Food Research International 112 (2018) 434–442

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Sapucaia ( Cambess) and its by-products: A promising and T underutilized source of bioactive compounds. Part II: Phenolic compounds profile

Fernanda Demolinera, Priscila de Britto Policarpia, Lúcio Flavo Lopes Vasconcelosb, ⁎ Luciano Vitalic, Gustavo Amadeu Mickec, Jane Mara Blocka, a Laboratory of Fats and Oils, Department of Food Science and Technology, Federal University of Santa Catarina, Florianópolis, SC, Brazil b Empresa Brasileira de Pesquisa Agropecuária do Meio-Norte (EMBRAPA Meio-Norte), Piauí, Brazil c Department of Chemistry, Federal University of Santa Catarina, Florianópolis, SC, Brazil

ARTICLE INFO ABSTRACT

Keywords: In this study, the profile of the bioactive compounds of sapucaia nut (Lecythis pisonis Cambess) and its by- Sapucaia nut shell products have been investigated. The phenolic profile by LC-ESI-MS/MS, the total phenolic content, the con- LC-ESI-MS/MS densed tannins and the antioxidant activity of the sapucaia nut and shell were determined. 14 phenolic com- Antioxidant pounds were identified in the sapucaia nut extract, primarily phenolic acids and flavonoids. Catechin, epica- FRAP techin, myricetin, ellagic acid and ferulic acid presented significant correlation to the antioxidant activity. The ABTS sapucaia shell contained 22 phenolic compounds, 13 of which were quantified. The sapucaia shell extract DPPH showed a high content of total phenolic compounds, a high condensed tannins content, and high antioxidant Chemical compounds: activity. The higher antioxidant activity of the shell can be associated with a higher content of phenolics. Aromadendrin (PubChem CID: 122850) Overall, it can be concluded that the sapucaia nut is a raw material rich in phenolic compounds that present high Catechin (PubChem CID: 73160) Ellagic acid (PubChem CID: 5281855) antioxidant activity. The nuts and the cake may be used as a promising raw material for the food industry, while Epicatechin (PubChem CID: 72276) the shells could be an alternative source of natural antioxidants. Further use in the cosmetics and pharmaceutical Epicatechingallate (PubChem CID: 107905) industry may also be envisaged. Epigallocatechin (PubChem CID: 72277) Ferulic acid (PubChem CID: 445858) Gallic acid (PubChem CID: 370) 4–hydroxymethylbenzoic acid (PubChem CID: 129720531) Kaempferol (PubChem CID: 5280863) Myricetin (PubChem CID: 5281672) Naringerin (PubChem CID: 42607905) p-coumaric acid (PubChem CID: 637542) Protocatechuic acid (PubChem CID: 72) Quercitin (PubChem CID: 5280343) Salicylic acid (PubChem CID: 338) Sinapaldehyde (PubChem CID: 5280802) Sinapic acid (PubChem CID: 637775) Siryngic (PubChem CID: 10742) Taxifolin (PubChem CID: 439533) Vanillic acid (PubChem CID: 8468) Vanillin (PubChem CID: 1183)

1. Introduction “cumbuca de macaco”, belongs to the family . This nut originates from the Brazilian Amazon and it is widely distributed Lecythis pisonis Cambess, popularly known as “sapucaia” or throughout Brazil, mainly in the Atlantic forest and in the Amazon

