Brazil), the residue was dissolved into 10 mL of HPLC grade methanol and divided into two equal parts. The first 5 mL (unhydrolysed extract containing soluble phenolic acids) was membrane-filtered (0.45 µm), and stored at −20 °C until analysis. The remaining 5 mL of the sample extract were used for a sequential hydrolysis experiment with base followed by acid as described below. The complete procedure was carried out with only the extraction solutions, as a blank, since a compound with antioxidant activity (BHA) was added into solvents.

2.3.

Phenolic acids’ hydrolysis

Phenolic acids were hydrolysed according to the method described by Mattila, Hellström, and Törrönen (2006), with slight modifications. Briefly, 6.67 mL of distilled water containing 1% ascorbic acid, 0.415% ethylenediaminetetraacetic acid tetrasodium salt dihydrate (Na4EDTA), and 2.78 mL of 10 M NaOH were added into the remaining 5 mL of the sample extract, sealed under N2 gas, to avoid compounds oxidation, and stirred overnight (about 16 h) at 20 °C using a magnetic stirrer. Next, the solution was adjusted to pH 2 with HCl 37% (w/w), and the liberated phenolic acid aglycones were extracted with a mixture of cold diethyl ether and ethyl acetate (1:1; 3 × 8.33 mL). The organic layers were then combined. An acid hydrolysis was performed by adding 2.5 mL of concentrated HCl into the test tube and incubating in a water bath (IKA, Campinas, São Paulo, Brazil) at 85 °C for 30 min. The sample was cooled, and further sample handling was performed in the same manner as after alkaline hydrolysis. The organic layers from the alkaline and acid hydrolyses were combined, evaporated to dryness, dissolved into 5 mL of HPLC grade methanol, filtered with a syringe filter (0.45 µm), and stored at −20 °C until analysis, and was called hydrolysed extract (total phenolic acids). Under this condition, the extracts were at a concentration of 50 mg of freeze-dried pulp per millilitre of methanol. For some analysis, the extracts were diluted 100-fold into HPLC grade methanol (0.5 mg/mL). The complete procedure was carried out with only the extraction solutions, as a blank, since compounds with antioxidant

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Journal of Functional Foods 18 (2015) 400–410

activity (BHA, EDTA, and ascorbic acid) were added into the solvents.

2.4.

Total flavonoids (TF) content

Flavonoid content was determined according to methods described by Gonzalez-Aguilar, Villegas-Ochoa, Martinez-Tellez, Gardea, and Ayala-Zavala (2007). Five hundred microlitres from each extract 100-fold diluted (0.5 mg/mL) were mixed and equilibrated with 2 mL of deionized water and 150 µL 5% NaNO2 for 5 min. After equilibrium, 150 µL of 10% AlCl3 (methanolic solution) were added; the mixture was allowed to stand for 1 min and then 1 mL of 1 M NaOH was added. The last volume was made to 5 mL with H2O, stirred, and readings were taken in a double beam UV–Vis spectrophotometer model LI-2800 (Lasany, Panchkula, Haryana, India) at 415 nm. Concentration of total flavonoids of fruits was calculated using a standard curve of quercetin (0–60 ppm) and expressed as milligram quercetin equivalents (QEs) per 100 g of fruit pulp on a dry weight (DW) basis.

2.5.

Total monomeric anthocyanin (TMA) content

TMA was determined by using a spectrophotometric pH differential method (Lee, Durst, & Wrolstad, 2005). Extracts (50 mg/mL) were either 10 or 20-fold diluted in buffers KCl (potassium chloride, 0.025 M, pH 1.0) and CH3COONa (sodium acetate, 0.4 M, pH 4.5), within the range of 20–3000 mg/L as cyanidin-3-glucoside equivalents. Absorbance was measured at 520 and 700 nm and TMA was calculated as follows: Monomeric anthocyanins = (A × MW × DF × 10 3 )/(ε × 1); where, A = (A 520 − A 700 ) pH 1.0 − (A 520 − A 700 ) pH 4.5 ; MW (molecular weight) = 449.2 g mol−1; DF = dilution factor; 103 = factor for conversion from g to mg; ε = molar extinction coefficient: 26,900 M−1 cm−1; and 1 = optical path of cuvette (cm). The results were expressed as milligrams of cyanidin-3-glucoside equivalents (C3G) per 100 g of fruit pulp on a dry weight (DW) basis.

2.6.

