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Received: 5 March 2017 Revised: 11 July 2017 Accepted article published: 19 July 2017 Published online in Wiley Online Library: 7 September 2017 (wileyonlinelibrary.com) DOI 10.1002/jsfa.8561

Chemical profile of pineapple cv. Vitória in

different maturation stages using electrospray

ionization mass spectrometry

Elizângela M Ogawa,

a

Helber B Costa,

a

José A Ventura,

b

Luiz CS Caetano,

b

Fernanda E Pinto,

a

Bruno G Oliveira,

a

Maria Eduarda S Barroso,

c

Rodrigo Scherer,

c

Denise C Endringer

c,d

and Wanderson Romão

a,d*

Abstract

BACKGROUND: Pineapple is the fruit of Ananas comosus var. comosus plant, being cultivated in tropical areas and has high energy content and nutritional value. Herein, 30 samples of pineapple cv. Vitória were analyzed as a function of the maturation stage (0–5) and their physico-chemical parameters monitored. In addition, negative-ion mode electrospray ionization mass spectrometry [ESI(−)FT-ICR MS] was used to identify and semi-quantify primary and secondary metabolites present in the crude and phenolic extracts of pineapple, respectively.

RESULTS: Physico-chemical tests show an increase in the total soluble solids (TSS) values and in the TSS/total titratable acidity ratio as a function of the maturity stage, where a maximum value was observed in stage 3 (3∕

4of the fruit is yellow, which

corresponds to the color of the fruit peel). ESI(−)FT-ICR MS analysis for crude extracts showed the presence mainly of sugars as primary metabolites present in deprotonated molecule form ([M− H]−and [2 M− H]−ions) whereas, for phenolic fractions, 11 compounds were detected, being the most abundant in the third stage of maturation. This behavior was confirmed by quantitative analysis of total polyphenols.

CONCLUSION: ESI-FT-ICR MS was efficient in identifying primary (carbohydrates and organic acids) and secondary metabolites (13 phenolic compounds) presents in the crude and phenolic extract of the samples, respectively.

© 2017 Society of Chemical Industry

Supporting information may be found in the online version of this article.

Keywords: ESI(−)FT-ICR MS; electrospray mass spectrometry; maturation stages; pineapple

INTRODUCTION

Pineapple is the fruit of Ananas comosus var. comosus plant, being cultivated in tropical areas. It is a perennial monocotyledon belonging to the Bromeliaceae family.1The fruit is used for

con-sumption in natura but also for industrial products such as juice, canned fruit, crystallized pieces, and jams.2It has high energy

con-tent and nutritional value due to its high concentration of carbo-hydrates, minerals (calcium, potassium, phosphorus, magnesium, sodium, copper and iodine) and vitamins, especially ascorbic acid, niacin, thiamine, and riboflavin.2

In medicine, pineapple is important because of the presence of bromelain, a proteolytic enzyme with pharmacological activi-ties such as anti-inflammatory, inhibition of platelet aggregation and cancer chemo-preventive properties.3,4In addition to this

pro-tease, polysaccharides are other macromolecules responsible for the benefits of pineapple. Fiber present in the fruit peel can reduce the gastrointestinal transit time and improve growth of probiotics in the intestine.5

Cultivar Vitória is a fruit originated from cross-breeding between two varieties of pineapple (female parent cv. Primavera and male parent cv. Smooth Cayenne), developed by the Capixaba Institute

of Research, Technical Assistance and Rural Extension (INCAPER) in partnership with the Brazilian Agricultural Research Corporation (EMBRAPA) breeding program.6Resistance to fusariosis is the main

characteristic of this fruit. Fusariosis is a disease caused by the fungus Fusarium guttiforme, the main phytosanitary problem of the crop in the country, causing an average of 30–40% losses in fruit production and 20% of propagative material. In addition, it

Correspondence to: W Romão, Laboratório de Petroleômica e Química Forense,

Departamento de Química, Universidade Federal do Espírito Santo (UFES), Avenida Fernando Ferrari, 514, Goiabeiras, Vitória-ES, CEP: 29075-910, Brazil. E-mail: wandersonromao@gmail.com

a Laboratório de Petroleômica e Química Forense, Departamento de Química,

Universidade Federal do Espírito Santo (UFES), Goiabeiras, Vitória, ES, Brazil

b Instituto Capixaba de Pesquisa, Assistência Técnica e Extensão Rural (INCAPER),

Vitória, ES, Brazil

c Universidade Vila Velha–UVV, Boa Vista, Espírito Santo, Brazil d Instituto Federal do Espírito Santo (IFES), Soteco, Vila Velha, ES, Brazil