⁎ Corresponding author at: University of Santa Catarina, Agricultural Science Center, Department of Food Science and Technology, Rodovia Admar Gonzaga, 1346, Itacorubi, CEP: 88034-001 Florianópolis, SC, Brazil. E-mail address: [email protected] (J.M. Block). https://doi.org/10.1016/j.foodres.2018.06.050 Received 28 March 2018; Received in revised form 18 June 2018; Accepted 20 June 2018 Available online 21 June 2018 0963-9969/ © 2018 Elsevier Ltd. All rights reserved. F. Demoliner et al. Food Research International 112 (2018) 434–442 region (Brandão et al., 2013; Vallilo, Tavares, Aued-Pimentel, Campos, shell were dried at 40 °C for 60 min in an oven equipped with air cir- & Moita Neto, 1999). Our previous study has demonstrated that the culation (model 400/D, Nova Ética®, Vargem Grande Paulista, Brazil) sapucaia nut and cake are an excellent source of proteins, dietary fiber, to reduce their moisture. Then, the samples were milled using an ana- and selenium. On the other hand, the sapucaia oil is a source of un- lytical laboratory grinder (model Q298A, Quimis®, Diadema, Brazil) saturated fatty acids (oleic and linoleic acids), γ-tocopherol and β-si- and sieved to 60-mesh size. The nuts and shells were defatted by tosterol (Demoliner et al., 2018). Several parts of sapucaia such blending with hexane (1:20 w/v, 30 min × 3) in a mechanical stirrer as the leaves, shell, and oil are also used in popular medicine (Agra, (model TE-139, Tecnal®, Piracicaba, Brazil) at a room temperature of Freitas, & Barbosa-Filho, 2007; Franco & Barros, 2006; Wickens, 1995). 22 °C. The defatted samples were air-dried until total evaporation of the The antipruritic effects and antinociceptive activity of Lecythis pisonis solvent and stored in amber bottles with a nitrogen atmosphere at leaves have been reported (Brandão et al., 2013; Silva et al., 2012). −24 °C for the preparation of the extracts. In the last decade, the phenolic compounds in natural nut products The extraction of phenolic compounds from the nut and shell was and by-products have been investigated (Gomes & Torres, 2016; Hilbig, performed under optimal operational conditions (solvent, time and Alves, Muller, Micke, Vitali, Pedrosa, and Block, 2018; John & Shahidi, temperature) determined in preliminary studies (data not shown). The 2010; Robbins, Gong, Wells, Greenspan, & Pegg, 2015). The phenolic defatted samples of sapucaia nut (1 g) and sapucaia nut shell (1 g) were compounds, including flavonoids, phenolic acids, tannins, stilbenes, placed into Erlenmeyer flasks, following the addition of 30 mL of the lignans, and phenolic aldehydes play an important role in human solvent (water for defatted samples of sapucaia nut; and methanol:- health, mainly due to their antioxidant properties (Taş & Gökmen, water −80:20 for defatted samples of sapucaia nut shell). Then the 2017). Several studies reported that the antioxidant activity of phenolic samples were sonicated at 80 °C for 60 min in an ultrasonic cleaning compounds is associated with the protection against diseases such as bath (EGS 5HD, 40 kHz, 300 W, Enge Solutions®, São Paulo, Brazil). diabetes, cancer, hypercholesterolemia, and cardiovascular diseases After sonication, the extracts were filtered with paper filter (Whatman (Alasalvar & Bolling, 2015; Hilbig, Policarpi, de Souza Grinevicius, no 541, 125 mm) and stored in amber bottles under nitrogen atmo- Santos Mota, Toaldo, Luiz, Pedrosa, and Block, 2018; Kaliora, sphere at −24 °C for further analysis. Kogiannou, Papassideri, & Kalogeropoulos, 2014; Shahidi & Ambigaipalan, 2015). 