Total phenolic compounds (TPC) content

TPC in both unhydrolysed and hydrolysed extracts was measured using a colorimetric Folin–Ciocalteu method according to Singleton and Rossi (1965). Briefly, 500 µL of the 100-fold diluted extract (0.5 mg/mL) were added to 2.5 mL of freshly diluted 10-fold Folin–Ciocalteu reagent. After 5 min of equilibrium, 2.0 mL of sodium carbonate solution (7.5%) were used to neutralize the mixture. The absorbance of the mixture was measured at 740 nm after 120 min of reaction in the dark and at room temperature. Gallic acid was used as a standard and the results were expressed as grams of gallic acid equivalents (GAE) per 100 g of fruit pulp on a dry weight (DW) basis.

2.7.

ABTS•+ assay

The ABTS•+ assay was based on a method developed by Miller et al. (1993) with modifications. ABTS radical cations were produced by reacting 7 mM ABTS stock solution with 140 mM potassium persulphate and allowing the mixture to stand in the dark at room temperature for 16 h before use. The ABTS•+ solution was diluted with ethanol to an absorbance of 0.70 ± 0.02

at 734 nm. After the addition of 30 µL of sample extracts, each one diluted in three different concentrations (500, 250, and 125 PPM), or trolox standard to 3 mL of diluted ABTS•+ solution, absorbances were recorded at 6 min after mixing. Ethanolic solutions of trolox ranging from 100 to 2000 µM were used for calibration and the results were expressed as µM trolox per 100 g of fruit pulp on a dry weight (DW) basis.

2.8.

DPPH• (free radical-scavenging) assay

The antioxidant capacity was determined by a modified DPPH• method (Brand-Williams, Cuvelier, & Berset, 1995) which is based on the quantification of free radical-scavenging with modifications. A methanol solution containing 0.06 mM DPPH• was prepared. After adjusting the blank with methanol, an aliquot of 100 µL of fruit extracts diluted in three different concentrations (500, 250, and 125 mg/L) was added to 3.9 mL of this solution. The decrease in absorbance at 515 nm was measured at 1 min intervals for the first 10 min, and then at 5 min intervals until stabilization (120 min). The antioxidant capacity was expressed as the concentration of antioxidant required to reduce the original amount of free radicals by 50% (EC50) and the values were expressed as gram fruit pulp per gram DPPH•, on a dry weight (DW) basis.

2.9.

Determination of ascorbic acid content

Ascorbic acid (AA) extraction was carried out with a solution of oxalic acid (4%). Fifty millilitres of this solution were added to 0.5 g of freeze-dried pulp. The mixture was stirred for 15 min, filtered, the solution was taken to an exact volume (volumetric flash of 50 mL) with the extraction solvent, and filtered through a 0.45 µm syringe filter before injection to the highperformance liquid chromatography (HPLC). A reversed phase HPLC method for the determination of AA content was used, with minor modifications, according to Mazurek and Jamroz (2015). Analyses were performed with the use of Agilent 1200 Series HPLC system (Wilmington, DE, USA) equipped with diode arrangement detector (DAD), controlled by Software EZChrom Elite (Agilent), with automatic liquid sampler (ALS), and quaternary pump. Separations were made using a Zorbax Eclipse XDB-C18 column (4.6 × 150 mm, 3 µm) (Agilent) connected with a guard-column Zorbax Eclipse XDBC18 (4.6 × 12.5 mm). The injection volume was 20 µL. The mobile phase was an aqueous solution of orthophosphoric acid at pH 2.5 pumped at a flow of 0.5 mL/min. Chromatograms were recorded at 244 nm. The concentration of AA was determined by the external calibration using ascorbic acid as standard reference. AA identification was performed on the basis of retention time and UV absorption spectrum of the standard ascorbic acid sample.

2.10. Liquid chromatograph analysis of phenolic compounds The same equipment and column, previously described for determination of ascorbic acid content, were used in the analysis of phenolic compounds by liquid chromatography.

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The separation of anthocyanins, phenolic acids and anthoxanthins was done under different conditions. Thus, there were three systems with different mobile phase compositions, gradient programme, total run time, injection volume of samples, wavelengths monitored, and flow rate.

2.10.1. System I (anthocyanins) The mobile phase consisted of water/formic acid/acetonitrile (95:2:3, v/v/v; eluent A) and water/formic acid/acetonitrile (48:2:50, v/v/v; eluent B) using a gradient programme as follows: from 10 to 25% B (10 min), from 25 to 31% B (5 min), from 31 to 40% B (5 min), from 40 to 46% B (5 min), from 46 to 90% B (1 min), from 90 to 10% B (2 min). Total run time was 30 min. The injection volume for all samples was 25 µL. Monitoring was performed at 520 nm at a flow rate of 0.8 mL/min.

