Characterization, phenolic profile, nitrogen compounds and antioxidant activity of Carignano wines

Accepted Manuscript Title: Characterization, phenolic profile, nitrogen compounds and antioxidant activity of Carignano wines Authors: Carlo Ignazio Giovanni Tuberoso, Gabriele Serreli, Francesca Congiu, Paola Montoro, Maurizio Antonio Fenu PII: DOI: Reference:

S0889-1575(17)30036-4 http://dx.doi.org/doi:10.1016/j.jfca.2017.01.017 YJFCA 2831

To appear in: Received date: Revised date: Accepted date:

18-1-2016 2-11-2016 23-1-2017

Please cite this article as: Tuberoso, Carlo Ignazio Giovanni., Serreli, Gabriele., Congiu, Francesca., Montoro, Paola., & Fenu, Maurizio Antonio., Characterization, phenolic profile, nitrogen compounds and antioxidant activity of Carignano wines.Journal of Food Composition and Analysis http://dx.doi.org/10.1016/j.jfca.2017.01.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights  Preliminary characterization of Carignano monovarietal red wine was performed.  High level of anthocyanins and other polyphenols were detected by HPLC-DAD  HPLC-FLD analysis revealed a low amount of biogenic amines.  Presence of xanthine was confirmed by LC/ESI/(LIT)MS and LC/ESI/QqQ/MS  The newly generated data can contribute to updating wine composition databases

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Original research article Characterization, phenolic profile, nitrogen compounds and antioxidant activity of Carignano wines Carlo Ignazio Giovanni Tuberosoa,*, Gabriele Serrelia, Francesca Congiua, Paola Montorob, Maurizio Antonio Fenua a

Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale

72, 09124 Cagliari, Italy b

Department of Pharmacy, University of Salerno, via Giovanni Paolo II, 132, 84084

Fisciano (SA), Italy * Corresponding author. Tel.: +39 0706758644; fax: +39 0706758612. E-mail address: [email protected] (C.I.G. Tuberoso).

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Abstract The aim of this study was to perform a preliminary chemical and physical characterization of the Carignano wine, an Italian monovarietal red wine mainly produced in the south-west of Sardinia (Italy). Technological parameters (alcohol, reducing sugars, total and volatile acidity, and pH), organic acid content, CIE L*C*abh°ab chromaticity coordinates, phenolic compound contents (with spectrophotometric assays and HPLCDAD), nitrogen compounds (with HPLC-FLD) and antioxidant capacity assessed by FRAP and DPPH assays were evaluated in 14 samples (vintage 2013) and compared with 3 aged samples. Carignano wines showed a significant level of phenolic compounds (2023 ± 435 mg GAE/L) and a good in vitro antioxidant capacity (31.6 ± 5.2 mmol Fe2+/L and 10.0 ± 1.4 mmol TEAC/L, respectively). The content of total polyphenols correlated significantly (p< 0.001) with the total reducing power and radical scavenging capacity. The nitrogen compounds found in samples were mainly amino acids, and among these, the content of essential amino acids was of 61.4 ± 22.5 mg/L. A relatively low amount of undesirable biogenic amines was also found (17.2 ± 6.9 mg/L). Xanthine was detected in all the samples in the range 48.0-101.4 mg/L. These data may help wineries improve their consumer safety procedures for Carignano wine production.

Keywords: Carignano; Phenolic compounds; Antioxidant activity; LC/ESI/QqQ/MS; Xanthine; Nitrogen compounds; Red wine; Food analysis; Food composition

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1.

Introduction Wine is an integral part of the Mediterranean diet. Moderate consumption of wine

has been linked to a variety of health benefits. Specifically, minor components, such as phenolic compounds, seem to be responsible for the protective effects of wine against oxidative stress (Biasi et al., 2014; Schrieks et al., 2013), and for the maintenance of healthy cardiovascular (Walzem, 2008) and nervous systems (Basli et al., 2012). Because the composition of wine is greatly influenced by the grape cultivars used along with the winemaking techniques used, it

therefore essential to know the chemical-physical

characteristics of each wine, especially the ones obtained from monovarietal grapes. “Carignano del Sulcis" became an Italian DOC (Controlled Origin Designation) wine in 1977. It is one of the most famous red wines of Sardinia and is made with grapes of the homonymous vine cultivated in Sulcis, in the southwestern region of Sardinia (Italy). Production of this wine is limited mainly to an area of about 1700 ha on the island of Sant’Antioco and to some adjacent areas (Sardegna Agricoltura, Regione Autonoma Sardegna, Area di distribuzione del Carignano). The origin of the Carignano wine is not fully known. There are some indications that Phoenicians or Carthaginians might have brought it to Sardinia ca. between 800 and 300 B.C.E. However, the first documented historical evidence of the presence of Carignano in Sardinia and in the Sulcis area is after 1946 (Nieddu, 2011). Genetic similarities of the Carignano vines with other Sardinian varieties, such as Bovale di Spagna, put them in the large group of Spanish and French varietals, such as Cariñena, Mazuela, and Carignan (Nieddu et al., 2007). It is therefore possible that the Carignano vine was imported from the Iberian Peninsula during the early Spanish domination of Sardinia in the 15th century. The latter hypothesis would also be supported by the local dialect name for the Carignano grape, “Axina de Spagna”, which means “Grape of Spain”.

