Italian Wild Rocket [Diplotaxis Tenuifolia (L.) DC.]: Influence of Agricultural Practices on Antioxidant Molecules and on

Wild rocket [ Diplotaxis tenuifolia (L.) DC.] belongs to the Brassicaceae family and has its origin in the Mediterranean region. The effect of conventional and integrated cultivation practices on the nutritional properties and benefits of wild rocket [ Diplotaxis tenuifolia (L.) DC.] were studied. Bioactive molecules content (vitamin C, quercetin, lutein), antioxidant properties and bioactivity of polyphenolic extracts from the edible part of rocket in Caco-2 cells were determined. Regarding antioxidant properties, FRAP (Ferric Reducing Antioxidant Power) values ranged from 4.44 ± 0.11 mmol/kg fw to 9.92 ± 0.46 mmol/kg fw for conventional rocket and from 4.13 ± 0.17 fw mmol/kg to 11.02 ± 0.45 mmol/kg fw for integrated rocket. The characteristics of wild rocket as a dietary source of antioxidants have been pointed out. Significant differences in the quality of conventional and integrated rocket have been shown, while no influence of agronomic practice on biological activity was reported. A significant accumulation of cells in G1 phase and a consequent reduction in the S and G2 + M phases were observed in Caco-2 cells treated with rocket polyphenol extract.


Introduction
Rocket includes different species belonging to Eruca and Diplotaxis genera.Wild rocket [Diplotaxis tenuifolia (L.) DC.] belongs to the Brassicaceae family and has its origin in the Mediterranean region [1].It is known for the pungent taste of its young leaves, which makes it appreciated for the salads and in cooked culinary preparations.Rocket has a long history of medicinal use for various purposes: antiphlogistic, astringent, depurative, diuretic, digestive, emollient, tonic, stimulant, laxative and rubefacient [2].
Rocket contains a range of health-promoting phytochemicals including carotenoids, vitamin C, fiber, polyphenols and glucosinolates [3,4].Glucosinolates represent the major class of compounds in rocket and their contents in this crop have been well documented in the literature [5,6].
Several investigations have shown the potential role of phytochemicals in Brassicaceae in the prevention of certain diseases and types of cancer [7]; the health-promoting activities, including tumorogenesis inhibition and hepatoprotective effects, are attributed to rocket plants [8][9][10].
It is well known that the content of bioactive compounds-hence the nutritional quality of food-can be influenced by several factors such as genetics, degree of ripeness, agronomic and environmental conditions during growth, food preparation and processing.
Growing conditions include a large number of variable agronomic and environmental factors: climate, soil type, hours of sunlight, irrigation, nutrient supply, pest control, growing location, cultivation methods, time of harvest and others [11].These variables can affect both crop productivity as well as the nutritional profile of plant.
Organic crop production systems are often contrasted to conventional crop production systems that typically rely on the application of broad-spectrum synthetic fertilizers and pesticides [12].On a wide scale in Europe, so-called conventional methods have been replaced by an integrated farming system that reduces the use of synthetic chemicals by a combination of organic and conventional techniques [13].The integrated farming system can be conceptualized as a "third way" or middle course for agriculture [14].Few studies are reported in literature on how integrated agricultural systems affect the nutrient composition in plants [15,16].
In this research the effects of conventional and integrated cultivation practices on the nutritional properties and benefits of wild rocket [Diplotaxis tenuifolia (L.) DC.] were studied.
In particular, we have studied antioxidant properties, bioactive molecules content (vitamin C, quercetin, lutein) and the cytotoxicity and antiproliferative effects of polyphenolic extracts from the edible part of rocket in Caco-2 cell models.
This paper describes changes in antioxidant composition of wild rocket in relationship to the agricultural practices utilized, assessing the optimal content of bioactive compounds and thus more healthful and nutritious food crops.

