Phytochemical and biological characterization of Italian “sedano bianco di Sperlonga” Protected Geographical Indication celery ecotype: A multimethodological approach

Journal Pre-proofs Phytochemical and biological characterization of Italian “sedano bianco di Sperlonga” Protected Geographical Indication celery ecotype: a multimethodological approach Cinzia Ingallina, Donatella Capitani, Luisa Mannina, Simone Carradori, Marcello Locatelli, Antonella Di Sotto, Silvia Di Giacomo, Chiara Toniolo, Gabriella Pasqua, Alessio Valletta, Giovanna Simonetti, Alessia Parroni, Marzia Beccaccioli, Giuliana Vinci, Mattia Rapa, Anna Maria Giusti, Caterina Fraschetti, Antonello Filippi, Alessandro Maccelli, Maria Elisa Crestoni, Simonetta Fornarini, Anatoly P. Sobolev PII: DOI: Reference:

S0308-8146(19)31775-3 https://doi.org/10.1016/j.foodchem.2019.125649 FOCH 125649

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

1 February 2019 24 September 2019 3 October 2019

Please cite this article as: Ingallina, C., Capitani, D., Mannina, L., Carradori, S., Locatelli, M., Di Sotto, A., Di Giacomo, S., Toniolo, C., Pasqua, G., Valletta, A., Simonetti, G., Parroni, A., Beccaccioli, M., Vinci, G., Rapa, M., Maria Giusti, A., Fraschetti, C., Filippi, A., Maccelli, A., Crestoni, M.E., Fornarini, S., Sobolev, A.P., Phytochemical and biological characterization of Italian “sedano bianco di Sperlonga” Protected Geographical Indication celery ecotype: a multimethodological approach, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem. 2019.125649

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Phytochemical and biological characterization of Italian “sedano bianco di Sperlonga” Protected Geographical Indication celery ecotype: a multimethodological approach Cinzia Ingallinaa, Donatella Capitanib*, Luisa Mannina a,b*, Simone Carradoric, Marcello Locatellic, Antonella Di Sottod, Silvia Di Giacomod, Chiara Tonioloe, Gabriella Pasquae, Alessio Vallettae, Giovanna Simonettif, Alessia Parronie, Marzia Beccacciolie, Giuliana Vincig, Mattia Rapag, Anna Maria Giustih, Caterina Fraschettib, Antonello Filippib, Alessandro Maccellib, Maria Elisa Crestonib, Simonetta Fornarinib, Anatoly P. Sobolevb aDipartimento

di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, P.le Aldo Moro

5,

Rome,

00185

Italy

[email protected];

[email protected];

[email protected]; [email protected];

[email protected]; [email protected] bIstituto

per i Sistemi Biologici, Laboratorio di Risonanza Magnetica “Annalaura Segre”, CNR,

00015 Monterotondo (Rome), Italy [email protected] cDipartimento di Farmacia, Università di Chieti-Pescara “G. d’Annunzio”, Via dei Vestini 31, 66100

Chieti, Italy [email protected]; [email protected] dDipartimento

di Fisiologia e Farmacologia “V. Ersparmer”, Sapienza Università di Roma, P.le

Aldo Moro 5, 00185 Rome, Italy [email protected]; [email protected] eDipartimento

di Biologia Ambientale, Sapienza Università di Roma, P.le Aldo Moro 5, 00185 Rome,

Italy [email protected]; [email protected]; [email protected]; [email protected]; [email protected] fDipartimento

di Sanità Pubblica e Malattie Infettive, Sapienza Università di Roma, P.le Aldo Moro

5, 00185 Rome, Italy [email protected] gDipartimento

di Management, Laboratorio di Merceologia, Sapienza Università di Roma, Via del

Castro Laurenziano 9, 00161 Rome, Italy [email protected]; [email protected] hDipartimento

di Medicina Sperimentale Sapienza Università di Roma, P.le Aldo Moro 5, 00185

Rome, Italy [email protected] *Corresponding authors at: Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, P.le Aldo Moro 5, 00185 Rome, Italy Tel: +39 06 49913735; e-mail: [email protected] (L. Mannina)

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Istituto di Metodologie Chimiche, Laboratorio di Risonanza Magnetica “Annalaura Segre”, CNR, 00015 Monterotondo (Rome), Italy Tel. +39 06 90672700; e-mail:[email protected] (D. Capitani).

Abstract Celery is a widely used vegetable known for peculiar sensorial and nutritional properties. Here, the white celery (Apium graveolens L.) “sedano bianco di Sperlonga” PGI ecotype was investigated to obtain the metabolic profile of celery edible parts (blade leaves and petioles) also related to quality, freshness and biological properties. A multi-methodological approach including NMR, MS, HPLCPDA, GC-MS and spectrophotometric analyses was proposed to analyse celery extracts. Sugars, polyalcohols, amino acids, organic acids, phenols, sterols, fatty acids, phthalides, chlorophylls, tannins and flavonoids were detected in different concentrations in blade leaf and petiole extracts indicating celery parts as nutraceutical sources. The presence of some phenols in celery extracts was here reported for the first time. Low contents of biogenic amine and mycotoxin confirmed celery quality and freshness. Regarding the biological properties, ethanolic celery extracts inhibited the oxidative-mediated DNA damage induced by tert-butylhydroperoxide and scavenged DPPH and ABTS radicals.

Keywords: White celery; NMR metabolic profiling; FT-ICR mass spectrometry; HPLC; Biogenic amines; Mycotoxins; Biological activity.

1 Introduction Celery (Apium graveolens L. – Fam. Apiaceae) is a daily consumption vegetable firstly cultivated in Europe, and then spread worldwide for its valuable sensorial and nutritional properties. Particularly, celery blade leaves and petioles are widely used in the Italian traditional cuisine being rich sources of proteins, vitamins, carotenoids, fibres, phenolics, flavonoids and tannins with flavouring and health properties (Ovodova et al., 2009; Li, Hou, Wang, Tan, Xu & Xiong, 2018; Kooti & Daraei, 2017; 2

Yusni, Zufry, Meutia & Sucipto, 2018). For instance, the presence of phenolics, was associated to the peculiar biological properties of celery products, including seeds, essential oil and leaves, used in traditional medicine for a variety of ailments, as antimicrobial, diuretic, spasmolytic, laxative and heart tonic remedies (Al-Asmari, Athar & Kadasah, 2017). The presence of antioxidant compounds such as apigenin, luteolin and quercetin was reported to be responsible for the ability of a celery leaf extract to decrease the pre- and post-prandial blood glucose levels in elderly pre-diabetic subjects without affecting plasmatic insulin levels, which could interfere with the oxidative stress, associated to hyperglycaemia (Yusni et al., 2018). Sedanolide reported as major responsible for celery aroma and present in essential oils from seeds and leaves was suggested to be a food preservative due to its antibacterial and antifungal activities (Marongiu et al., 2012; Torun, Biyik, Ercin & Coban, 2018). Petioles are also enriched in pectic polysaccharides (Al-Asmari et al., 2017) and celery has been proposed as a natural rich source of the soluble mannitol (Rupérez & Toledano, 2003) with prebiotic activity. In line with this evidence and in view of valorizing local products, in the present study the Italian white celery ecotype “sedano bianco di Sperlonga” (Apium graveolens variety dulce), certified by the Protected Geographical Indication (PGI) was studied. According to the production disciplinary (Commission Regulation (EU) No 222/2010; Official Gazette of the Italian Republic OG n. 86/2010), this ecotype is strictly cultivated in the Lazio region of Central Italy, at the charming seaside of Sperlonga-Fondi in the Ciociaria area. The cultivation land, characterized by a sandy-loamy soil with a high degree of salinity together with the Mediterranean climate with relatively dry and warm summers and humid winters bestows the characteristic flavour, sweet and moderately aromatic taste of this celery ecotype particularly well-suited to being eaten raw (OG n. 86/2010) Only partial chemical characterization of celery, focused on specific classes of compounds, has been reported up to now. Here, the whole metabolite profile of the edible part (petioles and blade leaves) of “sedano bianco di Sperlonga” ecotype has been investigated by means of a multimethological approach (Sobolev et al.,

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2018). This approach includes Nuclear Magnetic resonance (NMR) and mass spectrometry (MS) untarged methodologies widely used to obtain the metabolic profile of foodstuffs (Lindon & Nicholson, 2008) and targeted chromatographic and spectrophotometric methods focused on specific classes of compounds such as secondary metabolites as well as toxins produced by mycotoxigenic fungi able to colonize plant and biogenic amines (BAs). The combination of different complementary methodologies seems to be a suitable approach to analyze a complex food matrix such as celery overcoming limitations of any single technique. In order to complete the characterization of this typical local celery, the beneficial properties of the celery phenolic extracts were also investigated for the possible biological activities, including antifungal, antimutagenic, antioxidant and hypoglycaemic ones. Our hypothesis is that the multimethodological approach can highlight possible differences between petiole and blade leaf chemical profiles, thus suggesting specific nutritional and health properties of the different celery parts thus valorizing a product strictly linked to the production area.