2.4. Extraction, identification and quantification of phenolic compounds by The sapucaia nuts and its by-products are not well known yet and it LC–ESI-MS/MS is still an underutilized raw material. Studies on bioactive compounds and antioxidant activity of sapucaia nuts and its by-products are still 2.4.1. Samples preparation scarce in the literature. Therefore, the use of this native nut from Brazil The sample preparation was performed following the methodology in diet may be considered as an alternative to improve the diet of the of Schulz et al. (2015), with some modification. Aliquots of 1 mL of the Brazilian population and its by-products as well an alternative source extract solution were mixed with 5 mL of HPLC grade methanol − for natural antioxidants. Based on this context, in order to divulge the (99.9%) and 5 mL of hydrochloric acid (0.01 mol L 1) and were sub- phenolic and antioxidant potentialities of sapucaia nut and its by-pro- jected to acid hydrolysis in an oven (model 400/D200 °C, New Ethics®, ducts, the aim of this research was to identify and quantify the phenolic São Paulo, Brazil) at 85 °C for 30 min. The solution was adjusted to pH 2 − compounds of the extracts obtained from the sapucaia nuts and shell using 6 mol. L 1 NaOH. Then, a partition extraction (three times) with using LC-ESI-MS/MS. 10 mL of ethyl ether was carried out followed by centrifugation at 3000 g for 10 min. The supernatants were combined, and the organic 2. Materials and methods solvent was removed using a rotary evaporator (model 558, Fisatom®, São Paulo, Brazil). The dried extract was resuspended in 1 mL of me- 2.1. Samples thanol and diluted 10 times with methanol:water (70:30, v/v) for in- jection in the LC-ESI-MS/MS system. Sapucaia nuts (Lecythis pisonis Cambess) were obtained from dry ripe sapucaia fruit from Teresina, Piauí, Brazil (samples A1, A2 and A3) 2.4.2. Phenolic profile by LC-ESI-MS/MS and provided by the Brazilian Agricultural Research Corporation The identification and quantification of phenolic compounds were (EMBRAPA Meio-Norte). Another sample was sourced from native trees carried out using a high performance liquid chromatography (LC) in the city of Viçosa, Minas Gerais, Brazil (sample B1). About 600 g of system (model 1200 Series, Agilent Technologies®, Waldbronn, each sample (harvested in October 2016) were obtained. Germany) according to the method reported by Schulz et al. (2015). The analytes separation was performed in a Synergi™ Polar-RP 80 A 2.2. Chemical reagents column (4.0 μm particle size, 150 mm, 2.0 mm internal diameter, Phe- nomenex, USA) using a SecurityGuard Cartridges Polar-RP 80A Folin-Ciocalteau phenol reagent, gallic acid, vanillin, (+)-catechin (4.0 μm × 2.0 mm, Phenomenex, USA). Mobile phases were mixtures of hydrated, ABTS [2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic methanol 95% and water 5% (v/v) (channel A), and water and formic acid)], DPPH (2,2-diphenyl-1-picrylhydrazyl), 2,2′- 2,4,6-tripyridyl- acid 0.1% (v/v) (channel B). The separation was carried out at 30 °C, triazine (TPTZ) and Trolox were obtained from Sigma-Aldrich (St. using segmented gradient elution as follows: 0 - 5 min, 10% A; 5–7 min, − Louis, MO, USA). Stock solution (1000 mg L 1 in methanol 100%) of 90% A; 7–10 min, 90% A; 10–17 min, 10% A. Between the analyses, the ultra-pure phenolic standards (Sigma-Aldrich, St. Louis, MO, USA) were column was conditioned for 5 min with the proportion of the initial − prepared and stored in a freezer. They were used to prepare the cali- mobile phase. The mobile phase flow rate was 150 μL min 1, and the bration curves of phenolic compounds using appropriate dilution from injected sample quantity was 10 μL. the mixture. All other chemical reagents and solvents used in the ex- The LC system was coupled with a mass spectrometry system con- periment were of analytical grade (P.A.) and obtained from Vetec (Rio sisting of a hybrid triple quadrupole/linear ion trap mass spectrometer de Janeiro, Brazil) and Sigma-Aldrich (Darmstadt, Germany). Q Trap 3200 (Applied Biosystems/MDS Sciex, Concord, Canada). The mass spectrometer was operated in negative electrospray 2.3. Obtaining of extracts TurboIonSpray™ (Applied Biosystems/MDS Sciex, Canada) ionization mode. The MS/MS parameters were: capillary needle, maintained at The extracts obtained from the nuts and shells were prepared fol- −4500 V; curtain gas, 10 psi; temperature, 400 °C; gas 1, 45 psi; gas 2, lowing the methods reported by John and Shahidi (2010) and Hilbig, 45 psi; and CAD gas, medium. Other parameters for: DP - Declustering Alves, et al. (2018), with some modifications. The sapucaia nuts and Potential; EP - Entrance Potential; CEP - Collision Energy Potential; CE -