Bioactive Molecules and Antioxidant Properties
In this agronomic investigation, we have studied the antioxidants content of wild rockets cultivated following a conventional and integrated system during two years of cultivation.
Weckerle et al. [19] identified three glucopyranosides of quercetin in Eruca Sativa leaves using NMR techniques.In a study by Martínez-Sánchez et al. [20], important differences between flavonoid profiles of E. vesicaria and D. tenuifolia were observed: E. vesicaria contained kaempferol derivatives as principal compounds, whereas D. tenuifolia accumulated quercetin derivatives.
Leafy vegetables contain several types of photosynthetic pigments, such as chlorophylls and carotenoids [21,22].Znidarcic et al. [23], studying pigments content of leafy vegetables (chicory, dandelion, garden rocket and wild rocket) commonly consumed in Mediterranean area, have shown that leafy vegetables are almost the richest dietary sources of lutein among xanthophyll pigments.
In our research, the rocket lutein content ranged from 40.92 ± 5.30 μg/g fw to 54.48 ± 19.17 μg/g fw.No significant differences were detected between conventional and integrated rockets and between the first and second year of cultivation for both agronomic practices (Table 1).
Martínez-Sánchez et al. [18] found a high vitamin C content in rocket.As reported in Table 1, in both years of cultivation, the content of vitamin C was significantly higher in the integrated product compared with the conventional product (I year: P < 0.05; II year: P < 0.001), except for the first sampling of both years of cultivation.
A correlation between antioxidant properties and components such as phenolics, carotenoids and vitamin C has been reported [18,24].The determination of antioxidant properties could be envisaged as the first stage in studying the potential benefits of foods.
Table 2 shows FRAP (Ferric Reducing Antioxidant Power) values (mmol/Kg fw) of rocket distributed by agronomic practice and year of cultivation.Among the cruciferous vegetables, rocket represents a good source of antioxidants [25].In addition, several works showed that the antioxidant properties depend on the species, agronomic practices and storage [4,18,26].Heimler et al. [4], by comparing the antiradical activity (determined by the reaction with the DPPH• radical) of Lactuca sativa, Cicorium intybus, Plantago coronopus, Eruca sativa, Diplotaxis tenuifolia, have shown that Eruca sativa and Diplotaxis tenuifolia are the least antioxidant salads among those tested.

Table 2. FRAP values of rocket distributed by agronomic practice and year of cultivation .
Values are expressed as mean values ± SD; S1-S2-S3 = Samplings1-2-3; Means in rows within same year followed by different letters differed significantly (P < 0.001, P < 0.01 or P < 0.05); # By column, levels of significance are represented by * P < 0.05, ** P < 0.01 and NS not significant.FRAP values do not differ between conventional and integrated rocket, with the exception of the second sampling of both the first and the second year of cultivation where it was observed to be higher in integrated samples than in conventional samples.
In addition, FRAP values vary with respect to the times of harvest.In the first year of cultivation both agronomic practices exhibited FRAP values (mmol/Kg fw) that were lowest (P < 0.001) in wild rocket cultivated in June.In contrast, in the second year of cultivation, the products cultivated in September reached the lowest values.

Cytotoxicity and Antiproliferative Activity of Rocket Polyphenol Extract
The cytotoxicity and antiproliferative activity of rocket polyphenol extract were studied using human colon carcinoma (Caco-2) cells.The cytotoxic effect of the polyphenol rocket extract was evaluated measuring cell viability by the MTT test.Figure 1 shows cytotoxicity obtained using Caco-2 cell model and expressed as cell viability percentage: 0.5, 1, 5, 10 mL/L rocket polyphenol concentrations did not affect cell viability, while 50 and 100 mL/L rocket polyphenol extract concentrations reduced cell viability to values between 31% and 71% and from 6% to 29% respectively.The same behavior was observed for conventional and integrated extracts; in addition, for both conventional and integrated products, no differences were observed with respect to times of harvest, except for the second sampling of integrated rocket.Lamy et al. [9] have reported the antigenotoxic effect of Eruca sativa in benzo[ ]pyrene exposed human hepatoma (HepG2) cells.Recently, Jin et al. [27] have studied the expression of genes involved in glucosinolate metabolism and antigenotoxicity of rocket leaf glucosinolates extract: glucosinolate-rich extracts of Eruca sativa cv.Sky, but not Diplotaxis tenufolia cv.Voyager, confer significant resistance of oxidative stress.Melchini et al. [28] demonstrated anti-proliferative activity on human lung carcinoma A545 cells of erucin from rocket salads.
We investigated the effect of the rocket polyphenol extract on cell proliferation; the distribution of cells in each phase of the cell cycle was determined by flow cytometric analysis of DNA content.As reported in Figure 2, a significant accumulation (P < 0.05) of cells in G1 phase and a consequent reduction in the S and G2 + M phases were observed in Caco-2 cells treated with 10 mL/L rocket polyphenol extract.No difference in biological effect was observed between conventional and integrated samples and between the several times of harvest for both agronomic practices.