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has similar or superior agronomic characteristics of cvs. Pérola and Smooth Cayenne. The plant has leaves of light green color and has the advantage of no thorns, which facilitates the cultivation. The fruit has white flesh, cylindrical shape, and yellow skin when ripe and weighs around 1.5 kg. Furthermore, exhibit a high sugar content (average of 15.8∘Brix) and excellent flavor in sensorial analyses. Additionally, they are more resistant to transportation and postharvest than the reference varieties.7

In 2013, Brazil was the second largest world producer of pineapple.8The harvest of pineapple, in 2014, was greater than

to 1.7 billion of fruits, and, in 2015, 1.4% higher compared to the previous year.9 Pineapple production is composed of 14 poles

of fruit present in the Espírito Santo state, Brazil. Traditionally, pineapple cultivation is practiced in the southern state.10

Therefore, it is very important to develop studies that evaluate the influence of climatic conditions and maturity stage on fruit quality such as size, color and especially on its chemical compo-sition (total soluble solids content, acidity, and polyphenols).11,12

Physico-chemical analyses and the study of molecules that influ-ence fruit maturity and grown have been the aim of several works.13–19

Steingass and colleagues have published several works which evaluated the profile of volatile compounds from pineapple fruits at different maturity stages by gas chromatography–mass spec-trometry (GC–MS).14,15These studies were of great significance for

describing metabolite changes during the process of maturation, where they have identified more than 290 volatiles compounds. Phenolic compounds in pineapple have also been studied by Steingass et al.17They used electrospray ionization multiple-stage

mass spectrometry as well as GC–MS to identify and assign a vari-ety of phenolic compounds, like p-coumaric, ferulic acid, S-sinapyl derivatives of glutathione, caffeic acid, and more.17

Bataglion et al.16 determined the phenolic composition

of several fruits from Brazil, including pineapple fruit, by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). They observed that the

p-coumaric, ferulic and sinapinic acids were the most abundant

phenolic compound presented in the pineapple specie studied.16

Similar results were also found by the study of Ma et al.,18in which

they detected and proposed the chemical structures of 26 polar components such as p-coumaric, ferulic, caffeic acid, chlorogenic acid, and others.18

Khakimov et al.19 studied the non-volatile metabolites by

GC–MS of five fruits, including pineapple. They found that citric acid was the most abundant metabolite in the pineapple followed by 2-hydrohycitric acid. Carbohydrates (like fructose) were the dominant species, whereas palmitic and stearic acids were the most abundant fatty acid species reported.19

In general, the majority of studies investigate the volatile com-ponents of pineapple, leaving most of the primary and secondary metabolites unexplored.19In addition, a great number of the

stud-ies have applied GC–MS to investigate these components dur-ing the process of maturity and ripendur-ing. Fourier transform–ion cyclotron resonance mass spectrometry (FT-ICR MS) technique enables the analysis of molecules with a high mass resolution, accuracy and resolving power, being great tool to identify and semi quantify the molecules present in a variety of samples.

The main advantage of ESI(±)FT-ICR MS technique, in relation to GC–MS, is related to fact that it provides information on a molec-ular level of the polar species with wider molecmolec-ular weight range (Mw from 200 to 3000 Da) without the need of a pre-separation step (the analysis is done via direct infusion of the ESI source),

and is therefore able to identify thousands of compounds in a short analysis time (t ∼ 30 s). On the other hand, the GC–MS pro-vides information of low molecular weight molecules (<500 Da), where the molecules need to have thermal stability. Thus, this work aims to study the variations in the metabolite profiles of pineapple cv. Vitória. Hence, physico-chemical analysis such as total soluble solids (TSS), total titratable acidity (TA), TSS/TA ratio and quantification of total polyphenols were evaluated in the pulp or crude extract in six different maturation stages (ranging from 0 to 5). Furthermore, in order to identify primary and sec-ondary metabolites responsible for the physico-chemical proper-ties of the fruit, qualitative and semi-quantitative analyses using FT-ICR MS combined to negative-ion mode electrospray ionization (ESI(−)FT-ICR MS) were performed. Finally, the crude and phenol extracts of pineapple were evaluated as potential cancer chemo-preventives by employing the NAD(P)H: quinone reductase (QR) enzyme induction assay in cell culture.

MATERIAL AND METHODS

Samples and reagents

Thirty pineapple samples of the cultivar Vitória in maturation stages from 0 to 5 were collected at the Bananal do Norte Experimental Farm/INCAPER – Pacotuba, located in Cachoeiro de Itapemirin, ES, Brazil. Samples were stored in temperatures of −20 ∘C in order to conserve their physico-chemical properties before analysis. Different stages of maturation were determined by the variation of color observed among the fruits, which can be seen in Fig. S1 (Supporting Information), where the pineapple maturity can be divided into six stages according to the color of the fruit peel, as follows: stage 0 (zero) = color begins to change from green to yellow; stage 1 =1

4of the fruit is yellow; stage 2 =1∕2of the fruit

is yellow; stage 3 =3

4of the fruit is yellow; stage 4 = the fruit is

completely yellow; and stage 5 = the fruit is fully ripe with all the fruitlets yellow.2

