Article pubs.acs.org/JAFC
Phenolic Compounds and Sesquiterpene Lactones Profile in Leaves of Nineteen Artichoke Cultivars Youssef Rouphael,† Jamila Bernardi,‡ Mariateresa Cardarelli,§ Letizia Bernardo,⊥ David Kane,∥ Giuseppe Colla,# and Luigi Lucini*,⊥ †
Department of Agricultural Sciences, University of Naples Federico II, Portici, Italy Department of Sustainable Crop Production and ⊥Institute of Environmental and Agricultural Chemistry, Università Cattolica del Sacro Cuore, Piacenza, Italy § Consiglio per la Ricerca in Agricoltura e l’analisi dell’economia agraria, Centro di ricerca per lo studio delle Relazioni tra Pianta e Suolo, Rome, Italy ∥ Knoell Iberia S.L., Paseo de la Castellana 95, 28046 Madrid, Spain # Department of Agricultural and Forestry Sciences, University of Tuscia, Viterbo, Italy ‡
S Supporting Information *
ABSTRACT: Leaves of globe artichoke are food industry byproducts gaining interest due to their therapeutic and nutraceutical potential. The total phenolics, flavonoids, and flavonols content as well as radical scavenging capacity and reducing antioxidant power were determined in leaves of 19 artichoke cultivars. An untargeted analysis based on high-resolution mass spectrometry was then carried out to profile phenolic compounds and sesquiterpene lactones (STLs). The phenolic profile of leaf extracts from different cultivars was widely diverse and included flavonoids, hydroxycinnamic acids, tyrosols, and lignans. Grosheimin and its derivative were the most abundant STLs in all artichoke cultivars. Among the examined cultivars, “Campagnano”, “Grato 1”, and “Violetto di Provenza” were found to be the richest in polyphenols and presented the highest antioxidant activity, whereas “Blanca de Tudela” and “Carderas” were characterized by a high STLs content. Hence, specific artichoke cultivars can be selected as the source of natural antioxidants with a desired profile of nutraceutical compounds like phenolics and STLs. KEYWORDS: Cynara cardunculus, antioxidant activity, grosheimin, nutraceuticals, untargeted profiling, high-resolution MS
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INTRODUCTION Cynara cardunculus L. subsp. scolymus (L.) Hegi, commonly known as globe artichoke, is a herbaceous plant from the Asteraceae family, originating from the Mediterranean area, but is now grown widely around the world.1,2 Globe artichoke is mostly cultivated for its immature inflorescence due to its high concentration of health-promoting compounds.3 Besides the edible heads, industrial byproducts such as stems, outer bracts, and especially leaves (representing 80−85% of the biomass) may be used as raw material for the extraction of high-value compounds for the pharmaceutical and cosmetic industries.1 In numerous in vitro, preclinical, and clinical studies, artichoke leaf extracts have exhibited hepatoprotective, antiatherosclerotic, antioxidant, choleretic, antimicrobial, anticarcinogenic, and antiHIV activities as well as the ability to inhibit cholesterol biosynthesis and LDL oxidation.1,4−6 These therapeutic and pharmacological properties of artichoke leaf extracts can be ascribed to several phenolic substances1 such as caffeoylquinic acid derivates,1,7 flavones (apigenin, luteolin, and their conjugates), as well as flavanones.1,8 In addition, artichoke synthesizes and accumulates sesquiterpene lactones (STLs),9,10 of which cynaropicrin, grosheimin, and its derivatives (in much lower amounts) are most common.11,12 STLs, in particular, cynaropicrin, have various medicinal properties including antiinflammatory activity and cytotoxicity against several types of cancer cells.9,13,14 © 2016 American Chemical Society
The quantitative and qualitative variations of bioactive compounds in artichoke leaves depends upon many preharvest factors such as genetic materials, their physiological stage of development, and the environment during plant growth.15−17 However, genetic material is a key preharvest factor and the major determinant of variation in polyphenol profile and content.16 As Italy possesses the richest and most important artichoke germplasm, with numerous commercial varieties and landraces,18 the characterization and identification of potential bioactive compounds could allow selecting artichoke cultivars with fortified phytonutrient content. Previous studies19−23 have evaluated the leaf polyphenol profile of globe artichoke cultivars, but most of these studies were limited to few cultivars (3−10) or to a specific type (“Romanesco”)24 without considering the most common early cultivars belonging to the elongated types. In addition, all previous work has focused on target phenolics, with limited information on STLs. In this regard, metabolomics provides an efficient tool for investigating the phytochemical profile of globe artichoke due to its ability to quantify a large number of metabolites, including health-promoting compounds, in a single analysis.25 Received: Revised: Accepted: Published: 8540
August 29, 2016 October 21, 2016 October 28, 2016 October 28, 2016 DOI: 10.1021/acs.jafc.6b03856 J. Agric. Food Chem. 2016, 64, 8540−8548