2 Materials and methods 2.1 Celery Samples and Chemicals Celery plants of Apium graveolens, dulce variety, “sedano bianco di Sperlonga” ecotype certificated PGI and grown in greenhouse, were provided by a farm located at Sperlonga, a seaside town in the Lazio region (Central Italy). The whole plant is characterized by an average height, medium or large size (from 500 to more than 800 g), compact form, 10-15 light green leaves, and not very fibrous and white with a light-green tinge petioles (OG n. 86/2010): both petioles and leaves are used as food. The compliance of the collected samples with the PGI requirements was confirmed by the morphological analysis. Celery samples (Figure 1) were carefully divided in the three main edible parts, i.e. petioles, blade leaves, heart (both petioles and leaves), and analysed separately. Chemicals (reagents and solvents) are reported in the Supplementary material. 2.2 Extraction processes 4

2.2.1 Extraction for untargeted analyses Fresh samples of blade leaves and petioles coming from 4 celeries were collected, frozen in liquid nitrogen and ground to constitute a homogeneous sample. Repeating this procedure, 4 samples of each part were obtained. Extracts of each part were obtained using a mixture of methanol/chloroform (2:1 v/v) to optimize the extraction of the greater number of metabolites. In details, each part (2.0 g) was added sequentially with 3 mL methanol/chloroform (2:1 v/v) mixture, 1 mL of chloroform and 1.2 mL of distilled water. After each addition, the sample was carefully shacked. The obtained emulsion was stored at 4 °C for 40 min. After centrifugation (800 g for 15 min at 4 °C) the hydroalcoholic and organic phases were carefully separated. Using half of the solvent volumes, pellets were re-extracted and the separated fractions were pooled. Both fractions were dried under a N2 flow at room temperature until the solvent evaporation. The dried phases were stored at -20 °C until further analyses. 2.2.2 Classical hydroalcoholic maceration for targeted analyses Petioles, blade leaves, and heart (50 g) were subjected to maceration under acidified ethanolic solution (250 mL of EtOH 70% v/v) for 48 hours in the dark and at room temperature. The combining of appropriate solvent and pH has been reported to be an appropriate method for enhancing the phenolic recovery, as pH determines their solubility and solvent address the efficient contact (Ajila, Brar, Verma, Tyagi, Godbout & Valéro, 2011). After filtration, the extracts were dried in a rotary vacuum evaporator (Büchi, 461 Water Bath) at controlled temperature (< 40 °C). 2.2.3 Extracts for the analyses of antifungal activity The antifungal activity was evaluated on celery extracts obtained with acetone, ethanol and water with an increasing polarity. Petioles and blade leaves (each 50 g FW, fresh weight) were subjected to three extractions (each 24 h, preceded by 20 min sonication) using a ratio solvent/biomass of 50:1 (mL/g FW). The extracts were separated from the biomass under vacuum filtration. Acetone and ethanol extracts were dried by means of rotary evaporator, while water extract was freeze-dried. The extracts were kept dry before performing the antifungal test.

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2.3 Untargeted metabolite profile 2.3.1 NMR analysis All spectra were collected at 27 °C with an Avance600 NMR Bruker spectrometer operating at the proton frequency of 600.13 MHz and at the carbon frequency of 150.9 MHz. The organic fraction of each sample was solubilised in 0.7 mL of a CDCl3/CD3OD mixture (2:1 v/v) and then transferred in a 5 mm NMR tube that was flame sealed. The hydroalcoholic phase of each sample was solubilized in 0.7 mL of 400 mM phosphate buffer/D2O containing 1 mM solution of 3(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) used as internal standard, and then transferred in a 5 mm NMR tube. NMR spectra of all extracts were carried out under the experimental conditions previously reported (Sobolev et al., 2018). 1H–1H TOCSY experiments were carried out with a mixing time of 80 ms, 1H–13C

HSQC experiments with a coupling constant 1J(C-H) of 150 Hz, and 1H-13C HMBC

experiments with a delay of 80 ms for the evolution of long-range couplings. In spectral regions where the partial overlapping of signals impaired the quantitative analysis, spectral deconvolution was applied to obtain the integral of signals using dm2006 software package (Massiot et al., 2002). Deconvolution was performed assuming a Lorentzian lineshape and parameters for deconvolution were the amplitude, chemical shift, and line width at half height. In the case of organic extracts, resonances selected for quantitative analysis were labeled with I1-I13, where Ii indicated the resonance integral. All integrals were normalized with respect to the integrals of α-CH2 groups of all fatty acid chains, I5 (esterified fatty acids, α-CH2, 2.35 ppm) +I6 (free fatty acids, α-CH2, 2.29 ppm) set to 100%. 2.3.2 MS analysis Stock solutions (1 mg/mL) of the hydroalcoholic and organic extracts from petioles and blade leaves were prepared in 30:70 water:methanol (v/v), vortexed for 1 h, filtered through 0.45 μm hydrophobic polypropylene Acrodisc (Sigma–Aldrich, Milan, Italy), and then diluted to the final concentration of 0.03 g/L in methanol for direct infusion into the electrospray ionization (ESI) source of the mass

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spectrometer. A 5 μL amount of either NH4Cl (5 mM) or NaNO3 (5 mM) were added to 1.0 mL methanol solution in order to aid ionic detection in negative and positive mode, respectively (Cole, 2000). To avoid degradation of the samples, the stock solutions were kept at -20 °C. All the analyses and the Collision Induced Dissociation (CID) experiments were conducted using an ESI source coupled with either a hybrid triple-quadrupole linear ion trap mass spectrometer (2000 QTrap, Applied Biosystems/MDS SCIEX, Toronto, Canada), or an ion trap mass spectrometer (Esquire3000+, Bruker Daltonics, Bremen, Germany). ESI settings were: flow rate of 240 µL/h, capillary spray voltage at -4.0 kV (for the positive mode) or +3.8 kV (for the negative mode), nebulizer at 11 psi, drying gas flow at 6 L/min and drying gas temperature of 300 °C. High resolution mass analysis was obtained using a Fourier Transform-Ion Cyclotron Resonance (FTICR) mass spectrometer (Apex II, 4.7 Tesla, Bruker Daltonics, Bremen, Germany). The FT-ICR mass spectra were internally frequency-to-m/z calibrated with respect to ions of known elemental composition. All mass measurements are based on the “monoisotopic” ion. The components of the extracts were assigned by comparing the high-resolution m/z values with the theoretical exact mass. Further confirmation of the identity of ionic species was gained by CID experiments and tested by databases. 2.3.3 GC-MS analysis The dried extracts, arising from both the hydroalcoholic and organic phases, were dissolved in methanol (2 and 1 mg/mL for leaves and petioles, respectively) and centrifuged at 1000 g for 5 min, to finally obtain the analytical samples. 1 µL of the latter stock solution was injected into an Agilent Technologies 6850 gas chromatograph interfaced with an Agilent Technologies 5975 mass spectrometer. The following GC-MS set-up has been set: HP-5MS capillary column (5% Phenyl 95% Methylpolysiloxane, 30 m x 0.25 mm x 0.25 µm); injector temperature, 250 °C; flow rate of the helium carrier gas (99.995% purity), 1.0 mL/min; oven temperature, 40 °C for 5 min, then raised up to 200°C (at a 5 °C/min rate) and kept at this temperature for 60 min; energy of electron ionization, 70 eV; solvent delay, 6 min; source temperature, 230 °C; quadrupole temperature, 150 °C; and mass

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scan carried out over the 50-350 m/z range. The qualitative analysis has been carried out by exploiting both the EI mass spectrum and the Kovats indices (KI) of the analytes. A quality matching above 80% between the measured EI spectra and those collected in the commercial FFNSC3 software, as well as in the NIST 11 open access databases, has been used as a threshold criterion. The KI values were calculated through the Kovats equation solved by introducing the retention times obtained by analysing a C8-C24 n-alkane mixture in the same chromatographic conditions. Then a cross-reference with the values collected in the FFNSC3 database, the NIST website confirmed the MS based attribution. The chromatographic peaks were manually integrated and the relative abundances was calculated without any correction. 2.4 Targeted analyses 2.4.1 Spectrophotometric analysis of total phenolics, tannins and flavonoids The total amounts of phenolics, tannins, and flavonoids in the ethanolic extracts of celery petioles, blade leaves, and heart were determined by the Folin-Ciolcalteau and aluminium chloride methods and expressed as tannic acid (TAE) and quercetin equivalents (QE), respectively (Di Sotto et al., 2018). 2.4.2 HPLC-PDA analysis for phenolic compounds HPLC-PDA phenolics analyses were carried out on a Waters mod. 600 solvent pump coupled to a 2996 photodiode array detector. Empower v.2 Software (Waters Spa, Milford, MA) was used for the data elaboration. A C18 reversed-phase packing column (Prodigy ODS(3), 4.6x150 mm, 5 µm; Phenomenex, Torrance, CA) was used for the chromatographic resolution of the different phenolics and the column was thermostated at 30±1 °C using a Jetstream2 Plus column oven. The quantitative analyses were achieved at maximum wavelength for each compound, using an injection volume of 20 µL. The mobile phase was directly on line degassed by using Biotech DEGASi, mod. Compact (LabService, Anzola dell’Emilia, Italy), and the chromatographic analysis were carried out in gradient elution using the mobile phase water-acetonitrile (93:7, v:v, 3% acetic acid. All hydroalcoholic and organic and ethanolic extracts were weighted and dissolved in mobile phase (1