435 F. Demoliner et al. Food Research International 112 (2018) 434–442

− Collision Energy; CXP - Collision Cell Exit Potential of the 49 phenolic containing 0.1 mL of extract (0.033 g mL 1). The samples were kept in compounds tested are listed in supplementary material (Table S1). The the absence of light for 30 min, and then the absorbance was measured software Analyst version 1.5.1 was used for the LC–ESI-MS/MS system with a spectrophotometer (SP 2000 UV, Bel Photonics®, Piracicaba, São control and data analysis. Paulo, Brazil) at 515 nm. The radical inhibition percentage was calcu-

lated using the following equation: % radical inhibition = (1 − Af/ 2.5. Determination of total phenolic content (TFC) A0) × 100, where, “A0” is initial absorbance and “Af” is the final ab- sorbance. A standard curve of Trolox stock solution (80, 160, 320, 640 − The TFC was determined by the Folin-Ciocalteau method (Prado, and 1280 μmol mL 1) was used to quantify the results. The results were − Aragão, Fett, & Block, 2009; Singleton & Rossi, 1965), with some expressed in μmol TEAC g 1 of dry weight (μmol Trolox equivalent − modifications. For the oxidation reaction, aliquots of 0.1 mL of the antioxidant capacity g 1 of sample). − extract solution (0.033 g mL 1) were transferred to 10 mL volumetric flasks. Then, 0.5 mL of Folin–Ciocalteau reagent and 1.5 mL of satu- 2.7. Determination of condensed tannins (CT) − rated sodium carbonate (75 g L 1) were added, and the volume of each flask was completed with deionized water, shaken and left in the The determination of condensed tannins was carried out according darkness at room temperature for 2 h. The absorbance of the resulting to the method described by Price, Van Scoyoc, and Butler (1978) and blue solution was measured at 764 nm with a spectrophotometer (SP adapted by Villarreal-Lozoya, Lombardini, and Cisneros-Zevallos − 2000 UV, Bel Photonics®, Piracicaba, São Paulo, Brazil) and correlated (2007). Aliquots of 1 mL of the extract solution (0.033 g mL 1) were − to a calibration curve of gallic acid (50–1000 mg L 1). The analysis was collected and placed into two separate test tubes (one for the sample performed in triplicate and the results were expressed as gallic acid and one for the blank solution). Then, 5 mL of vanillin reagent (0.5 g − equivalent (mg GAE g 1) of dry weight. reagent and 200 mL of 4% HCl in methanol) was added to each sample, and 4% HCl in methanol was added to the blank solution. The test tubes 2.6. Antioxidant activity (AA) were kept in the absence of light for 20 min and the absorbance was measured at 500 nm using glass cuvettes in a spectrophotometer (SP 2.6.1. FRAP assay 2000 UV, Bel Photonics®, Piracicaba, São Paulo, Brazil). A standard FRAP assay was carried out according to the method described by curve of catechin solution (0, 10, 37.5, 75, 150, 300, 600 and − Benzie and Strain (1996) and Arnous, Makris, and Kefalas (2002), with 1200 mg L 1) was used for the quantification and the results were ex- − modifications. The method is based on the reduction of a ferric 2,4,6- pressed in cathechin equivalent (mg CE g 1) of dry weight. tripyridyl-s-triazine complex (Fe3+ − TPTZ) by antioxidants to the ferrous form (Fe2+ − TPTZ). Aliquots of 0.2 mL of the extract solution 2.8. Statistical analysis −1 −1 −1 (0.033 g mL ) and 0.2 mL of FeCl3 (3 mmol L in 5 mol L citric acid) were mixed in a tube and incubated for 30 min in a water bath at The normal distribution and homoscedasticity of the data was 37 °C. Then 3.6 mL of TPTZ solution (0.0780 g diluted in 250 mL of evaluated by the Brown-Forsythe tests. Analysis of variance (ANOVA) 0.05 M HCl) was added and the resulting solution was mixed by a and Tukey's test for comparison of means at a significance level of 5% vortex. After exactly 10 min, the absorbance (620 nm) was read using a (p < 0.05) were performed using the software Statistica® 7.0 (2008). spectrophotometer (SP 2000 UV, Bel Photonics®, Piracicaba, São Paulo, Correlations between the antioxidant capacity and phenolic content of Brazil). The Trolox standard curve (20–1000 μM) was used to quantify the samples were carried out using the Pearson's test. All analyses were − the results, which were expressed as μmol TEAC g 1 of dry weight carried out in triplicate and results expressed as the mean ± standard − (μmol Trolox equivalent antioxidant capacity g 1 of sample). deviation (SD).