2.10.2. System II (phenolic acids) The mobile phase consisted of 2% (v/v) acetic acid in water (eluent A) and 0.5% acetic acid in water and acetonitrile (50:50, v/v; eluent B) using a gradient programme as follows: from 10 to 15% B (10 min), 15% B isocratic (3 min), from 15 to 55% B (12 min), from 55 to 100% B (5 min), 100% B isocratic (4 min), from 100 to 10% B (3 min). Total run time was 40 min. The injection volume for all samples was 15 µL. Simultaneous monitoring was performed at 280 nm (hydroxybenzoic acids) and at 320 nm (hydroxycinnamic acids) at a flow rate of 1.0 mL/min.

2.10.3. System III (anthoxanthins) The mobile phase consisted of the same eluents as described for system II using a gradient programme as follows: from 10 to 24% B (20 min), from 24 to 30% B (20 min), from 30 to 55% B (20 min), from 55 to 100% B (15 min), 100% B isocratic (8 min), from 100 to 10% B (2 min). Total run time was 86 min. The injection volume for all samples was 15 µL. Simultaneous monitoring was performed at 280 nm (flavanols) and at 370 nm (flavonols) at a flow rate of 1.0 mL/min. The separated compounds were confirmed through the following combined information: elution order on the reversed phase column, co-chromatography with standards, and UV– Vis spectra (λmax), and calibration curves for each standard were prepared for quantification. The following individual phenolic standards were purchased from Sigma-Aldrich (St. Louis, MO, USA): cyanidin-3-glucoside, gallic acid, caffeic acid, p-coumaric acid, protocatechuic acid, p-hydroxybenzoic acid, quercetin, and catechin.

3.

Results and discussion

Ascorbic acid (AA), known as vitamin C, is one of the most important water-soluble vitamins, naturally present in foods, especially in fruits and vegetables (Maieves et al., 2015; Valente, Albuquerque, Sanches-Silva, & Costa, 2011). AA has important antioxidant and metabolic functions, making its incorporation into the human diet essential, since humans cannot synthesize AA (Cruz-Rus, Amaya, Sánchez-Sevilla, Botella, & Valpuesta, 2011). AA can act as an antioxidant in a wide range of enzymatic and non-enzymatic reactions in humans, and it is also active in the non-enzymatic regeneration of other antioxidant molecules, such as α-tocopherol (vitamin E) (Cocetta et al., 2012). The AA concentrations measured from all five stages of B. ligustrifolia ripening are shown in Table 1. RS1 showed the lowest AA content, and, in relation to fresh weight, during the ripening process, after RS2, the AA level remained relatively stable, without significant difference between ripening stages (p < 0.05). However, RS3 presented the highest value of AA content when the analysis was based on dry weight, being statistically different than RS1 and RS5. It suggests that the biosynthesis of this compound in the fruit increased with its development, since size and weight do not change after RS3 (the developmental stage when the fruit is mature). The AA level in fruits is mainly determined by genotype, but it is also influenced by maturation (Cocetta et al., 2012). Our results agree with the data obtained from grapes and strawberry, in which the AA levels were shown to increase during fruit development (Cruz-Rus et al., 2011; Cruz-Rus, Botella, & Gomez-Jimenez, 2010). Our result for RS5 (30.01 mg/100 g FW) can be compared to AA content of sweet cherry (Serrano, Guillén, Martínez-Romero, Castillho, & Valero, 2005), some raspberry cultivars (Pantelidis, Vasilakakis, Manganaris, & Diamantidis, 2007), apple (Franke, Custer, Arakaki, & Murphy, 2004), cape-gooseberry (Valente et al., 2011), grapefruit (Valente et al., 2011), and purple passion fruit (Valente et al., 2011; Vasco, Ruales, & Kamal-Eldin, 2008; Zeraik, Pereira, Zuin, & Yariwake, 2010). Phenolic compounds are the major group of phytochemicals in berry fruits that include the subgroups: flavonoids (anthocyanins, flavonols, and flavanols), stilbenes, tannins, and phenolic acids (Seeram, 2006).

Table 1 – Ascorbic acid content in Byrsonima ligustrifolia freeze-dried pulp at different ripening stages. Ripening stage

2.11.

Statistical analysis

All extraction assays were carried out in triplicate, and a duplicate of each extract was analysed. Results were expressed as means ± standard deviation (SD). One-way analysis of variance (ANOVA) for comparison of means and significant differences according to Tukey test at the 5% level was performed in Statistica 8.0 (StatSoft, Tulsa, OK, USA). Correlations amongst data obtained were calculated using Pearson’s correlation coefficient.