The materials used were: deionized water, methanol and hydrochloric acid (Dinâmica – HPLC grade), ammonium hydrox-ide (28%, NH4OH) and ethyl acetate (both supplied by Vetec Química Fina Ltda, Duque de Caxias, Brazil), standard glucose deuterated (D-glucose-1,2,3,4,5,6,6-d7; Sigma–Aldrich, St Louis, MO, USA) and Chromabond(r) C18ec SPE cartridge (1000 mg sorbent, 6 mL volume, pore width of 60 Å, and 45 μm particle size; Macherey Nagel, Bethlehem, PA, USA). Sodium trifluoroacetate (98%, NaTFA) was purchased from Sigma–Aldrich.

Sample preparation

Crude extract

To produce the crude extract, pineapple fruit was initially washed in chlorinated and distilled water, respectively. Then, 20 g of the pulp was obtained by peeling and cutting the fruit by longitudinal cuts from the bottom to the top of the pineapple crown. The sample obtained was mashed in a mortar with the aid of a pestle. Then it was filtered with Unifil® black stripe 125 mm filter paper.

Phenolic extract

Isolation of phenolic compounds present in the pulp sample of pineapple ‘Vitória’ was conducted using the methodology described by Alothman et al.20 and Steingass et al.,17 both with

modifications. Portions of 25 g of pulp were used and homog-enized in a domestic blender for 3 min. Then, 75 mL of a 70%

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methanol solution containing 0.1% (v/v) of HCl was added. The

solution obtained was placed in an amber glass vial and sub-jected to ultrasonic bath with a frequency of 40 kHz for 20 min. Subsequently, the solution was centrifuged at 3500 × g for 10 min to precipitate the pectin. The supernatant generated by cen-trifugation was evaporated using a rota-evaporator at 45 ∘C. The residue generated was resuspended in 5 mL of deionized water and loaded onto an SPE cartridge pre-activated by injection of 3 mL of methanol followed by 10 mL of deionized water. The methanolic extract was then washed with 20 mL of a HCl solu-tion of 0.01% (v/v) for the removal of organic acids and sugars.21

The acid solution was used to prevent the deprotonation of phe-nolic compounds, which could reduce the adsorption during the elution of polar fraction.22 The analytes were eluted using 6 mL

of methanol and subsequently 1 mL of ethyl acetate. The extract containing the phenolic compound was concentrated into the rota-evaporator at 25 ∘C and resuspended in 1.5 mL of a methanol solution of 50% (v/v).

Physico-chemical analysis

Titratable acidity (g citric acid equivalent per 100 g of pulp), TSS (∘Brix) and calculation of TSS/TA ratio were determined. The TSS analysis was performed using a benchtop refractometer (Abbé Model 2 WAJ with refractive ranging of 1.300 to 1.72 nD 0–95∘Brix; Jupiter, FL, USA). TA analysis was determined by titration using a NaOH 4 g L−1solution (g of citric acid equivalent per 100 g of

pulp). The TSS/TA ratio was calculated dividing the TSS values by the TA values. All procedures were performed according to the methodology described by Adolfo Lutz Institute.23

ESI(−)FT-ICR MS and ESI(−)MS/MS analysis

For crude extract analysis, 10 μL of the extract was doped with 5 μL of internal standard, deuterated glucose solution at ≈1.0 g L−1, and

basified with 4 μL of a NH4OH solution. Subsequently, 980 μL of

a solution of methanol/water (50% v/v) was added to the crude extract. For the phenolic extract analysis, 15 μL of the extract was doped with 3 μL of internal standard, deuterated glucose solution at 9.4 g L−1, and basified with 2 μL of NH

4OH solution. To the final

phenolic extract solution was added 483 μL of methanol/water solution (50% v/v).

The mass spectrometer (model 9.4 T Solarix; Bruker Daltonics, Bremen, Germany)24–27was set to ESI(−)FT-ICR MS analysis, over

a mass range of m/z 150–1250. The ESI source conditions were as follows: nebulizer gas pressure 1.4 bar, capillary voltage of 3.8 kV and transfer capillary temperature of 200 ∘C. Ion time accu-mulation was 0.010 s. ESI(−)FT-ICR mass spectra were acquired by accumulating 32 scans of time-domain transient signals at 4 mega-point time-domain data sets. All mass spectra were exter-nally calibrated using NaTFA solution (m/z from 200 to 1200). Resolving power, m/Δm50%= 540 000 (in which Δm50%is the full

peak width at the half-maximum peak height of m/z 400) and a mass accuracy of<1 ppm provided the unambiguous molecular formula assignments for singly charged molecular ions. Mass spec-tra were acquired and processed using the DataAnalysis software (Bruker Daltonics).

