Food Chemistry 145 (2014) 522–529

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Isolation by pressurised fluid extraction (PFE) and identification using CPC and HPLC/ESI/MS of phenolic compounds from Brazilian cherry seeds (Eugenia uniflora L.) Alessandra L. Oliveira a,⇑, Emilie Destandau b, Laëtitia Fougère b, Michel Lafosse b a Departamento de Engenharia de Alimentos, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Av. Duque de Caxias Norte, 225, 13635 900 Pirassununga, SP, Brazil b Institut de Chimie Organique et Analytique, Université d’Orléans, CNRS UMR 6005, 2 Rue de Chartres, 45067 Orléans, France

a r t i c l e

i n f o

Article history: Received 24 April 2013 Received in revised form 6 August 2013 Accepted 14 August 2013 Available online 29 August 2013 Keywords: Eugenia uniflora Pressurised fluid extraction High performance liquid chromatography Centrifugal partition chromatography High resolution mass spectrometry

a b s t r a c t Brazilian cherry seeds are a waste product from juice and frozen pulp production and, the seeds composition was investigated to valorize this by-product. Compounds separation was performed with ethanol by pressurised fluid extraction (PFE). Here we determine the effect of temperature (T), static time (ST), number of cycles (C), and flush volume (VF) on the yield, composition and total phenolic content (TPC) of the seed extracts. T, ST and their interaction positively influenced yield and TPC. Extracts were fractionated by high performance liquid chromatography (HPLC) and centrifugal partition chromatography (CPC). The collected fractions characterizations were made by electrospray ionisation mass spectrometry (ESI/MS) and high resolution mass spectrometry (HRMS) indicated the presence of ellagic acid pentoside and deoxyhexose, quercitrin and kaempferol pentoside. All of these compounds have antioxidant properties and normally are found in plant extracts. These results confirm that Brazilian cherry seed extract is a potentially valuable source of antioxidants. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The Brazilian cherry (Eugenia uniflora L.) is a traditional crop in Southern and Southeastern Brazil, but, in recent years, regular cultivation for commercial purposes has begun in the Northeast. It is a globular berry, 1.5–5.0 cm in diameter, with seven to ten longitudinal grooves. During maturation, the exocarp varies from green to yellow, orange, red or dark red (Bezerra, Silva and Lederman, 2000). The fruit has 69% pulp, 31% seeds and is about 85% water (Guimarães, Holanda, Maia, & Moura Fé, 1982; Silva, 2006). Among the tropical fruits, the Brazilian cherry has one of the higher levels of carotenoids (225.9 lg/g) and vitamin C content of 29.4 mg/ 100 g. It also contains large amounts of calcium phosphorus and anthocyanins. The carotenoids are phytofluene, b-carotene, f-carotene, b-cryptoxanthin, c-carotene, lycopene, and rubixanthin. Lycopene represents 32% of the total carotenoids. Among the phenolic compounds, the dark red Brazilian cherry has 22.50 mg/100 g of anthocyanins (Bezerra et al., 2000; Filho et al., 2008). Recently, the volatile compounds present in the Brazilian cherry leaves, whose biological effects have already been proved, were found in the fruit as selina-1,3,7(11)-trien-8-one for example (Oliveira, Lopes, Cabral, & Eberlin, 2006). An extract obtained by super⇑ Corresponding author. Tel.: +55 19 3565 4268; fax: +55 19 3565 4284. E-mail address: [email protected] (A.L. Oliveira). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.08.065

critical fluid extraction (SFE) is rich in lycopene and other phenolic compounds in considerable concentration (Malaman, Moraes, West, Ferreira, & Oliveira, 2011; Oliveira, Kamimura, & Rabi, 2009). However, for Brazilian cherry seeds, no data in the literature suggests potential biological activity. A recent study showed that the seed extract is rich in phenolic compounds with high antioxidant activity, twice as much as some fruits also rich in phenolic compounds (Bagetti, Facco, Rodrigues, Vizzotto, & Emanuelli, 2009). In the industrial processing of concentrated Brazilian cherry juice and frozen pulp, the seeds are treated as waste with negligible commercial value, with thousands of tonnes of solid waste produced annually. As numerous biological activities are attributed to Brazilian cherry leaves and as the characteristic odour of the leaves is also present in the seeds, it appears important to research the compounds present in the seeds in order to characterise this agro-food waste as a source of raw material for food or related industries. Another goal of this work was to develop processing systems that exploit potentially bioactive components. Beyond scientific research, PFE has several advantages for obtaining extracts from natural products (Vandenburg, Clifford, Bartle, Carroll, & Newton, 1999). PFE or accelerated solvent extraction (ASE) gave similar results to Soxhelt in terms of recovery, repeatability and selectivity. However, both extraction time and solvent consumption were dramatically reduced with PFE. In fact increased temperature