RS1 RS2 RS3 RS4 RS5

Ascorbic acid (mg/100 g) FW

DW

22.54 ± 1.87B 26.80 ± 0.77A 28.90 ± 0.57A 28.29 ± 1.64A 30.01 ± 0.42A

194.36 ± 16.10C 258.68 ± 7.40A 267.36 ± 5.26A 242.15 ± 14.05AB 228.18 ± 3.23B

Results are mean values (n = 3) plus standard deviations; FW = fresh weight; DW = dry weight. Different letters indicate significant differences (p < 0.05) according to the Tukey’s mean test.

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Journal of Functional Foods 18 (2015) 400–410

Although most of the yellow colour of foods is attributed to the presence of carotenoids, in some foods this colour is due to the presence of flavonoids, not the anthocyanin-type (Fennema, 2010). Flavonoids are the most common and widely distributed group of plant phenolic compounds (Bakar, Mohamed, Rahmat, & Fry, 2009). They have gained a great deal of attention due to their health-related properties, which are based on their antioxidant activity. Total flavonoids content can be determined in the sample extracts by reaction with sodium nitrite, followed by the development of coloured flavonoid–aluminium complex formation using aluminium chloride which can be monitored spectrophotometrically at 415 nm (Bakar et al., 2009). In B. ligustrifolia, total flavonoids showed a significant decrease during the maturation process (Table 2). To each ripening stages, there was no statistically significant difference between TF of hydrolysed and non-hydrolysed extracts. This denotes that the hydrolysis did not promote any significant loss of these compounds. The flavonoids content of hydrolysed fruit extract at RS5 (806 mg of quercetin/100 g DW) is similar to those reposted for red cashew (834 mg of quercetin/100 g DW), purple star apple (844 mg of quercetin/100 g DW) (Moo-Huchin et al., 2015), and red grape (881 mg of quercetin/100 g DW) (Molina-Quijada, Medina-Juárez, González-Aguilar, Robles-Sánchez, & Gámez-Meza, 2010). Anthocyanins are compounds responsible for much of the red, blue, and purple colouring of fruits, which have been extensively studied due to their wide range of bioactivities including antioxidant, anticancer, and anti-inflammatory properties (Seeram, 2008). They are especially abundant in berries that have recently generated considerable interest as a rich source of phenolic antioxidants (Reynertson, Yang, Jiang, Basile, & Kennelly, 2008). TMA content of B. ligustrifolia pulp extracts in five ripening stages is presented in Table 2. Statistical analysis revealed significant differences (p < 0.05) in the anthocyanin content between all five developmental stages. The results display a significant increase of TMA during the ripening. Fruit ripening is associated with important biochemical changes that modify colour, texture, taste and other quality

traits. The change in fruit colour is the most noticeable evidence of early maturation. The reddish colour of ripe fruits is a result of anthocyanin accumulation and a decrease in chlorophyll content (Kerbauy, 2012). Studying acerola fruits, Lima, Mélo, Maciel, and Lima (2003) found total anthocyanins content ranging between 3.79 and 59.74 mg/100 g FW, depending on the varieties which show different colour (from yellow to red), and Mezadri, Villaño, Fernández-Pachón, García-Parrilla, and Troncoso (2008), Rufino et al. (2010), and Ribeiro da Silva et al. (2014) presented values of total anthocyanins of 4.98 mg/100 g FW, 18.9 mg/100 g FW, and 144.27 mg/100 g DW, respectively. Despite being from the same family, B. ligustrifolia at ripe stage (RS5) presented values for anthocyanins content (1730 mg/100 g DW or 227.5 mg/100 g FW, considering moisture of 86.85%) much higher than acerola. So, our result for TMA at developmental RS5 can be compared to black currant (225 mg/100 g FW) (Contessa, Mellano, Beccaro, Giusiano, & Botta, 2013), Cornelian cherry (223 mg/100 g FW) (Pantelidis et al., 2007), highbush blueberry (222 mg/100 g FW) (Contessa et al., 2013), and juçara (192 mg/100 g FW) (Rufino et al., 2010). Fig. 1 shows the HPLC chromatogram (System I, at 520 nm) for the soluble extract of fruit pulp at ripening stage five (RS5 = purple-black peel). The peaks for the different anthocyanins were numbered according to their retention time (Rt). The major anthocyanin was cyanidin-3-O-glucoside (peak 1, Fig. 1), which was identified in all five developmental stages with a significant increase during the maturity stages that ranged from 16.98 to 1678.92 mg/100 g of DW, raising nearly 100 times its concentration (Table 3). This compound was confirmed by co-chromatography with cyanidin-3-O-glucoside standard and analysing the UV–vis spectra that was characterized by two major absorption bands: λmax 279 and 515 nm (Fig. 1). According to Seeram (2006) cyanidin is the most ubiquitous between the six most common anthocyanidins (cyanidin, delphinidin, pelargonidin, malvidin, petunidin, and peonidin), and according to Wu and Prior (2005), cyanidin is a major anthocyanidin in berries like black raspberry, blackberry, blueberry, Concord grape, cranberry, marionberry, raspberry, red grape, and sweet cherry, and glucose was the dominant