Tandem mass spectrometry (MS2) experiments were performed

on a quadrupole analyzer coupled to the FT-ICR mass spectrom-eter. ESI(−)MS/MS spectra were acquired using an infusion flow rate of 10 μL min−1, capillary voltage of 3.0 kV, nebulizing

tem-perature of 250 ∘C, argon as collision gas, ion accumulation time was raised from 0.02 to 1 s, isolation window of 1.0 (m/z units),

and 3–20 V of the collision energy. The spectra were acquired by accumulating 32 scans of time-domain transient signals in 500 k time domain.24,26

In order to semi-quantify sugar and phenolic compounds present in the samples, a ratio of the sum of the signal intensity of sugars or phenolic compounds signals were divided by the sum of the intensity of deuterated glucose compounds signals. This ratio was monitored as a function of maturity stages and the results were statistically analyzed using analysis of variance (ANOVA) and Tukey test (P< 0.05).24

Total polyphenolic content

Analysis of phenolic compounds in the extracts was performed using a UV–visible spectrophotometer (model 482; Femto, São Paulo, Brazil) according to the spectrophotometric method of Folin–Ciocalteu with adaptations.28 Absorbance was registered

at 715 nm. An analytical curve was built using five different con-centrations of gallic acid anhydrous standard solutions (ranging from 10 to 120 μg mL−1) (Fig. S2, Supporting Information).

Quan-tification of total polyphenols was obtained using the equation of the analytical curve [y = (8.1 × 10−4) + 0.07] containing

regres-sion coefficient of r2= 0.992. Results were expressed as μg of

phenols per 100 g of the fruit pulp. All analyses were performed in triplicate.

Determination of quinone reductase activity in cell cultures Activity of quinone reductase of crude and phenolic extracts was assessed in 96-well plates using Hepa1c1c7 (murine hepatoma cells, ATCC CRL-2026), as previously described by Prochaska and Santamaria,29 modified by Gerhäuser et al.30 Briefly, cells were

grown to a density of 2 × 104 cells mL−1, in 200 μL of MEM-a

containing 5% antibiotic–antimycotic (Gibco, Thermo-Fisher, Waltham, MA, USA) and 10% fetal bovine serum at 37 ∘C in a 5% CO2 atmosphere. After a 24-h pre incubation period, the

media were changed and cells were treated with the indicated sample or control concentrations. Cells were incubated with test samples for an additional 48 h. QR activity was measured as a function of the NADPH-dependent menadiol mediated reduction of 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a blue formazan. Protein content was determined via the crystal violet staining of identical plates. Specific activity is defined as nmol of formazan formed per mg protein per min. Induction ratio (IR) of QR activity represents the specific enzyme activity of agent-treated cells compared with a DMSO treated control. Concentration to double activity (CD) was determined through a dose response assay for active substances (IR> 2). Data presented are the results of three independent experi-ments run in duplicate. 4-Bromoflavone (CD = 0.01 nmol L−1)

was used as a positive control. The chemoprevention index (CI = IC50/CD) was also determined. Data were processed using

non-linear regression analysis (TableCurve 2D V4; Systat Software, San Jose, CA, USA)

Statistical analysis

Analysis of variance (ANOVA) and Tukey’s post hoc test was con-ducted to statistically evaluate the differences among mean val-ues of the relative intensity of signals obtained for the six stages of maturation. Differences were considered significant if P< 0.05. Statistical analyses were performed using Assistat program version 7.7 beta.24,31

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(a) (b) (c) (d) (e) (f)

Figure 1. ESI(−)FT-ICR mass spectra of pineapple cv. Vitória at various maturation stages: (a) 0, (b) 1, (c) 2, (d) 3, (e) 4 and (f ) 5. The signals identified highlighted with red color number correspond to the dopant that is detected in five different forms.

RESULTS AND DISCUSSION

Physico-chemical analysis

The results of physico-chemical analyses are shown in Fig. S3a–c (Supporting Information). The TSS values increase as a function of the ripening degree of the fruit (stages 0 to 5), ranging from 14.6 to 17.5∘Brix (Fig. S3a), resulting from the ripening process where starch (accumulated in the growth phase of the fruit) is converted into soluble sugars. The decrease in TSS observed in stage 4 (Fig. S3a), could be related to its use as an energy source by the fruit itself.32

Correlating the physico-chemical analysis with the fruit con-sumption (Fig. S3a), stages 2 to 4 did not differ substantially in TSS, indicating that, from stage 2, the fruit reached the ideal amount of sugar for consumption. These data are in accord with the results obtained by Costa,33who evaluated the same variety of fruit.