Article
Journal of Agricultural and Food Chemistry
rutin calibration curve, and results are expressed as rutin equivalents (RE). Flavonols were determined colorimetrically by mixing 1 mL of each extract with 1 mL of AlCl3 (20 g L−1) and 3 mL of sodium acetate (50 g L−1). Absorbance was then read at 440 nm after 2.5 h at 20 °C. A calibration curve was prepared by analyzing rutin standard solutions in ethanol, and flavonols content was expressed as RE. The coefficient of determination R2 of each calibration curve was calculated for phenolic compounds, flavonoids, and flavonols and accepted when >0.98. Assay of Antioxidant Capacity. The antioxidant capacity of our artichoke leaf extracts was assessed as previously reported.2,26 The 2,2diphenyl-l-picrylhydrazyl (DPPH, from Sigma-Aldrich) radical scavenging activity was determined from 1 mL of extract, placed in a cuvette with 1.5 mL of a 1.0 × 10−4 mol L−1 daily prepared ethanol solution of DPPH. Absorbance measurements were carried out at 517 nm using a PerkinElmer (Ontario, Canada) Lambda 12 spectrophotometer, at 5 min intervals, until the steady state. The results were expressed as trolox equivalents (TEAC) using a calibration curve from trolox solutions in 70% absolute ethanol. Assessments of the oxygen-radical absorbing capacity (ORAC) were carried out using a Synergy HT Multi-Detection Microplate Reader (BioTek Instruments, Inc. Winooski, VT) at 485 nm for excitation and at 528 nm for emission. The peroxyl radical generator 2,2′-azobis-2amidinopropane dihydrochloride (AAPH) and Trolox (both from Sigma-Aldrich), together with fluorescein (Invitrogen, Monza, Italy), were used for the assays. The wells in the plate were filled with 150 μL of fluorescein solution. Then 25 μL of extract was added, and the resulting solution was incubated for 30 min. Reactions were initiated by adding 25 μL of AAPH solution and fluorescence monitored continuously every minute to calculate the area under the curve. Results were expressed as TEAC. The ferric reducing antioxidant power (FRAP) assay was determined using a clinical autoanalyzer ILAB 600 (Instrumentation Laboratory, Lexington, MA). The FRAP working reagent was prepared at the time of use by mixing (a) acetate buffer 300 mM, pH 3.6, (b) TPTZ (2,4,6-tripyridyl-s-triazine, Sigma-Aldrich) 10 mM in 40 mM HCl, and (c) FeCl3 20 mM in a ratio of 10:1:1. The extract (10 μL) was mixed with 300 μL of working FRAP reagent. Absorbance (600 nm) was measured after 243 s of incubation at 37 °C. FRAP values were finally expressed as TEAC 100 g−1. Profiling of Phenolic Compounds and Sesquiterpene Lactones. High-resolution MS analyses were performed on a hybrid quadrupole-time-of-flight instrument coupled to an UHPLC chromatographic system (UHPLC/Q-TOF-MS) to investigate secondary metabolites profile in leaf extracts. A 1290 liquid chromatograph system, equipped with a binary pump and a dual electrospray jetstream ionization system, and coupled to a G6550 mass spectrometer detector (all from Agilent technologies Santa Clara, CA, USA) was used. The mass spectrometer was run in the positive scan mode to acquire spectra in the range of 50−1000 m/z, while chromatographic separation was performed using an Agilent Zorbax Extend-C18 column (75 × 2.1 mm i.d., 1.8 μm). The LC mobile phase A consisted of water (proteomic grade, VWR, Milan, Italy), while mobile phase B was methanol (LCMS grade, VWR, Milan, Italy). Formic acid 0.1% (v/v) and ammonium formate (5 mM) (both from SigmaAldrich) were added to both phases. The gradient was initiated with 5% B and increased to 90% B within 25 min and then held for 3 min. The LC mobile phase temperature was set to 35 °C, the injection volume was 4 μL, and the flow rate was 220 μL min−1. Source conditions were as follows: nitrogen was used for both sheath and drying gas (10 L min−1 at 350 °C and 8 L min−1 at 330 °C, respectively); nebulizer pressure was 60 psig, nozzle voltage 300 V, and capillary voltage was 3.5 kV. Lock masses were continuously infused to correct accurate mass values: purine at m/z 121.0509 and HP-0921 at m/z 922.0098 (both from Agilent technologies) were used. Raw data, as provided by the time-of-flight analyzer, were processed by the MassHunter Qualitative Analysis B.06 software (from Agilent Technologies) using the “find-by-formula” algorithm. Confidence of
The aims of the current study were (1) to depict the antioxidant and secondary metabolite profiles of leaves from 19 artichoke cultivars by focusing on polyphenol pattern, DPPH, FRAP, and ORAC radical scavenging activity and the sesquiterpene lactones content and (2) to assess the associations between these nutraceutical traits. With several cultivars profiled for the first time, these findings will allow a better understanding of the variation in nutraceutical quality of a wide collection of artichoke. The results will also assist pharmaceutical and cosmetic industries in selecting food industry byproducts (artichoke leaves) suitable for optimal extractable biocompounds.