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decreases the viscosity of the solvent enabling an improved penetration of the matrix and enhances diffusivity of the solvent. Moreover, high pressure keeps the solvent in the liquid state and forces the solvent through the matrix. The reduced time of extraction avoids a possible thermal degradation (Kaufmann & Christen, 2002; Wang & Weller, 2006). PFE can be performed using static or dynamic methods or a combination of both. This method is widely used as an extraction technique for sample preparation, to identify the presence of minor components. More recently, this extraction technique has also been used to obtain extracts enriched with bioactive compounds (Claude, Morin, Lafosse, Belmont, & Haupt, 2008; Pól et al., 2007). In this paper, we seeked to optimise the PFE procedure by examining the impact of altering four important variables: extraction temperature (T), static time (ST), number of cycles (C) and the volume of solvent in the extractor in each cycle (VF). In addition, chemical analyses were performed to detect biological activity, as well as analyse composition of these extracts using thin layer chromatography (TLC), high performance liquid chromatography (HPLC) coupled with UV, evaporative light scattering detectors (ELSD), and diode-array detection (DAD), centrifugal partition chromatography (CPC) and HPLC followed by mass spectrometry coupled with electrospray ionisation (ESI/MS).

2. Materials and methods 2.1. Feedstock and chemicals Native red Brazilian cherries collected from a local farm in Brazil were selected manually and were separated from the seeds. These seeds were washed to remove traces of pulp, and dried in forcedair at 50 °C for 54 h. Dried seeds were packed in waterproof bags and stored at 18 °C. The ground seeds had 12.73 ± 0.2% water. The average crushed seeds diameter (0.48 mm) was determined by shaking for 15 min in six-game series standard Tyler sieves. For the extraction process anhydrous ethanol was used (Carlo Erba, Val-de-Reuil, France) as solvent and sodium sulfate (99% anhydrous; Merck, Val de Fontenay, France) as adsorbent material. As reagents for preparation of purified extracts methanol and chloroform were used, from Carlo Erba (Val-de-Reuil, France). For spectrophotometric phenols detection the Folin–Ciocalteu reagent was used and gallic acid, which purchased from Sigma–Aldrich (Saint Quentin Fallavier, France). For TLC analysis Silica Gel 60 F254 plates were used (Merck, Darmstadt, Germany) for the stationary phase. Purified water was from Elgastat UHQ II system (Elga, Antony, France), acetonitrile and ethyl acetate from Carlo Erba (Val-de-Reuil, France); acetic and formic acid were purchased from Sigma–Aldrich (Saint Quentin Fallavier, France) and were used in the mobile phase preparation. The anysaldehide acid solution and ninhydrin were from Sigma–Aldrich (Saint Quentin Fallavier, France). The Molish reagent was prepared with Naphthol (Sigma–Aldrich, Saint Quentin Fallavier, France) and ethanol (Carlo Erba, Val-de-Reuil, France), the VS1 reagent solution was prepared with H2SO4 and ethanol both from (Carlo Erba, Val-de-Reuil, France) and Neu-PEG. The Neu reagent was prepared by mixing diphenyl boric acid ethylamino ester (Sigma–Aldrich, Saint Quentin Fallavier, France) and methanol (Carlo Erba, Val-de-Reuil, France), and the PEG reagent was a solution of polyethylene glycol (PEG) 4000 from Sigma– Aldrich (Saint Quentin Fallavier, France) and ethanol (Carlo Erba, Val-de-Reuil, France). The developer for determining the antioxidant activity of compounds was composed of 2,2-diphenyl1-(2,4,6 -trinitrophenyl) hydrazyl radical (DPPH) (Sigma–Aldrich, Saint Quentin Fallavier, France) solution in methanol (Carlo Erba, Val-de-Reuil, France). All organic solvents used in this work were chromatographic grade.