Plant Material
Greenhouse experiments were carried out over a period of two years at two farms, a conventional farm (Fogliamica Orticoltura F.lli Tebaldini) and an integrated farm (Orticoltura Ferrari Elio) located in the same vicinity to ensure similar climatic and soil conditions.Cultivation area was in the province of Brescia, Italy.At both farms, cold greenhouses were used, consisting of metal frames covered in plastic sheeting and having a sprinkler irrigation system.Greenhouse surface areas were 300 m 2 for the conventional farm and 500 m 2 for the integrated farm.For both farms, soil is calcareous, medium-to fine-textured, slightly alkaline; it has a low carbon-nitrogen ratio and a high Cation Exchange Capacity (CEC).
Fertilization for the integrated practice consisted of manure application at the rate of 70 q/ha at the beginning of each crop cycle.For the conventional practice, the fertilizer Nitrophoska 20-20-20 was applied at the rate of 17 q/ha at the beginning of each crop cycle; in addition, for the conventional practice, a fertigation treatment was carried out after each crop cutting.
For conventional practice the pest control consisted of two treatments of Cyfluthrin (Baythroid 5 EC)(0.5 L/ha) for each crop cycle, whereas for integrated practice a single treatment was conducted by spraying Deltamethrin (Bayer Crop Science) (2.5 L/ha) after each crop cycle.
For the conventional practice soil disinfestation was performed with Basamid once every two years, while for the integrated practice soil disinfestation treatment was not applied.
For each agronomic practice samplings were replicated three times each year.For each year, the first sampling was carried out in June (S1), the second in July (S2) and the third in September (S3).In addition for each sample, as a rule, both conventional and integrated vegetables were harvested on the same day.For each sampling, leaves from 10 plants per cultivar per treatment group were harvested together.

Chemicals and Standards
All the solvents and reagents were of HPLC or Optima grade; common reagents and standards were purchased from Sigma-Aldrich Srl (Milan, Italy), Extrasynthese (Genay, France), Carlo Erba (Milan, Italy) and BDH Laboratory Supplies (Poole, UK) and were of the highest grade available.Double-distilled water (Millipore, Milan, Italy) was used throughout the study.

Sample Preparation Method
The crops were delivered directly to the laboratory and sampled on receipt.Only the edible portion of the samples was utilized for analysis and injured samples were not selected.Fresh samples were homogenized in a Waring blender.The extraction of carotenoids, vitamin C, polyphenols and antioxidant activity were done on the same day.Three replicates were prepared from each sample.

Extraction and Quantification of Polyphenols
Phenolics were hydrolyzed to obtain total free forms and extracted as described by Hertog et al. [31].Briefly, polyphenols were extracted from about 3 g homogenized sample with methanol after acidic hydrolysis with HCl (6 M) at 90 °C for 2 h.Quantitative analysis was performed using an ESA series (MODEL 580) of HPLC solvent delivery module, an ESA 5600 eight-channel coulometric electrode array detector and an ESA coularray operating software that controls the equipment and performs data processing (ESA, Chemsford, MA, USA).A Supelcosil LC-18 column (25 cm × 4.6 cm, 5 μm) with a Perisorb Supelguard LC-18 (Supelco, Milan Italy) was used.The volume injected was 30 μL.Chromatography was performed at 30 °C and 1 mL min 1 flow rate using 0.02 M sodium phosphate adjusted with 85% ortophosphoric acid to pH 2.8 (solvent A) and methanol (solvent B).Eluent flow rate was maintained at 1 mL min 1 and the setting potentials were set at 60, 120, 200, 340, 480, 620, 760 and 900 mV [32].