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MATERIALS AND METHODS
Plant Material and Sampling. The trial was conducted at the experimental farm of Latium Regional Agency for development and the Innovation of Agriculture (ARSIAL) Cerveteri, central Italy (latitude 41° 59′ N, longitude 12° 01′ E, altitude, 81 m), on sandy clay loam soil (60% sand, 16% silt, 24% clay), with a pH of 6.1, electrical conductivity of 0.3 dS m−1, organic matter of 1.0% (w/w), total N at 0.1%, available P at 20 mg kg−1, and exchangeable K at 373 mg kg−1. Nineteen artichoke cultivars, “Blanca de Tudela”, “Brindisino”, “C3”, “Campagnano”, “Carderas”, “Castellamare”, “Catanese”, “Grato 1”, “Italo”, “Locale di Fano”, “Macau”, “Montelupone A”, “Pertosa”, “Spinoso di Palermo”, “Spinoso Sardo”, “Tondo rosso di Paestum”, “Violetto di Maremma”, “Violetto di Provenza”, and “Violetto di Toscana”, were considered in the present study. These cultivars were selected as the most representative cultivars grown in Europe. All artichoke cultivars were vegetatively propagated by offshoots and transplanted to the open field on August 18, 2014 at a crop density of 7700 plants ha−1. A randomized complete block design was used, with treatments replicated three times. Each experimental unit consisted of 20 plants. The open field experiment was conducted under low chemical inputs. Cultural practices and management procedures during the cropping period were performed according to the best farming practices used for artichoke in central Italy.24 A representative sample of 200 g of fresh weight of leaves was collected from 10 plants per cultivar at the end of harvesting period (May 11, 2015). A subsample of fresh leaves was used for determination of dry matter content, while the remaining subsample was instantly frozen in liquid nitrogen and stored at −80 °C for chemical analysis. Dry Matter Content. An amount of fresh sample was oven dried at 65 °C until a constant weight was achieved to determine the dry matter (DM) content. Leaf dry matter was expressed in percentage (%). Extraction and Preparation for Assays. Each frozen sample was homogenized using a pestle and mortar. A sample (0.5 g) was then extracted twice in 15 + 15 mL of 75% (v/v) methanol (LCMS grade) acidified with 10 mM LCMS-grade formic acid in an ice bath for 3 min and using an Ultra Turrax (Ika T-25, Staufen, Germany). Extracts were then centrifuged (8000 × g) and the supernatant filtered through a 0.2 μm cellulose syringe filter. Total Phenolic, Flavonoids, and Flavonols Content in Leaf Portions. All chemicals were of reagent grade (Sigma-Aldrich, USA). The total phenolic content in each extract was determined using the Folin-Ciocalteu assay. Aliquots (0.5 mL) of each extract were mixed with 1.5 mL of Folin-Ciocalteu reagent (diluted 5-fold) and with 2 mL (75 g L−1) of sodium carbonate. Absorbance was recorded at 765 nm, after 40 min at 20 °C in the dark. A calibration curve was prepared using gallic acid (Sigma-Aldrich, reagent grade) in 75% absolute ethanol, and the results are expressed as gallic acid equivalents (GAE). Flavonoids content was determined colorimetrically. Aliquots (0.5 mL) of each extract were mixed with 0.5 mL of AlCl3 in ethanol (20 g L−1) and then diluted with ethanol to 12 mL. Absorbance was read at 415 nm, after 40 min at 20 °C. Blank samples were prepared from 1 mL of plant extract and 1 drop of acetic acid and diluted to 12 mL. The concentration of flavonoids in the extracts was calculated from a 8541
DOI: 10.1021/acs.jafc.6b03856 J. Agric. Food Chem. 2016, 64, 8540−8548
± ± ± ± ± ± ± ± ± ± ± ± ± ±
148.0 202.0 226.9 225.7 171.7 204.7 204.5 224.6 264.5 228.4 182.0 248.7 210.1 141.7 199.0 ± 32.6a‑d 203.4 ± 32.8a‑d
10.2 ± 1.2 9.5 ± 0.5 10.6 ± 0.3 10.4 ± 1.0 9.6 ± 1.1 10.8 ± 1.0 10.6 ± 1.9 10.5 ± 0.3 10.2 ± 1.6 10.4 ± 1.0 9.8 ± 0.8 10.5 ± 1.0 9.5 ± 1.3 10.0 ± 0.6
10.1 ± 1.4 10.0 ± 0.5
Blanca de Tudela Brindisino C3 Campagnano Carderas Castellammare Catanese Grato 1 Italo Locale di Fano Macau Montelupone A Pertosa Spinoso di Palermo Spinoso Sardo Tondo rosso di Paestum Violetto di Maremma Violetto di Provenza Violetto di Toscana Significance
± ± ± ± ± ± ± ± ± ± ± ± ± ± 90.2e 196.3b 174.7ab 183.6a 115.5de 130.8cd 129.4b 149.2ab 101.6c‑e 61.0cd 139.5e 123.2ab 88.8e 66.2e
8542
2127.1 ± 128.3c ***
248.9 ± 39.4a 221.1 ± 32.0ab **
10.6 ± 1.1
9.8 ± 1.4
ns
± ± ± ± ± ± ± ± ± ± ± ± ± ± 206.0fg 462.8ab 386.0ab 359.0a‑c 260.2fg 305.2de 283.8bc 343.2a 253.5d 148.8d 338.3e‑g 267.4a‑c 200.4g 130.3h
***
4768.7 ± 287.2a‑c
4882.7 ± 294.4a‑c
4078.3 ± 386.2de
3896.7 ± 253.2d‑f 4017.7 ± 262.3de
3431.0 5152.0 5166.0 4784.3 3465.0 4082.0 4674.3 5242.0 4146.7 4334.7 3574.3 4840.0 3340.3 2900.3
± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.2 1.3 1.4 1.0 1.8 1.2 1.8 1.2 1.9 1.8 0.2 1.8 1.0 1.3
ns
19.2 ± 1.6
19.4 ± 1.5
19.1 ± 1.7
18.0 ± 1.6 19.8 ± 1.9
19.1 19.3 19.0 19.5 18.6 18.0 19.8 19.2 19.6 19.5 19.8 20.4 19.7 18.6
± ± ± ± ± ± ± ± ± ± ± ± ± ± 151.8f 76.2c‑e 124.2b‑d 107.4ab 126.0f 162.6de 198.0e 160.8ab 119.4a 99.0c‑e 111.6e 128.4ab 157.8de 60.6f
***
2091.0 ± 95.4
2372.4 ± 81.0a
2017.8 ± 62.4c‑e
1816.8 ± 70.2e 2017.8 ± 256.8c‑e
1335.0 1919.4 2099.4 2307.6 1309.8 1883.4 1828.2 2165.2 2356.2 1966.2 1782.0 2328.6 1882.2 1441.2
± ± ± ± ± ± ± ± ± ± ± ± ± ±
187.8ge 298.2b‑d 233.4c‑f 435.6b 219.0c‑f 350.4g 310.8f 264.6c‑e 316.8a 334.8d‑f 448.2b‑d 283.2bc 343.8c‑f 194.4g
***
5282.4 ± 110.4d‑f
3658.8 ± 521.4e
3145.2 ± 254.4f
5240.4 ± 250.2d‑f 5091.0 ± 67.2ef
3739.2 5723.4 5446.2 6156.0 4210.8 5388.6 4879.8 5467.2 6697.8 5220.6 5725.8 5932.8 5383.8 4236.6
a
All data are expressed as mean ± standard deviation (n = 3). Superscript letters denote Duncan’s multiple range tests from one-way ANOVA (p = 0.01). ns, **, ***: Nonsignificant or significant at p < 0.01 and 0.001, respectively.
