Abstract
Artichoke is one of the most important crop cultivated in the Mediterranean area and especially in Italy. Among all the different artichoke types available on the market, two typical genotypes grown in the Veneto basin in northern Italy, Violetto di S. Erasmo (VE) and Violetto di Chioggia (VC), both unknown from a nutritional point of view, were characterized. All the results obtained were compared with those of Violetto di Toscana (VT) that can be considered a standard for the oval purple type. Violetto di S. Erasmo (VE) and VC showed higher antioxidant capacity than VT. Chlorogenic acid content was very high, especially in VE and VC. Caffeic acid was also detected, whereas p-coumaric acid, ferulic acid, and gallic acid were not found. Moreover, Veneto genotypes showed a high ascorbic acid content leading us to consider them suitable also for the processing industry and a rather good source of functional compounds and healthy properties.
La alcachofa es uno de los cultivos más importantes en el área mediterránea y especialmente en Italia. Entre todos los tipos diferentes de alcachofa disponibles en el mercado, dos variedades típicas cultivadas en el Véneto, en el norte de Italia, Violetto di S. Erasmo (VE) y Violetto di Chioggia (VC), ambas no estudiadas desde un punto de vista nutricional, fueron caracterizadas. Los resultados obtenidos se compararon con los de Violetto di Toscana (VT), que se puede considerar el estándar del tipo púrpura oval. El contenido en ácido clorogénico fue muy alto, especialmente en VE y VC. También se detectó ácido cafeico, mientras ácido p-cumárico, ácido ferúlico y ácido gálico no fueron encontrados. Además, los genotipos del Véneto mostraron un alto contenido en ácido ascórbico, lo que indica que pueden ser consideradas apropiadas para la industria de procesado así como una buena fuente de componentes funcionales y propiedades saludables.
Introduction
Artichoke (Cynara cardunculus, subsp. scolymus), is an herbaceous perennial crop, widely cultivated in the Mediterranean area. The heads of the artichoke are used worldwide for their particular taste and health benefits (Lattanzio, Kroon, Linsalata & Cardinali, Citation2009). In the last years, many studies have focused on its antioxidant properties and beneficial effects against liver complaints (Fratianni, Tucci, De Palma, Pepe, & Nazzaro, 2007; Gil-Izquierdo, Gil, Conesa, & Ferreres, Citation2001); it seems that these actions could be strictly related to the polyphenolic fraction mainly composed of mono- and dicaffeoylquinic acids and flavonoids (Espin, Tudela, & Garcia-Canovas, Citation1997; Kukic et al., 2008; Wittemer et al., Citation2005).
This crop is mainly grown in Italy, with more than 50,000 ha ( http://www.istat.it), which accounts for 50% of world production, followed by Spain, France, and China ( http://www.faostat.fao.org). From an agronomical point of view, each region preserves its typical cultivars which may differ in chemical composition and exhibit different properties. The first reliable information on artichoke cultivation in Italy appeared in the fifteenth century, when, from the Naples area, it spread first to Tuscany and then elsewhere with favorable climatic conditions. This includes the Venice lagoon which is the only production area in north Italy. This paper deals with the characterization of two typical varieties cultivated in Veneto region: Violetto di S. Erasmo (VE) and Violetto di Chioggia (VC)(both are late varieties with violet-colored heads), which are usually considered an important artichoke germoplasm source but unknown from a nutritional point of view.
In this work, their antioxidant and phenolic composition, sugars and minerals were considered, an approach that could provide a first nutritional insight into these interesting genotypes. All the results obtained were compared with those of var. Violetto di Toscana (VT), which is widely used in Italy and has already been studied (Coinu et al., Citation2007; Romani, Pinelli, Cantini, Cimato, & Heimler, Citation2006).
Material and methods
Standards and reagents
Acetic acid (glacial) and sodium carbonate anhydrous werepurchased from Riedel–de Haën (Hanover, Germany). Gallicacid monohydrate was obtained from Fluka (Sigma-Aldrich, Italy); methanol from VWR Prolabo (France), Folin–Ciocalteau's (FCs) reagent from Labochimica (Padova, Italy). Chlorogenic acid and ferulic acid were purchased from Aldrich Chemical Company (Sigma-Aldrich, Italy); p-coumaric acid, formic acid, and caffeic acid from Sigma (Sigma-Aldrich, Italy); D-(+)-glucose, and sucrose from Carlo Erba (Milan, Italy); D-(−)-fructose from Fluka. Deionized water (18 ΩA) was prepared using an ultrapure water purification system (Sartorius, Italy). Sodium carbonate and sodium bicarbonate eluent concentrate and a solution containing seven anions standard are available from Dionex (Sunnyvale, CA, USA). All reagents and standards were of analytical or high performance liquid chromatography (HPLC) grade.
