1. Introduction
In recent decades, researchers and food manufacturers have become increasingly interested in bioactive compounds because of their abundance in our diet, as well as the recommendations to consume a diet rich in fruits and vegetables containing antioxidants that counteract the oxidative damage to biological targets [
1,
2,
3,
4]. Their consumption is associated with the prevention of human diseases, such as cardiovascular diseases, chronic inflammation, or cancer [
4,
5,
6].
Strawberry (
Fragaria x
ananassa Duch.) is one of the most usually consumed berries, eaten either fresh or processed [
7,
8], and is an important source of nutrients and bioactive compounds, such as organic acids, vitamins, or phenolic compounds [
9,
10]. The wide array of phenolic compounds in this fruit include hydroxycinnamic acids (
p-coumaric), hydroxybenzoic acids (gallic and ellagic acids), hydrolysable tannins (ellagitannins), flavan-3-ols (catechins), flavonols (quercetin, kaempferol, and myricetin), and anthocyanins, with pelargonidin–3–glycoside as the major compound [
11]. Among them, anthocyanins are quantitatively the main phenolic compounds in strawberries and are responsible for their bright red color and significantly affect therefore their acceptability and organoleptic characteristics such as visual quality (good color or undesirable browning) and taste (astringency or flavor) [
12,
13].
Several factors may affect the phenolic contents of fruit, including genotype, physiological status (e.g., ripeness at harvest), environmental parameters during growth, as well as postharvest processing and storage practices [
14,
15]. In general, the phenolic acid content of fruits may decrease during ripening, whereas anthocyanin levels may increase [
16]. Furthermore, these contents are also affected by environmental factors such as pedoclimatic or agronomic factors, including types of growth conditions under protected greenhouses or in open fields, or their cultivation under conventional production systems or ecological production, and hydroponics [
17].
Previous research has reported fruits being obtained that are enriched in bioactive compounds through genetic engineering [
18]. However, other alternatives to improve crops and fruit quality by means of enriching the edible organs with bioactive phytochemicals using agronomical practices and avoiding the genetic manipulation of the plants are of increasing interest for a more sustainable approach [
13,
19,
20,
21,
22,
23].
Saline stress is a common situation in the agroecosystems of the Mediterranean basin, where soil-water availability is a limiting factor. Meanwhile, mineral nutrients are essential for plant development; one of them, nitrogen (N), is among the most important components of amino acids, proteins, nucleic acids, chlorophylls, and other metabolites critical for plant survival [
24]. However, the nitrogen supply is limited due to the pollution of aquatic systems (aquifers) or soils, particularly in sensitive ecological areas as Almería (south-eastern Spain). Based on this situation, several studies have reported the effect of salt stress and nitrogen supply on nutritional and phytochemical content through cultivation practices. Changes in the nitrogen supply affected the organoleptic properties of strawberries, such as their firmness or soluble solid contents [
25]. Another study showed an increase in phenolic compounds induced by salt stress in aromatic plants [
26] and medicinal herbs [
27], while these compounds were higher in tomato fruits when they had a lower supply of nitrogen [
28].
Besides, it is essential to search for an alternative harvesting period for strawberries to avoid the production peaks in Spain from March to May while keeping a high-quality product on the market and improving the existing fruit offer for a longer time. In this context, the aim of this research was to evaluate the phenolic profile and the antioxidant capacity of strawberries (cv. Primoris) cultivated under conditions of reduced nitrogen and adverse irrigation (high salinity) similar to those prevailing in Almería (south-eastern Spain).
2. Materials and Methods
2.1. Fruit Samples
Strawberry samples of
Fragaria x
ananassa Duch cv. Primoris were collected from a cultivar area at the “La Mojonera” IFAPA (Almería, Spain, latitude 36°45′ N, longitude 2°42′ W). The plants were raised in a plastic-covered greenhouse (960 m
2) from October 2011 to April 2012. The conditions of the hydroponic system and irrigation used to cultivate the strawberry plants were a basic solution with an electrical conductivity (EC) of 1.7 dS/m, 9 mmol/L of nitrogen (N), by using potassium nitrate and calcium nitrate fertigation, and 2 mmol/L of NaCl (r.a.) (added to the irrigation water) during the first steps of development (60 days after transplanting, DAT) [
29]. After that period, four different treatments were applied, as shown in
Table 1.
Treatments were randomised in a factorial design with 2 factors (N and NaCl) and 2 levels, and 3 replicates were performed per treatment (with 10 strawberries per plot, collected on 3 sampling dates: 16 January, 22 March, and 29 April 2012). All the fruits were collected at commercial maturity, defined as a homogeneous (red) color for the strawberry, which was considered as optimum ripeness. Thus, the fruits were selected using a Minolta CR-400 colorimeter (Minolta, Osaka, Japan), setting the parameter b* in a range between 20 and 30 as an indicator of commercial maturity.
2.2. Chemicals and Reagents
We purchased 2,2–azino–bis (3–ethylbenzothiazoline–6–sulfonic acid) diammonium salt (ABTS), ellagic acid, rutin, and 2,2-Diphenyl-1-picrylhydrazyl (DPPH•) from Sigma-Aldrich (Steinheim, Germany). HPLC grade methanol and formic acid were supplied by Scharlau (Sentmenant, Spain). From Fluka Chemika (Neu-Ulm, Switzerland), 6–hydroxy–2,5,7,8–tetramethylchroman–2–carboxylic acid (Trolox) was obtained. Cyanidin–3–glucoside was acquired from Polyphenols AS (Sandnes, Norway).