2335.6 ± 140.8ab
210.6 ± 29.3a‑c
1843.8 ± 174.7cd
1895.0 ± 123.0c 1656.5 ± 108.1c‑e
1503.4 2184.8 2336.6 2443.5 1532.8 1749.2 2132.0 2277.9 1663.3 1772.6 1473.3 2228.8 1483.3 1474.0
flavonoids flavonols DPPH FRAP ORAC (mg rutins eq 100 g−1 fw) (mg rutins eq 100 g−1 fw) (mmol trolox eq 100 g−1 fw) (mmol trolox eq 100 g−1 fw) (mmol trolox eq 100 g−1 fw)
9.1 ± 0.3
23.7bc 33.2a‑d 38.3ab 35.9ab 27.3b‑d 36.6a‑d 33.0a‑d 33.5ab 44.7a 36.3ab 27.1b‑d 40.9a 30.6a‑c 20.5d
total phenolics (mg gallic acid eq 100 g−1 fw)
DM (%)
cultivar
Table 1. Dry Matter (DM) Percentage, Total Phenolic Compounds, Flavonoids, Flavonols Content, 2,2-Diphenyl-1-picrylhydrazyl Radical Scavenging Capacity (DPPH), Ferric Ion Reducing Antioxidant Power (FRAP), and Oxygen Radical Absorbance Capacity (ORAC) in Artichoke Leaves in Relation to Cultivara
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.6b03856 J. Agric. Food Chem. 2016, 64, 8540−8548
Article
Journal of Agricultural and Food Chemistry compound identification was based on accurate mass and isotope pattern and expressed as overall identification score, computed as a weighted score gained from the isotopic pattern (exact masses, relative abundances, and m/z spacing having a weight of 100, 70, and 60, respectively). Unidentified molecular features were subjected to a recursive analysis workflow using Mass Profiler Professional B.12.06 (from Agilent Technologies) for features alignment and filtering after initial deconvolution. Features that were not present in at least two out of three replications within at least one treatment were discarded. Filtered features were exported in MassHunter Qualitative Analysis, targeted using the “find-by-formula” algorithm and finally identified twice, using two different databases: (i) the database exported from Phenol-Explorer 3.027 and (ii) an in-house custom database of sesquiterpene lactones. A tolerance of 5 ppm was adopted for mass accuracy, and a threshold of 70/100 was chosen regarding identification scores. The compounds passing the thresholds were retained, and their abundance (peak area) was exported for statistics and chemometrics. Sesquiterpene lactones were finally expressed as cynaropicrin equivalents using a cynaropicrin pure reference standard (PhytoLab, Nürnberg, Germany) for calibration. Statistics. Colorimetric data were statistically analyzed through ANOVA using PASW Statistics 18.0 (SPSS Inc.) at 99% confidence level. Duncan’s multiple range tests were performed on each of the significant variables measured. Pearson’s correlation coefficients were also calculated using PASW Statistics 18.0. Leaf quality traits were subjected to principal component analysis (PCA) to explore relationships among variables and treatments and also to determine which traits were the most effective in discriminating between cultivars. The PCA outputs include variable loading to each selected component and treatment component scores. Interpretation of metabolomic analysis was carried out using Mass Profiler Professional B.12.06; the exported compounds were filtered (only those at >5000 counts as peak volume and appearing in 66% of samples in at least one condition were considered) and normalized at the 75th percentile. Thereafter, compound abundance values in each sample were baselined to the median of each compound in all samples. Statistics and interpretations were finally performed on the latter, filtered data set. Unsupervised hierarchical cluster analysis was carried out on both compounds and treatments (setting similarity measure as “Euclidean” and “Wards” as linkage rule). Furthermore, multivariate partial least square discriminant analysis (PLS-DA) was carried out: the PLS-DA variables loadings, used to build the class prediction model, were plotted according to their weight within the latent vectors, and the most relevant (i.e., those having the highest scores, after applying an N-fold validation with N = 4) were exported from the covariance structures in the PLS-DA hyperspace. Finally, the “find̈ Bayesian biomarker identification of minimal-entities”, i.e., a Naive Mass Profiler Professional, was carried out targeting the 25 compounds that were better able to explain differences among cultivars (forward selection algorithm; evaluation metric: overall accuracy = 100).