Plant material and location
Three artichoke genotypes were selected (): “Violetto di Chioggia”, “Violetto di S. Erasmo”, and “Violetto di Toscana” (Cynara cardunculus, subsp. scolymus) differing in their biological and morphological profiles. They were field grown in four different locations [Rosolina (45° 06′ 76′’ N, 12° 26′ 39′ E), Piovini (45° 24′ 44′ N, 12° 20′ 38′ E), Ca Lino (45° 16′ 09′ N, 12° 29′ 92′ E), and S. Erasmo (45° 45′ 39′ N, 12° 40′ 36′ E)] of Veneto region (north-east Italy) in order to cover a representative area and obtain reliable data. This trial was conducted in 2010 under standard growing conditions and management practices. These locations are near the Venice lagoon and are considered very suitable for vegetable production. The local climate is typical of the Po Valley, mitigated by the proximity to the sea: Average minimum temperature is 3°C and maximum 24°C. It can be considered a transition climate between the continental and Mediterranean one. Rainfall peaks in spring and autumn and thunderstorms are frequent in summer.
Table 1. Head characteristics of three varieties of artichoke: “Violetto di S. Erasmo” (VE), “Violetto di Chioggia” (VC), and “Violetto di Toscana” (VT).
Tabla 1. Características de la inflorescencia de las tres variedades de alcachofa: “Violetto di S. Erasmo” (VE), “Violetto di Chioggia” (VC) y “Violetto di Toscana” (VT).
Morphological description of the cultivars
In this study, only lateral inflorescences were considered sampled from three years old cultivated artichoke, propagated by division. Artichoke heads at commercial maturity were collected in April in three plots from at least 10 different plants for each variety in all locations. Heads were harvested with the floral stem including 2–3 leaves at marketing stage. After harvesting, the stem was removed from the flower by cutting 5 mm under the receptacle. All heads of each genotype coming from all locations were put together, then weighed and their maximum diameter and length measured. Head length/diameter ratio, an important index of head shape, was calculated. This ratio, as reported by Lombardo et al. (Citation2010), is a relative constant trait of each genotype, varying from 0.9 to 1.1 and ≥1.2 in spherical/subspherical and in long shaped types, respectively. The color of outer and inner bracts was also recorded. After these measurements, all heads for each genotype were immediately frozen with liquid nitrogen. Then, always using liquid nitrogen, samples were chopped and mixed in order to get an homogenous sample and stored at −80°C until extraction and analysis. Chemical analysis data were collected from three independent extractions of the chopped sample with three replication for each genotype, so each mean comes from nine data.
Extraction procedure
Head tissues (5 g) were homogenized in methanol (20 mL) with an Ultra Turrax T25 at 13500 rpm for 30 s until uniform consistency. Samples were filtered (filter paper, 589 Schleicher) and appropriate aliquots of extracts were assayed by the FC assay for total phenols (TP) content. For the determination of phenolic acid, extracts were further filtered through cellulose acetate syringe filters (0.45 μm) and analyzed by HPLC.
Anions extraction procedure
For the estimation of anions, dry matter (200 mg), obtained in a ventilated oven (model M80-VF; Instruments s.r.l.; Bernareggio, Milan) set at 65°C for 72 h, was extracted in water (50 mL) and shaken at 150 rpm for 20 min. Samples were filtered in sequence through filter paper (589 Schleicher) and the extracts were further filtered through cellulose acetate syringe filters (0.20 μm) before analysis by ion chromatography (IC). For each sample, triplicate extractions and analyses were performed following the method reported by Parente et al. (Citation2002).