2.6.2. ABTS assay 3. Results and discussion The ABTS [2,2′-azino-bis (3-ethylbenzotiazoline-6-sulphonic acid)] assay was carried out according to Re et al. (1999) with some mod- 3.1. Phenolic profile by LC-ESI-MS/MS ifications. The ABTS·+ radical (7 mM−0.03836 g ABTS dissolved in 10 mL deionized water) was mixed with 10 mL of potassium persulfate Table 1 shows the mass spectra limits of detection and quantifica- solution (2.45 mM−0,0331 g of potassium persulfate dissolved in tion, as well as the retention times, the fitting equation, and determi- 25 mL deionized water), homogenized and stored for 12–16 h at room nation coefficient obtained from the analysis of the phenolic com- temperature in the absence of light. For the sample analysis, the ABTS·+ pounds. 14 phenolic compounds were identified in the sapucaia nut radical solution was adjusted by mixing it with ethanol to achieve an extracts, including seven phenolic acids, six flavonoids and one phe- absorbance value of (0.700 ± 0.02) at 734 nm (A734 = A0). Then, nolic aldehyde (Table 2). Myricetin and, vanillic, ferulic, and ellagic − 20 μL of extracts solutions (0.033 g mL 1) and 980 μL of ABTS solution acids were the main compounds quantified in all samples of sapucaia were added to the test tubes, and after 6 min the absorbance was read nut. On the other hand, catechin was quantified in samples A1 and B1, by a spectrophotometer (SP 2000 UV, Bel Photonics®, Piracicaba, São and epicatechin only in sample A1. For sample A1, the concentration of

Paulo, Brazil) at 734 nm (A734 = Af), using glass cuvettes. The radical ellagic acid, myricetin, and catechin were significantly higher inhibition percentage was calculated using the following equation: % (p < 0.05) when compared to samples A2, A3, and B1. radical inhibition = (1–A/A0) × 100, where, “A0” is the initial absor- 22 phenolic compounds were identified in the sapucaia shell ex- bance and “Af” is the final absorbance. A standard curve of Trolox stock tracts, corresponding to ten phenolic acids, ten flavonoids, and two − solution (190, 390, 590, 790 and 1000 μmol mL 1) was used to quan- phenolic aldehydes (Table 2). The gallic, protocatechuic, vanillic, − tify the results. The results were expressed in μmol TEAC g 1 of dry ferulic, and ellagic acids, epigallocatechin, catechin, epicatechin, taxi- − weight (μmol Trolox equivalent antioxidant capacity g 1 of sample). folin, myricetin, and vanillin were quantified in all samples. Kaempferol was quantified only in samples A1, A3, and B1; and aromadendrin was 2.6.3. DPPH assay quantified in samples A1 and B1. Catechin was the most abundant The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was performed phenolic compound found in sapucaia shell. according to Brand-Williams, Cuvelier, and Berset (1995), with mod- Although the phenolic compounds have also been reported for other ifications, as described by Mensor et al. (2001) and Prado et al. (2009). Brazilian nuts, such as Brazil, , baru and nuts, it has not Initially, an aliquot of 2.9 mL of DPPH solution (0.1 mM–0,03943 g of been previously reported in the literature for sapucaia nuts (Table 3). DPPH dissolved in 10 mL of ethanol) was transferred to test tubes Both sapucaia and pecan nuts present a wide variety of phenolic

436 F. Demoliner et al. Food Research International 112 (2018) 434–442

Table 1 Parent and quantitative ion, retention times, limits of detection (LOD), limit of quantification (LOQ), curve equations and determination coefficients (R2) of the phenolic analysis of sapucaia nut and shell.

Phenolic compounds [M-H]/Quantitativeion (m/ Retention time LOD LOQ Fitting equation Determination coefficient − − z) (min.) (mg L 1) (mg L 1) (R2)

Gallic acid 168.8/124.1 3.98 0.023 0.076 y = 52,947x − 6035 1.0000 Protocatechuic acid 153.02/109 6.65 0.006 0.021 y = 1,000,000x + 7910 1.0000 Epigallocatechin 30