Overall dietary polyphenol intake in a bowl of strawberries: The influence of Fragaria spp. in nutritional studies

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Overall dietary polyphenol intake in a bowl of strawberries: The influence of Fragaria spp. in nutritional studies Mattia Gasperotti, Domenico Masuero, Fulvio Mattivi, Urska Vrhovsek * Department of Food Quality and Nutrition, Research and Innovation Centre, Fondazione Edmund Mach (FEM), via E. Mach 1, 38010 San Michele all’Adige, Italy

A R T I C L E

I N F O

A B S T R A C T

Article history:

Strawberry (Fragaria × ananassa Duch.) is an important source of polyphenols in the human

Received 29 May 2014

diet, and is considered a functional food. However, at the moment, it is not fully under-

Received in revised form 13 August

stood which compounds are involved. The aim was to present a comprehensive picture of

2014

the amount of polyphenols present in strawberry. A total of 56 individual compounds were

Accepted 25 August 2014

identified and quantified with a triple-quadrupole mass spectrometer, their concentration

Available online

ranging from 1 µg/100 g to 40 mg/100 g of fresh fruit. Furthermore, spectrophotometric assay for estimation of high-molecular mass proanthocyanidins was applied. Several com-

Keywords:

pounds were identified/quantified for the first time.

Strawberry

© 2014 Elsevier Ltd. All rights reserved.

Polyphenols Ellagitannins Anthocyanins Flavonoids Fruit composition

1.

Introduction

According to the statistical database of the Food and Agriculture Organization (FAO) of the United Nations (2013), strawberry (Fragaria × ananassa Duch.) represents one of the most important crops worldwide, with production and overall global trade value of 4.1 million tonnes. In terms of production, there has been a global increase of 20% from 2005 to 2011, with an increase of 50% in Europe and the United States alone. In the European market, average strawberry consumption is 2.16 kg/year per person, including raw fruit and processed food (Freshfel – European Fresh Produce Association, 2013). On

the basis of this figure, strawberries would clearly appear to be one of the most widely consumed berries. Furthermore, strawberries are one of the richest sources of healthful polyphenols in the human diet (Giampieri et al., 2012; Hannum, 2004). Regular consumption of strawberries in the diet contributes significantly to the intake of different phenolic compounds. Several classes of polyphenols are present in a bowl of strawberries (Bhagwat, Haytowitz, & Holden, 2013; Rothwell et al., 2013). Studies of different cultivars including profiling of polyphenols have already been performed in some cases, but without quantitative determination or detailed characterisation (Aaby, Ekeberg, & Skrede, 2007; Aaby, Mazur, Nes, & Skrede,

* Corresponding author. Tel.: +39 0461 615 140; fax: +39 0461 615 200. E-mail address: [email protected] (U. Vrhovsek). http://dx.doi.org/10.1016/j.jff.2014.08.013 1756-4646/© 2014 Elsevier Ltd. All rights reserved. Please cite this article in press as: Mattia Gasperotti, Domenico Masuero, Fulvio Mattivi, Urska Vrhovsek, Overall dietary polyphenol intake in a bowl of strawberries: The influence of Fragaria spp. in nutritional studies, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.08.013

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2012; Hanhineva et al., 2008; Josuttis et al., 2012; Seeram, Lee, Scheuller, & Heber, 2006). Furthermore, the chemical composition varies with the genotype, as already reported, but also depending on agricultural practices, the degree of maturity and environmental factors (Olsson et al., 2004; Tulipani et al., 2011). The most abundant polyphenols are proanthocyanidins and anthocyanins, followed by ellagitannins and flavonol glycosides (Giampieri et al., 2012; Hannum, 2004; Simirgiotis, Theoduloz, Caligari, & Schmeda-Hirschmann, 2009). Analysis of proanthocyanidins and ellagitannins, polymeric and oligomeric compounds, have not yet been fully studied, due to analytical problems and the complexity of their chemical characterisation. Only recently a complete profile for ellagitannins has been published, with identification and precise quantification of several compounds (Gasperotti et al., 2013), while similarly detailed information is still not available in the literature in relation to proanthocyanidins, although this class of compounds represents the main group of polyphenols in strawberries (Buendia et al., 2010). Strawberries are considered to be a functional food, with multiple health benefits, over and beyond nutritional needs, as demonstrated by extensive evidence regarding their antioxidant, anti-inflammatory, antihypertensive and antiproliferative properties (Basu, Nguyen, Betts, & Lyons, 2014). Epidemiological studies have shown an inverse correlation between the consumption of fruits rich in polyphenols and degenerative or proliferative, cardiovascular and chronic diseases (Basu et al., 2014; Giampieri et al., 2012; Scalbert, Manach, Morand, Rémésy, & Jiménez, 2005; Spencer, 2010). The potential effects of strawberry consumption on health have been well described in the literature for decades, but the precise mechanism involved in the healthy effect of polyphenols on the mammalian system is still generally unclear. It is not even fully understood which compounds are involved in rendering these biological effects. Comprehensive quantification of polyphenols in food matrices is an essential step in assessing the biological mechanism, nutritional properties and healthful effects. However, due to the huge variety of polyphenol compounds, their precise characterisation in fruits and vegetables, even the most widely consumed ones, is still unclear. To study the nutritional quality of any fruit it is essential to start with phytochemical profiling of the native compounds and then move on to more sophisticated bioavailability studies. Moreover, in bioavailability studies today the role of gut microflora also has to be taken into account and can improve the variability of the phytochemical compounds present in the fruits consumed (Del Rio, Costa, Lean, & Crozier, 2010; Tuohy, Conterno, Gasperotti, & Viola, 2012). The aim of this study was to present a comprehensive picture of the quantity of polyphenols present in the human diet. Attention was paid on the consumption of a standard bowl of different Fragaria spp. (100 g). Indeed, to assist long and expensive future nutritional studies on the effective healthy effects of strawberry, it is necessary to carry out detailed profiling of its polyphenolic composition. A total of 56 individual compounds were accurately identified and quantified with a triplequadrupole mass spectrometer, ranging from 1 µg/100 g, for low abundance polyphenols, to 40 mg/100 g of fresh fruit, along with precise estimation of high-molecular weight proanthocyanidins (HMWP).