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2.2. Pressurised fluid extraction PFE was performed using an ASE 100 accelerated solvent extraction system (Dionex, Voisins le Bretonneaux, France) with 34 mL stainless steel ASE vessels. Anhydrous ethanol was used as a solvent. Ethanol was chosen for two reasons: to use of a solvent generally recognised as safe (GRAS), and the Brazilian potential to produce this solvent. In the optimization process, four parameters were studied: T, ST, C and VF in a central composite design (CCD) 24, with five levels, 8 star points and triplicate at the central point (Table 1). Dried and crushed seeds (5 g) were packed in the vessel extractor with 5 g sodium sulfate as adsorbent material used to disperse the vegetal matrix in the extraction cell, allow a better contact with solvent and, clarify the extract. The ethanol extract obtained by PFE was named crude extract and was evaporated then prepared for chromatographic analysis. 2.3. Preparation of crude extracts The crude extracts were purified to eliminate tannins with a high degree of polymerization. First, crude extract (100 mg) was diluted in 2.5 mL methanol and 32.5 mL chloroform. This proportion was important; since preliminary studies showed that any modification may precipitate other phenolic compounds present in the extracts (Lhuiller et al., 2007). The diluted chloroform extract was stored at 4 °C for 3 h in the dark. This solution was centrifuged in a Jouan BR4i multifunction centrifuge (Thermo, Illkrich, France) at 4000 rpm and 5 °C for 10 min. The decanted supernatant was evaporated under N2 at room temperature in the dark. This solution was named purified ethanol extract. 2.4. Spectrophotometric detection of phenols The total phenolic content (TPC) in the PFE extracts was determined using the Folin–Ciocalteu colorimetric method (Singleton & Rossi, 1965). Crude extracts (1 mL) were diluted in methanol (1,000 ppm) with 5 mL Folin–Ciocalteu in water (1:10, v/v). After 10 min, 4 mL anhydrous sodium carbonate solution was added. After 2 h at room temperature in the dark, the absorbance was measured on a DU 640 spectrophotometer (Beckman, Villepinte, France) at wavelengths between 400 and 800 nm. Gallic acid was used for the calibration curve. The TPC is expressed as the g gallic acid equivalent (g GAE) per 100 g dry seeds. 2.5. TLC analysis As the composition of Brazilian cherry seed extract was totally unknown, TLC was used for preliminary analysis to identify families of compounds in the extracts. We used Silica Gel 60 F254 plates, different mobile phases, developers and standards. The samples were applied to the plates using a semi-automatic applicator Linomat IV (CAMAG, Muttenz, Switzerland) with 2 lL of each standard and 5 lL of samples, deposited on the plates in 6 cm bands separated by 4 cm. Anysaldehide acid solution was used as developer to analyse the presence of terpenes and sugars in the extract (Wagner & Bladt, 1996). As the presence of sugar was noted in the extracts, a new TLC method was adopted using three developers. After the chromatographic with acetonitrile:water (75:25, v/v), ninhydrin was sprinkled on the plate to view amino acid compounds, and then the plate was dried with a hairdryer and warmed with a heat pistol until the pink bands were visible. Then, Molish reagent (2 g Naphthol in 100 mL of ethanol) was sprinkled on the same plate, dried with hairdryer, and then the VS1 reagent solution (5% H2SO4 in ethanol) was applied. The plate was dried again and heated with a heat pistol until the violet bands that indicate the presence of sugars were visible.

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Table 1 Matrix of the central composite design (CCD) 24 to study the effect of T, ST, C and VF on the extract yield (Y) and TPC-coded and real variables.

a

Assay

T (°C)

C (no)

VF (%)

ST (min)

Y (%)

TPC (g GAE/100 g)

TPC (ppm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25a 26a 27a

1 (50) +1 (70) 1 (50) +1 (70) 1 (50) +1 (70) 1 (50) +1 (70) 1 (50) +1 (70) 1 (50) +1 (70) 1 (50) +1 (70) 1 (50) +1 (70) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) a (40) +a (80) 0 (60) 0 (60) 0 (60)