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This grape has no particular climatic requirements and is highly productive. The traditional farming system is the Latin sapling, installed on the very ancient dunes which quite frequent in Sant’ Antioco island. The clay and sandy soil of these dunes made it possible for the vines to survive the late-19th c. phylloxera epidemic in the Mediterranean, so that it is quite common to find saplings from 80-100 year-old vines, which were never grafted onto rootstock (Sardegna Agricoltura, Regione Autonoma della Sardegna, Carignano del Sulcis). The Carignano vine generates a red wine with high alcohol content: the “Carignano del Sulcis”, which is a ruby red wine, with a vinous odour and a dry fruity and harmonious taste. Its total alcoholic content must be at least 12% vol, the minimum total acidity should be 4.5 g tartaric acid/L and the minimum dry extract 25 g/L (Sardegna Agricoltura - Regione Autonoma Sardegna. Disciplinare del Carignano). To the best of our knowledge, no studies of the chemical-physical characteristics of the Carignano wines have been reported so far. Therefore, the aims of the present paper were (a) to characterize the chemical-physical composition of commercial Carignano wines, (b) to investigate the profile of phenolic and nitrogen compounds in these wines, and (c) to evaluate their in vitro antioxidant activity by FRAP and DPPH assays.

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Materials and methods 2.1. Chemicals and reagents Gallic

acid,

Folin-Ciocalteu’s

phenol

reagent,

Na2CO3,

1,1-diphenil-2-

picrylhydrazyl radical (DPPH), (±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carbossilic acid (Trolox), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), ferric chloride hexahydrate, ferrous sulphate heptahydrate, Na2B4O7•10H2O, CH3COONa, dansyl chloride, acetone, methanol,

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ethanol, orthophosphoric acid 85%, acetic acid and acetonitrile were obtained from Sigma-Aldrich (Milan, Italy). Hydrochloric acid (37% p/p) and Na2HPO4 were obtained from Carlo Erba (Milan, Italy). Citric acid was obtained from AnalaR Normapur (Milan, Italy). LC-MS grade acetonitrile and formic acid were purchased from Merck (Darmstadt, Germany). Standards of phenolic compounds were purchased from Extrasynthese (Genay, France). Standards of amino acids and biogenic amines (purity > 99.9%) were obtained from Sigma-Aldrich, Merck and Carlo Erba (Milan, Italy). All the chemicals used in this study were of analytical grade. Ultrapure water (18 MΩ•cm) was obtained with a Milli-Q Advantage A10 System apparatus (Millipore, Milan, Italy). 2.2. Samples The Carignano wine samples (n = 14) used in this study were commercially available, with certified origin and directly collected at the wineries (Table 1). Each specimen was sampled in triplicate. Wines were obtained from Carignano grapes harvested in 2013, processed according to the traditional oenological processing techniques of Carignano wines and following the sensorial parameters described in the Carignano DOC production specification (Sardegna Agricoltura - Regione Autonoma Sardegna. Disciplinare del Carignano). All the samples were provided from the wineries that together account for almost 95% of the production of Carignano wines in Sardinia. Samples were stored in dark glass bottles at 8 ± 1 °C and analysed within 3 months. Before analysis, samples were filtered through an Econofilter RC membrane (0.45 μm, Ø 25 mm, Agilent Technologies, Milan, Italy). Three additional aged Carignano samples (CAR2010, CAR2011, and CAR2012) were analysed to compare the analytical data. 2.3. Technological parameters The contents of alcohol, reducing sugars, total and volatile acidities, pH, and organic acids (tartaric acid, malic acid, lactic acid and citric acid) were determined using

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WineScan™ (Foss, Padua, Italy), which uses the FTIR technology (Fourier Transform Infrared Spectroscopy). 2.4. Chromaticity coordinates CIE L*C*abh°ab The transmittance was registered using a Varian Cary 50 Scan spectrophotometer (Varian, Leini, Turin, Italy) and 2 mm quartz cuvettes. The colour analysis was performed in the visible spectrum (380-780 nm) using D65 illuminant and a 10° observation angle. CIELAB L*, C*ab, and h°ab parameters were calculated using the Cary Win UV Color Application V. 2.00 software without transformations of the optical path. 2.5. Total polyphenols and total anthocyanins The total polyphenol content was measured spectrophotometrically with a modified Folin-Ciocalteu’s method according Tuberoso et al. (2013). The total anthocyanin content was determined using a spectrophotometric differential assay (Ribéreau-Gayon et al., 2006). Briefly, a solution containing 500 L of wine and 500 L of 1% chloridric ethanol was added with 5 mL of 2% HCl solution (solution d1) and a second solution containing 500 L of wine and 500 L of 1% chloridric ethanol was added with 5 mL of citric acidNa2HPO4 buffer pH 3.5 (solution d2). Next, the absorbance of the two samples was measured at 520 nm in a 10 mm optical polystyrene cuvette (Kartell® 01937, Kartell Spa, Noviglio, Milan, Italy) using a Varian Cary 50 Scan spectrophotometer, and the total anthocyanin content was calculated using the appropriate formula (Ribéreau-Gayon et al., 2006). The data were reported in equivalents of malvidin 3-O-glucoside (mg ME/L). 2.6. DPPH and FRAP assay The antiradical activity was evaluated with a spectrophotometric analysis using the DPPH assay (Tuberoso et al., 2010). A calibration curve in the range 0.05–1.0 mmol/L was used for the Trolox, and data were reported as Trolox equivalent antioxidant capacity