Colorimetric analysis of total phenolics
The TP content was determined by the FC method (Singleton & Rossi, Citation1965) using gallic acid as calibration standard, with a Shimadzu UV-1800 spectrophotometer (Columbia, MD, USA). The FC assay was conducted by pipetting 200 μL of artichoke extract into a 10 mL PP tube. This step was followed by addition of 1 mL of FCs reagent. The mixture was vortexed for 20–30 s. Eight hundred microlitre of sodium carbonate solution (20%) was added after 1 min the addition of the FC reagent. This was recorded as time zero; the mixture was then vortexed for 20–30 s after addition of sodium carbonate. After 2 h at room temperature, the absorbance of the colored reaction product was measured at 765 nm. The TP content in the extracts was calculated from a standard calibration curve, built with different concentrations of gallic acid, ranging from 0 to 600 μg mL−1 (correlation coefficient: R 2 = 0.9991). Results are expressed in mg of gallic acid equivalent per kg (GAE mg kg−1 fw) of fresh weight.
Determination of total antioxidant activity by ferric reducing antioxidant power
The assay was based on the methodology of Benzie and Strain (Citation1996). The ferric reducing antioxidant power (FRAP) reagent was prepared fresh so that it contained 1 mM 2,4,6-tripyridyl-2-triazine (TPTZ) and 2 mM ferric chloride in 0.25 M sodium acetate at pH 3.6. A 100 μL aliquot of the methanol extract prepared as above was added to 1900 μL of FRAP reagent and mixed. After leaving the mixture at 20°C for 4 min, the absorbance at 593 nm was determined (Shimadzu – UV 1800). Calibration was against a standard curve (0–1200 μg mL−1 ferrous ion; correlation coefficient: R 2 = 0.9893) produced by the addition of freshly prepared ammonium ferrous sulfate. Ferric reducing antioxidant power (FRAP) values were calculated as μg mL−1 ferrous ion (ferric reducing power) from three determinations and are presented as mg kg−1 of Fe2+E (ferrous ion equivalent).
Extraction and determination of ascorbic acid
Five-gram samples were homogenized until uniform consistency in a meta-phosphoric acid and acetic acid solution. Ascorbic acid was determined following the ISO 6557 method.
Quantitative determination of free phenolic acids by HPLC
Chlorogenic acid, ferulic acid, p-coumaric acid, caffeic acid, and gallic acid were separated and quantified using an HPLC diode array detector (DAD). The liquid chromatography apparatus utilized in these analysis was a Jasco X.LC system consisting of a model PU-2080 pump, a model MD-2015 multiwavelength detector, a model AS-2055 autosampler, and a model CO-2060 column oven. ChromNAV chromatography data system was used as software. The separation of phenolic acids was obtained on a Tracer Extrasil ODS2 (5 μm, 250 × 45 mm – Teknokroma) operating at 35°C. The flow rate was set to 1 mL min−1. The mobile phase consisted of 0.1% formic acid (A) and methanol (B). Elution gradient was as follows: 25–70% B over 15 min and 70–100% B over 5 min to clean the column. Two wavelengths (325 and 310 nm) were used to detect phenolic acids composition. High performance liquid chromatography (HPLC) analysis at 325 nm was used for identification of chlorogenic acid, caffeic acid, and ferulic acid. Identification of p-coumaric acid was performed at 310 nm. Phenolic acids were quantified following a calibration method. All standards were dissolved in methanol and the calibration curves were generated with concentrations ranging from 0.3 mg L−1 to 30 mg L−1.
Quantitative determination of sugars by HPLC
The liquid chromatography apparatus utilized in these analysis was the same reported at 2.9. The separation of sugars was achieved on a HyperRez XP Carbohydrate Pb++ analytical column (7.7 mm × 300 mm, ThermoScientific), operating at 80°C. Isocratic elution was effected using water at a flow rate of 0.6 mL min−1. D-(+)-glucose, D-(−)-fructose, and sucrose were quantified following a calibration method. Standards were dissolved in water and the calibration curves were generated with concentrations ranging from 100 mg L−1 to 1000 mg L−1.
Quantitative determination of anions by ion chromatography (IC)
The IC was performed using an ICS-900 system (Dionex Corporation) equipped with a dual piston pump, a model AS-DV autosampler, a column at room temperature, a DS5 conductivity detector, and a AMMS 300 suppressor. Chromeleon 6.5 chromatography management software was used for system control and data processing. A Dionex IonPac AS23 analytical column (4 mm × 250 mm) and a AG25 guard column (4 mm × 50 mm) were used for separations. The eluent consisted of 4.5 mM sodium carbonate and 0.8 mM sodium bicarbonate at a flow rate of 1 mL/min. Anions were quantified following a calibration method. A Dionex solution containing seven anions at different concentrations were taken as standards and the calibration curves were generated with concentrations ranging from 0.4 mg L−1 to 15 mg L−1.