1 (2) 1 (2) +1 (4) +1 (4) 1 (2) 1 (2) +1 (4) +1 (4) 1 (2) 1 (2) +1 (4) +1 (4) 1 (2) 1 (2) +1 (4) +1 (4) 0 (3) 0 (3) 0 (3) 0 (3) a (1) +a (5) 0 (3) 0 (3) 0 (3) 0 (3) 0 (3)

1 (80) 1 (80) 1 (80) 1 (80) +1 (120) +1 (120) +1 (120) +1 (120) 1 (80) 1 (80) 1 (80) 1 (80) +1 (120) +1 (120) +1 (120) +1 (120) 0 (100) 0 (100) a (60) +a (140) 0 (100) 0 (100) 0 (100) 0 (100) 0 (100) 0 (100) 0 (100)

1 (4) 1 (4) 1 (4) 1 (4) 1 (4) 1 (4) 1 (4) 1 (4) +1 (8) +1 (8) +1 (8) +1 (8) +1 (8) +1 (8) +1 (8) +1 (8) a (2) +a (10) 0 (6) 0 (6) 0 (6) 0 (6) 0 (6) 0 (6) 0 (6) 0 (6) 0 (6)

6.70 11.20 6.93 7.44 7.01 7.44 6.81 10.94 7.76 11.63 7.06 13.20 6.42 12.00 8.62 12.07 6.06 8.42 7.50 8.50 6.93 13.38 6.78 14.27 9.07 7.90 8.36

1.00 1.33 0.61 0.79 0.99 0.91 0.71 1.21 0.57 1.00 0.72 1.68 0.80 1.60 1.17 1.42 0.42 1.03 0.52 0.80 0.76 1.68 0.98 1.20 1.30 1.16 1.20

53.95 43.33 55.31 48.95 86.96 74.27 65.82 95.13 49.17 56.04 64.05 32.61 37.23 78.27 47.25 40.40 48.23 40.16 48.64 57.63 65.54 74.14 82.39 62.32 84.01 85.03 84.89

Central point of the experimental design (CCD), GAE = gallic acid equivalent, Y% ¼ extractðgÞ  100. seedsðgÞ

In order to verify the presence of phenolic compounds in the extracts, another TLC methodology was applied. The mobile phase was ethyl acetate:acetic acid:formic acid:water (100:11:11:26, v/v/v/v) with the same stationary phases used to identify sugars, the plate was developed with Neu-PEG (Wagner & Bladt, 1996). Neu reagent was obtained by mixing 1 g of diphenyl boric acid ethylamino ester in 100 mL of methanol and PEG reagent was a solution of polyethylene glycol (PEG) 4000 at 5% in ethanol. Moreover, TLC was used in order to determine the antioxidant activity of compounds. For this analysis, the developer used was a solution of 0.05% 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl radical (DPPH) in methanol sprinkled on the plate after elution and drying. The compounds with anti-oxidant properties reacted with DPPH radical generating white spots on the plate.

2.6. HPLC analysis Crude and purified extracts were analysed using a LaChrom HPLC (Merck Hitachi, Tokyo, Japan) with an L-7100 pump, an L7200 autosampler, an interface (D-7000) for the L-7400 UV-detector set at 254 nm. To determine the maximum absorption of the components present in the extracts, an Elite HPLC LaChrom (VWR Hitachi, Tokyo, Japan) consisting of a L-2130 pump, an L2200 autosampler, with a L-2455 diode array detector (DAD) was used. The UV detection was at 254 and 360 nm and peak spectra were recorded from 200 to 600 nm. For the stationary phase, LiChrospher 100 RP-18 (100  4.6 mm  5 lm; Agilent Technologies, Palo Alto, CA) and LiChrospher C18 (150  4.6 mm  5 lm; Bischoff Chromatography, Leonberg, Germany) columns were used. The mobile phase consisted of ultra pure water distilled with a Millipore Elix UV system (Millipore, Saint-Quentin-en-Yvelines, France) and then purified with an Elgastat UHQ II system (Elga, Antony, France) (A) and methanol (B), both acidified with 0.1% formic acid. The flow (1.0 mL/min) and T (25 °C) were constant. The solvent concentrations varied during

the chromatographic analysis according to the applied gradient of 90% A for 5 min, decreasing from 90 to 50% A for 15 min, from 50% to 30% A for 10 min, 30 to 0% A for 5 min, and then 0% for 5 min, from 0 to 90% for 5 min, and finally at 90% A for 5 min.