To the best of our knowledge this is the first time that such a comprehensive targeted profiling has been performed on strawberry polyphenols, in terms of the number of individual compounds identified and quantified at the same time with proper standard references.

2.

Materials and methods

2.1.

Chemicals and reagents

All chromatographic solvents were HPLC grade or LC-MS grade for MS experiments. Acetonitrile, acetone, methanol and formic acid were purchased from Sigma Aldrich (Milan, Italy). Detailed information regarding the standard references used and vendors is reported in Vrhovsek et al. (2012a). Sanguiin H6 was isolated as described in Gasperotti, Masuero, Vrhovsek, Guella, and Mattivi (2010), agrimoniin as described in Vrhovsek et al. (2012b), and casuarictin and methyl ellagic acid rhamnoside as described in Gasperotti et al. (2013).

2.2.

Plant material

Different Fragaria spp. 6 Fragaria × ananassa cultivars (Alba, Clery, Eva, Elsanta, Darselect, and Portola) and 2 accessions (one red and one white) of Fragaria vesca were used. The plants were grown in the same experimental field in Vigalzano (Trentino, Italy; 520 AMSL) during the 2011 season. All cultivars were grown in standardised conditions in order to minimise the effect of environmental and agronomic factors. For each Fragaria spp. analysed, 3 repeats of 6 plants (18 plants in total) were considered, in order to obtain 3 biological replicates. The plants were cultivated with the soilless technique. Plants of the same cultivar were obtained vegetatively. For each cultivar, 250 g of strawberries were harvested at commercial ripeness. Detailed information regarding the agronomical conditions is reported in Gasperotti et al. (2013). Once collected the samples were stored in a freezer at –20 °C until extraction.

2.3.

Extraction of phenolic compounds

Extraction was performed as reported by Mattivi, Tonon, and Sanchez (2002) with an acetone/water mixture (70:30, v/v), avoiding the addition of acids in order to prevent any possible chemical hydrolysis. Briefly, 60 g of fresh fruits were homogenised in a 847-86 model Osterizer blender at speed one, in 2 × 100 mL of mixture acetone/water (70:30, v/v) for 1 min and made up to 250 mL with the same solvent. The centrifuged extracts were stored at –20 °C until analysis.

2.4.

Sample preparation for UPLC-MS/MS analysis

An aliquot of the extract, 1.5 mL, was completely dried under a stream of nitrogen. Then, 150 µL of methanol/water (50:50, v/v), with 1 mg/L of rosmarinic acid as internal standard, was added to the dry sample and filtered (0.22 µm) into an HPLC vial. The samples, 10 times concentrated, were now ready for further UPLC-MS/MS analysis. The internal standard, rosmarinic

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acid, was added to monitor the analytical performance of the instrument.

2.5. UPLC-MS/MS method for anthocyanins and the main ellagitannins An UPLC-MS/MS method was developed by combining anthocyanins, reported in the literature (Wu & Prior, 2005), and the main ellagitannins detected (Gasperotti et al., 2013) in the strawberry. The analysis was performed with an ultra performance LC (UPLC) system coupled with a triple quadrupole (TQ) mass spectrometer. The UPLC system used was a Waters Acquity UPLC system (Milford, MA, USA). Separation was performed with a Waters Acquity UPLC column (Milford, MA, USA), BEH C18 (150 mm × 2.1 mm, 1.7 µm) equipped with the proper guard column (Acquity UPLC BEH C18 VanGuard Pre-column, 5 mm × 2.1 mm, 1.7 µm), maintained at 60 °C. The injection volume was 2 µL and solvent flow was 0.45 mL/min. Mobile phases of 2.5% formic acid in Milli-Q water (A) and 0.1% formic acid in acetonitrile (B) were used. Chromatographic separation was performed using a gradient as follows: 0 min, 5% B; 0–10 min, 5–25% B; 10–10.5 min; 25–90% B; 10.5–12.50 min, 90% B, 12.60–16.60 min, 5% as equilibration time. The TQ mass spectrometer used was a Waters Xevo TQ (Milford, MA, USA) coupled with an electrospray interface. Direct infusions of each individual compound were used to optimise the MRM conditions. This was done automatically by the Waters IntelliStart software, and then manually checked for selection of the quantifier and qualifier ions. The MRMs for each compound are listed in Table 1 with cone voltages and collision energies. Calibration curves were performed for each individual compound for precise quantification, using commercially available anthocyanins and the isolated ellagitannins. Calibration curves were prepared ranging from 0.0005 to 20 mg/L.

2.6. UPLC-MS/MS method for multiple classes of phenolics Quantitative analysis for the rest of the polyphenols, excluding anthocyanins and ellagitannins, was carried out with a

previously validated method for the rapid quantification of multiple classes of phenolics (Vrhovsek et al., 2012a). Briefly, the analysis was performed with the same ultra performance LC (UPLC) system, coupled with the triple quadrupole (TQ) mass spectrometer used in the previous method. Separation was performed with a Waters Acquity UPLC column (Milford, MA, USA), HSS T3 (100 mm × 2.1 mm, 1.8 µm) equipped with the proper guard column, maintained at 40 °C. The injection volume was 2 µL, with a flow of 0.4 mL/min. Mobile phases of 0.1% formic acid in Milli-Q water (A) and 0.1% formic acid in acetonitrile (B) were used. Chromatographic separation was performed using a gradient as follows: 0 min, 5% B; 0–3 min, 5–20% B; 3–4.30 min; 20% B; 4.30–9 min, 20–45% B, 9–11 min, 45–100% B, 11–14 min, 100%; and 14.01–17 min, 5% as equilibration time. Further information regarding the UPLC-MS/MS method and MRM quantification is described in Vrhovsek et al. (2012a). A total of 44 polyphenols were identified among the samples and proper calibration curves were obtained for each individual compound for precise quantification. Calibration curves were prepared, with appropriate standard references, ranging from 0.0005 to 20 mg/L.

