Influence of genotype and harvest time on Cynara cardunculus L

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Bioactive Constituents, Metabolites, and Functions

Influence of genotype and harvest time on Cynara cardunculus L. sesquiterpene lactone profile Aurelio Scavo, Carlos Rial, Rosa M. Varela, José M. G. Molinillo, Giovanni Mauromicale, and Francisco A. Macías J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02313 • Publication Date (Web): 16 May 2019 Downloaded from http://pubs.acs.org on May 16, 2019

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Journal of Agricultural and Food Chemistry

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Influence of genotype and harvest time on Cynara cardunculus L. sesquiterpene

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lactone profile

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Aurelio Scavoa, Carlos Rialb, Rosa Maria Varelab*, José Maria Gonzalez Molinillob,

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Giovanni Mauromicalea, Francisco Antonio Maciasb

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a)

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via Valdisavoia, 5, 95123 Catania, Italy.

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b)

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(INBIO), Campus de Excelencia Internacional (ceiA3), School of Science, University of

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Cadiz, C/ República Saharaui nº 7, 11510 Puerto Real, Cadiz, Spain.

Department of Agriculture, Food and Environment (Di3A), University of Catania,

Allelopathy Group, Department of Organic Chemistry, Institute of Biomolecules

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*Corresponding

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956012729; Fax: +34 9560161933.

author: Prof. Rosa María Varela, email: [email protected], phone: +34

1 ACS Paragon Plus Environment


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ABSTRACT

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The excessive and inappropriate application of herbicides has caused environmental

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pollution. The use of allelochemicals as bioherbicides could provide a solution to this

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problem. The allelopathic activity of Cynara cardunculus L. has been studied previously

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and sesquiterpene lactones (STLs) were identified as the most relevant allelochemicals.

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The goal of the study reported here was to investigate the effect of six genotypes and three

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harvest times on the quali-quantitative composition of STLs in C. cardunculus leaves

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through a new UHPLC-MS/MS analysis method and, thus, the effect on phytotoxicity.

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Overall, wild cardoon contained the highest levels of STLs of the three botanical varieties

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studied. Nevertheless, climatic conditions had a marked influence on the presence of

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STLs among the six genotypes, which was higher in the April harvest. Cynaropicrin was

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the most abundant STL detected. A close relationship was found between the STL profiles

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and the allelopathic activity, expressed as inhibition of wheat coleoptile elongation. The

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data provide a new and important contribution to our understanding of C. cardunculus

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allelopathy.

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Keywords: allelopathy; weed control; Cynara cardunculus; sesquiterpene lactones;

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cynaropicrin


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INTRODUCTION

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In recent decades the wide and inappropriate use of synthetic herbicides in intensive

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agriculture has led to a considerable impact on human, animal and environment health.

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For this reason, the scientific community, agricultural politics and society have demanded

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alternative and sustainable methods for weed control. An important and innovative

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solution is provided by allelopathy, which refers to the direct or indirect harmful or

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beneficial effects of a particular plant on another through the release of chemical

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compounds, commonly called allelochemicals, into the environment.1 The possible

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applications of allelopathy in weed management are numerous. The most common

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approach is provided by the selection of active allelochemicals and their use as

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bioherbicides.2

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According to Fiori’s classification, the Asteraceae member Cynara cardunculus L.,

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a typical C3 species of the Mediterranean Basin, includes three botanical varieties that are

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cross-pollinated and cross-compatible with one another: the globe artichoke [C.

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cardunculus L. var. scolymus (L.) Fiori], the cultivated cardoon (C. cardunculus L. var.

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altilis DC.) and their common progenitor wild cardoon [C. cardunculus L. var. sylvestris

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(Lamk) Fiori].3,4 Several studies have been carried on the allelopathic activity of C.

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cardunculus, especially on cultivated cardoon.5–7 The allelopathic potential of this plant

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is mainly due to the presence of sesquiterpene lactones (STLs), particularly cynaropicrin,

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grosheimin and aguerin B,5 as well as polyphenols such as caffeoylquinic acid, luteolin-

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and apigenin derivatives,7 which are present in high quantities in the leaves.8 In a previous

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study, Scavo et al. (2019)9 isolated three STLs (cynaratriol, deacylcynaropicrin and

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11,13-dihydro-deacylcynaropicrin) and a lignan (pinoresinol) from the ethyl acetate

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fraction of the aqueous extract of cultivated cardoon.9 Deacylcynaropicrin, 11,13-

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dihydro-deacylcynaropicrin and pinoresinol showed inhibitory effects on wheat



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coleoptile elongation, while cynaratriol was inactive. Although the leaf polyphenol