Monitoring the TA values as a function of the maturation stage (Fig. S3b) shows TA values ranged from 0.81 to 0.45 from stage 0 to 5, indicating an accentuated decrease in organic acid content. This could be a result of the widely use of organic acid as substrates, along with sugar, in the respiratory process, providing energy at different stages of the life cycle of plant products. The decrease in TA can also be attributed to the conversion of acids to sugars.32

The TSS/TA ratio (Fig. S3c) increases from the stage 0 to the stage 5. This ratio is one of the most used forms to evaluate the flavor, being more representative than the measurement of isolated sugars or acidity, showing the balance between these two components.32

ESI(−)FT-ICR MS and ESI(−)MS/MS

Sugar analysis

ESI(−)FT-ICR MS analysis was performed for crude extracts in the maturation stages from 0 to 5 of the pineapple cv. Vitória (Fig. 1).

ESI(−) was employed because of the simplicity in the interpre-tation of signals in relation to the ESI(+) mode. Additionally, the ESI(−) has a lower ion suppression, related to the presence of min-erals containing ions such as Na+, K+, Ca+, among others, normally

present in natural products.27In ESI(−) (Fig. 1a–f ) occurs

deproto-nation of the analyte ([M − H]−ions), and chemical species such as

acids and sugars are easily ionized.34

Figure 1a–f shows the ESI(−)FT-ICR mass spectra for pineapple samples as a function of the maturation stage (0–5). The high content of sugars in samples, confirmed by physico-chemical tests, can also be observed by the intense signals generated by the presence of deprotonated disaccharides such as [C12H22O11− H]−

ions of m/z 341, and also by adduct molecules, such as [2 M − H]

and [3 M − H]− ions of m/z 683 and 1025, respectively, where

M = C12H22O11. This behavior was also seen by Wan et al.,35who

studied the ratio of the intensity of disaccharides adducts by sugar concentration in the sample.

Sucrose, the principal sugar identified, was detected in the deprotonated form, ion of m/z 341, as well as adducts of water ([C12H24O12− H]− of m/z 359), and chlorine ([C12H22O11+ Cl]− of

m/z 377). Besides, lactic acid ([C15H28O14− H]−of m/z 431), glucose

or its isomers ([C18H34O17− H]−of m/z 521), deuterated glucose

([C18H26D7O17− H]−of m/z 528) and citric acid ([C18H30O18− H]−

of m/z 533) are also reported.35Signals of lower intensity were also

observed for glucose molecules or its isomers ([C6H12O6− H]−ion

of m/z 179, and [C6H12O6+ Cl]− ion of m/z 215), and malic acid

([C10H18O11− H]−ion of m/z 313) (Fig. 1a–f ).

The dopant was detected in five different forms (compounds 2, 5, 9, 10 and 15) such as: the deprotonated molecule, [C6H5D7O6− H]−

of m/z 186, as chlorine adduct, [C6H5D7O6+ Cl]− of m/z 222, as

dimer, [C12H9D14O12− H]−of m/z 373, as adduct composed of one

dopant molecule and one glucose molecule, [C12H17D7O12− H]−

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Table 1. Primary metabolites identified from ESI(−)FT-ICR MS data as a function of the maturation stages

Compound Molecular formula m/zMeasured m/zTheoretical Error (ppm) DBE Proposed structure

1 [C6H12O6-H]− 179.05599 179.05611 0.68 1

Glucose or isomers

2 [C6H5D7O6-H]− 186.09998 186.10005 0.35 1

Glucose D7 standard

3 [C6H8O7-H]− 191.01963 191.01973 0.05 3

Citric acid or isomers

4 [C6H12O6+ Cl]− 215.03272 215.03279 0.31 0

Glucose or isomers/Cl−

5 [C6H5D7O6+ Cl]− 222.07677 222.07673 −0.05 0

Glucose D7 standard/Cl−

6 [C10H18O11-H] 313.07773 313.07763 −0.31 2

Glucose or isomers/malic acid or isomer

7 [C12H22O11-H] 341.10904 341.10894 −0.30 3 Sucrose or isomers 8 [C12H24O12-H] 359.11957 359.11950 −0.21 1 Sucrose or isomers/H2O 9 [C12H17D7O12-H]− 366.16352 366.16344 −0.24 1 Sucrose or isomers/glucose D7 10 [C12H9D14O12-H]− 373.20755 373.20740 −0.46 1 Glucose D7/glucose D7 11 [C12H22O11+ Cl] 377.08567 377.08561 −0.24 1 Sucrose or isomers/Cl−

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Table 1. Continued

Compound Molecular formula m/zMeasured m/zTheoretical Error (ppm) DBE Proposed structure