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(TEAC, mmol/L). The total antioxidant activity was performed with a modified FRAP (ferric reducing antioxidant power) assay (Benzie and Strain, 1996; Tuberoso et al., 2010). Quantitative analysis was performed according to the external standard method (FeSO4, 0.1–2 mmol/L), correlating the absorbance with the concentration and results were reported as mmol/L of Fe2+. 2.7. HPLC-DAD determination of phenolic compounds and xanthine Detection and quantitative analysis of phenolic compounds were carried out using an HPLC-DAD method as described by Tuberoso et al. (2013). The same chromatographic conditions were used to detect and quantify xanthine. Chromatograms and spectra were elaborated with a ChromQuest V. 2.51 data system (ThermoQuest, Rodano, Milan, Italy). Anthocyanins were detected and quantified at 520 nm, flavonols at 360 nm, and all the other compounds at 280 nm. The calibration curves were built with the method of external standard, correlating the area of the peaks with the concentration. Standards of xanthine, gallic acid, protocatecuic acid, trans-resveratrol, p-coumaric acid, caffeic acid, trans-caftaric acis, procyanidin B1, procyanidin B2, (+)catechin, ()epicatechin, epigallocatechin, delphinidin 3-O-glucoside, cyanidin 3-O-glucoside, petunidin 3-O-glucoside, peonidin 3-O-glucoside, malvidin 3-O-glucoside, myricetin, myricetin 3-O-glucoside, quercetin, quercetin 3-O-glucoside, and kaempferol 3-Oglucoside were used. The correlation values were 0.9989–0.9998 in the range of 0.5–20 mg/L. The samples were diluted with ultrapure water (1:20 v/v) and injected into the HPLC column without any further purification. The established method was validated in agreement with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance note which describes validation of analytical methods (ICH Topic Q2 (R1), 2005) by

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determining linearity, limits of detection (LOD), limits of quantification (LOQ), precision and accuracy. Stock standard solutions were prepared in methanol for phenolic compounds and xanthine. The working standard solutions were prepared in ultrapure water. The linearity was evaluated by preparing standard mixtures at six different concentrations and analysing them by HPLC-DAD. The calibration curves for commercial standards were plotted with the method of external standard, correlating the peak area with the concentration by means of the least-squares method, with coefficient of determination (r2) > 0.998 for all compounds. The LODs and LOQs were calculated according to the equation LOD = 3.3 r/S and LOQ = 10 r/S, respectively (where r = standard deviation of the blank, and S = slope of the calibration curve). The precision of this method was evaluated testing intra- and interday repeatability. Six injections of the same standard containing all the phenolic compounds within one day and over three consecutive days, were performed. The relative standard deviation (RSD) for the area under the peak was determined as a measure of precision, and all RSDs were lower than 5%. The accuracy of the method was evaluated using recovery rates. Two Carignano samples (2 and 14) were spiked with two concentrations of gallic acid (20 and 100 mg/L), catechin (5 and 20 mg/L), cis-caftaric acid (5 and 20 mg/L), quercetin 3-O-glucoside (5 and 20 mg/L), malvidin 3-O-glucoside (20 and 100 mg/L), trans-resveratrol (5 and 20 mg/L) and xanthine (20 and 100 mg/L) and each spiked sample was analysed in triplicate. Recovery rates were between 94.2 and 102.4%. The matrix effect was evaluated comparing the response of a standards mix containing the previously listed compounds (each at 20 mg/L), prepared both in Carignano sample 2 and in water. No statistical differences were observed (p < 0.05). The specificity, intended as the lack of interference with other substances detected in the region of interest, was assessed by the ChromQuest purity calculation software index (total peak purity ≥ 0.99), and resulted to be specific

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with no any other peak interfering at the retention times of the dosed compounds in the HPLC-DAD detection mode. 2.8. LC/ESI/LIT MS and LC/ESI/QqQ/MS qualitative determination of xanthine The electrospray ionisation (ESI) source of a Thermo Scientific LTQ-XL mass spectrometer (Thermo Scientific, Dreieich, Germany) was tuned in positive ion mode with a standard solution of kaempferol-3-O-glucoside (l µg/mL) infused at a flow rate of 5 L/min with a syringe pump. The source voltage was 5 kV and capillary voltage was +35 kV, the tube lens offset 120 V and the capillary temperature was set at 280°C, auxiliary gas was set at 5 (arbitrary units). Qualitative LC/ESI/LIT MS was performed using a Finnigan Surveyor HPLC (Thermo Finnigan, San Jose, CA, USA) equipped with a Waters Xselect CSH C18 3.5 m column (150 mm × 2.1 mm i.d.) (Milford, MA, USA) and coupled to a Linear Ion Trap (LIT) mass spectrometer (Thermo Scientific). A linear gradient elution with a mobile phase comprising water acidified with 0.1% formic acid (solvent A) and acetonitrile acidified with 0.1% formic acid (solvent B) starting from 95% A, was converted in 65%A in 45 min, followed by 10 min of maintenance. The mobile phase was supplied at a flow rate of 200 µL/min keeping the column at room temperature, and the effluent was injected directly into the ESI source. The mass spectrometer was operated in the positive ion mode. Samples were prepared diluting 10

L into 990 L of H2O (a volume of 10 L was injected). Multiple Reaction Monitoring (MRM) analysis was performed on an Agilent 1100 HPLC system (Palo Alto, CA, USA) equipped with a Waters Xselect CSH C18 column (150 mm × 2.1 mm i.d., 3.5 m) (Milford, MA, USA) coupled to an Applied Biosystems API2000 triple quadrupole instrument. Separation was performed at room temperature by using a gradient system with two eluents: 20 mM ammonium formate adjusted with