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composition of C. cardunculus has been widely reported in the literature,8,10 only a few

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studies have been carried out on the STL profile.11–13 STLs are characteristic compounds

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of the Asteraceae family and they are derived from farnesyl diphosphate (FPP). These

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compounds are the major lipophilic constituents in cultivated cardoon leaves (95 g/kg dry

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weight).14 In addition to phytotoxic activity, these compounds show fungicidal,15

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antimicrobial16 and anti-cancer activity.17 STL-containing plants have long been known

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to induce a contact dermatitis in exposed farm workers.18 In our case, the immature

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inflorescence of cultivated cardoon is commonly used as a vegetable or the leaves have

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been used to obtain the Italian liqueur Cynar and in Germany and in Switzerland as

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remedy for indigestion, being STLs the responsible of their bitter taste.19

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Genotype and plant age are two important aspects that must be considered in order

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to understand allelopathic potential.2 The influence of genotype is widely reported in the

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literature for several crop species such as rice,20,21 sorghum,22 rye,23 sunflower,24 etc. A

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great deal of evidence has also been reported on the influence of plant or organ age on

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the allelopathic effects of plants.25,26 Pandino et al. (2013)27 studied the quali-quantitative

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composition of polyphenols in globe artichoke tissue from November to April and

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reported that the highest polyphenol content for leaves was detected in the February-April

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harvest period. Considering the effects of genotype and harvest time on C. cardunculus

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polyphenol profiles, our hypothesis is that these biotic factors could affect the STL

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concentration in C. cardunculus leaves and thus its phytotoxic activity.

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The goal of the work described here was to examine the quantity and composition of

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STLs in C. cardunculus leaves as affected by genotype and harvest time by applying a

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new UHPLC-MS/MS analysis method. Moreover, the leaf ethanolic extracts of six

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different genotypes obtained from three harvest times were evaluated on wheat coleoptile


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elongation in an effort to identify a correlation, if any, between the inhibitory activity and

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the STL profile.

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MATERIAL AND METHODS

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Experiment design, plant material, field sampling and crop management. The

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experiment was set up in a completely randomized design with four replications including

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the six genotypes reported in Table 1. 'Naro 2' is a landrace from small-holdings grown

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in central Sicily, 'VSB3' is a clone selected from the most popular Sicilian varietal type

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'Violetto di Sicilia', 'Marsala' and 'Valparaiso' were obtained from a native stand in

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Marsala (Western Sicily) and Valparaiso (Chile), respectively, 'Altilis 41' is a synthetic

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cultivar developed by the University of Catania for biomass and bioactive molecule

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production and 'Bianco gigante' is an Italian commercial cultivar. The latter two are

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cultivated cardoons.

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Plants were grown in the Catania experimental station [Southern Italy, 37° 25I N; 15°

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30I E; 10 m a.s.l.] in a typic vertic and/or xerochrept soil (Soil Survey Staff, 1999)28 with

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a clay texture. The soil characteristics were as follows: sand 27%, clay 45%, silt 28%,

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organic matter 1%, total nitrogen 0.1%, available P2O5 20 ppm, and pH 7.2. The local

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climate is semi-arid Mediterranean, with mild rainy winters and hot dry summers. Prior

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to planting, the field was ploughed to a depth of 30 cm, harrowed and a fertilizer rate of

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50 kg N/ha (as urea), 80 kg P2O5/ha (as double perphosphate) and 80 kg K2O/ha (as

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potassium sulfate) was given.

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One hundred seedlings, each bearing three leaves, of cultivated and wild cardoon or

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sprouting ‘ovoli’ (semi-dormant offshoots) of globe artichoke were transplanted into the

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field at the end of August at a rate of 1 plant/m2, using an inter- and intra-row spacing of

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1.25 and 0.80 m, respectively. The expanded plot was kept weed- and insect-free by

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spraying oxyfluorfen and imidacloprid, respectively, when required. Fifty fully expanded



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leaves were randomly sampled at the phenological growth stage reported in Table 2 in

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the centre of each plot. Leaves from each genotype were harvested three times during the

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growing season from November to April. The genotypes, the harvest times and the

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corresponding phenological growth stages according to the BBCH scale proposed by

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Archontoulis et al. (2010)29 are reported in Tables 1 and 2. These harvest times cover

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significantly different meteorological conditions (Figure S1) that could affect the

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concentration of sesquiterpene lactones in C. cardunculus leaves and thus, according to

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our hypothesis, they identify the most appropriate periods for a possible industrial

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collection of leaf biomass and the extraction of bioactive molecules, as reported by

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Pandino et al. (2013)27 for polyphenols.