Table 2 – Total phenolic compounds, total flavonoids, and total monomeric anthocyanin contents of Byrsonima ligustrifolia fruit pulp extracts in five ripening stages. Ripening stages RS1 RS2 RS3 RS4 RS5

TMAa (mg/100 g)

TFb (mg/100 g)

TPCc (g/100 g)

Unhydrolysed extract

Unhydrolysed extract

Hydrolysed extract

Unhydrolysed extract

Hydrolysed extract

21.6 ± 1.2E 113.1 ± 1.9D 375.4 ± 5.5C 720.7 ± 18.1B 1729.8 ± 30.2A

2126.5 ± 91.6Aa 1727.1 ± 75.2Ba 1687.6 ± 39.3Ba 1166.3 ± 94.5Ca 1002.8 ± 76.2Ca

1994.8 ± 135.5Aa 1510.4 ± 110.8Ba 1439.4 ± 48.5Ba 846.0 ± 55.4Ca 806.2 ± 49.2Ca

19.14 ± 0.91Aa 16.27 ± 0.40Ba 14.98 ± 0.28Ba 11.18 ± 0.29Ca 10.28 ± 0.39Ca

19.64 ± 0.79Aa 16.55 ± 0.75Ba 15.17 ± 0.17Ba 10.61 ± 0.13Ca 9.98 ± 0.49Ca

Results are mean values (n = 3) plus standard deviations. a TMA = total monomeric anthocyanins content expressed as mg of cyanidin-3-glucoside equivalent (C3G)/100 g of fruit pulp on a dry weight (DW) basis. b TF = total flavonoids content expressed as mg of quercetin equivalents (QEs)/100 g of fruit pulp on a dry weight (DW) basis. c TPC = total phenolic compounds content expressed as g of gallic acid equivalent (GAE)/100 g of fruit pulp on a dry weight (DW) basis. Same upper case letters in the column mean no significant differences at 95% of confidence (p < 0.05) by Tukey test between ripening stages. Same lower case letters in the line mean no significant differences at 95% of confidence (p < 0.05) by Tukey test between unhydrolysed and hydrolysed extracts at a single test.

Journal of Functional Foods 18 (2015) 400–410

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Fig. 1 – HPLC separation and UV–vis spectra of anthocyanins in non-hydrolysed extract of B. ligustrifolia at ripening stage five (RS5), monitored at 520 nm. Peaks: 1, cyanidin-3-glucoside; 2, pelargonidin-3-glucoside.

Table 3 – Anthocyanins in Byrsonima ligustrifolia fruit at five ripening stages.

RT (min) RS1 RS2 RS3 RS4 RS5

Cyanidin-3-Oglucoside

Pelargonidin-3-Oglucoside

9.27 ± 0.20 16.98 ± 0.90E 106.45 ± 6.02D 346.80 ± 14.69C 699.67 ± 10.70B 1678.92 ± 13.79A

11.04 ± 0.05 ND ND ND 9.57 ± 0.19B 40.43 ± 0.10A

Results are mean values (n = 3) plus standard deviations, mg/100 g of fruit pulp on a dry weight (DW) basis. ND = not detected. Same upper case letters in the column mean no significant differences at 95% of confidence (p < 0.05) by Tukey test.

monosaccharide linked to aglycons (anthocyanidins) to form anthocyanins. A large number of studies have quantified individual anthocyanins in berries, but it is difficult to find a fruit that presents value so high of one single anthocyanin like B. ligustrifolia. For example, Veberic, Slatnar, Bizjak, Stampar, and Mikulic-Petkovsek (2015) determined the anthocyanin composition of 24 berry species and only one species (Eastern shadbush) presented the amount of cyanidin-3-glucoside (219 mg/100 g FW) similar to our fruit at developmental RS5, and another (cultivated elderberry) whose value is greater than B. ligustrifolia. Chokeberry presented 248 mg/100 g FW of cyanidin-3-galactoside, and the other 21 berries presented values of individual anthocyanins much lower than the value of cyanidin-3-glucoside in our study (221 mg/100 g FW).