100 g fw, with the highest values recorded in Italo, Montelupone A, and Violetto di Provenza (Table 1). Moreover, the flavonoids and flavonols content ranged from to 1473.3 to 2443.5 and from 2900.3 to 5242.0 RE per 100 g fw, respectively (Table 1). The highest flavonoids contents were recorded in C3, Campagnano, Grato 1, Montelupone, and Violetto di Provenza, whereas, the lowest were observed in Blanca de Tudela, Macau, Pertosa, and Spinoso di Palermo (Table 1). Total phenolic content was significantly (p < 0.05) correlated to flavonoids (Pearson’s coefficient 0.620) and flavonols (Pearson’s coefficient 0.700). A significant correlation (p < 0.01) was also found between flavonoids and flavonols (Pearson’s coefficient 0.933). Among cultivars examined, Campagnano, Grato 1, and Violetto di Provenza were found to be rich in polyphenols (total phenolics, flavonoids, and flavonols), suggesting that the leaves of these specific cultivars could be used as raw material for pharmaceutical compounds extraction. Antioxidant Capacity. Three commonly used in vitro assays, i.e., DPPH·, FRAP, and ORAC, were used to evaluate the antioxidant activities in leaves of artichoke. The antioxidant capacity is an important parameter in assessing the quality of vegetables, since antioxidant molecules play an important role in (1) ensuring plant growth under unfavorable conditions and (2) promoting health effects in the human diet.31 No significant difference was observed between cultivars for the DPPH• radical scavenging activity (average 19.2 mmol of trolox eq 100 g−1 fw). The FRAP and ORAC scavenging activities of the selected artichoke cultivars ranged from 1309.8 to 2372.4, and from 3145.2 to 6697.8 mmol of trolox eq 100 g−1 fw, respectively (Table 1). The highest FRAP values were recorded in the cultivars Campagnano, Grato 1, Italo, Montelupone A, and Violetto di Provenza, whereas the highest level of ORAC was observed in Italo, indicating that these cultivars could be considered a good source of natural antioxidants. However, it must be stressed that the antioxidant activities of artichoke leaf extracts were not always consistent when measured by the three assay methods. Although a significant correlation (p < 0.05) was identified between FRAP and ORAC, no significant correlation was identified between other two pairs (DPPH• vs ORAC and DPPH• vs FRAP) (data not shown). Differences in antioxidant activity methods were not surprising, as radical scavenging values from different tests were strongly linked to oxidant species and the reaction mechanisms and conditions.32 The correlation between the phenolic compounds and the antioxidant activity in artichoke leaves supports the hypothesis that these compounds may play an important role in free radical scavenging.33 In this study, the correlation coefficient between total phenolics, flavonoids, flavonols, and FRAP scavenging activity was highly (p < 0.01) significant (0.80, 0.67, and 0.70, respectively), whereas the correlations between polyphenols and ORAC and DPPH• were weak and not correlated, respectively (data not shown). The same type of correlation between antioxidant activities and total phenolic compounds has been reported in globe artichoke as well as their byproducts.34,35 Phenolic Profile. The UHPLC/Q-TOF-MS untargeted screening analysis in the leaf extracts from different cultivars of artichoke resulted in a wide diversity of phenolic compounds that included flavonoids, hydroxycinnamic acids, tyrosols, and lignans. The full list of phenolic compounds identified in artichoke leaves is provided as Supporting Information. The unsupervised cluster analysis (linkage Ward), built on the basis
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RESULTS AND DISCUSSION Dry Matter, Total Phenolics, Flavonoids, and Flavonols Content. No significant difference among cultivars was observed on leaf dry matter percentage (average 10.1%; Table 1). The pharmaceutical properties of globe artichoke leaves are linked to their specific chemical composition in particular polyphenols, which are one of the main groups of compounds acting as primary antioxidants or free radicals terminators.1 In the current study, the total phenolics compounds, flavonoid, and flavonol contents expressed on fresh weight basis were cultivar dependent (Table 1). The strong influence of genetic materials on the phenolic profile has been previously demonstrated in other vegetables such as potato,28 garlic,29 and tomato.30 The total phenolic compounds of the selected artichoke cultivars ranged from 141.7 to 264.5 mg of GAE per 8543
DOI: 10.1021/acs.jafc.6b03856 J. Agric. Food Chem. 2016, 64, 8540−8548
Article
Journal of Agricultural and Food Chemistry
Figure 1. Not averaged unsupervised cluster analysis on the phenolic profile in artichoke leaves from different cultivars (similarity, Euclidean; linkage rule, Ward). Compound intensity was used to build up heat maps on the basis of which the clusters were generated.