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Meteorological conditions. Meteorological data were measured during leaf

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development (from October to April) by a meteorological station (Mod. Multirecorder

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2.40; EGT, Florence, Italy) sited within 250 m of the experimental field. Both maximum

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and minimum monthly average temperature, total monthly rainfall and global radiation

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were acquired. Air temperature (minimum, maximum and mean) and global radiation

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were recorded every hour. The mean air temperature fell in December and January (11

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and 12.8 °C, respectively) and then rose to 16.5°C in April (Figure S1). A similar trend

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was observed for global radiation, with the minimum and maximum values registered in

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the first decade of February (6.8 MJ/m2) and in the third decade of April (23.3 MJ/m2),

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respectively. The total precipitation during the growing season was less than 306 mm,

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with the lowest level occurring in April (5.4 mm).

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Reagents. The STLs cynaropicrin (6), aguerin B (8), grosheimin (5) and 11,13-

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dihydroxy-8-deoxygrosheimin (2) were obtained according to the procedure previously

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described

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deacylcynaropicrin (1) and cynaratriol (3) were isolated according the methodology

by

Rial

et

al.

(2014).5

Deacylcynaropicrin


(4),

11,13-dihydro-

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describe by Scavo et al. (2019).9 Compound 1 was used as a 1:1 epimeric mixture.

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Santamarine (7) was synthesized as reported previously by Lu and Fischer (1996).30 The

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molecular structures of compounds analyzed in this study are shown in Figure 1 and their

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purity have been checked by NMR spectroscopy and mass spectrometer. The solvents for

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UHPLC-MS/MS analysis and the extractions were UHPLC-grade. Methanol (MeOH),

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ethanol (EtOH), dichloromethane and formic acid were obtained from Fischer Chemicals

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(Geel, Belgium). Water for LC-MS/MS analysis was type I obtained from an Ultramatic

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system from Wasserlab (Barbatáin, Spain). Ammonium formate was obtained from

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Sigma-Aldrich (St. Louis, MO, USA).

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Leaf extract preparation. Leaf extracts were prepared according to the

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methodology proposed by Scavo et al. (2018)7 with some modifications. 12 g of powdered

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dried leaves were mixed in 250 mL of ethanol/water (80:20, v/v) in an agitator incubator

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(Thermo Fisher Scientific Inc., USA) at 100 rpm for 72 h at room temperature (25 °C ±

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1) in the dark, and then filtered through filter paper (Whatman No. 2). Solvents were

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evaporated using a rotary evaporator (Laborata 4000, Heidolph, Germany) at 35 °C and

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the residues were redissolved in water. Since EtOH extracts were very rich in

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chlorophylls, it was decided to remove them by solid phase extraction (SPE) to facilitate

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the UHPLC-MS/MS analysis. Extracts, previously dissolved in 1 mL of 100% MeOH

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and 1 mL of water, were passed through Strata-X 33 μm Polymeric Reversed Phase

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cartridges (200 mg/ 3 mL, Phenomenex, CA, USA) using a vacuum manifold system

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(Phenomenex, CA, USA). Cartridge polymers were activated with 4 mL of 100% MeOH

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and 4 mL of water. The samples were then loaded onto the cartridges and were eluted

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with 100% MeOH (4 mL).

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Wheat coleoptile bioassay. The inhibitory activities of the different extracts were

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evaluated by the wheat coleoptile bioassay at 800, 400 and 200 ppm. A negative control



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(0 ppm) represented by a phosphate-citrate buffer containing 2% sucrose31 at pH 5.6 and

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the commercial herbicide Logran® as positive control were also evaluated. The

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experiment was carried out by the method described by Macias et al. (2000),32 with three

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replicates per dilution in duplicate.

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UHPLC-MS/MS analysis

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Multiple Reaction Monitoring. The compound-dependent parameters for each

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standard and the internal standard (IS) santamarine were optimized by direct infusion to

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the mass spectrometer to obtain maximum multiple reaction monitoring (MRM) signal

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intensities for the ammonium adduct [M + NH4]+. The parent or precursor ions, the

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fragments obtained by MRM analysis and the collision energy to achieve each

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fragmentation are provided in Table 3.