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Peak 2 was detected only in stages four and five. This peak was identified as pelargonidin-3-O-glucoside, since the UV– vis spectra was typical of this compound, showing two major absorption bands: λmax 277 and 499 nm, as was indicated by Santiago et al. (2014). According to Veberic et al. (2015) raspberry presented values of pelargonidin-3-glucoside (3 mg/100 g FW) similar to this fruit at RS5 (5.3 mg/100 g FW). With this simple profile, B. ligustrifolia can be a good source of cyanidin-3-glucoside that is considered one of the most important anthocyanins because of its antioxidant capacity (Chen et al., 2014). We know that a diet rich in natural antioxidants improves health and prevents diseases because antioxidants may terminate the attack of free radicals (Li, Hydamaka, Lowry, & Beta, 2009). In this context, B. ligustrifolia has potential as a source of novel natural antioxidants for disease prevention and health promotion, and also as natural pigment to the food industry. Phenolic acids have been largely studied mainly due to their bioactivities, such as antioxidant, antitumour and antimicrobial properties (Heleno et al., 2015). They are present in plant foods mostly in bound form, as esters or glycosides conjugated with other natural compounds such as flavonoids, alcohols, hydroxyfatty acids, sterols, and glucosides (Mattila et al., 2006). Initially, the soluble phenolic acids (free and bound soluble forms) were extracted with a solution of methanol and acetone (80:20, v/v) acidified with acetic acid (2%). Next, the samples were sequentially hydrolysed by base and acid to determine the total phenolic acid content (sum of bound soluble and insoluble forms plus free phenolic acids as aglycones). In this paper, three phenolic acids were identified and quantified in freeze-dried pulp from B. ligustrifolia fruit. From these, two were hydroxybenzoic acids (gallic and p-hydroxybenzoic) and one was hydroxycinnamic acid (p-coumaric) (Table 4). Fig. 2 presents the chromatogram of phenolic acid standards (A), the chromatogram of phenolic acids in non-hydrolysed extract (B), and hydrolysed extract (C) of B. ligustrifolia at ripening stage one (RS1). Even when concentrations decreased with maturation, gallic acid was the most abundant phenolic acid in the five ripening stages, followed by p-coumaric and p-hydroxybenzoic acids. This gradual decrease can be connected to an increased polyphenol oxidase activity, transformations (polymerization, oxidation and conjugation reactions) of phenolic acids, and the reduction of primary metabolism in the over-ripe fruit, resulting in a lack of substrates necessary for the biosynthesis of phenolic compounds (Gruz, Ayaz, Torun, & Strnad, 2011).

Studying the phenolic acids profiles of six berry fruits, Zadernowski, Naczk, and Nesterowicz (2005) found values of gallic acid, p-hydroxybenzoic acid and p-coumaric acid much lower than our result for B. ligustrifolia at RS5. Besides, the amount of gallic acid in grape skins (from 56 to 146 mg/100 g DW) and goji berry (15.31 mg/100 g FW) is also much lower than ours (Donno, Beccaro, Mellano, Cerutti, & Bounous, 2014; Molina-Quijada et al., 2010). Gallic acid has several reported bioactivities, such as antineoplastic, bacteriostatic, antimelanogenic and antioxidant properties. p-Coumaric acid also revealed antioxidant activities against free radicals, some antitumour activities, and antimicrobial activity against several pathogenic bacteria and fungi. p-Hydroxybenzoic acid has been reported to have antioxidant activities against free radicals, antimicrobial activities against pathogenic bacteria and fungi, amongst other bioactivities, such as oestrogenic and antimutagenic properties (Heleno et al., 2015). According to Li et al. (2009), strawberry presents 12.4 mg of p-hydroxybenzoic acid per 100 g FW that is similar to that found in our study (13.26 mg/100 g FW). Our results for catechin is displayed at Table 4 and the HPLC chromatogram of anthoxanthins in non-hydrolysed extract of B. ligustrifolia at ripening stage one (RS1) is shown in Fig. S2. Tsanova-Savova, Ribarova, and Gerova (2005) analysed the presence of catechin in 15 fruits, amongst these were eight berries, and all results were much lower than the catechin value of our fruit. There was a significant decrease of TPC (Table 2) during the maturation process, which complies with other papers that studied changes in the total phenolic content throughout the developmental stages of Brazilian cherry (Celli, Pereira-Netto, & Beta, 2011), red raspberry (Wang, Chen, & Wang, 2009), blackberry, strawberry (Wang & Lin, 2000), and acerola (Lima et al., 2005), concluding that ripening reduces TPC in these fruits. In this paper, B. ligustrifolia fruit pulp at ripe stage (RS5) showed a high value of TPC (10283 mg GAE/100 g DW) compared to acerola (Paz et al., 2015; Rufino et al., 2010) and camucamu (Reynertson et al., 2008; Rufino et al., 2010). The TPC determination by Folin–Ciocalteu reagent is being increasingly considered as a reducing power assay due to the basis of this method, which is based on the phosphomolybdic– phosphotungstic acid reagent reducing to a blue-coloured complex in an alkaline solution. This reaction can be interfered by non-phenolic antioxidants and reducing substances such as ascorbic acid, glucose, fructose, sulphites, amino acids (tyrosine, tryptophan), and proteins containing these amino

Table 4 – Gallic acid, p-hydroxybenzoic acid, p-coumaric acid, and catechin in Byrsonima ligustrifolia fruit at five ripening stages.