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formic acid at pH 4 (solvent A) and acetonitrile (solvent B). The gradient started from 5% of eluent B to achieve the 50% of solvent B in 15 min and then reached 100% of solvent B in 1 min. The flow (200 L/min) generated by chromatographic separation was directly injected into the electrospray ion source. The mass spectrometer was operated in the positive ion mode under the following conditions: declustering potential 20 eV, focusing potential 200 eV, entrance potential 12 eV, collision energy 30 eV, collision cell exit potential 15 eV, ion spray voltage 4200, temperature 250 °C. Data were acquired using the MRM scanning mode involving the transition pair (153.3/109.9 amu). This is the common reaction observed for purine and pyrimidine derivatives, originated from the loss of HNCO (43 amu). The procedure was reproduced using the method described by Boudra et al. (2012). The samples were prepared diluting 10 L into 990 L of H2O (injected 10 L). Quantitative analysis of xanthine was carried out using the HPLC-DAD method described in 2.7. 2.9. HPLC-FLD determination of amino acids and biogenic amines Determination of amino acids (AA) and biogenic amines (BA) was done after derivatisation with DCl as described by Tuberoso et al. (2015). The HPLC determination of the AA and BA dansyl derivatives was performed with an HPLC-FLD Varian system ProStar (Varian Inc., Walnut Creek, CA, USA) fitted with a pump module 230, an autosampler module 410, and a Jasco 821-FP fluorimetric detector (Jasco Europe, Cremella, Lecco, Italy) with wavelengths set at 293 nm (Ex) and 492 nm (Em). Separation was obtained with a Phenomenex Gemini C18 110A column (150 x 4.60 mm, 3 μm, Chemtek Analitica, Anzola Emilia, Bologna, Italy) thermostated at 25 °C. Injection volume was 20 μL. Chromatograms and data were acquired with a HP Hewlett Packard 3396 series II integrator (Hewlett Packard, Cernusco sul Naviglio, Milan, Italy). The

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quantitative analysis was performed using calibration graphs built according to the internal standard method (100 mg/L norvaline), correlating the analyte/IS peak area ratios vs. the concentration. The full validation procedure in agreement with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance note which describes validation of analytical methods (ICH Topic Q2 (R1), 2005) is reported by Tuberoso et al. (2015). 2.10. Statistical analyses All measurements were conducted in triplicate. One-way analysis of variance (ANOVA) followed by Tukey’s test was performed in order to ascertain possible significant differences between groups using the Graph Pad Prism 5 software (GraphPad software, San Diego, CA, USA). Correlation analysis was performed and the evaluation of statistical significance of observed differences was performed by using Spearman coefficients of correlation.

3.

Results and discussion The 14 Carignano samples tested in this study were very similar, even though they

were made by different producers using different winemaking techniques. Tables 2-4 report the minimum, maximum and mean ± standard deviation (SD) values for all wines tested. The results for each sample are reported in Tables S1-S5 in the Supplementary Material. 3.1. Technological parameters and chromaticity coordinates Table 2 shows the percentage of alcohol, the content of reducing sugars, total and volatile acidity, pH and organic acids (tartaric acid, malic acid, lactic acid and citric acid) for the 14 wines tested. It should be noted that all the samples met the legal standards for alcohol content, total acidity, volatile acidity and citric acid (European Commission

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Regulation (EC) No. 479/2008; European Commission Regulation (EC) No. 606/2009). In addition, all alcohol values were within the parameters specific to the DOC Carignano, category “red” wine (Sardegna Agricoltura - Regione Autonoma Sardegna. Disciplinare del Carignano). All the tested samples had completed the malolactic fermentation process except for sample 11, which still showed significant malic acid content (0.58 ± 0.00 g/L). This sample also showed the highest total acidity (6.42 ± 0.06 g/L). Marked variations in the citric acid content were observed (from “not detectable” to 0.19 ± 0.01 g/L); variability such as this can result from different rates of production of this acid during malolactic fermentation. Overall, the technological parameter values were in agreement with those for monovarietal red wines (Heras-Roger et al., 2016). The CIE L*C*abh°ab chromaticity coordinates showed homogeneous distribution across the samples tested (Table 2), describing the typical purple-red colouring of young wines. Figure 1 shows the distribution of the 14 Carignano samples for the CIE L*C*ab and CIE L*h°ab coordinates compared with the three aged Carignano samples. Samples 3, 4, 13 and 14 had high C*ab values (ranging from 47.0 to 52.1) compared to the average (43.4), and low L* values (ranging from 42.8 to 52.4). Differences among samples can be due to several factors, such as vineyard soil, age of vines and oenological procedures. Reynolds et al. (2008) found a significant correlation between colour, content in phenolic compounds (including anthocyanins) and age of the vine from which the grapes were harvested. Furthermore, samples CAR2011 and CAR2012 showed the lowest C*ab values (33.3 and 36.1, respectively) in comparison with the average (43.4), and high L* values (range 63.4 to 65.9) compared with the average (56.7). Hue analysis of the 14 Carignano wines showed that all the samples had low h°ab values and displayed a very intense colour. Conversely, samples CAR2010, CAR2011 and CAR2012 had high h°ab values ranging between 16.4 and 27.6, when compared to the average of 3.4. The high hues

