Morphological characterization, biomass and pharmaceutical compounds in Italian globe artichoke genotypes

Industrial Crops and Products 49 (2013) 326–333

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Morphological characterization, biomass and pharmaceutical compounds in Italian globe artichoke genotypes Anna Ciancolini a,b , Marion Alignan c,d , Mario Augusto Pagnotta b , Julie Miquel c,d , Gérard Vilarem c,d , Paola Crinò a,∗ a

ENEA, CR Casaccia, Unit of Sustainable Development and Innovation of the Agro-Industrial System, Via Anguillarese 301, 00123 Roma, Italy Università degli Studi della Tuscia, Department of Science and Technologies for Agriculture, Forestry, Nature and Energy (DAFNE), Via S.C de Lellis snc, 01100 Viterbo, Italy c Université de Toulouse, INP-ENSIACET, LCA (Laboratoire de Chimie Agro industrielle), 4 allée Emile Monso, F 31030 Toulouse, France d INRA, UMR 1010 CAI, F 31030 Toulouse, France b

a r t i c l e

i n f o

Article history: Received 7 March 2013 Received in revised form 15 May 2013 Accepted 18 May 2013 Keywords: Cynara cardundulus var. scolymus Natural by-products Accelerated Solvent Extraction Genetic variability HPLC

a b s t r a c t Globe artichoke [Cynara cardunculus var. scolymus (L.) Fiori] is a perennial herbaceous plant cultivated principally in the Mediterranean basin for its immature inflorescences (heads). Among the other possible uses of this species, biomass production may be considered. In this work, 17 Italian globe artichoke genotypes have been studied for two years in the field in order to evaluate their biomass production for pharmaceutical active compound extraction and to select the genotypes more suitable for this purpose. Biomass has been characterized agro-morphologically, using five of the UPOV (International Union for the Protection of New Varieties of Plants) descriptors (i.e. plant height, number of lateral shoots, floral stem diameter, first fully developed leaf length and leaf lobe number) along with other six traits explaining biomass production (i.e. lateral shoot number, first fully developed leaf width, main floral stem leaf number, dry leaf number, plant diameter and plant dry weight), and biochemically to determine by HPLC analysis the phenolic compound content. Genotypes were significantly different for many of the morphological and biochemical traits evaluated. The results indicated that globe artichoke dry biomass yield of some Italian spring genotypes is worth considering (9.7 t ha−1 , as average value of all genotypes evaluated in the two growing seasons). Chlorogenic acid (ranging from 0.22 g kg−1 DM to 27.85 g kg−1 DM) and 1,5-O-dicaffeoylquinic acid (ranging from 0.42 g kg−1 DM to 2.10 g kg−1 DM) were the main phenolic compound detected using HPLC analysis. Two genotypes were selected for high biomass and phenolic compound production. This may open new horizons to the industrial use of the crop, which could represent a potential for the increase of the farmers’ income. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Since ancient time, globe artichoke [Cynara cardunculus var. scolymus (L.) Fiori] has been used in traditional medicine for its recognized therapeutic effects such as hepatoprotective, anticarcinogenic, antioxidative, antibacterial, urinative, anticholesterol, glycaemia reduction (Saénz Rodriguez et al., 2002; Coinu et al., 2007; Rondanelli et al., 2011; Fantini et al., 2011) linked principally to the high content of polyphenolic compounds, which include mono- and di-caffeoylquinic acids and flavonoids (Fratianni et al., 2007; Lattanzio et al., 2009; Lombardo et al., 2010; Pandino et al., 2010, 2011a, 2011b, 2012, 2013; Negro et al., 2012). In particular, within the caffeoylquinic acid derivatives, chlorogenic acid is

∗ Corresponding author. Tel.: +39 06 30 48 3634; fax: +39 06 30 48 6044. E-mail address: [email protected] (P. Crinò). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.05.015

the most abundant component (Lattanzio et al., 2009). Also the flavonoids apigenin and luteolin and their glycosides have been widely described in globe artichoke (Lombardo et al., 2010; Pandino et al., 2010, 2011b, 2012, 2013; Negro et al., 2012). All these compounds have strong antioxidant properties and protect low density lipoproteins from oxidative damages (Lattanzio et al., 2009). In this regard, some studies have been done to analyze biochemically globe artichoke germplasm suitable principally for fresh consumption or/and industrial processing of the heads (Fratianni et al., 2007; Bonasia et al., 2010; Lombardo et al., 2010; Pandino et al., 2010, 2011a, 2011b). In the last years, other possible applications of globe artichoke, alternative to the traditional ones, were envisaged. Different types of products can be harvested and utilized to obtain: (i) oil from seeds (Foti et al., 1999); (ii) inulin from roots (Raccuia and Melilli, 2004, 2010); (iii) energy from biomass (Ierna and Mauromicale, 2010; Ierna et al., 2012; Ledda et al., 2013); (iv) fiber as potential reinforcement in polymer composites

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Table 1 Globe artichoke spring genotypes evaluated. Genotypes

Type

Donor institute

Origin

S2 S3 S5 S11 S17 S18 Castellammare S23 Campagnano Grato 1 Ascolano Jesino Montelupone A Montelupone B Bianco di Pertosa Tondo Rosso di Paestum Pisa

Romanesco Romanesco Romanesco Romanesco Romanesco Romanesco Romanesco Romanesco Romanesco Romanesco Romanesco Romanesco Romanesco Romanesco Romanesco Romanesco Violetto

Enea (RM) – Tuscia University (VT) Enea (RM) – Tuscia University (VT) Enea (RM) – Tuscia University (VT) Enea (RM) – Tuscia University (VT) Enea (RM) – Tuscia University (VT) Enea (RM) – Tuscia University (VT) Enea (RM) – Tuscia University (VT) Enea (RM) – Tuscia University (VT) Enea (RM) – Tuscia University (VT) Enea (RM) – Tuscia University (VT) CRA-ORA Monsampolo del Tronto (AP) CRA-ORA Monsampolo del Tronto (AP) CRA-ORA Monsampolo del Tronto (AP) CRA-ORA Monsampolo del Tronto (AP) CRA-ORT Pontecagnano (SA) CRA-ORT Pontecagnano (SA) Pisa University (PI)

Latium, Italy Latium, Italy Latium, Italy Latium, Italy Latium, Italy Latium, Italy Latium, Italy Latium, Italy Latium, Italy Latium, Italy Marche, Italy Marche, Italy Marche, Italy Marche, Italy Campania, Italy Campania, Italy Tuscany, Italy

RM, Rome; VT, Viterbo; AP, Ascoli Piceno; SA, Salerno; PI, Pisa.