RT (min) RS1 RS2 RS3 RS4 RS5

Gallic acid

p-Hydroxybenzoic acid

p-Coumaric acid

Catechin

2.86 ± 0.02 3060.32 ± 58.77A 2722.11 ± 45.72B 2589.02 ± 30.12B 1630.70 ± 15.99C 1418.53 ± 16.99D

9.54 ± 0.05 282.41 ± 3.59A 205.92 ± 6.80B 200.75 ± 4.63B 127.27 ± 2.04C 100.86 ± 2.72D

20.46 ± 0.10 526.52 ± 16.74A 450.19 ± 16.31BC 421.91 ± 16.78CD 385.98 ± 9.56D 289.82 ± 10.40E

10.53 ± 0.50 293.81 ± 14.79A 233.78 ± 7.39B 232.66 ± 5.08B 229.18 ± 9.56B 163.13 ± 4.15C

Results are mean values (n = 3) plus standard deviations, mg/100 g of fruit pulp on a dry weight (DW) basis. Same upper case letters in the column mean no significant differences at 95% of confidence (p < 0.05) by Tukey test.

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5

A

700

1 600

mAU

500

400

300

4

2 3

200

100

0 0

2

4

6

8

10

12

14

16

18

20

22

24

26

Minutes 600

B

5 500

mAU

400

300

200

100

0 0

2

4

6

8

10

12

14

16

18

20

22

24

26

Minutes 2750

C

1

2500

2250

2000

1750

mAU

1500

1250

1000

750

500

3

250

0 0

2

4

6

8

10

12

14

16

18

20

22

24

26

Minutes

Fig. 2 – (A) HPLC chromatogram of phenolic acid standards. Peaks: 1, gallic acid; 2, protocatechuic acid; 3, p-hydroxybenzoic acid; 4, caffeic acid; 5, p-coumaric acid; (B) HPLC chromatogram of phenolic acids in nonhydrolysed extract of B. ligustrifolia at ripening stage one (RS1), monitored at 280 nm; (C) HPLC chromatogram of phenolic acids in hydrolysed extract of B. ligustrifolia at ripening stage one (RS1), monitored at 280 nm.

407

acids (Gruz et al., 2011; Medina, 2011; Singleton, Orthofer, & Lamuela-Raventos, 1999). Since the methodologies for the determination of antioxidant capacity can be subjected to interferences, nowadays the use of two or more techniques is proposed, as no single assay would reflect exactly the ‘total antioxidant capacity’ of a sample (Huang, Ou, & Prior, 2005). In this context, we determined the antioxidant capacity by employing also ABTS and DPPH methods, as shown in Table 5. In all methods studied, the higher antioxidant capacity was observed in the developmental RS1, and the lower value (measured by all antioxidant assays performed) corresponded to RS1. Therefore, the amount of pulp fruit required to reduce the DPPH radical by 50% (EC50) was progressively higher in each maturity stage. When evaluated by the ABTS method, our fruit ranged from 2543 to 1296 µM trolox equivalents/g DW (from RS1 to RS5, respectively). This result shows a significant decrease in the antioxidant capacity throughout ripening stages. Rufino et al. (2010) determined the antioxidant activity of 18 non-traditional Brazilian tropical fruits by ABTS assay. The results showed that Camu-camu was the fruit with the highest antioxidant capacity (1237 µmol TE/g DW). This fruit is the only one that can be compared to our result of developmental stage 5 (RS5). Since there is a large number of different types of antioxidant compounds that might contribute to the total antioxidant capacity, it is not clear which components are responsible for the antioxidant effect observed. Therefore, to explore the influence of the phytochemical constituents on antioxidant capacity in B. ligustrifolia fruits at different developmental stages, we determined the correlation between the antioxidant activity assays (TPC by Folin–Ciocalteu, ABTS, and DPPH) and the antioxidant substances (AA, TMA, and TF) (Table 6). The statistical analysis applied shows strong significant correlations (p < 0.01) between TPC and ABTS (0.963, for hydrolysed extracts), and TPC and DPPH (−0.973, for non-hydrolysed), which confirms the idea that TPC by Folin–Ciocalteu can be considered a reducing power assay. For all fruit extracts, the higher antioxidant capacity was observed in the first ripening stage (RS1), and the lower corresponded to RS5. This trend of decreasing in the antioxidant capacity can be closely related with the flavonoid contents as shown by the Pearson’s correlation coefficients: TF(1)TPC(1) = 0.995, TF(2)-ABTS(2) = 0.957, and TF(1)-DPPH(1) = −0.960, for example. In our study, antioxidant capacity was not correlated with vitamin C content and presented a negative correlation with TMA. This supports the observation that phenolic acids and flavonoid compounds may be the main phytochemicals responsible for the antioxidant capacity of B. ligustrifolia fruit. Finally, the antioxidant capacity of B. ligustrifolia appears to be largely influenced by phenolic acids since highly significant linear correlations (p < 0.01) were observed: gallic acid and TPC = 0.985; p-hydroxybenzoic acid and ABTS = 0.959; p-coumaric and DPPH = −0.959.