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mg/mL) and 20 µL directly injected into HPLC-PDA system. For over range samples, 1:10 dilution factor was applied. (Sobolev et al., 2018) 2.4.3 HPTLC and densitometric analysis The HPTLC analysis of the ethanolic extracts of petioles, blade leaves, and heart was performed by a CAMAG HPTLC system (Muttenz, Switzerland), controlled by the ‘WinCATS’ 1.4.4 Planar Chromatography Manager (CAMAG) software according to previous published method (Sobolev et al., 2018). 2.4.4 Spectrophotometric analysis of pigments (chlorophyll a, b and total carotenoids) Chlorophyll a, chlorophyll b and total carotenoid contents in petioles, blade leaves, and heart. organic extracts were determined through the use of absorption coefficients and equations according to Boutaoui et al. (2018). The data were reported as means of three replications and expressed as mg/g dry weight (DW) ± SD 2.5 Freshness and Quality 2.5.1 Determination of biogenic amines Petioles, blade leaves and hearts were subjected to extraction according to previous published method (Sobolev et al., 2018). The evaluation of precision (RSD <4.4%) and accuracy (recovery >96%) was assessed by using samples, coming from petioles, blade leaves and heart,

spiked at three

concentration levels of BAs. The internal standard (1-7 diaminoheptane) calibration method was used to carry out the quantification by a linear regression analysis (r ≥0.999). 2.5.2 Mycotoxins determination Mycotoxins were extracted from 0.5 g of homogenated petioles. HPLC-MS/MS analyses allowed to evaluate the mycotoxin content. A multi-mycotoxin method has provided the quantification of aflatoxins (AFLAB1, AFLAB2, AFLAG1, AFLAG2), alternariol, fumonisins (FB1, FB2), ochratoxin A, patulin and trichothecenes (DON, 3-AcDON, 15-AcDON, HT-2, T-2) as reported in Sobolev et al. (2018).

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2.6 Biological activities The antifungal activity of petiole and blade leaf extracts obtained with solvents with increasing polarity and with hydroalcoholic and organic extraction, was evaluated. The antifungal activity was assessed against Candida albicans ATCC10231 coming from American Type Culture Collection (ATCC, Rockville, MD, USA). The minimal inhibitory concentration (MIC) of the extracts, preventing the growth with respect to the untreated control, were determined by following the broth microdilution M27-A3 guidelines (CLSI, 2008). Dried extracts were dissolved in dimethyl sulfoxide at a concentration 100 times higher than that of the test, as indicated in the protocol (CLSI, 2008). Subsequently, the extracts were diluted in the in the culture medium or stored at -20 °C. The concentration of the extracts ranged from 1.78 µg/mL to 1000 µg/mL. For each extract, three independent experiments in duplicate was performed. The antimutagenic activity of petiole, blade leaf and heart ethanolic extracts was assessed towards the oxidative DNA damage of tBOOH by Ames test (Di Sotto et al., 2018). The bacterial strain Escherichia coli WP2uvrA/R (trpE65ΔuvrA pKM101), obtained by Research Toxicological Centre (Pomezia, Rome, Italy), was used as a sensitive model to the oxidative-induced DNA damage. The strain identity was preliminarily verified by the Strain Check assay (data not shown). The presence of pKM101 plasmid was confirmed in the ampicillin-resistance assay. To perform the experiments, the strain was treated with non-toxic concentrations of celery extracts and tBOOH, assessed at a concentration inducing a submaximal mutagenic response. An exogenous S9 metabolic activator (i.e. the supernatant of the post-mitochondrial liver fraction from phenobarbital/β-naphthoflavone-induced rats) was added to the mixture, in order to mimic a possible CYP450-mediated biotransformation of tested samples. Suitable control plates, treated with the vehicle alone or tBOOH, were included to obtain the lack of mutagenicity and the maximum mutagenicity, respectively. At least two biological replicates, in which each concentration was tested in triplicate, were made.

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The scavenger activity of DPPH and ABTS radicals and the inhibition of linoleic acid peroxidation were carried out according to Di Sotto et al. (2018). At least two repeated experiments and three replicates for each experiment were made. For each test, the vehicle (negative control) and a standard antioxidant agent (positive control), along with additional control of the possible test substance absorbance, were included. Data analyses were made as previously reported (Di Sotto et al., 2018). The ability of celery ethanolic extracts to affect in vitro α-amylase and lipase enzymes was evaluated according to Vitalone et al. (2017). Acarbose (250 μg/mL) and orlistat (25 μg/mL) were used as standard inhibitors for α-amylase and lipase, respectively. At least three repeated experiments and six replicates for each experiment were made. The inhibition was expressed respect to negative control.

3 RESULTS AND DISCUSSION 3.1 Untargeted metabolite profile NMR and MS methodologies were carried out as complementary untargeted methodologies for a comprehensive metabolomic characterization of celery blade leaves and petioles. 3.1.1 NMR analysis of hydroalcoholic and organic extracts 1D and 2D NMR experiments and literature data (Sobolev, Brosio, Gianferri, & Segre, 2005) allowed the assignment of 1H spectra of blade leaf and petiole hydroalcoholic and organic extracts, see Tables 1 and 2. Hydroalcoholic extracts The 1H NMR spectrum of a hydroalcoholic extract of celery blade leaves, Figure S1 and Table 1, showed in the high field spectral region (0.8-3.6 ppm) signals belonging to amino acids, namely alanine, asparagine, aspartate, γ-aminobutyrate, glutamate, leucine, lysine, isoleucine, proline, threonine and valine. Signals of malic, citric, acetic, succinic, lactic, and quinic acids were detected. Signals characteristic of pimelic acid were also detected at 1.30, 1.59, and 2.19 ppm, see Table 1. In the range 3.6-3.9 ppm, the 1H spectrum was dominated by the intense signals characteristic of mannitol, the main polyol present in celery (Rupérez & Toledano, 2003). However, between 3.2 and

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5.5 ppm, signals of other polyols (myo-inositol and scyllo-inositol), carbohydrates (α- and -glucose, sucrose, -D-fructopyranose, -D-fructofuranose, α-D-fructofuranose), choline and ascorbic acid were observed. In the low frequency spectral region, signals of amino acid aromatic groups (phenylalanine, tryptophan, and tyrosine) and formic, fumaric, shikimic and caffeoylquinic acids were detected. In the case of petioles, see Table 1, signals of glutamine were also observed, whereas signals of lysine and caffeoylquinic acid were not detected. Organic extracts The spectral regions of the 1H NMR spectrum of an organic extract of celery blade leaves are reported in Figure 2 along with the assignment of some selected resonances (I1-I15) used for the quantitative analysis, according to the equations reported in the Supplementary material. Between 0.55 and 0.73 ppm, the CH3-18 signals of some sterols were detected, namely stigmast-7-en-3-ol (I1) CH3 group at 0.55 ppm, -sitosterol + campesterol (I2) at 0.71 ppm, and stigmasterol (I3) at 0.73 ppm were quantified (Figure 2a). The resonance centred at 2.03 ppm was ascribed to allylic CH2 of all unsaturated fatty chains (I4), whereas resonances at 2.25 and 2.29 ppm were due to α-CH2 of free (I5) and esterified (I6) fatty acids, respectively. Resonances at 2.74 and 2.77 ppm were ascribed to diallylic CH2 of linoleic (I7) and linolenic acids (I8). Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were identified owing to characteristic 1H

signals of their head groups, namely, the multiplet at 3.11 ppm of CH2N of PE (I9) and the singlet

at 3.19 ppm of N(CH3)3 of PC (I10) (Figure 2b). The resonance at 4.87 ppm (I11) belongs to CH-1” of digalactosyldiacylglycerols (Figure 2c). All signals of senkyunolide A, Fig. 2c, assigned according to the literature (Leon, Del-Angel Avila & Delgado, 2017), were clearly detected and described in Table 2. Trans-neocnidilide, Fig. 2d, was also identified as reported in Table 2. Resonances at 9.33 and 9.67 ppm belong to CH-10 of pheophytin a (I12) and CH-10 of pheophytin b (I13), respectively (Fig. 2e). It was also possible to detect peaks belonging to mono-, di- and triacylglycerols, see Table 2, however, owing to the strong peaks overlapping it was not possible to perform a quantitative analysis. 12