12 [C15H28O14-H] 431.14076 431.14063 −0.31 2

Sucrose or isomers/lactic acid or isomers

13 [C16H28O16-H] 475.13057 475.13046 −0.24 3 Not identified

14 [C18H34O17-H] 521.17268 521.17245 −0.69 2

Sucrose or isomers/glucose or isomers

15 [C18H26D7O17-H]− 528.21622 528.21626 0.07 2

Sucrose or isomers/glucose D7

16 [C18H30O18-H] 533.13636 533.13540 −0.79 4

Sucrose or isomers/citric acid or isomers

17 [C24H44O22-H]− 683.22545 683.22515 −0.44 3

Sucrose or isomers/sucrose or isomers

18 [C36H66O33-H]− 1025.34267 1025.34136 −1.28 4

Sucrose or isomers/sucrose or isomers/sucrose or isomers

molecule and one molecule of sucrose, [C18H26D7O17− H]−ion of

m/z 528 (Fig. 1).24

ESI(−)FT-ICR mass spectra of the pulp also show signals gener-ated by the presence of citric acid or its isomers in their deproto-nated form, [C6H8O7− H]−ion of m/z 191, and as an adduct with

sucrose, [C18H30O18− H]−ion of m/z 533. It is possible to observe

the signals decrease from stage 0 to stage 5, thus corroborating the physico-chemical tests, which revealed a decrease in acidity with the progression of fruit ripening (Fig. 1b).

Table 1 summarizes primary metabolites detected from ESI(−)FT-ICR MS, being identified 18 signals where informa-tion such as minimum formulae, mass errors (ppm), double bond equivalent (DBE), m/z measured and theoretical m/z values are reported.

A semi-quantitative evaluation was performed using the ESI(−)FT-ICR MS data (Fig. 1) by monitoring the signals produced

by the presence of sugars in each stage of maturation in the pineapple cv. Vitória. Figure 2 shows the ratio of the sum of sugar signals found in the pineapple (ions of m/z 179, 215, 341, 359, 366, 377, 521, 528, 533, 683 and 1025) divided by the sum of the deuterated glucose signals (ions of m/z 186, 222, 366, 373 and 528) as a function of the maturation stage. In general, a slight increase in the sugar content is observed, thus corroborating the data of Fig. S3a (Supporting Information). However, this behavior, statistically results in no significant difference among the ripening stages (Fig. 2).

In order to verify the proposed chemical structure (Table 1 and Fig. 1) of the main sugars detected, CID experiments were carried out for the signals of m/z 341, 431, 521, 533, 683 and 1025, where the ESI(−)MS/MS spectra are shown in Fig. S4a–f (Supporting Information). Neutral losses of monosaccharide isomers such as glucose/fructose (C6H12O6, 180 Da), water (H2O, 18 Da), C3H6O3

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Figure 2. Ratio of the sum of sugar signals found in the pineapple divided by the sum of the deuterated glucose signals as a function of the maturation stage. Same letters indicate that no significant difference exist among the data (P< 0.05).

(a) (b) (c) (d) (e) (f)

Figure 3. ESI(−)-FT-ICR mass spectra of the pineapple cv. Vitória phenolic extracts at various maturation stages: (a) 0, (b) 1, (c) 2, (d) 3, (e) 4 and (f ) 5.

(90 Da) and disaccharides (341 Da) were mostly identified. These fragments were also observed by Wan et al.35who analyzed

disac-charide isomer patterns. In their work, they showed the formation of non-covalent compounds among disaccharides, thus justifying, the formation of dimers or a sequence of adducts, as shown in Fig. 1.

The presence of sucrose/glucose dimers and trimers during the ionization process using the ESI(−) source can be explained based on the cluster formation during electrospray ionization.24–26

This hypothesis was confirmed by Oliveira et al.,24who doped a

solution of sucrose standard of M = C12H22O11at 20% m/v with 5 μL

of deuterated glucose and analyzed by ESI(−)-FT-ICR MS. Ions of

m/z 521, 683, and 1025 that correspond to clusters of glucose were

again produced, similar to the behavior observed in Fig. 1.

Phenolic compounds analysis

Extracts obtained from the samples of pineapple cv. Vitória were doped with deuterated glucose solution and ana-lyzed by ESI(−)FT-ICR MS (Fig. 3a–f ). A total of 11 chemical species was detected, with mass errors lower than 1 ppm. The ultra-high accuracy of the FT-ICR MS technique, as well as double bond equivalent (DBE) values, and minimum formula (CcHhOoSsNn) confirms the chemical identify of

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Table 2. Phenolic compounds species proposed from ESI(−)FT-ICR MS data of the pineapple extract cv. Vitória

Compound Molecular formula m/zMeasured Error (ppm) DBE Proposed structure MS/MS

1 [C11H14O3-H] 193.08703 −0.08 5 4-Allyl-2,6-dimethoxyphenol or isomers – 2 [C14H20O9-H]− 331.10360 −0,42 5 3,4-Dihydroxyphenylethanol-5-𝛽-D-glucose or isomers 197, 226, and 282 3 [C15H14O9-H] 337.05666 −0.46 9 Coumaroyisocitrate or isomers 198, and 173 4 [C16H20O9-H]− 355.10384 −1.08 7 Feruloyl hexoside or isomers 193 5 [C15H20O10-H]− 359.09857 −0.55 6 Syringoyl hexoside or isomers 197, and 176