(Fiore et al., 2011); (v) green forage for ruminant feeding (Fateh et al., 2009); and (vi) natural rennet for traditional cheese making (Llorente et al., 2004). Globe artichoke can also be used as crop for metal-accumulation (Hernández Allica et al., 2008). In general, these new possible uses of globe artichoke are related principally to the European Union research support on new agricultural byproducts (industrial raw materials) and have led to an increasing interest in aboveground biomass of this species. This interest is due mainly to the great adaptation of the crop to Mediterranean climate, characterized by low annual rainfalls and hot dry summer, to the relatively low crop energy input and to the large biomass productivity (Angelini et al., 2009). Until now, several studies on globe artichoke as energy crop have been done (Ierna and Mauromicale, 2010; Ierna et al., 2012), but there are only few suggestions on the use of its biomass as raw industrial material to recover phenolic active compounds. In particular, not many data are available in the literature on the extraction from globe artichoke biomass of active biocompounds, which are of interest for the pharmaceutical industry. This interest could meet the increasing demand of natural antioxidants due both to health concerns, linked either to the present use of synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) or to consumers’ preference (Llorach et al., 2002). Therefore, in the present work, a sustainable production of globe artichoke biomass and biocompounds of interest for pharmaceutical industry have been evaluated and the possibility of using such biomass, without upsetting traditional agricultural practices, has been also considered to allow a possible increase of farmers’ income. Taking into account these preliminary remarks, the present work aimed at: (i) establishing the most appropriate plant traits capable of describing globe artichoke plant biomass production; (ii) characterizing the aboveground biomass of Italian globe artichoke spring genotypes both under the morphological and biochemical profiles; and (iii) selecting the genotypes more suitable for this purpose.

soil classification system, 1975). The soil characteristics were as follows: sand, 62%; clay, 23%; silt, 15%; pH 6.3; organic matter, 1.24%; total nitrogen, 0.08‰; P, 24 ppm; K, 355 ppm; CE 0.16 mS. All genotypes were vegetatively propagated by offshoots and assessed in a completely randomized block experimental design with three replications. The total area used for the experiment was about 0.20 ha. Each field plot (elementary unit area of 15.60 m2 ) totally consisted of 20 plants (planting density of about 7700 plants ha−1 , inter and intra-row distances of 1.30 and 1.00 m, respectively). The transplanting date was 17 August 2008. Field experiments were conducted under low chemical inputs (minimized fertilization using N 100 kg ha−1 , P2 O5 90 kg ha−1 , K2 O 125 kg ha−1 each year) for the crop agronomical management, taking into account the agronomic techniques traditionally used in the cultivation area of Central Italy. In the first year of the trial, one third of the nitrogen fertilizer was distributed as ammonium sulphate in autumn and two thirds as ammonium nitrate during the stem elongation phase. In the second year, half nitrogen was applied as ammonium sulphate at the plant sprouting stage in September and half as ammonium nitrate at the stem elongation stage. Weed control was performed manually three times during the first experimental year and twice during the second one. At the end of the first growing season (at the end of May), stalk removal operation has been performed manually. No evident crop diseases were detected. The crop was irrigated with 50 mm of water only twice per year, using the drip irrigation system. During the two-year experimental trials, daily temperature and rainfall were measured by a meteorological station in the experimental field. The collected data are shown in Fig. 1.

2. Materials and methods 2.1. Experimental field and plant material Seventeen Italian spring globe artichoke genotypes were considered in our study (Table 1). Field trials were conducted for two years, during the 2008–2009 and 2009–2010 growing seasons, at the experimental station of ARSIAL (Latium Regional Agency for the Development and the Innovation of Agriculture) in Cerveteri (41◦ 59 N 12◦ 01 E), Rome (Italy), in a sandy clay loam soil (USDA

Fig. 1. Rainfall and air temperature during the experimental growing seasons at Cerveteri experimental field station. Data reported are the temperature mean or the cumulative rainfall amount of 15 days.

Morphological characterization for biomass production was performed using five of the standard UPOV descriptors previously established for globe artichoke (i.e. plant height, number of lateral shoots, floral stem diameter, first fully developed leaf length and leaf lobe number) (Crinò et al., 2008; Ciancolini et al., 2012) along with a group of six complementary other descriptors capable of explaining biomass production (i.e. lateral shoot number, first fully developed leaf width, main floral stem leaf number, dry leaf number, plant diameter and plant dry weight) (Table 2). In total, during the two growing seasons, 11 morphological and agro-physiological data were recorded at the primary head harvest time on nine central plants per genotype (Table 2). The primary head harvesting dates of each genotype in the two growing season are reported in Table 3. In order to evaluate quantitatively biomass production at the harvest time, three central plants per each genotype were harvested after the primary head harvesting date, immediately weighed and ovendried at 103 ◦ C, until a constant weight was reached, to determine the dry matter content of the whole plant. 2.3. Pharmaceutical compound extraction and analysis 2.3.1. Sample preparation A representative sample (300 g FW) of biomass (leaves and floral stems) was collected from nine plants per genotype, at the primary head harvesting date, in 2009–2010 growing season. All samples were immediately weighed, freeze-dried and ground to 1 mm diameter fine powder. 2.3.2. Extraction procedure Extraction was performed using Accelerated Solvent Extraction (ASE). The instrument Accelerated Solvent Extractor ASE100 Dionex Corporation (Sunnyvale, CA, USA) was used for pressurizing solvent extraction. An amount (1 g) of powdered dried sample was packed into the extraction cell and an amount of washed sea sand was used to fill the cell. Ethanol 80% was used as solvent. The ASE was performed at 150 ◦ C and 100 bars for 5 min. After each extraction, the vessels were allowed to cool the samples at room