2.7. HPLC/ESI/MS and HRMS identification The main CPC fractions were analysed by both positive and negative HPLC/ESI/MS using an Agilent 1100 Series HPLC (Agilent) with an interface for a DAD detector and ESI/MS. HPLC conditions were used and the mass spectra were obtained on an API3000 triple quadrupole mass spectrometer (AB SCIEX, Foster City, CA, USA) equipped with a turbo ion spray source and analysis software version 1.4.2 (Applied Biosystem MDS Sciex). The 1 mL/min flow rate from the HPLC device was split to a flow rate of approximately 0.3 mL/min directed to the MS system. In the analysis of a single scan (Q1), the quadrupole was operated under the following conditions: flow rate of the curtain gas of 1.2 L/min, the nebulizer gas 1.2 L/min; with +4.2 kV ion spray voltage for positive mode and 4.0 kV for negative mode. The declustering potential (DP) was 70 V, 300 V focusing potential (FP), and 10 V entrance potential (EP) were used for positive mode, while a DP of 100 V, FP of 400 V and EP of 10 V were used for negative mode. Nitrogen was used as the curtain and nebulizer gas, and compressed air as auxiliary gas. The mass spectra were obtained in a scan range from 100 to 1000 m/z. In the analysis of precursor ions and product ion conditions were as previously described, but the collision energy (CE) varied over the analysis. High resolution mass spectrometry (HRMS) was performed on a maXis mass spectrometer (Bruker, Bremen, Germany). The mass spectrometer was operated in negative modes and acquired data in the mass range from m/z 50 to 1650. The capillary voltage was set at +4.5 kV, the end plate offset at 500 V, the drying N2 flow at 6 L/min at 200 °C, and nebulizer N2 at 1 bar. The accurate mass data of the molecular ions were processed though the Data

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Analysis 4.0 software (Bruker Daltonik), which provided a list of possible elemental formulas using the SmartFormula Editor tool. 2.8. Statistical analysis The influence of the independent variables studied in the PFE process using ethanol as the solvent in the extraction yield (Y), in the TPC, and in the concentration of the main component present in extracts, was verified using the STATISTICA software version 8 (StatSoft, Tulsa, OK) for Windows. 3. Results and discussion 3.1. PFE The extraction yield (Y) of the crude PFE extracts and TPC were the dependent variables analysed as responses in the CCD 24. Yields were high, considering the raw material, with a mean of 8.90 ± 2.45% (Table 1); consequently, ethanol is a suitable solvent to obtain large quantities of this material. The evaluation of four independent variables (T, C, VF and ST) and their interaction showed T, ST, and their interactions showed significant effects (P < 0.05) and positively influenced the yield. The influence of T was higher relatively to ST. Specifically, for this material; there was no influence of the number of cycles (C) and of the flush volume used in each cycle (VF). Considering the cost and ease of use, the fact that these variables (C and VF) do not affect the extraction is significant, because there is no need to use large solvent volumes to generate higher yields. The analysis of variance (ANOVA) for the 1st and 2nd order experimental designs to analyse the PFE efficiency with ethanol shows that, for both models (linear and quadratic), there was a significant effect of these variables on the yield. The coefficients of determination for linear (R2 = 0.85) and quadratic (R2 = 0.84) models suggest that both, when fitted to experimental data, have good predictive capacity for the extract yield. The regression equation to predict Y as a function of independent variables (T, C, VF and ST) are shown in equations 1 and 2 (Eqs. (1) and (2)).