Extraction and Quantification of Carotenoids
Carotenoids were determined as described by Sharpless et al. [33].This method is used by the National Institute of Standards and Technology (NIST, Gaithersburg, Maryland).Briefly, approximately 1 g of sample was combined with 3 mL THF (tetrahydrofuran) and 2.7 mL methanol.The mixture was saponified for 30 min in a water bath (40 °C) after adding 0.3 mL of a 40% (w/v) methanolic KOH solution and then 0.15 g ascorbic acid was added to neutralize the KOH.The analytes were extracted with three 15 mL portions of hexane-diethyl ether (50 + 50).The organic phases were combined and evaporated under a stream of nitrogen and the residue redissolved in 10 mL of n-hexane.For the analysis 1 mL of the sample was re-evaporated and reconstituted in 1 mL of mobile phase (50% methanol, 45% acetonitrile and 5% tetrahydrofuran).50 μL of reconstituted extract were injected on a Waters Nova Pack C18 column (3.9 mm × 150 mm, 4 μm) at a flow rate of 1 mL min 1 .The extracts were analyzed by a Perkin-Elmer ISS 200 series HPLC system.The eluents were methanol/acetonitrile/ tetrahydrofuran (50:45:5).The peaks were detected with a variable spectrophotometric detector (Perkin-Elmer LC-95, Norwalk, CO, USA) connected to a personal computer Pe Nelson mod 1020 (Perkin-Elmer).The detection wavelength was 450 nm for carotenoids [34].

Extraction and Quantification of Vitamin C
Total ascorbic acid was extracted and quantified by HPLC system according to the method of Margolis et al. [35], with some modifications [36].Briefly, 1.5 g of sample was suspended in 5 mL water.To the sample was added 1 mL of 0.5 mol L 1 of dibasic potassium phosphate containing 100 g L 1 of DTT (1,4-dithiothreitol).The sample was vortexed for 15 s, kept at room temperature for 30 min and 1 mL of aqueous MPA (400 g L 1 metaphosphoric acid) was added.After adding 2 mL of acetonitrile, the suspension was centrifuged (1000 g, 30 min, 5 °C) and the supernatant was transferred to 1.8-mL vials.Chromatographic separation was carried on a 250 × 4.6 mm Capcell Pak NH 2 column (Shiseido, Tokyo, Japan), using ESA series HPLC, equipped an eight-channel coulometric electrode array detector and an ESA CoulArray operating software that controls the equipment and performs data processing (ESA, Chemsford, MA, USA).The setting potential was 0, 100, 200, 300 and 400 mV.The column was equilibrated at 40 °C at a flow rate of 0.8 mL min 1 with a solvent composed of 0.680 g monobasic potassium phosphate, 200 mL water, 800 mL acetonitrile and 7.5 mL concentrated phosphoric acid.The injection volume was 30 μL.

Antioxidant Properties Evaluation
Antioxidant properties were evaluated using FRAP (Ferric Reducing Antioxidant Power). 1 g of the homogenized sample was extracted with 4 mL of water under agitation for 15 min at room temperature, centrifuged at 1000 g for 10 min and the supernatant (water soluble fraction) was collected.The extraction was repeated with 2 mL of water.The two supernatants were combined and directly used for assay.The residue was extracted using 4 mL of acetone under agitation for 15 min at room temperature, centrifuged at 1000 g for 10 min and the supernatant (liposoluble fraction) was collected.The extraction was repeated with 2 mL of acetone.The two supernatants were combined and directly used for assay [37].
Values of samples were obtained by summing the values of water and liposoluble fractions.The method followed Benzie and Strain [38] and Pulido et al. [39] through the use of a Tecan Sunrise® plate reader spectrophotometer.The method is based on the reduction of Fe 3+ -TPTZ (2,4,6-tripyridyl-striazine) complex to ferrous at low pH.