2.7.

Analysis of proanthocyanidins

The assay to determine the quantity of proanthocyanidins was based on their transformation into anthocyanidins, in a warm, acid environment (Porter, Hrstich, & Chan, 1985). An aliquot of the initial extract, 5 mL, was evaporated using a rotavapor in order to eliminate the acetone fraction and dissolved in a 5 mL flask with 0.05 M H2SO4. One millilitre of this solution was loaded onto a conditioned Sep-Pak C18 (Waters, Milford, MA, USA), which was previously washed with 2 mL of 5 mM H2SO4. Proanthocyanidins were eluted with 3 mL of MeOH and collected in a 50 mL flask, shielded from light with aluminium foil, containing 9.5 mL absolute EtOH. Twelve and a half millilitres of FeSO4 in concentrated HCl (300 mg/L) were added to the mixture and then immediately placed in a boiling water bath and refluxed for 50 min. After 10 min, the spectrum from 380 to 700 nm was recorded in a 10 mm cell, against a blank

Table 1 – MRM parameters of anthocyanins and ellagitannins. Compound

Anthocyanins Cyanidin-3-galactoside Cyanidin-3-sambioside Cyanidin-3-glucoside Cyanidin-3-arabinoside Cyanidin-3-rutinoside Pelargonidin-3-glucoside Peonidin-3-galactoside Pelargonidin-3-rutinoside Peonidin-3-rutinoside Ellagitannins Casuarictin Sanguiin H6 Agrimoniin Methyl ellagic acid rhamnoside

Quantifier ion

Qualifier ion

Rt (min.)

ES

Cone voltage (V)

Q1 m/z

Collision energy (V)

Q2 m/z

Collision energy (V)

Q2 m/z

2.54 2.81 2.83 3.09 3.15 3.45 3.63 3.94 4.37

+ + + + + + + + +

26 30 26 24 34 24 24 30 30

449.3 581.3 449.3 419.3 595.4 433.3 463.3 579.2 609.2

22 26 28 24 38 24 24 20 30

287.2 287.2 287.2 287.2 287.2 271.2 301.1 433.1 301.1

66 72 58 56 66 54 42 30 56

137.2 137.2 137.2 137.2 137.2 121.1 286.1 271.2 286.1

3.27 3.63 5.06 6.97

− − − −

66 34 32 30

934.7 934.1 934.1 460.8

26 22 22 18

632.7 632.8 632.8 314.8

46 38 46 20

300.8 300.8 300.8 299.8

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(water). The tangent from the minimum (450 nm) was drawn, and the absorbance between the maximum (550 nm) and the tangent was measured. To subtract natural anthocyanins present in the sample, which can interfere with the assay, one millilitre of the latter solution dissolved in 0.05 M H2SO4 was prepared under the same conditions. However, in this case the reaction was carried out in an ice bath and the absorbance obtained was then subtracted to obtain the net absorbance value. The proanthocyanidins concentration (mg/L) can conventionally be expressed as the cyanidin formed. Further information is available in the existing literature (Porter et al., 1985; Rigo et al., 2000; Vrhovsek, Mattivi, & Waterhouse, 2001).

2.8.

Explorative profiling of proanthocyanidins

An aliquot of the acetone extract was concentrated 4 times and dissolved in methanol/water (50:50, v/v) for explorative analysis using high definition mass spectrometry (HDMS), a Synapt Waters, coupled with UPLC and injected with a previously 60 min chromatographic method (Gasperotti et al., 2010). The molecular ions of the main oligomeric form were selected and extracted from the HDMS spectrum.

2.9.

Data analysis and statistical analysis

Data processing was carried out using Waters MassLynx 4.1 and TargetLynx software. All samples were in biological triplicates. The data collected were presented as average values and standard deviations. Principal component analysis (PCA) was performed with MassLynx Exstended Statistics by Umetrics.

3.

Results and discussion

3.1.

Variety

The varieties chosen for the dietary intake of polyphenols included 2 wild accessions of F. vesca, the red and white types, whereas the 6 main representative cultivars of Fragaria × ananassa were chosen: Portola, Eva, Elsanta, Clery, Darselect and Alba. The chosen cultivars were selected from those most widely consumed in both Italy and Europe. All the samples were grown and picked in Trentino, Italy under the same agronomical conditions and harvested at commercial maturity to avoid any environmental and agronomical effects on the polyphenolic composition (Pincemail, Kevers, Tabart, Defraigne, & Dommes, 2012). Due to the difference in the varieties, cultivars and accessions investigated, it was expected that the amount of polyphenols would make it possible to obtain a clear estimate of strawberry polyphenol intake in both Italy and Europe associated with consumption of a bowl of strawberries, 100 g, from the most widely used cultivars or wild species.

3.2.

Targeted profiling of strawberry polyphenols

The polyphenols in the fruit of the different Fragaria spp. considered were identified and quantified precisely with the use of a TQ mass spectrometer. With the use of the triple