of the heat-map produced according to fold-change analysis, resulted in two main clusters (Figure 1): the first was composed of Campagnano, Castellammare, Grato 1, Italo, Locale di Fano, Montelupone A, Pertosa, and Tondo rosso di Paestum, and the second was composed of Blanca de Tudela, Brindisino, Carderas, Catanese, Macau, Spinoso Sardo, Spinoso di Palermo, Violetto di Maremma, and Violetto di Provenza. The first cluster, which belongs to the Romaneschi type, was characterized by the presence of anthocyanins, which were abundant with respect to the second. Indeed, a total of 68 anthocyanin compounds derived from delphinidin, malvidin, petunidin, cyanidin, pelargonidin, and peonidin were detected. In previous studies, only cyanidin, peonidin, and delphinidin were identified in artichoke,36 suggesting a more complex profile and a higher degree of diversity regarding anthocyanins in leaf extracts from cultivars in the current study. Concerning hydroxycinnamic acids, 63 different compounds were identified in the leaf extract from the 19 cultivars. The most abundant hydroxycinnamics were caffeoylquinic and coumaric acid derivatives, although many others were found, in particular, ferulic acid derivatives. Caffeoylquinic acids and flavonoids are known to be among the more abundant
phenolics in both leaves and heads followed by bracts and receptacle.37 Results of this study demonstrate that caffeoylquinic and dicaffeoylquinic acids are most abundant hydroxycinnamic acids in artichoke, which is consistent with other studies.7,37 Regarding flavonoids, the flavones apigenin and luteolin and their glycosyl derivatives were found in most of the cultivars analyzed. Results are consistent with a recent review assessing the phytochemical profile of artichoke.38 However, several other flavones such as chrysin and its derivatives were common in our cultivars as well as additional flavones which are reported for the first time (neodiosmin, rhoifolin and rhoifolin 4′-Oglucoside, scutellarein, and tetramethylscutellarein). Flavonols were also common in cultivars in the current study, as they represented the second most populated class, with 67 different compounds. The most abundant were kaempferol and quercetin derivatives, followed by isorhamnetin and miricetin. Several lignans were also identified for the first time in artichoke; the only lignan identified before was pinoresinol.37 Overall, 24 additional lignans were detected. Matairesinol, secoisolariciresinol, and todolactol A were the most common and detectable in all cultivars. Furthermore, several secoiridoid tyrosols were also identified in artichoke leaves; overall, 16 8544
DOI: 10.1021/acs.jafc.6b03856 J. Agric. Food Chem. 2016, 64, 8540−8548
Article
Journal of Agricultural and Food Chemistry
Table 2. Sesquiterpene Lactones Abundance in Artichoke Leaves in Relation to Cultivar, as Resulted from UHPLC/Q-TOF Screeninga cultivar
grosheimin
cynaratriol
8-deoxy-11,13-dihydroxygrosheimin
dihydrocynaropicrin
cynaropicrin
Blanca de Tudela Brindisino C3 Campagnano Carderas Castellamare Catanese Grato Italo Locale di Fano Macau Montelupone A Pertosa Spinoso di Palermo Spinoso Sardo Tondo rosso di Paestum Violetto di Toscana Violetto di Maremma Violetto di Provenza significance
1606.9 ± 7.2b 975.0 ± 30.2c 7.9 ± 7.7e 9.8 ± 3.9e 1952.7 ± 11.3a 6.8 ± 0.6e 870.2 ± 20.8c 30.6 ± 3.3e 7.3 ± 1.2e 340.6 ± 30.6c 556.2 ± 58.3d 16.9 ± 11.5e 18.5 ± 12.6e 1092.2 ± 17.8c 1433.8 ± 13.1b 39.1 ± 15.7e 111.8 ± 25.2e 433.8 ± 14.3de 483.1 ± 22.8d ***
300.2 ± 3.6a 72.1 ± 1.7bc 7.1 ± 4.2c 5.4 ± 0.1c 185.8 ± 4.6b 6.3 ± 1.9c 78.9 ± 4.9bc 22.9 ± 7.6c 26.6 ± 7.9c 66.2 ± 0.9bc 108.8 ± 6.8bc 7.1 ± 0.3c 101.3 ± 9.8bc 39.1 ± 6.5c 60.7 ± 10.8bc 9.2 ± 3.2c 19.8 ± 3.4c 115.4 ± 7.5bc 60.2 ± 8.3bc ***
1652.7 ± 9.1a 274.2 ± 17.5c 7.6 ± 6.9d 28.5 ± 4.3d 1941.5 ± 48.2a 9.7 ± 7.9d 167.7 ± 11.6cd 132.1 ± 18.6cd 7.6 ± 1.0d 356.8 ± 61.6c 329.3 ± 57.7c 58.7 ± 4.3d 26.7 ± 3.1d 1093.8 ± 138.3b 7.0 ± 0.1d 13.4 ± 3.3d 8.4 ± 1.8d 446.1 ± 5.4c 484.3 ± 14.2c ***
25.6 ± 9.6a 7.4 ± 0.4bc 8.2 ± 0.5bc 3.3 ± 1.9c 19.6 ± 1.1b 7.5 ± 0.9bc 7.8 ± 0.9bc 3.3 ± 2.4c 8.5 ± 0.9bc 10.0 ± 4.5bc 9.1 ± 0.5bc 9.3 ± 0.8bc 8.4 ± 0.9bc 8.2 ± 0.5bc 25.3 ± 0.7a 3.4 ± 0.3c 9.3 ± 1.1bc 10.2 ± 2.5bc 10.3 ± 0.7bc ***
16.5 ± 1.1c 11.1 ± 0.6c 21.2 ± 2.2c 9.5 ± 2.1c 10.6 ± 0.2c 5.0 ± 2.7c 4.5 ± 0.7c 8.0 ± 0.9c 9.4 ± 4.3c 9.5 ± 2.7c 12.0 ± 0.5c 16.2 ± 2.9c 8.8 ± 0.9c 10.3 ± 4.2c 6.2 ± 0.5c 22.4 ± 3.4c 34.4 ± 2.6c 1274.2 ± 2.5a 799.2 ± 13.1b ***
Results are reported as mg cynaropicrin equivalents 100 g−1 fw and expressed as mean ± standard deviation (n = 3). Superscript letters denote Duncan’s multiple range tests from one-way ANOVA (p = 0.01). ***: Significance at p < 0.001.