Cell Culture
The human colon adenocarcinoma Caco-2 cells were obtained from European Tissue Culture Collection and cultured in D-MEM with GlutaMAX TM -I supplemented with 10% fetal bovine serum and 100 IU/mL penicillin and 100 μg/mL streptomycin (all from Gibco-Invitrogen).Cells were grown at 37 °C in a humidified atmosphere containing 5% CO 2 .This investigation has been carried out utilizing polyphenol fractions from rocket hydrolyzed extracts derived from the first year of cultivation' rocket grown in conventional and integrated regimes in the three samplings.

Antiproliferative Activity
In order to study the effect of the rocket polyphenol extracts on cell proliferation, we performed a cell cycle distribution analysis by determining bromodeoxyuridine (BrdU) incorporation versus DNA content.Caco-2 cells were incubated with 30 μM BrdU during the last hour of a 24-h treatment with 10 mL/L rocket polyphenol extracts, fixed in cold 70% ethanol.Fixed cells were washed in PBS, resuspended in 2 N HCl for 30 min at room temperature, pelleted, and then resuspended in 0.1 N sodium tetraborate for 15 min.The samples were then washed in PBS, incubated for 15 min in PBS containing 1% bovine serum albumin and 0.2% Tween-20 (PBT), and then for 60 min in 100 μL of anti-BrdU monoclonal antibody (Becton Dickinson) diluted 1:20 in PBT.After two washes with PBT, cells were incubated for 30 min with 100 μL of FITC-conjugated anti-mouse antibody (Amersham) dilute 1:100 in PBT, then washed twice and resuspended in PBS containing 5 μg/mL Propidium Iodide (PI) and 1 mg/mL of RNase A. Cells were analyzed with a Coulter Epics XL flow cytometer (Coulter Corporation).Ten thousand cells were measured for each sample.Computer statistical analysis of Mean Fluorescence Intensity (MFI) and graphic representation were performed with the XL2 software (Coulter Corporation) [42].

Statistics
All analyses were performed in triplicate.Data are given as the mean and Standard Deviation (SD).Statistical analysis was performed using Statistica for Windows statistical package (release 4.5; StatSoft Inc., Vigonza PD, Italy).
One-way Analysis of Variance (ANOVA) and a Least Significant Difference (LSD) test at a 95% confidence level (p < 0.05) was used to identify differences between samplings within each year.
P-values were calculated to determine the significance of observed differences between conventional and integrated treatments by Student's t-test.
For cytotoxicity and antiproliferative experiments, mean values of the percentage of cells in each phase of cell cycle were obtained from three independent experiments; the significance was calculated in comparison to the control, using the Student's t-test.

Conclusions
Our results confirm as phytochemicals content are strongly dependent by environmental factors and growing factors in wild rocket.This agronomic research have shown that there are significant differences in the quality of rockets of the two cultivation systems compared, while no influence of agronomic practice on biological activity in vitro was reported.Our findings could be useful in the selection of agronomic practices to produce healthful and more nutritious food crops, potentially enhancing the commercial value of this crop.
Results obtained could contribute to a better understanding of the potential health benefits of cruciferous vegetables.The characteristics of wild rocket as a dietary source of antioxidants have been pointed out.The apoptotic and antiproliferative effects of rocket polyphenol extracts could be promising with respect to the potential beneficial activity associated with rocket consumption.The potential protective effect observed in vitro needs to be clarified and verified by in vivo animal model studies as well as pharmacokinetics studies.Thus, future research is needed to assess the biological role of rocket extracts in vivo.

Figure 2 .
Figure 2. Effect of 10 mL/L rocket polyphenol extract on cell cycle # .

Figure 3 .Figure 3 .
Figure 3. (a) incorporation of BrdU in control cells; (b) Incorporation of BrdU in cells treated with 10mL/L polyphenol of CR S1.

Table 1 .
Bioactive molecules in rocket by agronomic practice and year of cultivation .