Y ¼ 8:88 þ 1:79T  þ 0:89ST þ 0:59T ST

ð1Þ

Y ¼ 8:47 þ 1:82T  þ 0:66C  þ 0:79ST þ 0:59T ST

ð2Þ

The regression coefficients, which showed no statistical significance (P > 0.05), were not considered in the model, but were added to the pure error. For the linear model (Eq. (1)), T, ST and the

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interaction between them (ST  T) were significant variables. The same is observed in the quadratic model (Eq. (2)). However, the number of cycles (C) was also significant. The response surfaces analysis (RSA) of the linear and quadratic model fitted to the experimental values of PFE extract yields for T and ST showed that elevated T raised Y (Fig. 1). The same is observed for ST, as longer contact time between the solvent and matrix resulted in better performance. 3.2. TPC The TPC, expressed as gGAE per 100 g seeds, ranged from 0.42 to 1.68 (mean, 1.02 ± 0.34), showing that the ethanol extracts obtained by PFE had a high concentration of phenolic compounds compared to the concentration in other tropical fruits seeds (Roesler et al., 2007). All 27 tests (Table 1) presented a similar profile with maximal absorbance around 750 nm. The concentration of phenolic compounds in the extracts was a mean of 61.55 ± 17.66 ppm. Statistical analysis also showed that the independent variables T and ST significantly influenced the TPC in the extracts. These variables were also relevant to Y, as the extracts with higher Y were also rich in phenolic compounds. Unlike for Y, TPC was also influenced by C interaction with ST. This is an indication that phenolic compounds strongly attached to seeds need more contact time and a greater amount of solvent to be extracted properly. For the TPC, ANOVA showed that, although there was a significant effect of T and ST on TPC, the low coefficients of determination calculated suggest that linear (R2 = 0.74) and quadratic (R2 = 0.71) models fitted to experimental data do not have good predictive ability. 3.3. Extract characterization using TLC A preliminary TLC analysis identified the compound classes present in the PFE extracts. The first compound classes identified in the extracts were amino acids and carbohydrates (Fig. 2a). Similar fingerprints were observed in ultrasonic methanol extraction and PFE with methanol, water and formic acid (8:0.5:1.5, v/v/v) mixture (Bagetti et al., 2009) (samples 3 and 4) while sample 5 highlights other compounds more reactive on silica in the crude ethanol PFE extract. Some spots have retention close to the phenolic compounds (phloridizin and avicularin used as standards in samples 6 and 8). Chromatographic plates stained with Neu-PEG show the presence of phenolic compounds detected at 366 nm (Fig. 2b). In Fig. 2c, the Neu-PEG reagent enables to show the

Fig. 1. T and ST RSA for the PFE extract yield for the linear and quadratic models (CCD 24).

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Fig. 2. Analysis of Brazilian cherry seed extract by TLC. TLC was performed with a Silica Gel 60 F254 plate using Brazilian cherry seed extract as the source material as described in the Materials and Methods section. (a) Ninhydrin, Molish, and VS1 revelation. The mobile phase was acetonitrile:water (75:25, v/v). 1: Sucrose, 2: Raffinose, 3: Ext. Ultra sonic (water/methanol/formic acid, 8:0.5:1.5, v/v/v), 4: PFE extracted with water/methanol/formic acid (8:0.5:1.5, v/v/v), 5: PFE extracted with ethanol, 6: Phloridzin dehydrate, 7: Rutin, 8: Avicularin. (b) Neu-PEG revelation. The mobile phase was ethyl acetate:acetic acid:formic acid:water (100:11:11:26, v/v/v/v), 366 nm wavelength. 1: Tannins isolated from crude ethanol PFE (5000 ppm), 2: Crude ethanol PFE (5000 ppm), 3: Purified ethanol PFE (10,000 ppm), 5: Purified ethanol PFE (10,000 ppm), 4, 6 and 7: HPLC F2 of purified ethanol PFE (1000 ppm), (c) and (d): Same mobile phase and samples used in (b) Neu-PEG revelation (track 1, 2, 3, 4 and 5) and DPPH (0.05%) revelation (track 10 , 20 , 30 , 50 ).

presence of tannins in crude ethanol PFE (samples 1 and 2) and phenolic compounds in purified ethanol PFE (samples 3 and 5), and in F2, fraction collected in HPLC from purified ethanol PFE

(sample 4) both in Fig. 2d. The antioxidant activity of the crude (sample 10 and 20 ) (Fig. 2c) and purified extracts (samples 30 and 50 ) (Fig. 2d) was shown using DPPH (2,2-diphenyl-1-(2,4,6-trinitro-