a
discriminate among cultivars because they are not typically characteristic of artichoke, thus only represented in some artichoke cultivars. The impact of such a diverse phenolic profile is important; several of the identified differential metabolites play an important role as functional and health-promoting compounds.42 Ellagic acid, 24-methylcholestanol ferulate, daidzein, and carnosic acid have been also described to possess anticancer activity.43−45 Oleuropein was reported as an anticancer, cardioprotective, gastroprotective, and hepatoprotective compound.46 Two oleuropein aglycones, i.e., 3,4DHPEA-EA and 3,4-DHPEA-EDA (the mono- and dialdehyde forms, respectively),47 were found among discriminating compounds. In addition to the role of cultivar-discriminating compounds, UHPLC/Q-TOF-MS analysis (whole phenolic profile) shows that artichoke leaf extracts provide an excellent source of health-promoting compounds, regardless the cultivar considered. Our results are in agreement with the findings from Dabbou and co-workers,35 which reported a phenolic content in leaves from two Tunisian artichoke cultivars (“Violet d”Hyéres and “Blanc d’Oran”) at levels comparable to bracts or floral stem (i.e., comparable to the commonly edible fractions). Romani and co-workers,48 while characterizing the composition of “Violetto di Toscana” and “Terom” cultivars, provided similar conclusions. Despite consideration of a larger population of genotypes, to account for a wider biodiversity among cultivars, the promising polyphenols profile of leaves has been confirmed. Comprehensive information has been already reviewed regarding the role phenolic antioxidants play in lowering the risk of health disorders49 and counteracting cancer cells proliferation.50 Sesquiterpene Lactones. Different sesquiterpene lactones (STLs) were screened in our cultivars through UHPLC/ QTOF-MS mass spectrometry using a custom reference database for compounds identification. To be retained within the data set, STLs had to be identified with minimum scores
compounds related to DHPEA, HPEA, and ligostroside structures were identified for the first time in this species. The same considerations can be applied to phenolic terpenes (9 phenolic compounds identified). The PLS-DA analysis provided discrimination between the cultivars considered, according to their phenolic profile. The different biological replications of each cultivar were clustered together, and different cultivars were separated in the PLS-DA hyperspace. These results were consistent with the unsupervised cluster analysis, thus confirming the potential of phenolic profile to characterize our artichoke cultivars. Compounds with a higher score in the class prediction model (scores > 0.01 or < −0.01 in either the first or the second PLSDA latent vector) were exported to better support the differential phenolics identified from the Naiv̈ e Bayesian “find-minimal-entities” biomarker analysis. The aim was to identify the phenolics most representative of differences among the artichoke cultivars. The phenolic compounds which best discriminated specific artichoke cultivars are reported in the Supporting Information, grouped according to their chemical classes. The most abundant class was the hydroxycinnamic acids (6 compounds), followed by flavones, flavonols, and anthocyanins (3 compounds each). Luteolin 7-O-glucuronide, which is the most common flavone in artichoke,7,37 was highlighted by PLS-DA. Apigenin derivatives were not found to be discriminant. In addition to the compounds abundant and common in artichoke which have already been emphasized, several other differential flavonoids and hydroxycinnamic acids were highlighted among differential phenolics. A good example of this is the differential anthocyanins in a Supporting Information table; it is likely that this is due to their potential to discriminate between colored and green cultivars. Most “minor” phenolics were not previously described in artichoke but were described in other botanically different plant species.39−41 It can be postulated that these phenolics could more efficiently 8545
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Journal of Agricultural and Food Chemistry (>70/100) and to pass the frequency filters (i.e., to be found in minimum 66% of replications in at least one cultivar). The profile of STLs in the leaves of the selected artichoke cultivars is provided in Table 2 and expressed as mg of cynaropicrin eq 100 g−1 fw. Grosheimin and its deoxydihydroxy derivative, cynaratriol, cynaropicrin, and dihydroxy cynaropicrin were the only STLs detected in assessed leaf samples. Although STLs biosynthetic pathway is not fully clear in artichoke, the biochemical basis for their presence in artichoke leaves has been postulated as follows: (+)-germacrene A synthase (i.e., one of the main upstream synthetic enzyme) expressed in leaf10 and particularly in mature and old leaves.12 Eljounaidi and coworkers10 also reported the detection of cynaropicrin and grosheimin (in even greater quantities) in leaves from two cultivars of artichoke (Concerto and C3). Findings from the ninteen cultivars tested in the current study are in agreement with Eljounaidi and co-workers,10 where grosheimin and 8deoxy-11,13-dihydroxygrosheimin were the most abundant STLs, followed by cynaropicrin, with marked differences among cultivars (up to 2−3 orders of magnitude). Regarding cynaropicrin, only Violetto di Maremma and Violetto di Provenza were significantly more abundant (Table 2). However, cynaratriol and dihydroxy cynaropicrin could be detected at very low abundances across all cultivars. Although the compounds were the same across different cultivars, their quantitative profiles were significantly different. C3, Campagnano, Italo, and Castellamare had a lower amount of STLs, whereas Blanca de Tudela and Carderas followed by Spinoso di Palermo, Violetto di Maremma, and Violetto di Provenza had the highest. Cynaropicrin is probably the most widely known STL in artichoke; however, it was not the most abundant in leaves in the current study. The highest differences in abundance were detected in grosheimin and its derivative and with cynaropicrin. No significant