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observed for these samples may indicate oxidation of the samples due to aging, which was also described by Sen and Tokatli (2016). 3.2. Content of total polyphenols, total anthocyanins and antioxidant capacity (total reducing power and radical scavenging capacity) Following the preliminary characterization described above, the total polyphenol and anthocyanin contents and the antioxidant properties of these wines were investigated. Table 2 shows the content of total polyphenols (TP), total anthocyanins (TA) and the antioxidant capacity (total reducing power and radical scavenging) in the 14 samples of Carignano wine. The overall TP content was high, with an average of 2023 ± 435 mg GAE/L. The observed variability is possibly due to several factors such as vineyard soil, the age of the vineyard and the winemaking techniques used to make the different wines. Overall, the concentration of total polyphenols is comparable with that of other red wines (Hosu et al., 2014; Radovanovic et al., 2011). Interestingly, samples 3 and 13 showed the highest TP amount, while sample 2, which came from a younger vineyard, had the lowest concentration of TP (1313 ± 50 mg GAE /L). The content of TP was correlated significantly (p < 0.001) with the total reducing power (r

TP/FRAP

= 0.930) and radical

scavenging capacity (r TP/DPPH = 0.846) (Table 3). Carignano wines had an average FRAP value of 31.6 ± 5.2 mmol Fe2+/L and of DPPH of 10.0 ± 1.6 mmol TEAC/L with sample 3 registering the highest (44.9 Fe2+ ± 3.2 mmol/L and 13.2 ± 1.2 mmol TEAC/L) and sample 2 the lowest (26.7 ± 1.3 mmol Fe2+/L and 8.6 ± 0.2 mmol TEAC/L) values. The radical scavenging activity of all the Carignano samples was found to be higher than that of red wines such as Syrah, Merlot and Cabernet-Sauvignon (Van Leeuw et al., 2014). The analysis of the TA levels provided additional information about the freshness of wines tested in this study. Some wines, such as samples 1, 2, 3 and 13 had values significantly above average (290.3 ± 55.4 mg ME/L). It can be observed that sample 3,

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which came from grapes grown from ungrafted vines cultivated in sandy soil dating back to the early 1900s, had the highest concentration of TA (431.3 ± 22.4 mg ME/L). Overall, all of the 14 analysed samples had a high level of total anthocyanins, typical of young wine samples and higher than other red wines produced using different varieties of grapes (Nixdorf and Hermosín-Gutiérrez, 2010; Van Leeuw et al., 2014). On the other hand, the aged wines (CAR2010, CAR2011 and CAR2012), had a lower concentration of anthocyanins (respectively 110.1 ± 6.9, 98.6 ± 6.0, and 88.0 ± 3.9 mg ME/L). It is interesting to note that no statistically significant correlation (p < 0.05) was found between the content of TA and antioxidant activity (total reducing power and radical scavenging) in the wines (Table 3). 3.3. Quali-quantitative determination of phenolic compounds and xanthine Table 4 shows the polar compound content assessed in the wines by HPLC-DAD. The results showed that the majority of these compounds were polyphenols. Among anthocyanins, the most abundant was malvidin 3-O-glucoside (120.0 ± 23.5 mg/L), of which the acetylated and p-coumarate forms were also detected. These compounds, besides being scavengers of free radicals, have the ability to modulate some intracellular signals involved in inflammation. They are therefore useful at preventing some diseases related to inflammation and oxidative stress, such as diabetes and obesity (Bognar et al., 2013). Among the flavanols, epicatechin was the most concentrated (62.7 ± 16.9 mg/L) with values higher than in other red wines such as Shiraz and Tempranillo (Fanzone et al., 2011). Within the flavonols category, it was possible to accurately measure only quercetin 3-O-glucoside (4.3 ± 2.3 mg/L). Other compounds present in relevant concentrations were cis-caftaric acid (52.9 ± 22.0 mg/L), which is a hydroxycinnamic acid, and gallic acid (179.9 ± 47.8 mg/L), which is a hydroxybenzoic acid. Gallic acid had higher value

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compared to other red wines, which tend to be lower than 70 mg/L (Anli and Vural, 2009; Fanzone et al., 2011). Samples 1, 3, 5 and 13 showed high values for all phenolic compounds, while samples CAR2011 and CAR2012 showed lower concentrations. These findings confirm the data obtained with the TP analysis. Furthermore, no trans-resveratrol was measured in any of the samples. Based on recent reports (Xiang et al., 2014) which indicate a lack of antioxidant activity of resveratrol compared to other polyphenols in wine, it is likely that the absence of trans-resveratrol does not diminish the potential health benefits of Carignano wines. In all the samples tested, the presence of xanthine was detected. This purinic compound is produced from hypoxanthine during malolactic fermentation (Arapitsas et al., 2012). Since, as far as we know, there are no quantitative studies about this compound in wine, xanthine content in Carignano wines was explored using LC/ESI/LIT and further confirmed using LC/ESI/QqQ/MS. To detect the presence of xanthine, a sample of wine diluted 1:100 with water was analysed by LC/ESI/LIT in positive ion mode. The Reconstructed

Ion

Chromatogram

(RIC)

at

m/z

153,

corresponding

to

the

pseudomolecular ion of xanthine [M+H]+ gave a peak at retention time of 1.28 min tentatively identified as xanthine (for chromatogram, see Fig. S1 in the Supplementary Material). To confirm the nature of the compound, an LC/ESI/QqQ/MS method working in Multiple Reaction Monitoring with a specific and selective ion transition for xanthine was developed, according to the method described by Boudra et al. (2012). An MRM transition in positive ion mode was selected, for the parent ion m/z 153.3 to the daughter ion m/z 109.9. Fragmentation involved the common reaction observed for purine and pyrimidine derivatives, originated from the loss of HNCO (43 amu). The peak corresponding to xanthine was identified at retention time of 2.72 (Fig. S1). The proposed