6 [C16H18O10-H]− 369.08304 −0.86 8 Not identified 337, 223, 198, and 176

7 [C21H16O7-H]− 379.08308 −2.00 14 Not identified 198, and 176

8 [C17H22O10-H]− 385.11419 −0.44 7

Sinapic acid-hexoside or isomers

279, 267, 247, 223, 205, and 176

9 [C19H26O9-H]− 397.15058 −0.43 7 Not identified 198, and 176

10 [C18H26O12-H]− 433.13538 −0.54 6 Not identified 331, 289, and 176

11 [C27H22O6-H]− 441.13391 1.03 17 Not identified 249, 231, and 193

4-allyl-2,6-dimethoxyphenol or isomers, [C14H20O9− H]− ion

of m/z 193; 3,4-dihydroxyphenylethanol-5-𝛽-D-glucose or iso-mers, [C14H20O9− H]− ion of m/z 331; coumaroyl isocitrate or

isomers, [C15H14O9− H]− ion of m/z 337; feruloyl hexoside or

isomers, [C16H20O9− H]− ion of m/z 355; syringoyl hexoside or

isomers, [C15H20O10− H]−ion of m/z 359; sinapic acid-hexoside or

isomers, [C17H22O10− H]−ion of m/z 385 (Table 2).

MS/MS experiments were performed for the most phenolic compounds identified (Fig. S5, Supporting Information) with the aim of confirming its chemical structure and connectivity. The 4-allyl-2,6-dimethoxyphenol compound was detected with a mass error of −0.08 ppm. FT-ICR MS is capable of detecting ions of

m/z greater than 150 Da, therefore the ion fragmentation profile

of m/z 193 could not be acquired. However, this compound has already been detected in pineapple.36The fragmentation profile

of 3,4-dihydroxyphenylethanol-5-𝛽-D-glucose compound, already detected in Umbu,37is presented in Fig. S5a. A hexoside loss is

observed, yielding ions of m/z 197.8 (−134 Da) and m/z 225.6 (−105 Da), revealing a hexoside moiety in the molecule. Another compound detected in the pineapple Vitória is the coumaroy isocitrate, also previously described in the literature.17,38,39 The

fragmentation profile of this ion (Fig. S5b) indicates the loss of the coumaroyl group with subsequent dehydration of isoc-itric acid (m/z 173).39 Feruloyl hexoside has been detected in

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Figure 4. Variation content of the phenolic compounds in the pineapple

cv. Vitória as a function of the maturation stages. Different letters above the bars indicate that exist significant difference among the data (P< 0.05). this cultivar, being also reported by Steingass.17Experiments of

ESI(−) MS/MS (Fig. S5c) generated the fragment of m/z 193.1, due to a loss of the hexoside moiety, suggesting the structure of the present compound. Syringoyl hexoside (Fig. S5d) was also detected producing fragment of m/z 197.8, from the loss of hex-oside moiety.17Sinapic acid-hexoside (Fig. S5e) had already been

reported in pineapple,17,40 presenting ions at m/z 279.1, 267.1,

247.1, 223.1, 205.1 and 176.1 as main fragments. Our results are in accord with the fragmentation profile found in other papers41

and with the MassBank North America database for natural products.

In general, the relative intensity of these compounds increases as a function of the maturation stage, where a higher suppres-sion of the signals corresponding to the internal standard (red signal) is mainly observed from stage 3 (Fig. 3d). Values of m/z measured, m/z theoretical, DBE, mass error, structures of proposal phenolic compounds and fragmentation ions are summarized in Table 2.

Steingass et al.17detected more than 60 phenolic compounds in

several parts of the pineapple fruit (genotype MD2 ‘Extra Sweet’). Thirty-nine of these compounds were found in pulp. In the liter-ature, the compounds 1, 4 and 8 are reported in pineapple fruits. Compound 1 (Mw = 193 Da) was found by Wu et al.36when

ana-lyzing compounds responsible for the aroma of pineapple. Com-pounds 4 and 8 are part of the phenolic composition already described in the literature for pineapple42and were also found in

bananas.41

Compounds 2 and 5 were found in umbu fruit and previous studies showed they have a high antioxidant activity and can also inhibit acetylcholinesterase. The presence of these compounds in the fruit makes it possible to be active ingredients in functional food.37

The ESI(−)FT-ICR MS data were statistically evaluated by ANOVA and the Tukey test. When comparing the intensity ratio of all pos-sible phenolic compounds over the sum of the signals generated by the presence of the internal standard signals (ions of m/z 186, 222, 366, 373 and 528), a greater abundance of these species is observed in stages 2 to 4, with a maximum observed at stage 3 (Fig. 4).