quadrupole mass spectrometer several compounds were found in trace amounts, leading to quantification of some polyphenols that were not previously reported in strawberry. Each compound was identified and confirmed in the samples by checking whether both transitions (MRMs), qualifier and quantifier ions prepared for the method of quantification with the appropriate standard references, were present in the chromatograms. With the use of the two complementary UPLCMS/MS methods (Fig. 1), a total of 56 compounds were separated, identified and quantified with standard references in the samples. The list of all the compounds identified is presented in Table 2, together with their contents in each sample. The values are reported in µg/100 g, as the average value of the 3 replicates (and their standard deviation). The amounts found ranged from a few µg/100 g to many mg/100 g of fresh fruit. As the main outcome, a list of polyphenols was produced for the creation of databases reporting on polyphenol composition in fruits and vegetables (Bhagwat et al., 2013; Rothwell et al., 2013) providing more information also as compared to similar studies involving targeted profiling of strawberry polyphenols (Aaby et al., 2007, 2012; Maatta-Riihinen, Kamal-Eldin, & Torronen, 2004; Seeram et al., 2006; Simirgiotis & Schmeda-Hirschmann, 2010; Simirgiotis et al., 2009). Moreover, in comparison to untargeted profiling studies on the strawberry (Aaby et al., 2007; Fait et al., 2008; Hanhineva et al., 2008) a similar number of polyphenolic compounds was reported but with a major improvement in identification/ quantification and not only with tentative identification on the basis of fragmentation patterns. The average relative compositions (percent) of the main classes or groups of classes are listed in Table 3. In order to simplify the table, 5 groups were highlighted: proanthocyanidins (HMWP), ellagitannins, anthocyanins, flavonoids (adding together flavones, flavanones, flavan3-ols and flavonols) and other polyphenols (adding together benzoic acid derivatives, phenylpropanoids, stilbenes, phenol glycosides and dihydrochalcones). From Table 3 it is clear that proanthocyanidins are the most abundant polyphenols in strawberry (Buendia et al., 2010). The other two main groups are anthocyanins and ellagitannins, followed by flavonoids and traces of other polyphenols. Considering the total amounts in the strawberry, the highest values were found for the two accessions of F. vesca, 324 and 448 mg/100 g, respectively, for the red and white types. The amounts in the cultivars were lower, falling between 201 and 289 mg/100 g, respectively, for the Clery cv. and the Darselect cv. The data on total polyphenol content are in accordance with the average values calculated in a different manner, i.e. Follin assay for total polyphenol content, and present in databases, but of course with more detailed characterisation of the individual amounts (Bhagwat et al., 2013; Rothwell et al., 2013).

3.3.

Classes of polyphenols

Using 3 rapid methods, 2 instrumental and one spectrophotometric assay, it was possible to identify the main classes of polyphenols present in the strawberry, but it was also possible to detect many minor polyphenols present in traces. A total of 56 polyphenols are listed in Table 2, all identified and quantified with the proper standard references, with the

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5

Fig. 1 – UPLC profile of the polyphenols detected in strawberry.

exclusion of proanthocyanidins. In this way it was possible to build up a comprehensive picture of the presence and type of different classes of polyphenols in a bowl of strawberries, along with the quantity of proanthocyanidins, not described in detail at molecular level, that may be consumed in the human diet.

3.3.1.

Proanthocyanidins

Precise quantification and characterisation of these are still an open problem in terms of polyphenol analysis, due to their complexity, given that they are compounds with a high molecular weight. A number of strategies have been reported for the study of high molecular weight proanthocyanidins (HMWPs), including the use of spectrophotometric assay (Rigo et al., 2000),

normal phase separation coupled with fluorescent detector (Gu et al., 2002), phloroglucinol adducts or the thiolysis method (Kennedy & Jones, 2001). Interestingly, in terms of estimation, good correlation with the accepted Bate–Smith assay was shown as compared to more laborious instrumental methods (i.e. phloroglucinol adducts or thiolysis) (Vrhovsek et al., 2001). For this reason Bate–Smith assay was used for the analysis of HMWPs, given that other more precise methods are not reliable for their characterisation and quantification. However, as an exploratory example, the HDMS spectrum of a sample of the red type F. vesca showed extracted ions of the main tentative proanthocyanidins reported in strawberry (Fig. 2). HDMS analysis confirmed the presence of some peaks at m/z

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µg/100g of fresh fruit

F. vesca, red type

Strawberry, cv. Portola

Strawberry, cv. Eva

Strawberry, cv. Elsanta

Strawberry, cv. Clery

Strawberry, cv. Darselect

Strawberry, cv. Alba

1.3 18.5

0 5

5.5 22.8

0 4

n.d. 16.6

0 2

0.1 17.5

0 2

1.7 26.9

1 8

0.1 19.5

0 2

0.1 32.8

0 4

n.d. 24.3

0 5

29.5 7.3 3.6 1.2

6 1 0 1

59.9 5.6 3.7 5.0

9 0 0 1

2.1 1.6 0.8 3.8

1 0 0 1

19.4 1.3 1.4 6.9

12 0 0 2

63.7 1.5 0.4 9.4

5 0 0 5

18.5 1.1 1.0 16.3

4 0 0 3

44.5 2.3 0.5 7.0

24 1 0 1

27.4 2.0 0.6 6.7

30 0 0 1

1.1 13.1 75.6

0 4

0.8 7.6 110.8

0 5

1.1 4.0 30.0

0 3

1.6 8.2 56.4

1 1

1.2 7.7 112.6

0 1

1.5 1.8 59.6

1 0

0.9 13.9 101.9

0 4

1.3 5.2 67.5

0 3

2.2 1.7 20.1 9.5 2.3 35.8 1.6 5.4 7.0 2.2 2.2 0.1 2.9 0.0 3.0 15.8 0.8 0.6 17.3 2.2 2.2 4515.6 48.1 381.5 2468.6 7413.8 1.9 6.4 8497.3 194.1

0 1 0 1 1

2.0 1.5 7.1 13.9 2.4 27.0 1.6 3.6 5.3 1.8 1.8 0.1 7.1 1.9 9.1 11.8 0.9 1.2 13.9 2.1 2.1 3810.5 43.4 298.4 2565.2 6717.5 5.3 17.6 9678.7 195.0

0 0 1 0 0

3.8 1.2 0.9 13.8 0.5 20.3 0.5 2.9 3.4 2.6 2.6 0.5 46.5 5.8 52.7 2.0 1.7 0.6 4.2 0.2 0.2 1810.3 19.2 203.1 1233.6 3266.3 5.8 3.4 2.3 97.8