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LC/ESI/LIT and LC/ESI/QqQ MS methods proved to be efficient at verifying the presence of xanthine in wine. The xanthine levels measured by LC-DAD ranged between 48.0 and 101.4 mg/L, with an average value of 63.4 ± 15.8 mg/L. The quantification of xanthine in foods is necessary because of its implication in some human disorders: as a matter of fact, at high concentrations, it can lead to hyperuricemia and gout and it can also could be a risk factor for cardiovascular disease, kidney disease, and metabolic syndrome (Kaneko et al., 2014). However, there are no legal limits set for xanthine in commercial wines. A daily intake of dietary purines has been recommended in Japan: this is up to 400 mg (Kaneko et al., 2014), a very higher concentration respect to that found in Carignano wines. Overall, it is our opinion that xanthine quantification should always be done for consumer’s health safety reasons. 3.4. Concentration of amino acids and biogenic amines Thirty-four nitrogen compounds (amino acids, AA, and biogenic amines, BA) were identified within the sample set of 14 wines. The results show significant differences between wines, particularly within the levels of amino acids and biogenic amines (Table 5). The total concentration of nitrogen compounds (AA + BA) was 1156.2 ± 292.3 mg/L, much lower than other Sardinian wines such as Vermentino (1879 ± 365 mg/L) and Cannonau (1796 ± 338 mg/L) (Tuberoso et al., 2015). Considering only the AA fraction, the levels were 1139.0 ± 292.9 mg/L, with Pro being the most concentrated (578.0 ± 229.3 mg/L), followed by Glu (250.9 ± 38.8 mg/L), Asp and GABA. This latter is an amino acid formed from Glu by GAD (glutamic acid decarboxylase) and its average concentration in Carignano wines was 40.0 ± 13.9 mg/L. The average content of essential amino acids in these wines was 61.4 ± 22.5 mg/L, and the most abundant essential amino acids were Leu + Ile (13.7 ± 5.7 mg/L). The total amino acid contents in samples 6, 7, 8

17

and 9 (all coming from stainless steel tanks) and aged samples CAR2010 and CAR2011, were the highest. Carignano wine has an average content of BA of 17.2 ± 6.9 mg/L, with the individual BA values generally <10 mg/L, on the average. The CAR2010 and CAR2011 samples, as observed with AAs, had high levels of BAs, which suggests the occurrence of collateral fermentative processes during winemaking (Ribéreau-Gayon et al., 2006). Among the BAs, PUT was always present in Carignano wines, and it showed the highest levels with a mean value of 7.8 ± 4.5 mg/L, which was much lower than in other highquality red wines such as Cabernet-Sauvignon and Merlot (Konakovsky et al., 2011). The only BA for which a higher value was measured was HIM (average 13.8 ± 0.8 mg/L). However, this was detected in only 3 samples (two from 2013, 10 and 11, with average values of 13.3 ± 0.2 mg/L and 14.4 ± 0.2 mg/L, respectively, and in CAR2012, 13.0 ± 0.1 mg/L); the other Carignano wines showed no trace of HIM. It is also important to note the presence of the TYM with mean values of 1.2 ± 1.0 mg/L, lower than those of another Sardinian red wine, Cannonau (Tuberoso et al., 2015), and lower than the average of 296 red wines evaluated by EFSA (2011). Along with HIM, TYM is one of the most well-known BAs, studied for its adverse effects. One of these is associated with excessive intake of TYM through certain foods (such as cheese, processed meat, alcoholic beverages, etc.) that have undergone fermentation or microbial decomposition that causes a syndrome known as cheese reaction, characterized by a sympathomimetic effect of acute hypertension with headache (Ladero et al., 2010). Despite these obvious health concerns, currently there are no established limits for the maximum levels of BAs tolerated in wines, not even by the OIV (Organisation Internationale de la Vigne et du Vin). However, some European countries have set maximum level limitations for HIM levels, ranging between 2 and 10 mg/L.

18

In the case of the Carignano samples with the highest BA levels, perhaps a change in some of the parameters of winemaking, such as the strain of yeast used, temperature and steeping time, could help reduce the BA levels. An overall strategy aimed at producing wines that are safer for human consumption by reducing the BA levels is warranted by the increased scrutiny on BA content and their possible accumulation in the food chain, which has become a crucial point for food safety (EFSA, 2010; EFSA, 2011).

4.

Conclusion This study provides preliminary information about the chemical composition of

Carignano wines. It showed that Carignano is a wine rich in phenolic substances, with high in vitro antioxidant capacity, particularly as free radical scavengers. The chemical and physical characterization of these mono-varietal wines will help to promote their commercial exploitation, and since the wines are labelled as DOC at the European level (E-Bacchus, 2007 the potential consumer group extends far beyond Italy. Testing in ex vivo and in vivo models is needed to further evaluate the antioxidant activity and other potential health benefits of Carignano wines. Furthermore, although undesirable substances such as biogenic amines were present in some of the wines tested, the levels were low, which is indicative of good winemaking techniques. The quantification of xanthine, although present in concentrations lower than ca. 100 mg/L, should not be neglected, and its production should be monitored and limited during the winemaking process.