2008/9

1.4 cf 0.5 i 1.4 cf 1.4 cf 1.8 b 0.8 hi 1.1 eh 0.7 hi 1.2 dg 1.5 be 2.5 a 0.9 fi 1.6 bd 1.1 eh 1.7 bc 1.7 bc 1.2 dg 1.3 cd 0.5 d 2.0 ab 1.3 cd 1.8 ab 0.8 d 1.1 d 0.8 d 1.0 d 1.1 d 2.0 ab 1.0 d 1.3 cd 1.0 d 1.8 ab 2.1 a 1.1 d 165.1 ab 152.7 ab 173.1 ab 167.4 ab 174.9 ab 159.1 ab 169.8 ab 166.8 ab 161.7 ab 169.6 ab 176.7 a 161.8 ab 159.5 ab 143.7 b 184.3 a 180.3 a 153.7 ab

2009/10 2008/9

167.3 bc 178.4 bc 201.8 ab 178.6 bc 215.1 a 188.1 ac 189.0 ac 181.0 ac 172.1 bc 179.6 ac 184.5 ac 161.0 c 162.7 c 155.0 c 184.9 ac 185.8 ac 155.2 c 12.7 bc 8.1 gh 13.0 bc 13.6 bc 12.4 bc 9.4 dh 8.6 fh 7.2 h 11.4 bf 13.2 bc 12.2 bd 11.7 be 12.8 bc 18.0 a 10.8 cg 14.0 b 9.0 eh

2009/10 2008/9

10.9 df 11.0 df 13.1 bd 11.8 cf 12.6 ce 11.1 df 9.4 e 10.2 de 11.4 df 11.9 cf 12.5 ce 12.8 be 14.3 ac 17.2 a 12.7 ce 15.7 ab 9.2 e 47.8 ab 32.0 cd 35.4 bd 45.6 ac 48.0 ab 36.5 bd 52.8 a 35.1 bd 40.7 ad 49.4 ab 54.3 a 31.0 cd 41.3 ad 27.7 d 43.3 ac 37.0 bd 36.3 bd

2009/10

Values are the means of measurements on 9 plants. Means followed by different letters for each parameter are significantly different at P < 0.05 (Tukey test).

2.2. Morphological characterization and biomass production

37.8 be 39.7 ae 46.9 ab 43.8 ad 53.8 a 50.2 ab 42.0 ae 43.7 ad 40.1 ae 46.2 ac 50.3 ab 31.7 de 41.0 ae 28.8 e 40.0 ae 32.2 ce 30.2 de

In both experimental years, the site was mainly characterized by increasing air temperatures from March to August. In the first growing season, the maximum and minimum air temperatures, expressed as average values, ranged from 20.9 and 11.9 ◦ C from August to May while, in the second one, the corresponding values were 21.3 and 12.1 ◦ C. Variability in rainfall, both as amount and distribution, has been observed between years. The amount of rainfall observed from August to May, in the first growing season, was 550 mm while, in the second one, was 971 mm.

Main floral stem leaf number (no.)

ns, not significant. Significant differences at P ≤ 0.05.

Table 3 Agro-morphological characterization of the globe artichoke spring genotypes under biomass profile (2008–2009 and 2009–2010 growing seasons).

*

2008/9

*

a

91.8 ac 78.8 cd 102.5 ab 99.1 ab 110.7 a 88.0 bc 104.1 ab 88.2 bc 94.7 ac 104.9 ab 95.3 ac 65.5 de 98.8 ab 51.7 e 102.0 ab 103.2 ab 98.0 ac

*

2009/10

*

*

99.1 ns 108.3 ns 104.4 ns 94.1 ns 105.3 ns 98.9 ns 87.7 ns 98.8 ns 94.9 ns 92.2 ns 96.3 ns 89.2 ns 86.7 ns 91.0 ns 98.5 ns 102.0 ns 94.4 ns

*

2008/9

*

*

*

2.7 ce 2.8 be 2.9 be 2.6 de 3.0 be 3.4 ad 3.2 bd 3.6 ac 2.9 be 2.2 e 3.2 bd 2.8 be 2.7 ce 3.0 be 3.7 ab 3.3 ad 4.2 a

*

2009/10

*

2.6 ns 3.1 ns 2.7 ns 3.0 ns 2.7 ns 2.7 ns 2.9 ns 2.7 ns 2.8 ns 3.7 ns 3.3 ns 2.8 ns 3.5 ns 3.2 ns 3.3 ns 3.2 ns 3.0 ns

*

*

2008/9

ns ns

76.2 bf 74.0 df 89.3 bd 77.3 bf 90.3 b 82.6 be 77.8 bf 68.5 ef 77.8 bf 89.6 bc 74.3 cf 62.7 f 63.3 f 68.5 ef 107.0 a 91.5 ab 70.8 ef

*

*

2009/10

*

69.1 be 71.7 be 79.0 b 74.1 bd 77.6 bc 75.9 bd 77.3 bc 54.9 e 63.9 be 60.9 ce 67.0 be 58.5 de 56.3 e 70.4 be 110.5 a 68.0 be 66.6 be

*

*

2008/9

ns

04/23 03/17 04/14 04/20 04/20 04/02 04/02 04/07 04/23 04/20 04/07 04/23 04/16 04/23 04/23 04/02 04/15

*

*

2009/10

*

04/10 03/18 04/07 04/15 04/10 03/31 03/20 03/26 04/10 04/10 04/14 04/27 04/14 04/27 04/27 04/23 04/20

*

*

2008/9

*

S11 S17 S18 S2 Campagnano Grato 1 Castellammare S23 S3 S5 Ascolano Jesino Montelupone A Montelupone B Bianco di Pertosa Pisa Tondo Rosso di Paestum

*

*

Genotypes

ns

Plant diameter (cm)

*

ns

First fully developed leaf width (cm)

Genotype*Year

*

First fully developed leaf length (cm)

Year

*

Lateral shoot number (no.)