relationship was identified between the profile of STLs and phenolic compounds or with antioxidant activity values. However, they are derived from different biosynthetic pathways, and their role as antioxidants has not yet been elucidated. However, the antihyperlipidaemia effect of STLs is well known,9 thereby strengthening the functional role of artichoke leaves in addition to the phenolicsrelated health promotion. Unlike polyphenols, little information has been reported to date regarding STLs abundance across artichoke leaves. In this study, results on STLs allow the same considerations made for phenolics. While confirming the nutraceutical potential of artichoke leaves, it is fundamental to consider those cultivars that are the most appropriate as a source of health-related compounds. Principal Component Analysis. Principal component analysis (PCA) was performed to interpret the variation in the nutraceutical leaf composition of artichoke cultivars. The first three PCs were associated with Eigen values higher than 1 and explained 79.7% of the total variance, with PC1 accounting for 49.6%, PC2 for 17.6%, and PC3 for 12.5% (Table 3). PC1 was correlated positively and strongly (>0.6) with FRAP scavenging activity, total phenolics, flavonoids, and flavonols and negatively correlated with STLs (grosheimin, cynaratriol, dihydrocinaropicrin, and 8-deoxy-11,13-dihydroxygrosheimin). Moreover, PC2 was positively correlated with cynaropicrin and negatively associated with ORAC, whereas PC3 was negatively correlated with cynaropicrin (Table 3). The loading plot in Figure 2A illustrates the relationships between nutraceutical traits. The variation in dihydrocinaropicrin was most closely aligned to that of grosheimin,
Table 3. Eigen Values, Relative and Cumulative Proportion of Total Variance, and Correlation Coefficients for Each Nutraceutical Trait with Respect to the Three Principal Componentsa principal components Eigen value percentage of variance cumulative variance Eigen vectors FRAP grosheimin total phenolics cynaratriol dihydrocynaropicrin 8-deoxy-11, 13-dihydroxygrosheimin flavonols flavonoids DPPH cynaropicrin ORAC
PC1
PC2
PC3
5.4 49.6 49.6
1.9 17.6 67.2
1.3 12.5 79.7
0.925 −0.837 0.832 −0.774 −0.751 −0.757 0.805 0.758 −0.042 0.130 0.482
0.113 0.206 0.358 0.277 0.175 0.364 0.345 0.445 −0.553 0.649 −0.686
−0.118 0.266 0.220 −0.027 0.242 0.262 0.247 0.225 −0.570 −0.705 0.428
a
Bold face factor loadings indicate the most relevant characters for each principal component.
Figure 2. Loading plot (A) and score plot (B) of the first and second principal components after PCA analysis on nutraceutical traits in leaves from 19 artichoke cultivars.
and variation in total phenolics content was more strongly correlated to flavonols (i.e., narrower angle between the 8546
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Journal of Agricultural and Food Chemistry corresponding vectors) rather than flavonoids content. Similarly, FRAP was more strongly correlated to total phenolics content than to flavonoids, whereas FRAP was not correlated to DPPH (angle > 90°). The two PCs score plot separates and categorizes treatment populations (i.e., cultivars) into four groups, enabling interpretation of results on the basis of all parameters examined (Figure 2B). The upper right quadrant in the positive side of PC1 included Violetto di Provenza, Castellamare, Grato 1, and Campagnano. Violetto di Provenza was characterized by high cynaropicrin content, whereas the three Romanesco-type cultivars (Castellamare, Grato 1, and Campagnano) were mostly characterized by high FRAP scavenging activity but also high total phenols, flavonoids, and flavonols. The cluster in the lower right quadrant represents artichoke cultivars characterized by ORAC scavenging activity. The upper left quadrant (negative side of PC1), which clustered four artichoke cultivars (Blanca de Tudela, Carderas, Spinoso Sardo, and Violetto di Maremma), depicted the treatments with the highest STLs content, especially the Green-type cultivars (i.e., Blanca de Tudela and Carderas). Furthermore, the artichoke cultivars of the lower left quadrant (Spinoso di Palermo, Macau, Pertosa, and Locale di Fano depicted the treatments of lowest nutraceutical quality. The results of the PCA may provide the basis for a more in-depth approach to elucidate the effects of genetic variation on the pharmaceutical and nutraceutical quality of artichoke. In conclusion, this study demonstrated that the polyphenol profile, antioxidant activities, and sesquiterpene lactones content in artichoke leaves appear to be strongly influenced by genetic factors, indicating that specific cultivars can be selected to obtain the desired profile of nutraceutical compounds. Among cultivars examined, Campagnano, Grato 1, and Violetto di Provenza were found to be rich in polyphenols but also characterized by high antioxidant activity, whereas Blanca de Tudela and Carderas were characterized by high STLs content. Accordingly, more focus should be directed toward promoting the use of these cultivars as raw material for pharmaceutical use.
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performance liquid chromatography; QTOF-MS, quadrupoletime-of-flight high-resolution mass spectrometry; STL, sesquiterpene lactone; PLS-DA, partial least squares discriminant analysis; PCA, principal component analysis
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b03856. Whole list of phenolic compounds identified in artichoke leaves with annotations, together with the list of phenolic compounds resulting differential among different cultivars of artichoke, and PLS-DA model outcome (compounds plot and loadings plot in upper and lower pane, respectively) (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*Phone: +39 0523 599156. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED DPPH, 2,2-diphenyl-1-picrylhydrazyl; FRAP, ferric ion reducing antioxidant power; ORAC, oxygen radical absorbance capacity; GAE, gallic acid equivalents; RE, rutin equivalents; TEAC, trolox-equivalent antioxidant capacity; AAPH, 2,2′azobis-2-amidinopropane dihydrochloride; UHPLC, ultrahigh8547
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