In order to evaluate the influence of each phenolic compound as a function of the maturation stages of the fruit, a ratio of each compound signal by the sum of the absolute intensities of the internal standard was evaluated (Fig. 5). According to the statistical tests performed, there are no differences between the species represented in Fig. 5c–f as a function of stages, indicating

that the concentration of these compounds during the ripening process of fruits remains constant. The compounds present in Fig. 5a, b and d shows higher concentrations at intermediate stages (1 to 4), most of them, having a maximum concentra-tion during maturaconcentra-tion stage 3 (Fig. 5a–c, and f ), whereas for compound identified in Fig. 5g, a higher abundance is observed in stage 4.

Total polyphenolic analysis by Folin–Ciocalteu

The total phenolic compounds content in the phenolic extract were determined and the values are represented in Fig. S6 (Sup-porting Information). The results were statistically analyzed by ANOVA and the Tukey test. The results confirm that stage 3 (941.68 μg phenols per 100 g pulp) had higher values of pheno-lic content. Additionally, stages 1 (772.77 μg phenols per 100 g pulp), 2 (763.88 μg phenols per 100 g pulp), 4 (769.83 μg phenols per 100 g pulp) and 5 (861.64 μg phenols per 100 g pulp) did not differ statistically. These results corroborate with the data of ESI(−)FT-ICR MS (Figs 3–5). The total polyphenolic analysis shows an increase of these species with the ripening process through the stage 3, followed by a decreased until stabilization. This suggests physiological maturation process in the pineapple directly affects the presence of these species and their antioxidant activity. One hypothesis is that the cellular activity would undergo a slight increase, causing the metabolization of biomolecules.43Thus, in

this period, the fruits need a greater amount of energy to support all physiological pathways of the cell.44 The generation of this

energy by the respiratory system would produce free radicals and therefore, the fruits may need to activate defense mechanisms producing antioxidants to prevent damage that would be caused by oxidative stress.45

Determination of quinone reductase activity

Crude extracts and phenolic extract samples in the six matura-tion stages (0–5) were evaluated to determine the inducmatura-tion of quinone reductase. In all cases, i.e. for the six stages studied, we observed an induction rate (IR) lower than 2 in the crude extracts (Fig. S7a, Supporting Information) as well as in the phenolic extract (Fig. S7b).

Although the concentration of phenolic compounds is consider-ably higher in crude extracts (Fig. S7a and b), the hypothesis that the Folin–Ciocalteu reagent could have reacted with the organic acids present in the crude extracts in superior quantities cannot be omitted. The fact that the IR in the phenolic extracts was low could be attributed to the low concentration of phenolic compounds, showing that the amount of fruit used for the preparation of the extract has not been ideal (≈ 25 of fruit were used for the phenolic extract preparation).

A useful strategy for cancer chemoprevention is the evaluation of compounds present in elements of the human diet that can induce the detoxification potential phase II enzyme, such as QR induction. However, as can be seen, these compounds may not be present in this product of natural origin. Kang and Pezzuto46tested

2475 plant extracts and only 136 showed induction of QR activity. Li et al.47analyzed 162 samples of 61 fruits and only nine of this

samples showed activity in the induction of QR.

CONCLUSION

The physico-chemical tests (TSS, TA and TSS/TA ratio) performed on the samples showed the variation of sugars and acids presents

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(a) (b)

(c) (d)

(e) (f)

(g)

Figure 5. Ratio of the absolute intensity of the phenolic compounds by the sum of the absolute intensity of the internal standard signals as a function of maturation stages. Different letters above the bars indicate that exist significant difference among the data (P< 0.05).

in the cv. Vitória pineapple fruit as a function of their maturity stage. A maximum value was observed in stage 5. The ESI-FT-ICR MS technique was efficient in identifying of primary (carbohy-drates and organic acids) and secondary metabolites (13 phenolic compounds) present in the crude and phenolic extract of the sam-ples, respectively. Moreover, these species were quantified, and the majority was present in stage 3. This result corroborates the total polyphenol data. The induction of QR enzyme was tested for

both crude and phenolic extracts, and no induction of QR enzyme was observed.

ACKNOWLEDGEMENTS

The authors thank FAPES (65921208/2014), CAPES

(23038.007083/2014-40 and PROSUP), and CNPq (445987/2014-6, 401409/2014-7 and 314453/2014/8) for their financial support.

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SUPPORTING INFORMATION

Supporting information may be found in the online version of this article.

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Figure

Figure 1. ESI(−)FT-ICR mass spectra of pineapple cv. Vitória at various maturation stages: (a) 0, (b) 1, (c) 2, (d) 3, (e) 4 and (f ) 5
Table 1. Primary metabolites identified from ESI(−)FT-ICR MS data as a function of the maturation stages
Table 1. Continued
Figure 2. Ratio of the sum of sugar signals found in the pineapple divided by the sum of the deuterated glucose signals as a function of the maturation stage
+4

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