Genotype

Plant height Number of lateral shoots Main floral stem diameter First fully developed leaf length First fully developed leaf width Main floral stem leaf number Lateral shoot leaf number Dry leaf number Plant diameter Number of leaf lobes Plant dry weight

Plant height (cm)

Descriptor

PlH NLSh MFSD Llength Lwidth MFSL LSL DL PD Llob PDW

Primary head harvesting date (mm/dd)

Code

Plant dry weight (kg)

Table 2 Morphological traits used for biomass characterization of globe artichoke spring genotypes and level of significance from the ANOVA analysis.a

2009/10

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Descriptors

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temperature and, then, each extract was filtered under vacuum and diluted up to 30 mL using ethanol 80%. All samples were stored at −20 ◦ C until the further analysis. All extractions were performed in duplicate. 2.3.3. Solvents and reagents Ethanol solvent and reagent grade formic acid (96%) were purchased from Scharlau Co. (Barcelona, Spain). Deionized water was home-made using a Milli-Q water purifying system purchased from Millipore Co. (Bedford, MA, USA). Apigenin, luteolin, luteolin-7-Oglucoside (cynaroside). The 1,3-O-dicaffeoylquinic acid (cynarin), and 1,5-O-dicaffeoylquinic acid were purchased from Extrasynthese (Lyon, France) while 3-O-caffeoylquinic acid (chlorogenic acid) was obtained from Sigma Chemical Co. (St. Louise, MO, USA). 2.3.4. HPLC analysis Polyphenol analysis was carried out using a Dionex HPLC chromatography equipped with an UV detector HP 1100. HPLC separation was performed at room temperature on a Pursuit C18 AQ18 (250 mm × 4.6 mm, 5 ␮m) column. The mobile phase was formed by 0.1% formic acid in water (solvent A) and in acetonitrile (solvent B) at a flow rate of 1 mL min−1 . The gradient program started with 6% solvent B to reach 30% from the 30th to the 35th min and then decreased to 6% solvent B from the 37th to the 40th min. The injection volume was 20 ␮l. Calibration curves for each available standard was generated with concentrations ranging from 1 to 200 mg l−1 . Identification of single compounds was done using retention time and UV spectrum, while quantification was performed by standard calibration curves. Chromatograms were recorded at 325 nm for chlorogenic acid, 1,3-O-dicaffeoylquinic acid and 1,5-O-dicaffeoylquinic acid and at 350 nm for luteolin, apigenin, and cynaroside. All samples were analyzed in duplicate. 2.4. Statistical analyses All data were analyzed by ANOVA with the Generalized Linear Model (GLM) procedure, Principal Component Analysis (PCA), Correlation and Cluster analyses using SPSS software version 15.0. Shapiro–Wilk and Kurtosis tests were used to assess normality of the observations. Mean separations were performed by Tukey test. Significance was accepted per P ≤ 0.05 level. 3. Results 3.1. Morphological characterization and biomass production evaluation Eleven agro-morphological traits were totally assessed to characterize biomass production of the 17 genotypes. The results of ANOVA analysis with the statistical significant differences related to each morphological trait among genotypes, between years and for genotype per year interaction have been reported in Table 2. Significant genotype per year interactions have been found for all traits, whereby morphological profile expression of each genotype varied across the different two-year environmental conditions. The most relevant biometric parameters recorded on the plants per each genotype and year have been shown in Table 3. Significant differences among genotypes have been found for all traits evaluated, except for the number of lateral shoots and leaf length both in the 2008–2009 growing season. In the same growing season, Bianco di Pertosa showed the greatest plant height while, in 2009–2010, Bianco di Pertosa and Pisa revealed the highest value for this trait. Only in the second experimental year, Tondo Rosso di Paestum, Bianco di Pertosa, S23, Grato 1 and Pisa had the highest number of lateral shoots. The width of the first fully developed leaf of the main floral stem ranged between about 30 to more than 50 cm in both

Fig. 2. Similarity dendrogram constructed according to the morphological biomass characterization of the 17 genotypes analyzed using agglomerative hierarchical cluster analysis.