Statistical analysis
Data were analyzed by mean of ANOVA. In the case of a significant F-value, the means were compared by Tukey's HSD test at the significance level of P ≤ 0.01.
Results and discussion
lists some inorganic components of artichoke heads (i.e. dry matter content and main inorganic mineral elements content). Data on “Violetto di Toscana”, which can be considered as a control genotype in this study, partially agree with those reported in the literature (Romani et al., Citation2006), although inflorescences cultivated in Veneto showed higher moisture content and sodium concentration. This latter parameter is probably due to high sodium chloride concentrations in the soil because of proximity to the sea. Among genotypes, “Violetto di Toscana” exhibited higher sulfates content, “Violetto di Chioggia” and “Violetto di S. Erasmo” the higher sodium and phosphates concentration, whereas no significant differences between varieties were found for the other parameters. Nitrates content was very low in all varieties and this result agrees with values found by Santamaria, Gonnella, and Valenzano (Citation2002).
Table 2. Mineral composition and dry matter content of heads of three artichoke varieties: “Violetto di S. Erasmo” (VE), “Violetto di Chioggia” (VC), and “Violetto di Toscana” (VT).
Tabla 2. Composición mineral y contenido de materia seca de los tres genotipos de la alcachofa: “Violetto di S. Erasmo” (VE), “Violetto di Chioggia” (VC) y “Violetto di Toscana” (VT).
From a qualitative point of view, it is interesting to note that “Violetto di S. Erasmo” and “Violetto di Chioggia” showed higher antioxidant capacity than “Violetto di Toscana” (). This trend is confirmed also by total polyphenols content, which are important for their role as a protective pool against oxidative damage caused by free radicals and for their health promoting effects (Lattanzio et al., Citation2009; Lombardo et al., Citation2010).
Table 3. Antioxidant capacity, total polyphenols, ascorbic acid, phenolic, and organic acids content of heads of three artichoke varieties: “Violetto di S. Erasmo” (VE), “Violetto di Chioggia” (VC), and “Violetto di Toscana” (VT).
Tabla 3. Activitad antioxidante, fenoles totals, acido ascorbico, contenido de algunos acidos fenolicos, y organicos en los tres genotipos de la alcachofa: “Violetto di S. Erasmo” (VE), “Violetto di Chioggia” (VC) y “Violetto di Toscana” (VT).
The most abundant phenolic substances reported in artichoke heads are caffeoylquinic acid derivatives (Lattanzio, Cardinali, Di Venere, Linsalata, & Palmieri, Citation1994), particularly chlorogenic acid (5-O-caffeoylquinic acid), and this result has been confirmed in this study. The HPLC chromatogram of the methanol extracts of artichoke heads is shown in . The peaks are of poor symmetrical shape, indicating possible poor resolution with other mono-caffeoylquinic acids (Schütz, Kammerer, Carle, & Schieber, Citation2004) which would lead to overestimation of the chlorogenic acid content. Even though the chlorogenic acid content was very high, especially in “Violetto di S. Erasmo” and “Violetto di Chioggia”, which showed relevant accumulations if compared with the “Violetto di Toscana” (). This result is very important since previous studies have demonstrated the antioxidant and anticarcinogenic properties of chlorogenic acid (Gonthier, Verny, Besson, Remesy, & Scalbert, Citation2003). This acid is usually weakly absorbed in the small intestine, but capable of providing higher yields of microbial metabolites, active compounds responsible for biological properties attributed to dietary polyphenols (Rechner et al., Citation2002). The high level of chlorogenic acid found in artichoke is probably due to its central role as a substrate for phenolic acids production (Wittemer et al., Citation2005). Caffeic acid was also detected, whereas p-coumaric acid, ferulic acid, and gallic acid were not found. This is in contrast with a previous findings (Fratianni et al., Citation2007) in which these phenolic acids were detected. If we compare “Violetto di S. Erasmo” and “Violetto di Chioggia” with Violetto di Provenza, Violetto di Sicilia, and other varieties characterized by green bracts (Curadi, Ceccarelli, Pinciarelli, & Graifenberg, Citation2005; Gil-Izquierdo et al., Citation2001; Lombardo et al., Citation2010), they presented more chlorogenic acid. Therefore, varieties cultivated in Veneto region can be considered a rather good source of functional compounds and healthy properties.