1 0 0 4 0

18.9 2.4 0.8 23.3 0.5 45.8 2.0 3.2 5.2 3.3 3.3 0.2 40.7 1.9 42.8 1.7 1.3 0.5 3.5 0.2 0.2 4245.8 35.6 432.2 1633.1 6346.7 13.9 3.1 0.9 265.3

5 0 0 3 0

11.0 1.6 1.0 75.4 1.3 90.2 1.4 3.4 4.8 3.6 3.6 1.5 80.1 14.1 95.7 5.5 2.3 0.5 8.2 0.2 0.2 5991.8 53.3 409.3 2837.5 9291.9 22.0 13.2 2.1 160.1

1 1 0 10 0

13.3 3.4 0.9 32.0 0.6 50.2 1.6 2.5 4.2 1.6 1.6 0.4 76.1 2.7 79.3 2.8 1.0 0.3 4.1 0.2 0.2 2069.9 18.4 174.7 1094.6 3357.5 14.3 2.5 0.9 100.8

4 2 0 3 0

9.7 0.8 1.3 12.8 0.6 25.1 2.2 4.1 6.2 3.5 3.5 1.0 98.2 12.0 111.2 2.0 1.5 0.4 3.9 0.3 0.3 8065.7 86.4 581.6 2883.6 11,617.3 19.7 6.0 1.1 227.5

2 0 0 5 0

11.9 3.9 1.0 30.2 0.3 47.3 1.6 2.8 4.4 1.1 1.1 0.3 83.0 4.5 87.8 1.8 1.3 0.5 3.6 0.2 0.2 3659.7 22.4 179.9 1341.5 5203.5 19.7 2.6 3.7 103.0

7 3 0 5 0

0 1 1 0 1 0 2 0 0 0 976 11 54 529 0 1 810 30

0 1 0 0 1 1 2 0 1 0 572 5 60 186 0 1 1224 20

0 0 1 0 5 1 0 0 0 0 906 3 37 151 1 0 1 4

0 0 1 0 1 0 0 0 0 0 788 9 92 435 1 0 0 46

1 0 0 1 7 3 1 1 0 0 438 8 32 442 7 2 1 12

0 1 1 0 9 1 1 0 0 0 139 5 13 91 1 1 0 31

1 1 0 0 21 2 0 0 0 0 1502 18 171 726 5 1 0 35

0 0 0 0 21 1 1 0 0 0 936 7 44 226 2 1 0 15

(continued on next page)

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1 Anthranilic acid 2 p-Hydroxybenzoic acid 3 Cinnamic acid 4 Vanillin 5 Vanillic acid 6 2,6-Dihydroxy benzoic acid 7 Methyl gallate 8 Catechol Total benzoic acid derivatives 9 p-Coumaric acid 10 Caffeic acid 11 Ferulic acid 12 Chlorogenic acid 13 trans-Coutaric acid Total phenylpropanoids 14 trans-Piceide 15 cis-Piceide Total stilbens 16 Arbutin Total phenol glycosides 17 Phloretin 18 Phlorizin 19 Trilobatin Total dihydrochalcones 20 Luteolin 21 Luteolin-7-O-Glc 22 Hesperidin Total flavones 23 Naringenin Total flavanones 24 Catechin 25 Epicatechin 26 Procyanidin B1 27 Procyanidin B3 Total flavan-3-ols 28 Kaempferol 29 Quercetin 30 Taxifolin 31 Kaempferol-3glucoside

F. vesca, white type

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Table 2 – Quantitative results of all the polyphenols detected in Fragaria spp. Data are the average of the three replicates and their standard deviations are in italics.

µg/100g of fresh fruit

F. vesca, red type

Strawberry, cv. Portola

Strawberry, cv. Eva

Strawberry, cv. Elsanta

154.3 71.9

62 14

406.9 178.7

3 13

47.8 2.9

8 0

37.7 4.5

9 1

35.9

14

92.2

5

1.5

1

7.0

2

n.d.

0

0

15.8

2

63.7

9

0.1

0

9

2.1

1

1.4

1

1.4

n.d. 35.7

0 17

n.d. 75.5

46.7 8.8

Strawberry, cv. Clery 9 2

40.9 4.1

Strawberry, cv. Darselect

Strawberry, cv. Alba

19 2

32.6 4.7

8 1

73.8 11.2

14 2

0

4.6

1

11.9

3

24.8

13

63.6

14

37.0

10

1

1.2

1

0.7

0

1.3

0

n.d.

n.d. n.d.

0 0

n.d. 0.3

0 0

8.4 1.1

2 0

3.3 1.4

1 1

0.2 n.d.

0 0

6.1 1.8

5 1

2.8 0.6

1 0

16.4 6.5

6 3

3.5

1

5.8

2

1.3

0

1.5

1

1.0

0

1.1

0

1.6

1

1.7

0

3.0 296.7 56.7

1 78 31

20.4 540.1 78.3

1 36 14

5.2 928.7 384.7

1 181 18

11.3 604.2 484.7

4 149 43

21.1 420.8 280.7

11 147 73

9.6 507.4 480.3

1 180 125

11.9 580.3 667.2

3 130 110

16.5 772.3 301.2

0 261 65

9357.3 n.d. 3.5 1.6 n.d. n.d. n.d.

0 2 1 0 0 0

11,294.7 1.3 12793.1 567.5 21.4 n.d. 28006.9

0 1381 25 3 0 4952

n.d.

0

n.d.