years. In both growing seasons, Montelupone A, Montelupone B and Pisa had the highest number of leaves, while only Montelupone B has been evidenced in the second year. Plant diameter varied from 143.7 cm for Montelupone B in the second year to 215.1 cm for Campagnano in the first growing season. In the 2nd year, the plants were generally higher and more vigorous. For the dry weight of the whole plant (leaves and floral stem), only Ascolano confirmed the highest value in both years; S18, Campagnano, Pisa and Bianco di Pertosa provided interesting values only in the first year. A dendrogram of the similarity index, based on these morphological traits describing plant biomass, was constructed using an agglomerative hierarchical cluster analysis. On the basis of the similarity dendrogram, our genotypes could be divided into five major clusters. Cluster 1 consisted of six genotypes: S3, S11, S17, S23, Montelupone A and Tondo Rosso di Paestum. Cluster 2 consisted of S2, S5, Grato 1, Castellammare and Ascolano. In cluster 3, S18, Pisa and Campagnano genotypes were grouped, while cluster 4 consisted of Jesino and Montelupone B. Finally, only Bianco di Pertosa genotype was grouped in cluster 5 (Fig. 2). An among-genotype pairwise similarity matrix was generated with the quantitative morphological traits explaining biomass production for the two growing seasons and a PCA was then applied. The first PC factor (39.75% of variance explained) included contributions from the following primary traits: plant height, first fully developed leaf length and width, number of shoot leaves, plant diameter and dry weight. The second factor (24.35% of the variance explained) involved the main floral stem leaf number. The third factor (15.97% of variance explained) considered the number of lateral shoots. These first three functions explained 80.01% of the variance and each genotype was plotted against these three functions (Fig. 3). PCA showed that four groups could be identified: one on the upper right side with the genotypes S18, Campagnano, Pisa, Bianco di Pertosa and Ascolano, one on lower right side with the genotypes Jesino and Montelupone B from Marche region, one on the lower left side including Tondo Rosso di Paestum, S17 and S23 genotypes and the last one, on the center of the graphic, with all other genotypes. A correlation matrix among the morphological traits analyzed was generated. A certain correlation was found between plant height and diameter (Pearson’s correlation coefficient r = 0.632, P ≤ 0.05), plant diameter and leaf length and width (Pearson’s correlation coefficient r = 0.781 and 0.681, P ≤ 0.05, respectively), leaf length and width (Pearson’s correlation coefficient r = 0.519,

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Fig. 3. Distribution on the basis of biomass production against the three discriminant functions (on the left side) of the 17 globe artichoke spring genotypes and their growing areas in Italian regions (on the right side).

P ≤ 0.05), plant diameter and plant dry weight (Pearson’s correlation coefficient r = 0.588, P ≤ 0.05).

3.2. Biochemical characterization of the biomass The biomass polyphenol content of all genotypes, determined by HPLC analysis, has been reported in Table 4. In our experiments, cynarin, luteolin and apigenin were not detected in all samples analyzed. As regard the other biocompounds, statistical differences among genotypes were found on biomass (leaves and floral stems) biochemical composition. In particular, S3, S5, S17, S18, S23, Ascolano, Campagnano and Grato 1 showed the highest significant content of 1,5-O-dicaffeoylquinic acid, while S3, S5, S17, S18, S23, Ascolano, Campagnano and Montelupone A showed the highest content of chlorogenic acid (Table 4). The genotypes S3, S5, S17, S23, Ascolano, Campagnano, Castellammare, Grato 1 and Montelupone A resulted the genotypes with the highest concentration of cynaroside. The content in total caffeoylquinic acids and measured polyphenols reflected the same trend of the biochemical parameters above described.

For a large-scale field biocompound production, the polyphenol yield per hectare and per genotype was estimated, considering the biomass dry matter accumulated per plant at the planting density used of about 7700 plant ha−1 (Table 5). The genotypes S5, Ascolano, Campagnano and S18 showed the highest total polyphenols per hectare with a yield of 1,5-O-dicaffeoylquinic acid (21.82–30.93 kg ha−1 ), chlorogenic acid (263.95–436.95 kg ha−1 ) and cynaroside (131.73–174.13 kg ha−1 ) (Table 5). In order to determine the connection among the polyphenolic compounds analyzed, a correlation analysis was performed. The 1,5-Odicaffeoylquinic acid and chlorogenic acid contents were positively related in the genotype biomass analyzed (Pearson’s correlation coefficient r = 0.915, P ≤ 0.05). On the contrary, there was no significant correlation among morphological and biochemical traits considered for genotype biomass characterization.

4. Discussion Italian spring genotypes here studied have been previously classified by head morphology in ‘Romanesco’ and ‘Violetto’ groups (Crinò et al., 2008; Ciancolini et al., 2012). This germplasm

Table 4 Phenolic (g kg−1 of DM) contents of globe artichoke spring genotype biomass (leaves and floral stem). Genotype

1,5-O-dicaffeoylquinic acid (g kg−1 DM)

Chlorogenic acid (g kg−1 DM)

Cynaroside (g kg−1 DM)

Total caffeoylquinic acids (g kg−1 DM)

Total measured polyphenols (g kg−1 DM)

Ascolano Campagnano Grato 1 Jesino Montelupone A Montelupone B Bianco di Pertosa Pisa S11 S17 S18 S2 Castellammare S23 S3 S5 Tondo Rosso di Paestum

1.46 ac 1.53 ac 1.36 ad 0.66 de 1.10 be 0.80 ce 0.50 e 0.42 e n.d. 1.56 ac 1.57 ab 0.55 e 0.67 de 1.52 ac 1.96 a 2.10 a n.d.

22.41 ab 18.99 ac 15.57 bc 1.87 de 16.83 ac 1.60 de 0.22 e 0.25 e n.d. 20.94 ab 27.85 a 7.59 ce 13.38 bd 20.61 ab 17.75 ac 21.18 ab n.d.

8.93 a 8.27 ab 5.65 ac 3.00 ce 5.57 ad 2.16 de 0.53 e 1.09 e 1.17 e 6.93 ab 5.10 bd 2.18 de 6.87 ab 8.35 ab 6.14 ac 8.97 a 0.72 e

23.87 ab 20.51 ac 16.93 bc 2.53 d 17.93 ac 2.40 d 0.72 d 0.68 d n.d. 22.50 ab 29.41 a 8.14 cd 13.05 bd 22.13 ab 19.71 ac 23.28 ab n.d.

32.80 ab 28.78 ab 22.58 ac 5.53 de 23.51 ac 4.57 e 1.15 e 1.53 e 1.17 e 29.42 ab 36.44 a 13.98 ce 19.91 bd 30.48 ab 25.85 ac 32.25 ab 0.72 e

n.d., not detected. Values are the means of measurements on 9 plants. Means followed by different letters for each parameter are significantly different at P ≤ 0.05 (Tukey test).