Quantitative analysis In Table 1 and 2 the content of metabolites in blade leaf and petiole extracts is reported. Amino acids. Among the 12 amino acids detected in blade leaves, Glu and Asp were the most abundant, whereas, with respect to other amino acids, Asn and Glu were the most abundant in petioles. Comparing the levels of specific amino acids in blade leaves and in petioles, the amount of all amino acids except Asn and Gln was higher in blade leaves than in petioles. Organic acids. In leaves as well as petioles, malic acid was present in the highest concentration with respect to the other organic acids, followed by citric and succinic acids; moreover, the concentration of these acids was higher in leaves than in petioles. The amount of lactic, acetic, and formic acids was higher in petioles than in leaves. Sugars. Glucose and fructose concentrations were higher in petioles than in blade leaves, whereas the concentration of sucrose was found to be definitely higher in blade leaves than in petioles (Figure S2). Polyols. Both in leaves and petioles mannitol was the polyol present in the highest concentration and its signals dominated the 1H NMR spectrum. The concentration of myo- and scyllo-inositols was higher in leaves than in petioles. Other metabolites. The concentration of choline and ethanolamine was higher in leaves than in petioles. Caffeoylquinic acid was detected only in leaves. Organic extracts The content of -sitosterol + campesterol was higher in petioles than in leaves, whereas the level of stigmast-7-en-3-ol was higher in leaves than in petioles, see Table 2. The content of stigmasterol in leaves and petioles was comparable. Most of the fatty acids were esterified in both leaves and petioles. As regards to fatty acid composition, high content of SFA and linoleic fatty chain was found in both leaves and petioles, however their level was higher in petioles than in leaves. On the contrary, linolenic acid content was higher in leaves than in petioles (Figure S3) as well as the level of MUFA.

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PE and PC were also detected in organic extracts, however PE content was higher in petioles than in leaves, whereas the PC level was well comparable in both. A rather high content of DGDG was detected in leaves, whereas it was much less in petioles. A considerable amount of other lipids was detected in both blade leaves and petioles. Pheophytin a and b were present in leaves, whereas they were not detected at all in petioles. Finally, senkyunolide A and trans-neocnidilide present in both blade leaves and petioles were more abundant in leaves. It is noteworthy that this novel approach based on NMR was applied here for the first time to characterize this local ecoptype not previously studied by means of other techniques. As a consequence, a direct comparison with the NMR data from other ecotypes was not possible. 3.1.2 Mass spectrometry analysis The results of full-scan mass spectra (m/z 50-1200 Da) of hydroalcoholic extracts from celery blade leaves and petioles are listed in Table 3. Examples of ESI FT-ICR mass spectra are displayed in Fig. S4 showing peaks in the positive ion mode, corresponding to either protonated molecules or alkali metal adducts. Fig. S5 reports the mass spectrum of the hydroalcoholic extract from blade leaves in ESI positive mode. Peaks at m/z 203.05225/ 219.02616, 205.06774/ 221.04221, and 365.10565/381.07865, due to sodium/potassium adducts of monosaccharides, polyalcohols (C6H14O6) and disaccharides (C12H22O11), respectively, confirmed the presence of glucose, mannitol and sucrose, as detected by 1H

NMR spectroscopy. Several other compounds were also detected, including valine at m/z

140.06822, asparagine at m/z 155.04285 and arginine at m/z 188.06782, as well as small peptide units, such as cysteinyl-asparagine at m/z 236.07032, reported to be inhibitors of foodstuff enzymatic browning (Aydemir & Akkanli, 2006). In the negative ion mode, ions were observed as deprotonated species [M-H]- or as chloride adducts [M+Cl]-. Fig. S6 shows the ESI MS analysis of blade leaf extract confirming the presence of numerous organic acids, including succinic, malic, quinic and caffeoylquinic acids at m/z 117.01953, 133.01426, 191.05659 and 353.08842, respectively, identified also by 1H NMR spectroscopy. The

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presence of maleic acid at m/z 115.00337 was also detected. Fine tuning of the ESI conditions in the m/z 150-200 mass range allowed detection of deprotonated ascorbic acid at m/z 175.02497. Fatty acids, such as myristic, palmitic and oleic acid, at m/z 227.20136, 255.23345 and 281.24794 respectively, and some of their derivatives, e.g. methylpalmitate at m/z 269.24894, were revealed, along with final compounds of the fatty acids metabolism, e.g. hexenol/hexanal at m/z 99.08181, a flavor-contributing agent (Ruiz, Alonso, Garcia-Martinez, Valero, Blasco, & Ruiz-Bevia, 2005) In agreement with previous reports about the presence of glycosylated compounds in celery seeds (Lin, Lu & Harnly, 2007), luteolin 7-O-glucoside (m/z 449.10732) and chrysoeriol 7-Oapiosylglucoside (m/z 595.16454) species were revealed. In agreement with previous reports (Hostetler, Riedl & Schwartz, 2012), aglycone compounds were found such as apigenin (sodium adduct at m/z 293.06509), together with its diglycoside derivative, apiin (deprotonated species at m/z 563.14122), known to possess anti-inflammatory and antioxidative properties (Mencherini, Cau, Bianco, Della Loggia, Aquino & Autore, 2007). The organic extract from blade leaves showed characteristic peaks in the m/z 850-950 range, including protonated pheophytin a (C55H74N4O5) at m/z 871.57502 (sodium and potassium adducts at m/z 893.55331 and 909.53110, respectively) (Fig. S7). The sodium complex of chlorophyll a [C55H72N4O5Mg+Na]+ was found at m/z 915.52681 in the blade leaf extract. Several other metabolites were detected in both leaf and petiole organic extracts, including sodiated adducts of: i) methylerythritol at m/z 159.06246; ii) senkyunolide A (sedanenolide) and neocnidilide (sedanolide) at m/z 215.10380 and 217.12035; iii) limonene-1,2-diol at m/z 193.11936. 3.1.3 GC-MS analysis GC-MS analysis of the organic extract from celery blade leaves pointed to the presence of different classes of phytochemicals, see Table 4, including phthalides (70.6%), phthalates (6.5%), and fatty acids esters (FAEs: 6.9%). The most abundant component was senkyunolide A (38.0%) also detected in the 1H NMR spectrum of celery organic extract, two unidentified species related to senkyunolide (19.3% in total), neocnidilide (9.1%), also detected in the 1H NMR spectrum, trans- and cis-ligustilide

15

(3.6% and 0.6%, respectively). An examination of Table 4 also revealed the presence of 1-methoxy3-(2-hydroxyethyl)nonane as an abundant component (11.8%). It is noteworthy that, differently from the composition of the analyzed organic extracts, terpenes have been reported to be the most abundant phytochemicals in the celery essential oils. (Baananou, Bouftira, Mahmoud, Boukef, Marongiu & Boughattas, 2013; Shankaracharya, Rao, Nagalakshmi & Naik, 2000). The fatty acid ester fraction was about 7%. On the other side, the composition of the organic extract from celery petioles partially overlapped with the one obtained for the blade leaf sample. Indeed, Table 4 shows that more than 65% of the detected compounds were fatty acid esters: methyl palmitate (47.5%), methyl stearate (11.1%), methyl linoleate (5.0%), and methyl linolenate (1.6%). Finally, phthalide-like compounds were not detectable. In contrast to the broad variety of volatile compounds contained in the organic phase, the hydroalcoholic one did not exhibit an appreciable amount of detectable species in the GCMS analysis. 3.2 Targeted analyses 3.2.1 Total phenolics, tannins and flavonoids Higher levels of total phenolics were found in the ethanolic extracts of blade leaves and heart with respect to petioles (Table S1). Similarly, blade leaves contained the highest levels of tannins followed by heart samples, and then petioles. Total flavonoids showed a similar trend of total phenolics. Particularly, the heart parts and blade leaves were the samples with high flavonoid levels, whereas a low content was estimated in petioles. Taking into account the solid:liquid ratio of extracts from heart petioles, heart leaves, blade leaves and petioles (i.e. 16:1, 20:1, 50:1 and 210:1) and despite the limitations of the detection method, a flavonoid amount in the fresh material of about 625, 950, 100 and 0.48 mg/Kg, respectively, can be estimated. According to the Peterson and Dwyer classification (Di Sotto et al., 2018), celery leaves resulted to be a possible nutraceutical source of flavonoids. Our results agree with literature, which highlighted leaves to be the most abundant part of celery in polyphenols and flavonoids, despite differences among crops and extraction method (Lin et al., 2007; Yao, Sang, Zhou, & Ren, 2009; Aydemir & Becerik, 2011; Hostetler et al., 2012; Liu, Zhuang, Song,