0

431.9

29

362.4

53

n.d. n.d. 5.1 5529.4 10091.2 1505.2 84329.1 28349.6

0 0

n.d. n.d. 41,390.2 7772.6 11022.3 2202.5 61627.6 35969.6

0 0

17.8 0.2 24,134.7 814.0 11360.8 1144.1 7635.6 10.9

3 0

22.3 0.4 37,112.2 711.9 19926.9 1086.3 19094.5 2.8

7 0

129,804.4 177537 177,537 324.3

570 686 177 5145 2276

10003

118,594.6 270012 270,012 448.2

1232 1974 62 6469 3659

13853

1508.8 n.d. 414.1 34.3 0.0 9.7 23226.6

20,965.4 171307 171,307 221.3

0 51 2 0 2 927

260 2946 127 554 3

6105

1503.8 n.d. 234.9 23.1 0.6 2.2 36466.1

40,822.5 186431 186,431 272.4

0 28 3 0 0 1554

226 2970 103 2874 0

7819

978.2 n.d. 182.3 17.8 n.d. n.d. 34614.8 0.4 31.1 n.d. 34,846.3 316.7 3038.1 209.1 17760.1 9.8 21,333.7 165253 165,253 232.0

0 40 4 0 0 6642

1195.9 n.d. 162.2 15.1 n.d. 2.8 36021.7

0 1 0 0 0 5209

1624.9 n.d. 193.9 19.3 n.d. 3.5 43612.0

0 56 4 0 1 2288

1378.8 n.d. 621.0 56.4 n.d. 27.9 34045.3

0 43 4 0 4 5058

0

365.7

42

600.1

62

663.3

86

14 0

16.2 0.8 36,584.5 692.3 9063.2 446.1 13334.8 8.7

4 0

45.5 0.4 44,474.6 551.0 9375.3 263.6 15510.8 11.5

20 0

35.3 4.5 35,453.7 1132.0 5046.0 244.5 9271.7 3.2

13 1

276 850 24 1388 2

12214

23,545.1 135706 135,706 200.6

174 1700 45 2626 1

11656

25,712.3 205187 205,187 288.9

74 1692 4 861 1

34189

15,697.5 177470 177,470 235.4

185 1094 7 1490 0

16583

ARTICLE IN PRESS

32 Quercetin-3-glucoside 33 Isorhamnetin-3glucoside 34 Quercetin-3acetylglucoside 35 Kaempferol-3rutinoside 36 Quercetin-3glucosilarabinoside 37 Rutin 38 Isorhamnetin-3rutinoside 39 Quercetin-3,4diglucoside 40 Dihydrokaempferol 41 Quercetin-3-glucuronide 42 Kaempferol-3glucuronide Total flavonols 43 Cyanidin-3-arabinoside 44 Cyanidin-3-glucoside 45 Cyanidin-3-galactoside 46 Cyanidin-3-sambioside 47 Cyanidin-3-rutinoside 48 Pelargonidin-3glucoside 49 Pelargonidin-3rutinoside 50 Peonidin-3-galactoside 51 Peonidin-3-rutinoside Total anthocyanins 52 Ellagic acid 53 Casuarictin 54 Sanguiin H-6 55 Agrimoniin 56 Methyl ellagic acid rhamnoside Total ellagitannins Proanthocyanidins (HMWP) Total proanthocyanidins Total polyphenols (mg/ 100g)

F. vesca, white type

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Table 2 – (continued)

7

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Table 3 – Average relative composition (percent) of different Fragaria spp. F. vesca, F. vesca, Strawberry, Strawberry, Strawberry, Strawberry, Strawberry, Strawberry, white type red type cv. Portola cv. Eva cv. Elsanta cv. Clery cv. Darselect cv. Alba High molecular weight proantocyanidins Ellagitannins Anthocyanins Flavonoids Other polyphenols

54.8

60.2

77.4

68.4

71.2

66.5

71.0

75.3

40.0 0.0 5.2 0.0

26.5 9.2 4.0 0.1

9.5 10.9 2.2 0.0

15.0 13.6 2.9 0.1

9.2 15.1 4.4 0.1

11.0 20.2 2.2 0.1

8.9 15.4 4.6 0.1

6.7 15.1 2.8 0.1

577 (dimers), 865 (trimers), 1153 (tetramers) and 720 (bicharged pentamers) corresponding to (epi)catechin-based proanthocyanidins. Representing the most abundant polyphenols in the strawberry (Table 3), the amount consumed in a bowl of strawberries is between 135 and 205 mg/100 g, respectively, for the Clery cv. and the Darselect cv., while the amount is higher in the red type of wild strawberry, namely 270 mg/100 g. As a major class

of strawberry polyphenols, they represent between 54.8 and 77.4% of polyphenolic compounds. The cultivar with the highest relative value is the Portola cv.

3.3.2.

Anthocyanins

The red colour of strawberries comes from the presence of anthocyanins. In strawberries, anthocyanins are made up of a mixture of cyanidin, pelargonidin and peonidin glycosides,

Fig. 2 – Preliminary high definition MS analysis of proanthocyanidins. Please cite this article in press as: Mattia Gasperotti, Domenico Masuero, Fulvio Mattivi, Urska Vrhovsek, Overall dietary polyphenol intake in a bowl of strawberries: The influence of Fragaria spp. in nutritional studies, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.08.013

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mainly glucosides, galactosides and rutinosides (Wu & Prior, 2005). On the contrary, anthocyanins are not present in the white type of F. vesca, with the exception of some traces of cyanidin glucoside and galactoside, and as a consequence the fruit is white/green. The main anthocyanin in the red strawberry is pelargonidin-3-glucoside (Koponen, Happonen, Mattila, & Torronen, 2007; Wu & Prior, 2005) (up to 43 mg/100 g in the Darselect cv.), followed by cyanidin-3-glucoside (up to 12 mg/ 100 g in the red type of F. vesca and up to 0.4 mg/100 g in the cultivars) and cyanidin-3-galctoside. Cyanidin-3-rutinoside, pelargonidin-3-rutinoside and peonidin-3-galactoside were also found in the cultivars, while they were not present at all in F. vesca. A certain variability in terms of the compounds identified and the amounts can be observed in the cultivars. The Elsanta cv. had the lowest number of different anthocyanins, although the total amount recovered was nearly 35 mg/100 g, while the Portola cv., with the largest number of different anthocyanins, was the cultivar with the lowest total amount, 24 mg/100 g.

3.3.3.