Acknowledgments The authors thank the wineries Calasetta, Giba, Mesa, Santadi, Sant’Antioco and Sella&Mosca for supplying samples, Dr. Christina D. Orrù, Rocky Mountain

19

Laboratories (National Institutes of Health, National Institute of Allergy and Infectious Diseases, USA) and Dr. Martina Foddis (University of Cagliari) for helpful discussion. This work has been partially supported by the Croatian Science Foundation under the project (IP-11-2013-8547) “Research of Natural Products and Flavours: Chemical Fingerprinting and Unlocking the Potential”.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/XXXX

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25

Figure caption Fig. 1. Correlation between CIE L*Cab* and L*hab° coordinates

26

Fig. 1.

27

Table 1 List of Carignano wines and their production locations

Code

Vinery

Area of production

Year

1

A

Sant’Anna Arresi

2013

2

A

Sant’Anna Arresi

2013

3

A

Sant’Anna Arresi

2013

4

B

Santadi

2013

5

B

Santadi

2013

6

C

Sant’Antioco

2013

7

C

Sant’Antioco

2013

8

C

Sant’Antioco

2013

9

C

Sant’Antioco

2013

10

D

Calasetta

2013

11

D

Calasetta

2013

12

E

San Giovanni Suergiu

2013

13

F

Sant’Antioco

2013

14

F

Sant’Antioco

2013

CAR2010

E

San Giovanni Suergiu

2010

29

CAR2011

E

San Giovanni Suergiu

2011

CAR2012

E

San Giovanni Suergiu

2012

30

Table 2 Technological parameters, chromaticity coordinates, total polyphenol and total anthocyanin contents, and antioxidant capacity (total reducing power and radical scavenging) in Carignano samples (min, max, mean ± SD, n = 14) Carignano wines Parameters min

max

mean

± SD

12.55

14.59

13.34

0.63

Reducing sugars (g/L)

1.07

2.70

1.94

0.41

Total acidityb (g tartaric acid/L)

4.62

6.42

5.40

0.49

Volatile acidityc (g/L)

0.44

0.94

0.64

0.17

pH

3.61

3.93

3.74

0.10

Malic acid (g/L)

nd

0.58

0.08

0.16

Lactic acid (g/L)

0.84

2.04

1.05

0.31

Tartaric acid (g/L)

1.70

2.76

2.25

0.36

nd

0.19

0.09

0.07

L*

42.8

63.3

56.7

6.7

C*ab

38.4

52.1

43.4

4.5

h° ab

1.3

7.4

3.4

2.1

1313

2764

2023

435

Alcohola (% v/v)

Citric acidd (g/L) Chromaticity coordinates CIE

Total polyphenols (mg GAE/L)

31

Total antocyanins (mg ME/L)

203.8

431.3

290.4

55.4

26.7

44.9

31.6

5.2

8.4

13.2

10.0

1.6

FRAPe (mmol Fe2+/L) DPPHf (mmol TEAC/L) a

minimum 9% (Regulation (EC) No 479/2008 of 29 April 2008. All IV) and 12% for DOC Carignano minimum 3.5 g/L (Regulation (EC) No 479/2008 of 29 April 2008. All IV) c maximum 1.2 g/L (20 mEq / L, Regulation (EC) No 606/2009 of 10 July 2009, App. IC) d maximum 1 g/L (Regulation (EC) No 606/2009 of 10 July 2009, App. IA) e FRAP value is expressed as Fe2+ millimolar concentration, obtained from a FeSO4 solution having an antioxidant capacity equivalent to that of the dilution of the wine. f DPPH values are expressed as TEAC millimolar concentration, obtained from a Trolox solution having an antiradical capacity equivalent to that of the dilution of the wine. b

32

Table 3 Spearman correlation coefficients for the correlation between total polyphenols (TP), total anthocyanins (TA), antioxidant activity (radical scavenging capacity, DPPH, and total reducing power, FRAP), CIE L*C*abh°ab coordinates, and malvidin 3-O-glucoside (M 3-glu).

TA

DPPH

FRAP

TP

0.305

0.846***

0.930***

-0.829*** 0.609*

0.255

-0.073

0.257

TA

-

0.487

0.349

-0.515

-0.407

0.710**

0.579*

-

0.811***

-0.761** 0.544*

DPPH FRAP

-

L*

L*

C*ab

0.451 ***

-0.856 -

C*ab

h°ab

M 3-glu Xanthine

0.159

0.073

0.416

0.741

**

0.090

0.015

0.343

0.849

***

0.022

-0.090

-0.469

-0.317

0.156

0.231

-

-0.737** 0.126

-

h°ab M 3-glu

-

0.337

Xanthine

-

* significant at p<0.05; ** significant at p< 0.01; *** significant at p<0.001.

Table 4 Concentration of phenolic compounds (mg/L) in Carignano wines (n = 14) Compound

Rt

Anthocyanins Delphinidin 3-O-glucoside 16.5 Cyanidin 3-O-glucoside 17.5 Petunidin 3-O-glucoside 18.5

ID* Rt /UV-VIS Rt /UV-VIS Rt /UV-VIS

LOD

LOQ

0.7 0.6 0.5

2.0 1.9 1.6

33

min 9.3 nd 12.7

max 22.1 3.8 28. 7

mean 16.8 3.7 20.9

± SD 4.4 0.1 4.3

Peonidin 3-O-glucoside 19.5 Malvidin 3-O-glucoside 19.9 b Malvidin 3-O-glucoside acetate 24.2 Malvidin 3-O-glucoside p34.7 b b coumarate Others Total Flavonols Quercetin 3-O-glucoside 23.3 c Others Total Flavanols Procyanidin B1 14.9 Catechin 15.8 Procyanidin B2 17.8 Epicatechin 18.4 Epigallocatechin 19.5 Total Hydroxycinnamics acids d cis-Caftaric 15.3 trans-Caftaric 17.3 e cis-Coutaric 16.9 e trans-Coutaric 18.2 d Others 22.6 Total Hydroxybenzoic acids Gallic acid 6.60 Protocatecuic acid 11.1