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Lu & Xu, 2017; Han, Gao, Xia, Zhang, Li & Gao, 2019). Accordingly, flavonoids and tannins were higher in celery herb than root (Iswantini, Ramdhani & Darusman, 2012). Also, Justesen, Knuthsen & Leth (1998) found a 50 fold-higher amount of apigenin and luteolin in celery leaves respect to petioles. However, the true recovering of phenolics and flavonoids of the celery leaves depends on several factors, such as meteorological conditions, cooking methods, drying, harvesting practices and sanitizing procedures, as also reported for other species (Pérez-Gregorio, González-Barreiro, RialOtero & Simal-Gándara, 2011; Pérez-Gregorio, Regueiro, Simal-Gándara, Rodrigues & Almeida, 2014). Moreover, simulated gastric digestion has been found to increase their levels, despite a lowering during intestine digestion (pH > 5) (Han et al., 2019). 3.2.2 HPLC-DAD and HPTLC analysis of phenolic compounds In order to study in more details the phenolic component, HPLC-PDA analyses of both hydroalcoholic celery and ethanolic extracts were carried out (Sobolev et al., 2018). A different pattern among celery blade leaves and petioles confirmed the data observed in the corresponding untargeted NMR and MS analyses (Table S2). Collectively, hydroalcoholic extract from blade leaves was characterized by a predominant presence of caffeoylquinic acid with respect to petioles; rutin was also detected. Conversely, the extract from petioles showed appreciable amounts of caffeoylquinic acid and small quantities of catechin, rutin, benzoic acid, naringenin and carvacrol. The organic extract of blade leaves revealed the presence of caffeoylquinic acid, naringin, benzoic acid and carvacrol, whereas petioles were enriched in carvacrol along with naringenin and benzoic acid. In the ethanolic extracts, it was possible to note the abundant presence of secondary metabolites such as 3-hydroxybenzoic acid, rutin and benzoic acid in both heart and blade leaves. Syringic acid and p-coumaric acid were the only ones detected in heart leaves. Petioles and heart petioles were only characterized by small amounts of 3-hydroxybenzoic acid and benzoic acid. This match was performed by means of a fully validated and selective HPLC-PDA procedure (Sobolev et al., 2018) using the retention times and UV/Vis spectra of each chemical standard. This procedure could be

17

performed because the chemical standards of 3-hydroxybenzoic acid and benzoic acid (just all the other chemicals) show the same retention times, and the same spectra. Additionally, during the validation procedure, no interferences (related to the use of PDA detector) were observed, and the titled compounds were fully resolved respect to the other compounds and the other matrix components, selectively. Most of these compounds were detected for the first time in celery. Data from HPLC-DAD analysis of the ethanolic extracts were also confirmed by HPTLC analysis: which highlighted the presence of specific phenolics, distributed predominantly in the lower part of the chromatogram (Figure S8). The extracts of heart and blade leaves were the most abundant samples in phenolics, among which, catechin, rutin and traces of quercetin and caffeoylquinic acid were identified, while gallic acid, kaempferol and caffeic acid were not revealed. On the basis of the literature, glycosylated flavonoids, such as those of apigenin (e.g. apiin), luteolin, kaempferol and chrysoeriol, as well as phenolic acids (e.g. caffeoylquinic, caffeic, ferulic and pcoumaric acids) are the most common phenolics identified in leaves of different celery varieties (Mencherini et al., 2007; Yao et al., 2010; Hostetler et al., 2012; Liu et al., 2017; Han et al., 2019).yao Although it is known that extraction techniques can greatly influence both extraction yield and chemical profile, the presence of naringin, benzoic acid, carvacrol, rutin, catechin, naringenin, and 3hydroxybenzoic, as detected by HPLC-DAD analysis in our samples, is not reported for other celery varieties (Momin & Nair, 2002; Yao et al., 2010; Baananou et al., 2013) 3.2.4 Chlorophyll a, chlorophyll b and total carotenoids analysis The amounts of chlorophyll a (Chl a), chlorophyll b (Chl b) and total carotenoids in the organic extracts from petioles, blade leaves and heart are reported in Table S3. It is possible to notice that the highest Chl a content was found in blade leaf extracts (10.333 ± 0.032 mg/g) and it was about 10 times higher than that measured in other parts of celery. Chl b also, in blade leaf extracts was detected in the highest concentration (4.905 ± 0.018 mg/g). Its level was about 2.3-3.5 times greater than that found in the hearth leaves (1.407 ± 0.005 mg/g) and petioles (2.133 ± 0.009 mg/g), respectively. The value of the Chl a/b ratio was 2.11 for the blade leaf extracts, that was on average 3.5 times higher

18

than that found in the extracts from other celery parts under study, to confirm the high content of Chl a characteristic of sun-exposed plants. In fact, the value of Chl a/b ratio tends to decrease in shade plants (Sobolev et al., 2018). As regards the total carotenoids content displayed in Table S3, the highest level was detected in the extract from blade leaves (0.602 ± 0.002 mg/g) and the minimum content was found in heart leaves (0.128 ± 0.001). The ratio of Chl a and b to total carotenoids is an indicator of the plant greenness: the maximum value of this ratio was determined in the blade leaf extracts (25.32), while the minimum was detected in the heart petiole sample (7.12). Both heart leaves and petioles showed a high Chl a and b to total carotenoids ratio (17.85 and 15.62 respectively) even if lower than that found in blade leaves, resulting in yellowish colour due to a higher percentage of carotenoids in these parts of celery. One of the distinctive characteristic of “sedano bianco di Sperlonga” is to have yellow-green leaves and lighter petiole colour. Different studies examined the beneficial effect of natural chlorophylls in foods (Katalin & Beata, 2017). Some authors reported a relationship between chlorophylls content in various vegetable extracts and its antimutagenic activity (Katalin & Beata, 2017). In vitro and animal studies showed that chlorophylls and chlorophylls derivatives (chlorophyll a, pheophytin a, pyropheophytin, and pheophorbide), common to canned green vegetables, are potential chemopreventive agents (Katalin & Beata, 2017). 3.4 Freshness and Quality 3.4.1 Biogenic amines The results of BAs determination in celery extracts are shown in Figure S9 and Table S4. The highest BAs concentration was found in the hearts (12.30 mg/100 g), followed by blade leaves (7.04 mg/100 g) and petioles (2.71 mg/100 g). These differences were mainly attributable to the higher concentration of spermidine and spermine in the different celery portions. Furthermore, spermidine and spermine were the only two BAs that had a statistically significative difference in the three investigated portions. It is noteworthy that spermine and spermidine are reported in literature as

19

regulators of plant growth and elongation in vegetables (Takahashi & Kakehi, 2010). Heart and blade leaves are in constant growth, suggesting, as reported in literature, the role of spermidine in regulating plant growth and of spermine in stem elongation (Takahashi & Kakehi, 2010). Agmatine was the only biogenic amine present in the highest concentration in petioles, whereas methylamine and putrescine were detected in major concentrations in the blade leaves, probably because they are more susceptible to rotting than the other part of the vegetable. No samples show the presence of serotonin, β-phenylethylamine, histamine, and tyramine; ethylamine was detected in only one sample, with no significant differences among the three parts considered. Previously, the BAs presence in vegetables has been reported (Moret, Smela, Populin & Conte, 2005; Sobolev et al., 2018), no one dealing on celery. In comparison with other vegetables, “sedano bianco di Sperlonga” ecotype shows a low content of putrescine, and the absence of tyramine and histamine, BAs related to health risks. 3.4.2 Mycotoxin determination An important aspect to be monitored for the evaluation of the safety and the quality of foods is the presence of mycotoxins under the limits imposed by EU legislation. The analysis of mycotoxins was performed by a very sensitive and unambiguous technical approach. The simultaneous content of different mycotoxins was investigated on petioles extracts and was generally low and mainly focused on Fusarium toxins, as shown in Table S5. Notably, toxins belonging to thrichothecenes type A and B (e.g DON and T-2) and fumonisins were found: their level was well below the legal limits or recommendations imposed by EU (EU No. 1881/2006 and EU No. 165/2013). 3.5 Biological activities Petiole and leaf extracts (i.e. hydroalcoholic and organic, aqueous, ethanolic, in acetone) were tested against Candida albicans, the main yeast, responsible for mucocutaneous and systemic infections. The results (data not reported) showed that all the extracts, at concentration up to 1000 µg/mL, have no activity against Candida albicans ATCC10231.