Ellagitannins

In the strawberry ellagitannins are the third most concentrated class of polyphenols, the main ellagitannins having recently been isolated for their clear identification and precise quantification (Gasperotti et al., 2013; Vrhovsek et al., 2012b). From previous publications, only the 3 main ellagitannins – agrimoniin, casuarictin and sanguiin H-6 – and ellagic acid with the methyl ellagic acid rhamnoside, isolated and characterised using NMR (Gasperotti et al., 2010, 2013; Vrhovsek et al., 2012b), were considered for the purpose of having a picture of the overall intake of polyphenols present in a bowl of strawberries. More in-depth profiling with many other ellagitannins has already been proposed for the same cultivars (Gasperotti et al., 2013). As already shown, agrimoniin was the main ellagitannin in the strawberry (from 7.6 to 84 mg/100 g), followed by casuarictin (from 3 to 20 mg/100 g) and then sanguiin H-6 (from 0.2 to 2.2 mg/100 g. The concentration of ellagic acid was from 0.3 to 7 mg/100 g. Methyl ellagic acid rhamnoside was present in large amounts in F. vesca, with a concentration of 28 and 35 mg/100 g, respectively, for the white and red types. Interestingly, traces of these ellagic conjugates were also found in the cultivars (2.8–11 µg/ 100 g), while in previous publications they were not reported (Gasperotti et al., 2013).

3.3.4.

Flavonoids

The fourth most characteristic group of polyphenols (see Table 3) in the strawberry is the flavonoid family (without considering the anthocyanins described above as an independent class of polyphenols). The flavonoids found in strawberry were flavones, flavanones (naringenin alone), flavan-3-ols and flavonols and dihydroflavonols. In the literature considered for this publication, no information regarding the survey of flavanones, naringenin or flavones, namely luteolin or hesperidin, was found. However, some traces of luteolin, luteolin-7-glucoside, hesperidin and naringenin were detected in the samples chosen. The main flavan-3-ol detected in the strawberry was catechin (1.8–8 mg/100 g), while epicatechin was about 10 times

9

lower in all the Fragaria spp. The dimers of the flavan-3-ols identified were instead procyanidin B1 and B3, in accordance with the literature (Aaby et al., 2007). In this case procyanidin B3 was about one grade of magnitude higher then procyanidin B1 (1–2.8 as compared to 0.17–0.58 mg/100 g). These flavan-3-ols are not listed in the HMWP group, since they do not respond so well to the Bate–Smith assay, being more selective for high molecules (Vrhovsek et al., 2001) and due to the fact that there are standard references available for quantification of these molecules. The content of overall flavonols, i.e. quercetin, unbound kaempferol and glycosides and isorhamnetin glycosides, varied in the samples from 0.9 mg/100 g (Portola) to 11 mg/100 g (F. vesca, red type). Of the glycosides, quercetin-3-glucuronide was most abundant in all cultivars and wild types, in accordance with the literature for the cultivars (Aaby et al., 2012; Buendia et al., 2010) but not reported in relation to the wild types (Mikulic-Petkovsek, Slatnar, Stampar, & Veberic, 2012). Quercetin-3-glucuronide was followed by kaempferol-3glucuronide, which was the second most abundant (from 0.28 to 0.66 mg/100 g) and kaempferol-3-glucoside (from 0.09 to 0.26 mg/100 g) in the cultivars selected. In the wild type, quercetin-3-glucuronide was followed by quercetin-3-glucoside and kaempferol-3-glucoside, in accordance with the literature (Mikulic-Petkovsek et al., 2012). Interestingly, five different adducts made with rutinose were present in almost all the cultivars considered, but have never been reported in the literature (Aaby et al., 2012; Buendia et al., 2010; Maatta-Riihinen et al., 2004; Mikulic-Petkovsek et al., 2012; Simirgiotis & Schmeda-Hirschmann, 2010), with the exception of isorhamnetin rutinoside (Hanhineva et al., 2008) and quercetin rutinoside (rutin) (Fait et al., 2008; Seeram et al., 2006), but with no information regarding their concentration. Other rutinose adducts clearly found for the first time was kaempferol3-rutinoside. Likewise, the free forms of flavonols, i.e. quercetin and kaempferol, have never been reported in the literature, or in untargeted profiling studies of the strawberry (Fait et al., 2008), and some traces were detected in the samples. The dihydroflavonol family (added to flavonols in Table 2), i.e. dihydroquercetin (taxifolin) and dihydrokaempferol in the Fragaria spp. considered, was present. Quantitative amounts are proposed for the first time in this publication, since their presence was only tentatively reported in an untargeted study (Aharoni et al., 2002). Remarkably, taxifolin appears to be the most abundant flavonoid, after anthocyanins, with a concentration of 9.5 and 8.5 mg/100 g, respectively, in the red and white types of wild strawberry.

3.3.5.

Other polyphenols

In this work it was possible to significantly increase the number of compounds detected with the use of a TQ mass spectrometer and also to characterise in detail many other polyphenols which were not as abundant as the previous polyphenols discussed above. These minor polyphenols were included in the category of other polyphenols (incorporating benzoic acid derivatives, phenylpropanoids, stilbenes, phenol glycosides and dihydrochalcones) in Table 3 and they represent less than 0.1% of the total amount of polyphenols that may be consumed in a bowl of strawberries. The individual concentration was no more than 100 µg/100 g in any of the samples.

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8

Hotelling’s T2 Ellipse (95%) = (13,32; 8,123) R2X[1] = 0,4247 R2X[2] = 0,158

A. Darselect

6

Elsanta Elsanta Darselect Darselect

4

Elsanta 2

t[2]

V. Red Eva Eva

0

-2

V. Red V. Red

V. V. White White

Eva

V. White

Clery Alba Clery Clery Portola Portola Alba Alba Portola

-4

-6

-8 -14

-13

-12

-11

-10

-9

-8

-7

-6

-5

-4

-3

-2

-1

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

t[1]

7 Hotelling’s T2 Ellipse (95%) = (8,123; 6,675) R2X[2] = 0,158 6 R2X[3] = 0,1067

B.