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Table 5 Biocompound yield from globe artichoke spring genotype aerial biomass (planting density 7700 plants ha−1 ). Genotype

1,5-O-dicaffeoylquinic acid (kg ha−1 )

Chlorogenic acid (kg ha−1 )

Cynaroside (kg ha−1 )

Total measured polyphenols (kg ha−1 )

Ascolano Campagnano Grato 1 Jesino Montelupone A Montelupone B Bianco di Pertosa Pisa S11 S17 S18 S2 Castellammare S23 S3 S5 Tondo Rosso di Paestum

28.55 a 27.91 a 10.41 c 4.79 c 14.63 bc 8.24 c 9.20 c 8.69 c n.d. 8.17 c 21.82 ab 8.36 c 7.18 c 10.31 c 24.23 ab 30.93 a n.d.

436.95 a 347.50 ab 119.15 de 13.55 e 223.01 bd 16.39 e 6.50 e 7.86 e 9.05 e 109.89 de 263.95 ad 40.17 e 132.70 de 140.19 ce 219.40 bd 311.20 ac 1.69 e

174.13 a 151.32 a 43.20 bd 21.75 cd 73.83 b 22.18 cd 9.68 d 22.32 cd 16.54 cd 36.35 bd 71.04 b 32.95 bd 73.64 b 56.81 bc 75.91 b 131.73 a 9.68 d

639.63 a 526.73 ab 172.75 de 40.08 e 311.46 cd 46.81 e 20.95 e 31.44 e 25.59 e 154.42 de 507.29 ac 210.95 de 213.52 de 207.30 de 319.54 bd 473.86 ac 11.37 e

n.d., not detected. Means followed by different letters for each parameter are significantly different at P ≤ 0.05 (Tukey test).

was selected by local farmers taking into account both climatic adaptation of the genotype and culinary purposes. In the traditional agricultural management of these genotypes, the flower heads were traditionally harvested in March-April and the aboveground biomass was left in the field without any use and profit. In the last years, the possible alternative application of globe artichoke, consisting in recovering phenolic active compounds from biomass as raw industrial material, has been investigated (Negro et al., 2012; Pandino et al., 2013). Several studies were focused on the polyphenolic composition of the head as a functional food and on the possibility of using outer bracts, floral stem and other head waste products obtained during the globe artichoke industrial processing (Llorach et al., 2002; Lattanzio et al., 2009; Bonasia et al., 2010; Lombardo et al., 2010; Pandino et al., 2010, 2011b). Although some globe artichoke genotypes have been studied for biomass production (Ierna and Mauromicale, 2010; Cravero et al., 2012; Ierna et al., 2012), there is lack of literature data about the selection of germplasm more suitable for both biomass and biocompound profiles. Taking into account the aforementioned remarks, in our work globe artichoke germplasm has been analyzed and characterized both morphologically and biochemically to select the genotypes most appropriate for large-scale biomass and pharmaceutical compound production. The agro-morphological descriptors used allowed a clear identification of the genotypes analyzed and significant differences among genotypes have been found for all the agro-morphological traits evaluated. In particular, the aboveground dry biomass yield provided useful information for industrial uses. In line with previous works (Raccuia and Melilli, 2010; Angelini et al., 2009; Ierna and Mauromicale, 2010), the aerial biomass yield, expressed in kg of dry matter (DM) produced per genotype, is considered an important trait to discriminate the most interesting genotypes under this profile. In our experiments, the field dry biomass yield was estimated 9.7 t ha−1 , as average value of all genotypes evaluated in the two growing seasons, in accordance with other works (Foti et al., 1999; Raccuia and Melilli, 2004). Also plant height and diameter, which both were considered in several studies on Cynara spp. biomass (Piscioneri et al., 2000; Angelini et al., 2009; Gominho et al., 2011), emerge as very useful traits to describe plant size. Other descriptors might be considered of low interest and not directly applicable for germplasm characterization and selection. However, it is interesting to notice that aerial biomass yield (expressed as kg DM plant−1 ) and many agro-morphological traits were also affected by the growing season (environment). In fact, the genotype per-year-interaction was significant for all traits evaluated, showing that environmental

conditions have a different influence on the morphological profile expression of each genotype. In particular, in the second growing season (2009–2010), all genotypes showed the most vigorous plants and the highest dry matter accumulation; this could be related to the general highest rainfall observed in the second growing season (Fig. 1). The high plant vigor of the 2nd year is also in agreement with the results obtained in previous works, which reported an increasing biomass dry yield from the second year of cultivation onwards (Angelini et al., 2009; Raccuia and Melilli, 2010; Gominho et al., 2011). In fact, in the first growing season, the Cynara spp. plants have a general stabilization stage investing mostly in root system development while, in the second year of cultivation, there is a great aerial biomass production and leaf expansion (Angelini et al., 2009; Gominho et al., 2011). In agreement with previous observations (Ciancolini et al., 2013), aerial fresh biomass was collected at the time when primary heads are collected for edible destination in order to take into account the possibility of the dual-valence of the crop (food and no-food). As reported by several authors (Wang et al., 2003; Lattanzio et al., 2009; Pandino et al., 2010, 2011b; Lombardo et al., 2012), also in our experiments, globe artichoke biomass (leaf and floral stem) showed a high content of caffeoylquinic acid and flavonoid derivatives. In particular, the main caffeic acid derivatives detected are chlorogenic acid (from 0.22 g kg−1 DM in Bianco di Pertosa to 27.85 g kg−1 DM in S18) and 1,5-O-dicaffeoylquinic acid (from 0.42 g kg−1 DM in Pisa to 2.10 g kg−1 DM in S5), in agreement with Pandino et al. (2011b). Nevertheless, limited to the flavonoids apigenin and luteolin, our results are in contrast with other work (Pandino et al., 2010); in fact, these compounds have not been here detected by HPLC analysis in biomass extracts, and only cynaroside (from 0.53 g kg−1 DM in Bianco di Pertosa to 8.97 g kg−1 DM in S5) has been quantified. This could be linked to genetic aspects, harvesting time and plant parts analyzed (Lombardo et al., 2010, 2012), but also to environment, agricultural management and abiotic/biotic stress. In addition, differences in the biochemical profile could depend on the different extraction methods used. Indeed, it is important to consider that each compound shows different polarities and thermal stabilities and is characterized by a different optimal set of extraction conditions. For example, the relative abundance of caffeoylquinic acid derivatives, identified in globe artichoke extracts, is strongly related to the solvent, pH and temperature used for their extraction (Lattanzio et al., 2009). In our case, 80% ethanol as extraction solvent fitted as more suitable for nutraceutical or cosmetic applications. The biocompound yield of biomass obtained per hectare, ranging from 11.37 kg ha−1 in Tondo Rosso di Paestum to 639.63 kg ha−1 in Ascolano, was very