The Milli-Q water was obtained using an Elix3 Millipore water purification system (Molsheim, France).
2.3. Extraction of Phenolic Compounds
The extraction of the phenolic compounds was performed following the method described by Sánchez-Rodríguez et al. [
13], with some modifications. Thus, 0.1 g of lyophilised sample was extracted with 1.5 mL of the mixed solvent of methanol/water/formic acid (50:48:2
v/
v/
v) by 1h of sonication. After, samples were macerated overnight and were sonicated for 1 h. The extract was centrifuged at 12,000 rpm for 5 min (model EBA 21; Hettich Zentrifugen) and filtered through a 0.22 µm PVDF membrane (Millipore Millex–GV syringe filter). The extracts were transferred into amber glass vials and stored at −80 °C until their analysis.
2.4. HPLC-DAD-ESI/MSn Identification and Quantification of Phenolic Compounds
The phenolic compounds were analysed following the method reported by Gironés-Vilaplana et al. [
30]. The HPLC-DAD-ESI/MS
n analyses were performed using an Agilent HPLC 1100 series model equipped with a photodiode array detector and a mass detector in series (Agilent Technologies, Waldbronn, Germany). The equipment consisted of a binary pump (model G1312A), an autosampler (model G1313A), a degasser (model G1322A), and a photodiode array detector (model G1315B). The HPLC system was controlled by ChemStation software (Agilent, version 08.03). The chromatographic analyses were performed on a Luna C18 column (250 × 4.6 mm, 5 mm particle size; Phenomenex, Macclesfield, UK). Water/formic acid (99:1,
v/
v) and acetonitrile were used as the mobile phases A and B, respectively, with a flow rate of 1 mL/min. The linear gradient started with 8% solvent B, reaching 15% at 25 min, 22% at 55 min, and 40% at 60 min, which was maintained to 70 min. The injection volume was 20 µL.
The mass detector was an ion trap spectrometer (model G2445A) equipped with an electrospray ionization interface controlled by LCMSD software (Agilent, version 4.1). The ionization conditions were a capillary temperature of 350 °C and voltage of 4 kV. The nebulizer pressure and nitrogen flow rate were 65.0 psi and 11 L/min, respectively. The full-scan mass covered the range of m/z from 100 to 1200. Collision-induced fragmentation experiments were performed in the ion trap using helium as the collision gas, with voltage ramping cycles from 0.3 to 2 V. The mass spectrometry data were acquired in the positive ionization mode for anthocyanins and in the negative ionization mode for the other phenolic compounds. The MSn was carried out in the automatic mode on the more abundant fragment ion in MS(n−1). Tentative identification was based on full-scan, data-dependent MS scanning from 100 to 1200 m/z, MSn fragmentation, and comparisons of the retention time and absorption with available standards.
For the quantification, all samples were also centrifuged for 5 min at 10,500 rpm. Each supernatant was filtered through a 0.45-μm PVDF filter (Millex HV13, Millipore, Bedford, Mass., USA) before injection into the HPLC system, as described by Gironés-Vilaplana et al. [
30]. Chromatograms were recorded at 280, 320, 360, and 520 nm. Anthocyanins were quantified as cyanidin 3–
O–glucoside at 520 nm, flavonols and xanthone derivatives as quercetin 3-O-glucoside at 360nm, ellagic acid derivatives as ellagic acid 3–
O–glucoside at 360 nm, and cinnamic acids as 5–
O–caffeoylquinic acid at 320 nm [
31,
32].
2.5. DPPH, ABTS, and Superoxide Radical (O2) Scavenging Activity, Assays of Antioxidant Capacity
Antioxidant capacity methods were determined spectrophotometrically in 96-well micro-plates (Nunc, Roskilde, Denmark) using an Infinite® M200 microplate reader (Tecan, Grödig, Austria). The assays were performed in triplicate.
2.5.1. DPPH• Assay
The determination of antioxidant capacity using DPPH radical was performed by measuring the absorption at 515 nm after 50 min of reaction [
33,
34]. The results were expressed as μmol equivalent of Trolox per 1 g of dried weight.
2.5.2. ABTS+ Assay
The analysis of antioxidant capacity using the ABTS+ radical was performed as described by Espín et al. [
33], evaluating the decrease of the absorbance at 414 nm for 50 min at 25 °C. The results were expressed as μmol equivalent of Trolox per 1 g of dried weight.
2.5.3. Superoxide Radical (O2−) Assay
This antiradical activity was evaluated by monitoring the effect of the sample extracts on the O
2− induced reduction of NBT at 560 nm. Superoxide radicals were generated by the NADH/PMS system following the method proposed by Ferreres et al. [
35]. The results were expressed as μmol equivalent of Trolox per 1 g of dried weight.
2.6. Statistical Analysis
The data were subjected to ANOVA analysis, with a factorial design with two factors (N and NaCl) and two levels, followed by an LSD test. Values were considered statistically significant at a p-value ≤ 0.05. The statistical software Statistix 9.0 was used.