20

Previously, Khudhur et al demonstrated the anti-Candida activity of the leave extracts of Apium graveolens collected in HanaraeSarw region in Erbil governorate of Iraq (Khudhur, Bakir, Rahman & Ismael 2019). Moreover, some metabolites found in the tested celery extracts have a demonstrated antifungal activity against C. albicans. Lee, Woo, & Lee (2018) have demonstrated that Apigenin induces cell shrinkage in C. albicans by membrane perturbation. The lack of activity could be attributed to the high concentration of sugars, detected in the above analyses, which could have been interfered with the antifungal activity. In regard to the antimutagenicity, celery leaves and petioles produced a moderate inhibition of the tBOOH-mutagenicity in the absence of S9 (about 33% and 39% respectively) (Table S6 and Figure S10). Conversely, in the presence of S9, a strong antimutagenicity was obtained with petiole extracts, while blade leaves exhibited a moderate activity (about 45% and 32% inhibition of tBOOHmutagenicity at the concentration of 100 μg/mL; Table S6 and Figure S10). The positive control rutin produced a 80% inhibition of tBOOH-mutagenicity at the highest concentration of 16 μg/mL (Table S6). According to literature (Geetha, Garg, Chopra, & Pal, 2004), the exctracts could act by preventing the oxidative DNA-damage of tBOOH, through antioxidant or other desmutagenic mechanisms (i.e. inhibition of mutagen uptake, interference with metabolic activation, chemical neutralization). In support, the essential oil of celery seeds was reported to inhibit the carbon tetrachloride (CCl4) genotoxicity, likely due to antioxidant phytoconstituents, such as D-limonene or phenolic acids (Sobti, Mittal, Sachdeva & Gill, 1991). On the basis of the phytochemical profile of our samples and according to the literature (Di Sotto et al., 2018; Katalin & Beata, 2017; Okai & Higashi-Okai, 1997; Liu et al., 2017), the contribution of different phytochemicals (e.g. catechin, rutin, caffeoylquinic acid, chlorophylls and phthalides) could be hypothesized. Further studies are required to better identify the bioactive phytochemicals and the in vivo activity. Under our experimental conditions, blade and heart leaves also exerted the highest DPPH radical scavenging activity (Figure S11), with IC50 values of 0.78 mg/mL (C.L., confidential limits, 0.48-

21

1.27) and 1.13 mg/mL (C.L. 0.87-1.47), respectively. The standard antioxidant agent trolox possessed the highest radical scavenger potency, being the IC50 value of 4.89 µg/mL (C.L. 3.67-6.46). Similarly, the extracts from the blade and heart leaves were the most effective samples towards ABTS radical (Figure S12), with IC50 values of 0.28 mg/mL (C.L. 0.21-0.39) and 0.48 mg/mL (C.L. 0.420.54), respectively. Despite the highest potency of blade leaf extract, as expected, trolox was the most potent antioxidant agent (IC50 of 1.89 - C.L. 1.68-2.18 µg/mL). Conversely, a weak inhibition of linoleic acid peroxidation (lower than 40% inhibition) was found (Fig. S13). Antioxidant properties of different celery varieties, widely investigated both in vitro and in vivo, have been mainly ascribed to flavone glycosides and other phenolics (e.g. apigenin, luteolin, caffeic acid), due to their antioxidant and reducing power (Yao et al 2010; Kooti & Daraei, 2017). However, these properties can differ due to the extraction method, which could affect the phenolic amounts: for instance, a greater antioxidant activity has been reported for a methanolic extract of celery respect to ethanolic and hexane ones (Kooti & Daraei, 2017). Along with phenolics, other components (e.g. phenolic terpenes) have been considered involved in the activity of the volatile oils from celery leaves and seeds (Kooti & Daraei, 2017). In line with this evidence, although our samples were effective at high concentrations, their activity suggest the presence of bioactive compounds, likely in lower concentrations. A better recovering of bioactive phytochemicals of our celery ecotype could be obtained by improvement of extraction methodologies. Celery by-products have been reported to possess hypoglycaemic and hypocholesterolaemic properties (Al-Asmari et al., 2017), thus suggesting that celery supplementation could be useful in metabolism regulation. In line with this evidence, we assessed the ability of our samples to inhibit the pancreatic s α-amylase and lipase enzymes, as a possible strategy to reduce the absorption of carbohydrates and fats. However, under our experimental conditions, tested samples produced only a weak or null inhibition of both α-amylase (maximum inhibition of about 23.3% by the petiole extract at the concentration of 1000 μg/ml) and lipase (maximum inhibition of about 21.6% by the heart petiole extract at the concentration of 500 μg/ml), in spite of a strong activity of the positive

22

controls (Table S7). Based on present results and taking into account that, to the best of our knowledge, α-amylase and lipase inhibition by celery has been not previously reported, we hypothesize that the claimed hypoglycaemic and hypocholesterolaemic properties of celery should be due to other mechanisms. Indeed, celery is a source of fibers and water (Ovodova et al., 2009), which can reduce glucose and fat absorption and favour intestinal microbiota homeostasis. Moreover, a recent study highlighted that a methanolic extracts of celery leaves inhibited to inhibit the formation of advanced glycation end products (AGE), with hyperglycemia complications (Perez-Gutierrez, Muñiz-Ramirez, Campoy, Flores and Flores, 2018). Furthermore, another extract of leaves reduced the pre- and post-prandial blood glucose levels in elderly pre-diabetics, by acting through multiple mechanisms, among which antioxidant activity and glucose uptake (Yusni et al., 2018). Further studies should clarify the true usefulness of celery supplementation in the management of metabolic disorders.

4. Conclusions The proposed multimethodological approach has allowed the holistic chemical characterization of “sedano bianco di Sperlonga” local ecotype. Differences in the metabolic profile of blade leaves and petioles have been found that can be reflected in different possible use of celery parts as nutraceutical sources. This novel approach is applied here for the first time to characterize a celery ecotype, allowing to record, for instance, the presence of several phenolic compounds that previously went unnoticed in any part of this plant (leaves or other). This could mean that these compounds are markers of the investigated ecotype or that different experimental conditions were used greatly influencing not only the yield of extraction but also the extract chemical profile. As a consequence a direct comparison with earlier reports is thus not easily drawn. Therefore, the proposed analytical protocol could be recommended to the study of other celery ecotype with the aim to underline possible differences in metabolite profile.

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Regarding the biological activities, the ethanolic extracts of blade leaves and petioles exhibited scavenging properties and antimutagenic activity towards the oxidative DNA-damage of tBOOH, thus suggesting the contribution of antioxidant compounds to the protective effects. Further studies are required to correlate the biological effects to specific compounds and to find out the best extraction method for exploiting these properties. Altogether the here applied integrated phytochemical and biological methodology allowed us not only to characterize special traits and nutritional properties of “sedano bianco di Sperlonga” ecotype, but also to highlight a possible interest for its by-products as nutraceutical sources for further development.

Acknowledgments This work has been realized with funds received from the following agencies: Italian Ministry of Education, Universities and Research - Dipartimenti di Eccellenza - L. 232/2016; “e-ALIERB” Project (Regione Lazio LR13/2008 – Dipartimento di Chimica e Tecnologie del Farmaco). Authors wish to thank the Società Cooperativa Agricola “San Leone” (Fondi-Sperlonga, Lazio region, Central Italy) for supplying the fresh raw material. Fellowship of Dr. Cinzia Ingallina and Dr. Silvia Di Giacomo was financed by “e-ALIERB” Project, while Dr. Antonella Di Sotto was supported by “Enrico and Enrica Sovena” Foundation. The Authors thank Dott. Martina Vecchiato for her technical assistance.

Conflict of interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at References

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31

Caption to the figures: Figure 1. “Sedano bianco di Sperlonga” PGI local ecotype: petioles, blade leaves and heart are indicated by an arrow. Figure 2. Regions of the 1H NMR spectrum of organic extract in CDCl3/CD3OD mixture from leaves of “sedano bianco di Sperlonga” PGI local ecotype, signals used for the quantitative analysis have been labeled: Ii, CH3, Stigmast-7-en-3-ol; I2, CH3, β-sitosterol/campesterol; I3, CH3, stigmasterol; I4, allylic CH2, all unsaturated fatty acids; I5, α-CH2, free fatty acids; I6, α-CH2, esterified fatty acids; I7, diallylic CH2, linoleic acid; I8, diallylic CH2, linolenic acid; I9, CH2N, phosphatidylethanolamine; I10, N(CH3)3, phosphatidylcholine; I11, CH, digalactosyldiacylglycerols; I12, CH, pheophytin a; I13, CH, pheophytin b; I14, CH, senkyunolide A; I15, CH, trans-neocnidilide. Sketches of the chemical structure of senkyunolide A and trans-noecnilidide are reported in the figure.

32

Table 1. Metabolites identified and quantified in the 600.13 MHz 1H spectra (27 °C) of the hydroalcoholic extracts from “Bianco di Sperlonga” celery.