Figure 1. Chromatographic profile registered at 325 nm of methanol extracts obtained from artichoke heads of three genotypes [“Violetto di Sant'Erasmo” (VE), “Violetto di Chioggia” (VC), and “Violetto di Toscana” (VT)]. Rt indicated in parenthesis means retention time.
Figura 1. Perfil cromatográfico registrado a 325 nm de extractos de metanol obtenidos de inflorescencias de alcachofa de tres genotipos: “Violetto di Sant'Erasmo” (VE), “Violetto di Chioggia” (VC) and “Violetto di Toscana” (VT). Rt indicado entre paréntesis significa tiempo de retención.
![Figure 1. Chromatographic profile registered at 325 nm of methanol extracts obtained from artichoke heads of three genotypes [“Violetto di Sant'Erasmo” (VE), “Violetto di Chioggia” (VC), and “Violetto di Toscana” (VT)]. Rt indicated in parenthesis means retention time. Figura 1. Perfil cromatográfico registrado a 325 nm de extractos de metanol obtenidos de inflorescencias de alcachofa de tres genotipos: “Violetto di Sant'Erasmo” (VE), “Violetto di Chioggia” (VC) and “Violetto di Toscana” (VT). Rt indicado entre paréntesis significa tiempo de retención.](/cms/asset/dad34a0d-5679-4723-a7c7-ffe6e5995075/tcyt_a_700951_o_f0001g.gif)
Ascorbic acid concentration was higher in “Violetto di Toscana”, with 835 mg kg−1 fw (5913 mg kg−1 dw), exceeding “Violetto di Chioggia” and “Violetto di S. Erasmo” by 34.6 and 43.3%, respectively. Anyway, Veneto genotypes showed a higher ascorbic acid content than others (Cabezas-Serrano, Amodio, Cornacchia, Rinaldi, & Colelli, Citation2009; Gil-Izquierdo et al., Citation2001) and this result led us to consider them suitable also for the processing industry. Several studies, in fact, reported the role of ascorbic acid in preventing browning, as found for lettuce (Heimdal, Kuhn, Poll, & Larsen, Citation1995) and other vegetables (Reyes, Villareal, & Cisneros-Zevallos, Citation2007), and browning is one of the most important problems of artichoke processing. Among organic acids no significant differences were found for malic acid, whereas oxalic acid was higher in the typical varieties cultivated in Veneto. The higher oxalic acid content does not pose problems in terms of toxicity since these values are very low (0.035%), if compared with other vegetables such as spinach (0.97%) or parsley (1.70%) ( http://ndb.nal.usda.gov/ndb/foods/list).
Concerning free carbohydrates (), glucose was the predominant sugar in the heads of all artichoke genotypes, showing the highest concentration in “Violetto di Toscana” (2875 mg kg−1 fw). Fructose and sucrose were detected mainly in “Violetto di Chioggia” (640 and 189 mg kg−1 fw, respectively). All values were in accord with Leroy, Grongnet, Mabeau, Le Corre, & Baty-Julien, (2010).
Table 4. Free carbohydrates content of heads of three artichoke varieties: “Violetto di S. Erasmo” (VE), “Violetto di Chioggia” (VC), and “Violetto di Toscana” (VT).
Tabla 4. Contenido de carbohidratos livres en los tres genotipos de la alcachofa: “Violetto di S. Erasmo” (VE), “Violetto di Chioggia” (VC) y “Violetto di Toscana” (VT).
In conclusion, our results showed that “Violetto di Chioggia” and “Violetto di Sant'Erasmo” are higher in phenolics and antioxidant activity than “Violetto di Toscana”. From this point of view, genotypes cultivated in Veneto would be suitable for fresh consumption, giving a high amount of functional compounds to the consumer. Further studies are required to investigate the phenolic profile of other artichokes and also consider environmental factors. Finally, considering these local types could be useful to ensure biodiversity and improve consumer knowledge about typical products in order to encourage wider cultivation and to identify germplasm with a high biosynthetic potential of bioactive substances.
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