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interesting, also considering that this material has never been selected for the double biomass and biochemical purpose. In fact, as observed by Cravero et al. (2012), in globe artichoke almost all the aerial biomass is up to now destined toward the flower heads, due to the domestication and selection criteria adopted by farmers aimed at these edible organs. In particular, significant differences have been shown among genotypes for all biochemical compounds analyzed, this highlighted the genetic variability existing in globe artichoke germplasm and enhanced the possibility to run successfully selections. For this reason, selection of genotypes of interest under both biochemical and biomass profiles is an important step in opening new horizons in globe artichoke breeding programs. 5. Conclusions In Italy, globe artichoke is up now cultivated as horticultural crop for food production. Results obtained in this work indicated that aboveground biomass of the species is worth considering, taking also into account the low energy input used for crop management. In addition, the possibility of using globe artichoke biomass for pharmaceutical compound extraction has been investigated and the real opportunity for a no-food production (biomass and biocompound) of the crop has been confirmed by the morphological and biochemical analyses of the genotypes Ascolano and Campagnano selected in our experiments. Both clones are two autochthonous genotypes, traditionally grown in Italian Marche and Latium regions, that may be valorized either for food (Ciancolini et al., 2012) or no-food use. The dual productive valence, with the possibility of biomass sale after that of the central and primary heads, can be of interest to increase the farmer’s income. In addition, the selection of this genetic material could represent an important basis for the development of new varieties addressed specifically to double head and biomass production purpose, up to now not yet considered. Efforts should be made to arouse the interest of industries toward these genotypes capable of optimizing the production of phenolic compounds. The fact that selection has favored these traditional genotypes enhances the potential of these genetic resources that, in the past, represented a source of sustenance for local people. Valorizing this germplasm is of paramount importance to preserve it. Further investigations should be also performed to study the effect of planting density and different agricultural management techniques (i.e. fertilization, irrigation) on biomass and biocompound production. Acknowledgements The authors acknowledge the CYNARES (EC) Nos. 870/2004, CAR-VARVI and EFDR 30159 projects, which received financial support of the AGRI GEN RES Community Programme (European Commission, Directorate-General for Agriculture and Rural Development), Italian Ministry of Agriculture, Food and Forestry Policies and European founds, respectively. The authors are grateful to ARSIAL (Cerveteri, Rome Italy) for having provided field facilities. Many thanks are addressed also to Dr. Rudolph Hupperts for the final linguistic revision of the manuscript. References Angelini, L.G., Ceccarini, L., Nassi o Di Nasso, N., Bonari, E., 2009. Long-term evaluation of biomass production and quality of two cardoon (Cynara cardunculus L.) cultivars for energy use. Biomass Bioenergy 33, 810–816. Bonasia, A., Conversa, G., Lazzizera, C., Gambacorta, G., Elia, A., 2010. Morphological and qualitative characterization of globe artichoke head from new seed-propagated cultivars. J. S. Food Agric. 90, 2689–2693.