Rt /UV-VIS Rt /UV-VIS UV-VIS UV-VIS UV-VIS

0.5 0.4 -

1.4 1.3 -

4.6 80.0 7.2 nd 8.9 134.5

8.2 165.7 29.9 17.6 32.7 266.6

6.5 120.0 10.3 12.6 16.5 206.1

1.0 23.5 5.7 2.6 5.9 37.4

Rt /UV-VIS UV-VIS

0.6 -

1.7 -

nd nd nd

11.5 42.9 54.5

4.3 21.8 26.2

2.3 9.9 11.8

Rt /UV-VIS Rt /UV-VIS Rt /UV-VIS Rt /UV-VIS Rt /UV-VIS

0.4 0.5 0.6 0.5 0.7

1.1 1.5 1.9 1.6 2.2

10.6 10.6 nd nd nd 38.1

29.8 30.9 49.5 81.1 21.1 184.8

17.2 21.6 30.5 62.7 11.2 113.6

6.5 5.2 16.3 16.9 3.8 52.2

UV-VIS UV-VIS UV-VIS UV-VIS UV-VIS

0.5 -

1.4 -

31.4 5.8 3.1 5.6 nd 58.9

107.4 28.3 16.9 18.7 6.2 158.7

52.9 12.6 5.5 10.9 4.5 86.4

22.0 6.3 3.5 4.5 0.9 30.6

Rt /UV-VIS Rt /UV-VIS

0.4 0.5

1.1 1.6

123.6 nd

277.2 65.8

179.9 34.9

47.8 19.0

34

Total

142.2

334.3

214.8

61.9

nd 48.0

nd 101.4

nd 63.4

nd 15.8

Other compounds trans-Resveratrol Xanthine

32.3 4.5

0.5 0.4

Rt /UV-VIS Rt /UV-VIS/ MS

1.5 1.3

nd: not detected (
Table 4 Concentration of AA and BA (mg/L) in Carignano wines (n=14) Compound

Rt

LOD

LOQ

min

max

mean

± SD

Amino acids Arginine Asparagine Glutamine Citrulline Serine 4-Hydroxyproline Aspartic acid Threonine Glycine Alanine Tyrosine Glutamic acid γ-Aminobutyric acid Proline Methionine Valine

Arg Asn Gln Cit Ser Hyp Asp Thr Gly Ala Tyr Glu GABA Pro Met Val

0.2 0.2 0.1 0.1 0.2 0.1 0.2 0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.1

7.2 9.0 10.8 10.6 12.0 12.5 13.5 0.7 17.1 20.9 21.5 23.3 26.6 27.7 28.5 30.4 35

0.6 0.7 0.2 0.2 0.5 0.2 0.5 0.4 0.2 0.2 0.2 0.5 0.2 0.2 0.1 0.2

nd 0.5 6.9 1.5 7.1 nd 38.5 3.1 9.5 13.7 4.8 153.1 23.6 203.6 nd 3.0

29.3 11.8 38.8 4.1 17.6 8.6 112.4 12.8 23.5 36.9 21.3 296.6 72.4 891.7 3.0 10.2

19.6 7.3 17.7 2.8 11.6 6.2 67.5 7.3 16.9 25.5 10.6 250.9 40.0 578.0 2.0 5.8

6.4 3.4 8.6 0.7 3.2 2.8 26.9 2.7 4.2 8.1 4.6 38.8 13.9 229.3 1.4 2.2

JFCA-D-16-00056

Phenylalanine*+ Tryptophan Leucine*+Isoleucine Cysteine Ornithine Histidine Lysine Biogenic amines Methylamine Ethylamine Tryptamine Phenylethylamine Isopentylamine Putrescine Cadaverine Histamine Tyramine Spermidine Spermine

Tuberoso et al.

Phe*+Trp Leu*+Ile Cys Orn His Lys

34.0 35.0 39.9 42.1 43.3 43.6

0.1 0.1 0.1 0.1 0.2 0.1

0.2 0.3 0.2 0.2 0.5 0.2

7.4 7.6 0.2 nd 14.3 11.1

25.4 26.5 1.3 0.4 47.6 36.2

14.3 13.7 0.6 0.4 37.2 19.9

5.2 5.7 0.3 0.0 9.8 7.4

MTA ETA TRM PEA IPA PUT CAD HIM TYM SPD SPM

32.1 37.0 47.0 48.7 49.4 50.2 51.3 51.9 55.8 56.8 59.3

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

0.2 1.1 nd 0.9 tr 3.7 0.1 nd nd nd nd min 539.3 525.8 34.4 11.2

0.8 2.8 nd 3.3 0.4 18.6 2.9 14.4 2.9 2.4 2.5 max 1538.4 1525.4 111.0 32.3

0.5 2.1 nd 1.8 0.2 7.8 0.7 13.8 1.2 1.8 1.4 mean 1156.2 1139.0 61.4 17.2

0.1 0.5

Total nitrogen compounds Total amino acids Total essential amino acids# Total biogenic amines

nd: not detected (
36

0.7 0.1 4.5 0.9 0.8 1.0 0.9 0.7 ± SD 292.3 292.9 22.5 6.9