4.

Conclusion

This is the first study on antioxidant capacity and content of individual phytochemical of B. ligustrifolia fruit throughout

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Table 5 – Antioxidant capacity of Byrsonima ligustrifolia fruit extracts at five ripening stages. Ripening stage

RS1 RS2 RS3 RS4 RS5

ABTS (µmol TE/g DW)

DPPH EC50 (g DW/g DPPH)

Unhydrolysed extract

Hydrolysed extract

Unhydrolysed extract

Hydrolysed extract

2543 ± 82.2Aa 2147 ± 26.0Ba 1477 ± 26.7Ca 1391 ± 21.5CDa 1296 ± 23.2Da

490 ± 27.4Ab 317 ± 44.8Bb 177 ± 20.6Cb 98.3 ± 19.2CDb 41.0 ± 11.3Db

29.3 ± 3.7Db 64.4 ± 3.8Cb 81.9 ± 2.8Bb 97.3 ± 5.4ABb 111.3 ± 4.8Ab

83.8 ± 10.9Ba 101.9 ± 9.1Ba 103.7 ± 11.2Ba 142.8 ± 12.2Ba 222.0 ± 23.5Aa

Results are mean values (n = 3) plus standard deviations; TE = Trolox equivalents; DW = dry weight; EC50 = Concentration of antioxidant required to reduce the original amount of free radicals by 50%. Same upper case letters in the column mean no significant differences at 95% of confidence (p < 0.05) by Tukey test between ripening stages. Same lower case letters in the line mean no significant differences at 95% of confidence (p < 0.05) by Tukey test between unhydrolysed and hydrolysed extracts at a single test.

Table 6 – Pearson’s correlation coefficients (r) between bioactive compounds and antioxidant capacity of Byrsonima ligustrifolia fruit extracts at five ripening stages.

TMA TF(1) TF(2) TPC(1) TPC(2) ABTS(1) ABTS(2) DPPH(1) DPPH(2)

AA

TMA

TF(1)

TF(2)

TPC(1)

TPC(2)

ABTS(1)

ABTS(2)

DPPH(1)

−0.025 −0.261 −0.330 −0.295 −0.285 −0.500 −0.470 0.469 −0.029

−0.886* −0.832 −0.871* −0.860* −0.759 −0.820 0.839 0.992**

0.994** 0.995** 0.993** 0.882* 0.947* −0.960** −0.898*

0.995** 0.996** 0.901* 0.957* −0.965** −0.843

0.999** 0.922* 0.969** −0.973** −0.872

0.916* 0.963** −0.967** −0.862

0.984** −0.964** −0.721

−0.996** −0.803

0.831

(1) = non-hydrolysed extract; (2) = hydrolysed extract. * p < 0.05. ** p < 0.01.

maturation process. The immature fruit (RS1) is a better source of phenolic acids than the mature ones, and the content of anthocyanins increased during ripening. Cyanidin-3-glucoside evolves from 17 mg/100 g DW in RS1 to 1679 mg/100 g DW in RS5, becoming 100 times more concentrated. Even at ripening stage five (RS5), the amount of gallic acid (1419 mg/100 g DW) is very expressive. The highest antioxidant capacity was found in the first stage (RS1) for all assays employed (Folin– Ciocalteu, ABTS, and DPPH). This was correlated with the levels of total flavonoids and phenolic acid individuals. In our fruit, vitamin C did not show correlation with antioxidant activity, and anthocyanins showed a negative correlation. Therefore, B. ligustrifolia is an underutilized fruit, although it is a rich source of cyanidin-3-glucoside and phenolic acids, especially gallic acid, which could be exploited to obtain pigments that might be used as food coluorants as well as a promising source of phenolic nutraceuticals.

Acknowledgements The authors are thankful to Lua Maria Crespo Anastácio and Gilson Crespo Anastácio for supplying the sample used in this work, and to Danilo Ramos Pinto Sampaio for English revision.

Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.jff.2015.08.004.

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