Fig. 3. Components in Brazilian cherry seed extracts detected by HPLC monitored with a UV detector. (a): crude (blue line) and purified (red line) extracts, LiChrospher RP-18 (100  4.6 mm  5 lm), mobile phase water (A) /methanol (B) both acidified with 0.1% formic acid, at 95% A for 5 min, 95–50% A in 5 min, 50–0% A in 20 min, 0% A for 5 min, 0–95% A in 5 min, 95% A for 10 min, with UV detection at 254 nm, (b): purified extract from Brazilian cherry seeds, LiChrospher C18 (150  4.6 mm  5 lm), mobile phase water (A) /methanol (B) both acidified with 0.1% formic acid, at 95% A for 5 min, 95–50% A in 5 min, 50–0% A in 20 min, 0% A for 5 min, UV detection at 360 nm (blue line) and 254 nm (red line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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phenyl) hydrazyl radical) as the developer for TLC. DPPH is a stable free radical and has been used widely to assess the ability of natural antioxidants to bind free radicals. This qualitative analysis shows that tannins with a high degree of polymerization present in the alcohol extract had antioxidant activity (Fig. 2c), as well as the purified extract (Fig. 2d).

the ion at m/z 255, also typical of quercetin (Sandhu and Gu, 2010). The phenolic compound quercetin 3-O-b-rhamnoside may be suggested in this fraction. Unfortunately, due to inadequate separation of the peaks by HPLC, mass spectrometry was not effective. Thus, it was decided to separate the peaks by CPC and then perform HPLC/HRMS analysis.

3.4. Extract characterization using HPLC

3.5. CPC analysis and HPLC/ESI/HRMS

Fig. 3a shows the analysis by HPLC at 254 nm of crude ethanol PFE (blue line) and Purified ethanol PFE (red line). The two chromatograms indicate a common peak at about 19 min while the tannins eliminated by purification were found in the dead volume (blue line). The shape of the common peak eluted at about 19 min showed the co-elution of two compounds. Therefore, the same analysis was carried out using a longer column Lichrospher C18 (150  4.6 mm  5 lm) that enables to split the peak of the purified extract in two peaks collected in fractions F1 and F2 (Fig. 3b shows the chromatogram at 254 and 360 nm). The spectral analysis of F1 by DAD showed two absorbance maxima at 251–253 nm and 360–361 nm while the one of F2 was 253–255 nm and 366 nm. The asymmetry of the two peaks corresponding at F1 and F2 suggested other co-eluted compounds. ESI/MS analyses were performed by direct infusion of F1 and F2 in negative mode to identify main compounds of these HPLC fractions. The fragmentation pattern shows a same base peak in F1 and F2 at m/z 263. In the F2 analysis, the ion at m/z 301 has as a precursor the ion at m/z 447, which could be from the loss of 146 that suggest a rhamnoside. The ion at m/z 301 is a typical fragment of quercetin that, with the loss of a fragment at m/z 46, generates

The purified ethanol PFE extract was fractionated by CPC using the Arizona C system which could provide better separation of these main compounds (Berthod, Hassoun, & Ruiz-Angel, 2005; Michel, Destandau, & Elfakir, 2011). The fractions collected in the tubes 12–14 were joined together in the fraction named CPC F3 and the fractions in tubes 49–54 in the fraction CPC F6 (Fig. 4). These two fractions contain the compounds observed in the fractions F1 and F2 obtained previously by HPLC; they were analysed by HPLC/ESI/HRMS. Using the Smart Formula Editor tool from the Data Analysis 4.0 software, we identified the principal compounds presents in these fractions (Table 2). The two peaks observed at 19.3 and 19.5 min in fraction CPC F3 showed a maximum absorbance of 253 and 357 nm, and 253 and 360 nm, respectively. The first peak at 19.3 min has been identified as ellagic acid pentoside, a phenolic acid in conjunction with a sugar (Table 2). This compound, present in plants, has antioxidant properties. In addition to the tentative identification by HRMS presented here, the literature revealed the presence of this acid in strawberries, with the same absorbance values and the ion MS/MS at m/z 301 as the principal ion (Aaby, Ekeberg, & Skrede, 2007) the aglycone formed by loss of a pentoside [MH132] (Liang, Jin, Feng, & Ke, 2011). In

Fig. 4. HPLC chromatogram of the purified ethanol extract of Brazilian cherry seeds (F1 and F2 HPLC fractions) and of its two CPC fractions (CPC F3 tubes 12–14, and CPC F6 tubes 49–54). Column: LiChrospher C18 (150  4.6 mm  5 lm), with a mobile phase water (A) /methanol (B) both acidified with 0.1% formic acid, at 95% A for 5 min, 95–50% A in 5 min, 50–0% A in 20 min, 0% A for 5 min, UV detection at 254 nm.