5

V. White V. White V. White

4 3 2

Eva Eva

t[3]

1

Eva

Portola Portola Portola Clery Clery Clery

0

Darselect Darselect Elsanta

Darselect

Elsanta

-1 Alba

-2

Elsanta

Alba

-3

Alba

V. Red V. Red

-4

V. Red

-5 -6 -7 -8

-7

-6

-5

-4

-3

-2

-1

0

1

2

3

4

5

6

7

8

t[2]

Fig. 3 – Principal component analysis (PCA) plots. PCA score plot for different Fragaria spp. on PC1 and PC2 (A) and on PC2 and PC3 (B). V. Red and V. White are referred to Fragaria vesca, red and white type.

Compounds derived from benzoic acid, i.e. p-hydroxybenzoic acid, cinnamic acid, vanillin, vanillic acid, 2–6-dihydroxybenzoic acid methyl gallate or cathecol, were present with a total concentration spanning from 30 to 112 µg/100 g. They have already been identified as putative metabolites but with no information about their concentration (Aharoni et al., 2002). Caffeic acid, ferulic acid, p-coumaric acid and chlorogenic acid – classified as phenylpropanoids – have already been

reported in the literature but only confirmed with the photodiode array system (Maatta-Riihinen et al., 2004), while chlorogenic acid was detected in untargeted data (Hanhineva et al., 2008) as a putative metabolite with no information about concentration, although chlorogenic acid was the most abundant among the latter compounds (75 µg/100 g in cv. Elsanta). Traces of trans-coutaric acid were also found for the first time.

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Some stilbenes were also detected, in particular transpiceid and cis-piceid, which have never been detected in the strawberry, due to their extremely low concentrations. The total concentration of stilbenes was from 3.4 to 7 µg/100 g in the Portola cv. and white type of F. vesca, respectively. Phloretin and phlorizin, belonging to the dihydrochalcones family, were reported for the first time by Hilt et al. (2003), but no evidence was found in other publications. The presence of another dihydrochalcone, namely trilobatin, detected in traces in the samples considered, has also never been reported in the literature.

3.4.

11

complete list of polyphenols present in the strawberry, but to our knowledge, at the moment they offer the most up-to-date list of clearly identified compounds, without any tentative identification, since they were compared with their reference compounds. Furthermore, quantitative data are provided for all the compounds and estimation only for the complex HMWP family. Several compounds were identified or quantified for the first time, not only because they are present in traces. Indeed, taxifolin was quantified for the first time in F. vesca, but represents one of the most concentrated polyphenols after anthocyanins and ellagitannins.

Principal component analysis

Acknowledgments PCA was applied to the entire data set collected for the 6 different strawberry cultivars and the 2 different types of F. vesca, with 3 biological replicates for each of them. The values of each variable were scaled automatically with unit variance scaling (UV). After scaling each parameter contributes equally to data variance and all variables are given equal weight for the principal component. PC1 represented 42% of total variance while PC2 represented 16%. The sample score plot for PC1 and PC2 is shown graphically in Fig. 3A and some distinctions between the different Fragaria spp. may be observed. Clearly, both types of F. vesca are located alone and distinguished by the strawberry cultivar. Red and white types of F. vesca show how the presence of anthocyanins, for the red type, or the absence of anthocyanins, for the white type, and the main cause of the red colour (due by the presence of anthocyanins) are not so relevant for PC1 and PC2 in terms of separation, along with all the other variables. The cultivars are instead grouped in the left part of the plot and two subgroups can be easily detected. In the upper part, Darselect and Elsanta cultivars represent one subgroup and one explanation may be due to the fact that Darselect is a cultivar deriving from the cross between the Parker × Elsanta cultivars. In the lower part of the plot the Alba, Clery, Portola and Eva cultivars are instead grouped together. However, the Eva cultivar may also be considered to be intermediate between the upper and lower subgroups of cultivars. To find out more about the relationships between variables and samples, other PCs were considered. PC3, which represents 11% of total data variance, was also considered. The sample score plot for PC2 and PC3 is shown graphically in Fig. 3B. From this plot it can be clearly observed that the F. vesca types are now at the opposite of the plot, proving that for this plot the presence of anthocyanins is strongly relevant in their relationship. On the contrary, separation is similar to the other plot for the cultivars, with the presence of 2 subgroups.

4.

Conclusion

This work offers some important new aspects and improvements in the study of strawberry polyphenols, which may also apply to the study of other polyphenol-based foods. The 56 polyphenols listed in this publication certainly do not represent a

This research was supported by the GMPF International PhD programme founded by the Autonomous Province of Trento, Italy. We thank Luisa Palmieri and Paolo Martinatti for supplying the strawberry samples.

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Please cite this article in press as: Mattia Gasperotti, Domenico Masuero, Fulvio Mattivi, Urska Vrhovsek, Overall dietary polyphenol intake in a bowl of strawberries: The influence of Fragaria spp. in nutritional studies, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.08.013

ARTICLE IN PRESS journal of functional foods ■■ (2014) ■■–■■

Vrhovsek, U., Mattivi, F., & Waterhouse, A. L. (2001). Analysis of red wine phenolics: Comparison of HPLC and spectrophotometric methods. Vitis, 40(2), 87–91. Wu, X., & Prior, R. L. (2005). Systematic identification and characterization of anthocyanins by HPLC-ESI-MS/MS in

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common foods in the United States: Fruits and berries. Journal of Agricultural and Food Chemistry, 53(7), 2589–2599. doi:10.1021/ jf048068b.

Please cite this article in press as: Mattia Gasperotti, Domenico Masuero, Fulvio Mattivi, Urska Vrhovsek, Overall dietary polyphenol intake in a bowl of strawberries: The influence of Fragaria spp. in nutritional studies, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.08.013