Ciancolini, A., Rey, N.A., Pagnotta, M.A., Crinò, P., 2012. Characterization of Italian spring globe artichoke germplasm: morphological and molecular profiles. Euphytica 186, 433–443. Ciancolini, A., Alignan, M., Miquel, J., Vilarem, G., Pagnotta, M.A., Crinò, P., 2013. Biomass and biocompound production for valorizing Italian spring globe artichoke genotypes. Acta Hortic. (ISHS) 983, 55–61. Coinu, R., Cart, S., Urgeghe, P., Mulinacci, N., Pinelli, P., Franconi, F., Romani, A., 2007. Dose-effect study on the antioxidant properties of leaves and outer bracts of extracts obtained from Violetto di Toscana artichoke. Food Chem. 101, 524–531. Cravero, V., Martin, E., Crippa, I., Lopez Anido, F., Maris Garcia, S., 2012. Fresh biomass production and partitioning of aboveground growth in the three botanical varieties of Cynara cardunculus L. Ind. Crop Prod. 37, 253–258. Crinò, P., Tavazza, R., Rey, N.A., Trionfetti Nisini, P., Saccardo, F., Ancora, G., Pagnotta, M.A., 2008. Recovery, morphological and molecular characterization of globe artichoke ‘Romanesco’ landraces. Genet. Resour. Crop Evol. 55, 823–833. Fantini, N., Colombo, G., Giori, A., Riva, A., Morazzoni, P., Bombardelli, E., Carai, M.A.M., 2011. Evidence of glycemia-lowering effect by a Cynara scolymus L. extract in normal and obese rat. Phytother. Res. 25, 463–466. Fateh, E., Chaichi, M.R., Sharifi Ashorabadi, E., Mazaheri, D., Jafari, A.A., Rengel, Z., 2009. Effects of organic and chemical fertilizers on forage yield and quality of globe artichoke (Cynara scolymus L.). Asian J. Crop. Sci. 1, 40–48. Fiore, V., Valenza, A., Di Bella, G., 2011. Artichoke (Cynara cardunculus L.) fibres as potential reinforcement of composite structures. Compos. Sci. Technol. 71, 1138–1144. Foti, S., Mauromicale, G., Raccuia, S.A., Fallico, B., Fanella, F., Maccarone, E., 1999. Possible alternative utilization of Cynara spp. biomass, grain yield and chemical composition of grain. Ind. Crop Prod. 10, 219–228. Fratianni, F., Tucci, M., De Palma, M., Pepe, R., Nazzaro, F., 2007. Polyphenolic composition in different parts of some cultivars of globe artichoke (Cynara cardunculus L. var. scolymus (L.) Fiori). Food Chem. 104, 1282–1286. Gominho, J., Lourenc¸o, A., Palma, P., Lourenc¸o, M.E., Curt, M.D., Fernández, J., Pereira, H., 2011. Large scale cultivation of Cynara cardunculus L., for biomass production—a case of study. Ind. Crop Prod. 33, 1–6. Hernández Allica, J., Becerril, J.M., Garbisu, C., 2008. Assessment of the phytoextraction potential of high biomass crop plants. Environ. Pollut. 152, 32–40. Ierna, A., Mauromicale, G., 2010. Cynara cardunculus L. genotypes as a crop for energy purposes in a Mediterranean environment. Biomass Bioenergy 34, 754–760. Ierna, A., Mauro, R.P., Mauromicale, G., 2012. Biomass, grain and energy yield in Cynara cardunculus L. as affected by fertilization, genotype and harvest time. Biomass Bioenergy 36, 404–410. Lattanzio, V., Kroon, P.A., Linsalata, V., Cardinali, A., 2009. Globe artichoke: a functional food and a source of nutraceutical ingredients. J. Funct. Food 1, 131–144. Ledda, L., Deligios, P.A., Farci, R., Sulas, L., 2013. Biomass supply for energetic purposes from some Cardueae species grown in Mediterranean farming systems. Ind. Crop Prod. 47, 218–226. Llorach, R., Espin, J.C., Tomás Barberán, F.A., Ferreres, F., 2002. Artichoke (Cynara scolymus L.) byproducts as a potential source of health-promoting antioxidant phenolics. J. Agric. Food Chem. 50, 3458–3464. Llorente, B.E., Brutti, C.B., Caffini, N.O., 2004. Purification and characterization of a milk-clotting aspartic proteinase from globe artichoke (Cynara scolymus L.). J. Agric. Food Chem. 52 (26), 8182–8189. Lombardo, S., Pandino, G., Mauromicale, G., Knödler, M., Carle, R., Schieber, A., 2010. Influence of genotype, harvest time and plant part on polyphenolic composition of globe artichoke [Cynara cardunculus L. var. scolymus (L.) Fiori]. Food Chem. 119, 1175–1181. Lombardo, S., Pandino, G., Ierna, A., Mauromicale, G., 2012. Variation of polyphenols in a germplasm collection of globe artichoke. Food Res. Int. 46, 544–553. Negro, D., Montesano, V., Grieco, S., Crupi, P., Sarli, G., De Lisi, A., Sonnante, G., 2012. Polyphenol compounds in artichoke plant tissue and varieties. J. Food Sci. 77 (2), 244–252. Pandino, G., Courts, F.L., Lombardo, S., Mauromicale, G., Williamson, G., 2010. Caffeoylquinic acids and flavonoids in the immature inflorescence of globe artichoke, wild cardoon, and cultivated cardoon. J. Agric. Food Chem. 58, 1026–1031. Pandino, G., Lombardo, S., Mauromicale, G., 2011a. Chemical and morphological characteristics of new clones and commercial varieties of globe artichoke (Cynara cardunculus var. scolymus). Plant Food Hum. Nutr. 66, 291–297. Pandino, G., Lombardo, S., Mauromicale, G., Williamson, G., 2011b. Profile of poyphenols and phenolic acids in bracts and receptacles of globe artichoke (Cynara cardunculus var. scolymus), germplasm. J. Food Compos. Anal. 24, 148–153. Pandino, G., Lombardo, S., Mauro, R.P., Mauromicale, G., 2012. Variation in polyphenol profile and head morphology among clones of globe artichoke selected from a landrace. Sci. Hortic. 138, 259–265. Pandino, G., Lombardo, S., Mauromicale, G., 2013. Globe artichoke leaves and floral stems as a source of bioactive compounds. Ind. Crop Prod. 44, 44–49. Piscioneri, I., Sharma, N., Baviello, G., Orlandini, S., 2000. Promising industrial energy crop, Cynara cardunculus: a potential source for biomass production and alternative energy. Energ. Convers. Manage. 41 (10), 1091–1105. Raccuia, S.A., Melilli, M.G., 2004. Cynara cardunculus L., a potential source of inulin in Mediterranean environment: screening of genetic variability. Aust. J. Agric. Res. 55, 693–698.

A. Ciancolini et al. / Industrial Crops and Products 49 (2013) 326–333 Raccuia, S.A., Melilli, M.G., 2010. Seasonal dynamics of biomass, inulin and watersoluble sugars in roots of Cynara cardunculus L. Field Crops Res. 116, 147–153. Rondanelli, M., Giacosa, A., Orsini, F., Opizzi, A., Villani, S., 2011. Appetite control and glycaemia reduction in overweight subjects treated with a combination of two highly standardized extracts from Phaseolus vulgaris and Cynara scolymus. Phytother. Res. 25, 1275–1282.

333

Saénz Rodriguez, T., García Giménez, D., De la Puerta Vázquez, R., 2002. Choleretic activity and biliary elimination of lipids and bile acids induced by an artichoke leaf extract in rats. Phytomedicine 9 (8), 687–693. Wang, M., Simon, J.E., Aviles, I.F., He, K., Zheng, Q.Y., Tadmor, Y., 2003. Analysis of antioxidative phenolic compounds in artichoke (Cynara scolymus L.). J. Agric. Food Chem. 51 (3), 601–608.