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Calibration curves. Each standard was dissolved in MeOH to obtain a stock standard

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solution concentration of 1000 mg/L. The internal calibration curve for each of the

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standards was prepared by serial dilution of these stock solutions as follows: 11,13-

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dihydro-deacylcynaropicrin from 50 to 0.025 mg/L (11 levels), 11,13-dihydroxy-8-

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deoxygrosheimin from 50 to 0.25 mg/L (8 levels), cynaratriol from 50 to 0.1 mg/L (9

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levels), deacylcynaropicrin from 50 to 0.05 mg/L (10 levels), grosheimin from 50 to 0.05

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mg/L (10 levels), cynaropicrin from 50 to 0.25 mg/L (8 levels) and aguerin B from 50 to

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0.1 mg/L (9 levels). Santamarine (the IS) was dissolved in MeOH to obtain a

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concentration of 1000 mg/L and this was added to all samples to give a final concentration

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of 1 mg/L. Santamarine is a characteristic sesquiterpene lactone of Asteraceae12,33,34 that

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has a structure and molecular weight similar to those of the sesquiterpene lactones of

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interest. The synthesis of santamarine has been described30 and this compound is not

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present in C. cardunculus extract. For these reasons santamarine was selected as the IS

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for the quantitation. A 5-μL aliquot of each standard solution was injected three times


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onto the UHPLC column. The calibration curve was obtained by plotting the peak area

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ratio (y) of standard to IS versus the ratio of their concentrations (x). The curve was fitted

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to a linear function with a weight of 1/nx (R2 > 0.99), where ‘n’ is the calibration level.

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The compounds in the sample were determined by their peak area ratio with respect to

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the internal standard and by reference to the standard curve. All samples were filtered

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through a PTFE syringe filter (0.22 µm) prior to analysis and they were stored at –80 ºC.

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Sample preparation. Extracts were dissolved in MeOH to give a ratio of 1/1 g/L and

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IS was also added to give a final concentration of 1 mg/L. A 5-μL aliquot of each sample

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was injected three times onto the UHPLC column. All samples were filtered through a

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PTFE syringe filter (0.22 µm) prior to analysis and samples were stored at –80 ºC.

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UHPLC-MS/MS method. Samples were analysed on a Bruker EVOQ Triple

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Quadrupole Mass Spectrometer with an atmospheric pressure chemical ionization source

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(APCI) in positive mode. Samples were separated using a Kinetex 1.7 µm C18 column

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(100 × 2.1 mm, 1.7 µm particle size) (Phenomenex, Torrance, CA, USA) maintained at

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40 ºC. The mobile phase consisted of solvent A (water, 0.1% formic acid, 2 mM

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ammonium formate) and solvent B (MeOH, 0.1% formic acid, 2 mM ammonium

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formate) and the flow rate was set to 0.3 mL/min. The optimized linear gradient system

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was as follows: 0−1 min, 20% B; 1−3 min, to 25% B; 3−7 min, 25% B; 7−8 min, to 70%

199

B; 8−9 min, to 100% B, 9–12 min, 100% B, 12–12.5 min, to 20% B, 12.5–15.5 min, 20%

200

B. The autosampler was set at 7 °C to preserve the samples. The injection volume was 5

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μL. The instrument parameters were as follows: spray current 100 µA, cone temperature

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300 ºC, cone gas flow 20 psi, heated probe temperature 250 ºC, heated probe gas flow 40

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psi, nebulizer gas flow 60 psi and collision pressure 2.0 mTorr.

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Method validation. The validation of the method was carried out following ICH

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recommendations35 (selectivity, linearity, precision of the instrumental system,



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repeatability and intermediate precision, detection and quantitation limits, and stability).

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The analytical properties of the calibration curves prepared are provided in Table 4. The

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quantitation (LOQ) and detection (LOD) limits were determined by nine replicate

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analyses and were measured as 10 and 3 times the standard deviation of baseline noise

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respectively. The precision of the method was studied in an intra- and interday assay (n

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= 9) for each compound. Repeatability was assessed by analysing replicates of the same

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sample with all standards. The method was found to be precise with RSD values below

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8% in all cases. Intermediate precisions were below 10% for all analytes injected on three

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different days. The chromatographic separation of standards is shown in Figure 2.

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Data and statistical analysis. Calibration curves and data acquisition of STLs were

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processed with the software MS Data Review (Bruker Chemical Analysis). The

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homogeneity of variance was verified by Bartlett’s test and the independence of cases by

218

graphical inspection of the residuals. All data were subsequently subjected to analysis of

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variance (ANOVA). A two-way ANOVA (6 genotype × 3 harvest time) for STL profile

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and a three-way ANOVA (6 genotype × 3 harvest time × 4 extract concentration) for the

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wheat coleoptile bioassay were applied. In both cases, means were separated using the

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Student–Neuman–Keuls test only when the F test of the ANOVA for treatments and

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interactions was significant at the P

Influence of genotype and harvest time on Cynara cardunculus L

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