33

1H

13C

(ppm)

Multiplicity: J[Hz]

α-CH β-CH β'-CH α,γ-CH α',γ'-CH

4.30* 2.66 2.39* 2.54* 2.69

dd [9.9, 3.2] dd [3.2, 15.4] dd [9.9, 15.4] d [15.0] d [15.0]

Formic acid (FA)

HCOOH

8.46*

s

Shikimic acid (SHA)

CH2-7 CH-6 CH-5 CH-4 CH-3

2.20 2.76 3.99 3.72 4.41 6.45*

Acetic acid (ACA)

CH3

1.93*

s

Fumaric acid (FUA)

CH=CH

6.52*

s

Ascorbic acid

CH2-6 CH-5 CH-4

3.75 4.03 4.53

d [1.8]

79.4

2.41*

s

35.2

Compound Malic acid (MA) Citric acid (CA)

Assignment

Succinic acid CH2-1,1’ Quinic acid (QA)

Lactic acid Pimelic acid Asparagine (ASN) Aspartate (ASP)

Isoleucine (ILE)

CH-2 CH-3 CH-4 CH2-5,5’ α-CH CH3 CH2-1 CH2-2 CH2-3 α-CH β-CH β'-CH α-CH β-CH β'-CH α-CH β-CH γ-CH γ'-CH γ-CH3

1.88* 2.08 4.31 3.56 4.16 1.99 2.06 4.12 1.33* 2.19 1.54 1.30 4.02 2.91* 2.95 3.92 2.72 2.79* 1.97 1.27 1.47 1.01*

(ppm) 71.4 43.6 43.6 46.4 46.4

Quantification (mM) 44.02 BL 31.55 P 5.25 BL 1.05 P 0.12 BL 0.18 P 0.07 BL 0.097 P

24.4

0.08 BL 0.17 P 0.25 BL 0.11 P

2.42 BL 0.79 P

41.8 0.44 BL 0.42 P 71.4 38.6 d(7)

dd [16.9;7.2] dd [16.9;4.4]

20.5 38.7 26.8 29.6 52.3 35.6 35.6

dd [3.9;17.4]

37.6 37.6

0.07 BL 0.15 P

0.67 BL 1.87 P 1.74 BL 058 P

0.32 BL 0.067 P

34

Alanine (ALA) Threonine (THR)

Triptophane (TRP)

Tyrosine

Phenylalanine (PHE)

Proline (PRO)

GABA

Glutamate

Glutamine

Lysine

Leucine (LEU)

Valine (VAL)

β-D-Fructopyranose (βFRUpy)

β-D-Fructofuranose (βFRUfu)

α-D-Fructofuranose (αFRUfu)

δ-CH3 α-CH β-CH3 α-CH β-CH γ-CH3 CH-4 CH-7 CH-6 CH-5 α-CH β-CH2 CH-2,6 CH-3,5 CH-2,6, ring CH-3,5, ring CH-4, ring α-CH β-CH ’-CH -CH2 -CH ’-CH α-CH2 β-CH2 γ-CH2 α-CH β-CH β’-CH γ-CH2 α-CH β-CH2 γ-CH α-CH β-CH2 γ-CH2 -CH2 ε-CH2 α-CH β-CH2 δ-CH3 δ'-CH3 α-CH β-CH γ-CH3 γ'-CH3 CH-1,1' CH-3 CH-4 CH-5 CH2-6,6' CH-1,1' C-2 CH-3 CH-4 CH-5 CH2-6,6' CH-3 CH-5

0.94 3.79 1.49* 3.62 4.27 1.34* 7.72 7.54 7.27 7.19 3.95 3.06 (dd) 3.18 (dd) 7.20* 6.91 7.35 7.43 7.39* 4.15 2.36 2.07 2.02 3.42 3.35 2.31 1.92 3.03* 3.77 2.08 2.12 2.34* 3.79 2.15 2.46* 3.75 1.92 1.50 1.74 3.04* 3.75 1.74 0.97* 0.92 3.62 2.28 1.00 1.03* 3.57; 3.72 3.81 3.90 4.00 3.72;4.03 3.60;3.57 4.12* 4.12* 3.83 3.81;3.68 4.13* 4.07

d [7.3]

51.5 17.2

d(6.4)

67.0 21.2

0.41 BL 0.079 P 0.44 BL 0.11 P

d(8.6) d(8.6)

d(8) d(8)

131.8 116.2 130.5 130.3 128.8 62.3

0.34 BL 0.086 P

0.27 BL 0.064 P

0.10 BL 0.057 P

m m

t [7.7] d [6.0] d [6.0] d [7.1] d [7.1]

55.5 27.9 27.9 34.4 55.2 27.3 31.9 54.5 31.1 27.5 40.1 54.4 40.9 23.1 21.6 61.3 30.1 17.7 19.0 64.9 68.5 70.6 70.1 64.4 63.8 102.6 76.4 75.4 81.6 63.3 82.8 82.3

3.33 BL 1.45 P n.d.a BL 0.88 P

0.29 BL n.d.a P

1.00 BL 0.17 P

0.60 BL 0.20 P

13.71 BL 50.00 P

35

CH-1 (Glc) CH-2 CH-3 CH-4 CH-5 CH2-6 CH2-1' (Fru) C-2' CH-3' CH-4' CH-5' CH2-6' CH-1 CH-2 CH-3 CH-4 CH-5 CH2-6,6' CH-1 CH-2 CH-3 CH-4 CH-5 CH2-6,6' CH-1 CH-2,5 CH-3,6 CH-4

4.23 4.06 3.90 3.81 5.25* 3.56 3.72 3.43 3.84 3.84;3.78 4.66* 3.26 3.49 3.43 3.48 3.91;3.74 4.08 3.54 3.63 3.29*

Scyllo-inositol

CH 1-6

3.36*

Mannitol

CH-1,6 CH-1’,6’ CH-2,5 CH-3,4

Sucrose (SUCR)

α-Glucose

β-Glucose

Myo-inositol (MI)

Caffeoylquinic acid

Choline (CHO) Ethanolamine

CH1’ CH2’ CH3’ CH4’ CH-5’ CH-8 CH-7 CH-2 CH-5 CH-6 N-CH3 N-CH2 CH2-OH N-CH2 OH-CH2

5.42* 3.58 3.78 3.49 3.85 3.83 3.70

d [3.8]

d [3.8] dd [9.8;3.8]

d [7.9] dd [9.3;8.0] t [9.1]

93.2 72.2 73.6 70.3 73.5 61.2 62.3 104.8 77.5 75.1 82.4 63.5 93.2 72.4 73.8 70.7 72.5 61.7 96.7 75.3 76.9 70.7 77.0 61.8 73.5

13.34 BL 3.91 P

3.67 BL 15.80 P

7.00 BL 29.23 P

2.75 BL 0.41 P t [9.38]

3.87* dd(11.8, 2.9) 3.69 dd(11.8, 6.2) 3.81 d(8.8) 3.78 ddd(8.6, 6.2, 29) Miscellanous metabolites 2.02, 2.19 2.04, 2.13 5.32 3.88 4.24 6.35* d(16) 7.61 d(16) 7.16 d(2) 6.93 d(8.2) 7.08 dd(8.2, 2) 3.21* s 3.52 4.07 3.16* 3.84

75.4 74.7

0.93 BL 0.11 P

64.3 64.3 70.2 71.9

53.26 BL 71.37 P

39.6 38.4 72.2 71.8 115.7 147.2 116.2 117.4 123,8 55.0 685 56.6 42.4 58.7

1.53 BL n.d.a P

1.42 BL 0.36 P 0.53 BL 0.19 P

BL= blade leaves P= Petioles a not detected

Table 2. Metabolites identified and quantified in the 600.13 MHz 1H spectra (27 °C) of organic extracts from “Bianco di Sperlonga” celery. 36

Compound

Pheophytin a (Phe-a)

Pheophytin b (Phe-b)

Sitosterol

Campesterol

Assignment

1H

(ppm) Multiplicity: J(Hz)

CH-5 CH-10 CH-20 CH-31 CH-32 CH-32’ CH3-21 CH3-71 CH2-81 CH3-82 CH3-121 CH-17 CH-18 CH-181 CH3-134 CH2-P1 CH-P2 CH-P31 CH-71 CH-5 CH-10

9.31 9.47 8.53 7.92 6.13 6.22 3.33 3.14 3.61 1.61 3.61 4.12 4.41 1.74 3.82 4.26; 4.37 4.95 1.45 11.09 9.89 9.60

CH-31

7.93

CH2-1 CH2-2 CH-3 CH2-4 CH-6 CH-8 CH-9 CH2-11 CH2-12 CH-14 CH2-16 CH-17 CH3-18 CH3-19 CH-20 CH3-21 CH2-22 CH2-23 CH-24 CH3-26 CH3-27 CH2-28 CH3-29 CH3-18

1.09, 1.87 1.82 3.46 2.26 5.35 1.49 0.96 1.53 1.19, 2.00 0.96 1.89 1.14 0.71 1.03 1.40 0.95 1.33, 104 1.2 0.96 0.86 0.86 1.32 0.86 0.71

s s s dd [17.8; 11.5] dd [11.5; 1.2] dd[17.8; 1.2] s s t [7.7] s

d [7.3] s Td [6.5, 0.6] s s s s

13C

(ppm)

97.9 105.0 93.8 129.3 123.3 123.3 12.2 11.3 19.7 17.6 12.2 51.5 50.3 23.3 53.1 62.0 117.9 16.4

Quantification (molar %) 6.50 BL n.d.a P

1.33 BL n.d.a P 37.4 31.2 71.7 42.0 121.9 32.3 50.5 21.2 39.8 57.1 28.3 56.4 12.0 19.4 36.4 18.9 34.5 26.6 46.1 20.6 19.9 22.8 12.0 12.0

1.22 BL 4.30 P

37

Stigmasterol

CH3-18

0.73

11.9

Stigmast-7-en-3-ol

CH3-18

0.55

12.0

CH sn2 CH2 sn1 CH2 sn3 CH-1' CH-2' CH-3' CH-4' CH-1'' CH-2'' CH-3'' CH-4'' CH sn2 CH2 sn1 CH2 sn3 CH2OP CH2N N(CH3)3 CH sn2 CH2 sn1 CH2 sn3 CH2OP CH2N COO-1 CH2-2 CH2-3 CH2-4,7 CH2-8 CH=CH 9-10 CH2-11 CH2-12,15 CH2-16 CH2-17 CH2-18 CH2-1 CH2-2 CH2-3 CH2-4,7 CH2-8 CH-9 CH-10 CH2-11 CH-12 CH-13 CH2-14 CH2-15 CH2-16 CH2-17