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Table 2 HPLC/ESI/HRMS data and tentative identification of the main compounds present in the PFE extracts of Brazilian cherry seeds. HPLC/CPC

tR (min)

UV–vis kmax (nm)

[M-H] (amu)

MS/MS

[M] formula

Tentative identification

F1/F3

19.3 19.5

253, 357 253, 360

20.3 20.8 22.2

254, 365 253, 367 263, 316, 353

433.04151 463.08849 447.05704 300.99951 447.09391 417.08456

301 301 301 229 300, 301 285

C19H14O12 C21H20O12 C20H16O12 C14H6O8 C21H20O11 C20H18O10

Ellagic acid pentoside Quercetin hexoside Ellagic acid deoxyhexoside Ellagic acid Quercitrin Kaempferol pentoside

F2/F6

the second peak at 19.5 min, there were two compounds, a derivative of quercetin hexose and an ellagic acid deoxyhexose with the same retention time and both with the same maximum absorbance (253 and 360 nm). The quercetin hexoside is a flavonol glycoside, also an antioxidant compound present in plants (Lin, Chen, & Harnly, 2008; Määttä, Kamal-Eldin, & Törrönen, 2003). The product ion at m/z 301 is the quercetin aglycone formed by loss of a hexoside [MH162] (Yu et al., 2008). Ellagic acid deoxyhexoside, another ellagic acid derivative with the parent ion at m/z 447 that was identified as an antioxidant compound in chestnut (Sanz et al., 2010). For this compound, the product ion at m/z 301 is the ellagic acid aglycone formed by loss of a deoxyhexoside [MH146]. Even if preliminary analysis of HPLC F1 by HPLC/ESI/ MS showed more than one component in the single chromatographic peak, the most intense ion spectra at m/z 301 can correspond to the quercetin or to ellagic acid structure. So, this analysis was insufficient to propose a structure for the different compounds. The detection of these several compounds in the same peak was only possible thanks to the sensitivity and precision of the HRMS which allow to detect several [MH] ions at different m/z that all lead after fragmentation to the m/z 301 product ion. According to its exact molecular mass obtained by HRMS, the corresponding molecular formula peak at 20.3 min could be identified as ellagic acid. In CPC F6 fraction, two compounds were detected at 20.8 and 22.2 min, quercitrin and a kaempferol pentoside (Table 2). Both compounds have antioxidant properties and are known constituents of plant extracts. In the HPLC/ESI/MS analysis of HPLC F2, the ion at m/z 301 has a precursor ion [M-H] at m/z 447, indicating that it was derived from the loss of a rhamnoside (a deoxyhexose) [M-H-146]. It could be that the phenolic compound quercetin 3-O-b-rhamnoside or quercitrin with [M-H] ion at m/z 447 is present in the Brazilian cherry seed PFE extract, which was confirmed by the HRMS analysis (Table 2) and by HPLC/MS analysis of quercitrin standard in the same conditions. The flavonol kaempferol, also present in plants, has antioxidant activity. Like the others, this flavonol glycoside is also present in other seeds, such as in green beans (Price, Colquhoun, Barnes, & Rodhes, 1998) and lotus seeds (Husam et al., 2010). Its presence in Brazilian cherry seed extracts was identified tentatively by UV spectrum (263 and 353 nm) and HRMS analysis. The abundant product ion at m/z 285 is the kaempferol aglycone that lost a pentoside [M-H-132] (Table 2). The tentative identification of the composition of Brazilian cherry seed extracts, obtained by PFE with ethanol as solvent, indicated the presence of flavonol and acids conjugated with sugars. These components have antioxidant properties, which were confirmed by the high antioxidant activity of these extracts. Furthermore, the tannins with a high degree of polymerization, removed from the crude extract, may also have the same action. Experiments are now being conducted to examine their possible bio-applications. Also in this study, PFE was optimised at laboratory scale; it will be interesting to demonstrate that this process, can be used at large scale to obtain extracts rich in bioactive compounds and

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