5.20 3.75; 3.98 4.25; 4.41 4.26 3.56 3.53 3.91 4.93 3.83 3.76 3.98 5.25 4.42; 4.20 4.02 4.29 3.65 3.25 5.25 4.42; 4.19 4.01 4.20 3.16

70.5 67.8 63.0 104.1 71.3 73.5 68.8 99.5 69.1 70.3 69.9 70.5 62.9 63.9 59.3 66.5 54.2 70.9 63.2 63.6 63.1 40.4 174.9 34.4 25.3 29.4-30.2 27.4 130.1 27.4 29.4-30.2 32.1 22.8 14.3 174.2 34.3 25.0 29.3; 30.2 27.3 130.2 128.4 25.8 128.4 130.2 27.3 29.3 31.8 22.9

Digalactosyldiacylglycerols (DGDGs)

Phosphatidylcholine (PC)

Phosphatidylethanolamine (PE)

Oleic fatty chain

Linoleic fatty chain

2.35 1.63 1.34 2.08 5.35 2.08 1.34 1.29 1.32 0.88 2.30 1.59 1.29 2.03 5.34 5.34 2.74 5.34 5.34 2.03 1.29 1.29 1.29

d [3.8]

s

m m

m m m t(7)

t m m q

1.47 BL 0.20 P 1.12 BL 0.18 P 18.39 BL 2.32 P

13.68 BL 9.87 P

2.57 BL 6.91 P

19.40 BL 40.49 P

38

Linolenic fatty chain

Free fatty chains

Saturated fatty chains (SFA)

Monoacylglycerol (MAG) Diacylgliycerol (DAG) Triacylglycerol (TAG)

Senkyunolide A

Trans-neocnidilide

Mono-unsaturated Fatty Acids (MUFAs) b Esterified Fatty Acids b Other lipids b

CH2-18 CH2-1 CH2-2 CH2-3 CH2-4-7 CH2-8 CH-9 CH-10 CH2-11, 14 CH2-13, CH-12 CH-15 CH-16 CH2-17 CH2-18 CH2-2 CH2-3 CH2-2 CH2-3 CH2 (n-3) CH2 (n-2) CH2 (n-1) CH3 CH2-sn1 CH2-sn1 CH-sn2 CH2-sn3 CH-sn2 CH-3 CH2-4 CH2-5 CH-6 CH-7 CH2-10 CH2-11 CH2-12 CH2-13 CH-3 CH3a CH2-4 CH2-5 CH2-6 CH-7 CH2-8 CH2-9 CH2-10 CH3-11

0.93 2.30 1.61 1.36 2.09 5.35 5.35 2.83 5.35 5.33 5.41 2.10 1.0 2.29 1.61 2.33 1.61 1.29 1.27 1.27 0.89 3.56, 3.64 4.20 4.36 5.12 3.71 5.28 4.96 2.43 2.50 5.92 6.13 1.54 1.88 1.36 0.88 3.99 1.92, 2.05 1.16, 1.52 2.19, 2.33 6.75 1.75 1.48 1.37 0.91

t(7.4)

t(7.6) t

m m m t [7.20]

dt [9.7; 2.1]

q [3.5]

14.1 174.2 34.5 25.2 29.5 27.3 130.2 128.1 25.7 128.4 127.2 132.0 20.6 14.2 34.7 25.2 34.7 25.4 29.2; 29.7 32.1 23.2 14.1 63.3 62.6 72.2 60.7 70.5 83.3 20.8 22.2 128.9 116.4 27.6 31.9 22.4 13.9 86.3 43.1

136.5 34.2

42.89 BL 7.94 P

2.08 BL 7.06 P

30.83 BL 48.74 P

7.75 BL 2.69 P

2.47 BL 0.21 P

14.0 6.88 BL 2.82 P 97.92 BL 92.94 P 63.29 BL 39

73.83 P BL= blade leaves P= Petioles a not detected b quantified as described in Materials and Methods

Table 3. Major metabolites in hydroalcoholic and organic extracts of blade leaves of “Bianco di Sperlonga” celery revealed by ESI FT-ICR MS.a ESI(+) FT-ICR Plausible Assignment Carbohydrates Hexose* Polyalcohol* Disaccharide* Trisaccharide* Amino acids and derivatives Glicine Choline* Isoleucine/Leucine* Valine* Asparagine* Phenylalanine* Lisine* Arginine* Cysteinil-Asparagine* Cystine Fatty Acids and derivatives Octenoic acid* Decenedioic acid* Palmitic Acida* Oleic Acid Octadienoic acid, methyl ester Oleic acid methyl ester Octadecanoic acid, methyl ester Arachidonic acid* Other compounds Pyruvic acid Acetoacetic acid

Formula(M)

Adduct

Theor. m/zb

Exp. m/zc

Δppmd

C6H12O6 C6H12O6 C6H14O6 C6H14O6 C12H22O11 C12H22O11 C18H32O16

[M+Na]+ [M+K]+ [M+Na]+ [M+K]+ [M+Na]+ [M+K]+ [M+Na]+

203.05261 219.02655 205.06826 221.04220 365.10543 381.07937 527.15826

203.05225 219.02616 205.06774 221.04221 365.10565 381.07865 527.15868

1.7 1.8 2.5 -0.04 -0.6 1.8 -0.8

C2H5NO2 C5H14NO C6H13NO2 C6H13NO2 C5H11NO2 C4H8N2O3 C9H11NO2 C9H11NO2 C6H14N2O2 C6H14N4O2 C7H13N3O4S C6H12N2O4S2

[M+H]+ [M]+ [M+H]+ [M+Na]+ [M+Na]+ [M+Na]+ [M+H]+ [M+Na]+ [M+Na]+ [M+Na]+ [M+H]+ [M+Na]+

76.03931 104.10699 132.10191 154.08385 140.06820 155.04271 166.08625 188.06820 169.09475 197.10090 236.06995 263.01307

76.03936 104.10684 132.10198 154.08379 140.06822 155.04285 166.08591 188.06782 169.09427 197.10032 236.07032 263.01340

-0.6 1.6 -0.5 0.4 -1.4 -0.9 2.0 2.0 2.8 2.9 -1.6 -1.2

C8H14O2 C10H16O4 C16H32O2 C18H34O2 C19H34O2 C19H36O2 C19H38O2 C19H38O2 C20H32O2

[M+Na]+ [M+Na]+ [M+Na]+ [M+Na]+ [M+Na]+ [M+Na]+ [M+Na]+ [M+K]+ [M+Na]+

165.08860 223.09408 279.22845 305.24510 317.24510 319.26075 321.27640 337.25034 327.22945

165.08884 223.09409 279.22798 305.24595 317.24574 319.25991 321.27702 337.24942 327.22950

-1.4 -0.4 1.6 -2.8 -2.0 2.6 -1.9 2.7 -1.5

C3H4O3 C4H6O3

[M+Na]+ [M+Na]+

111.00526 125.02091

111.00548 125.02124

-2.0 -2.6 40

Valeric acid* Methylerythritol* Nicotine Glucosamine Dimethyl-glutaric acid* Limonene-1,2-diol Senkyunolide* Neocnidilide* Methylcitric acid L-Uridine* Aspidinol Amylmercaptopurine Apigenin* C23H32N2O3 C23H38N2O3 Pentahydroxytrimethoxyflavone* Luteolin 7-O-glucoside Methoxy-dihydroxyvitamin D3 Chrysoeriol 7-Oapiosylglucoside* Pheophytin ae Pheophytin ae Pheophytin ae Chlorophyll ae Chlorophyll ae

C5H10O2 C5H12O4 C10H14N2 C6H13NO5 C7H12O4 C10H18O2 C12H16O2 C12H18O2 C7H10O7 C9H12N2O6 C12H16O4 C10H14N4S C15H10O5 C23H32N2O3 C23H38N2O3 C18H16O10

[M+Na]+ [M+Na]+ [M+H]+ [M+H]+ [M+Na]+ [M+Na]+ [M+Na]+ [M+Na]+ [M+Na]+ [M+H]+ [M+Na]+ [M+K]+ [M+Na]+ [M+H]+ [M+H]+ [M+H]+

125.05730 159.06278 163.12280 180.08665 183.06278 193.11990 215.10425 217.11990 229.03187 245.07681 247.09408 261.05705 293.04205 385.24857 391.29552 393.29994

125.05747 159.06246 163.12297 180.08699 183.06272 193.11936 215.10380 217.12035 229.03149 245.07741 247.09441 261.05767 293.04164 385.24765 391.29464 393.29889

-1.4 2.0 -1.0 -1.8 0.3 2.8 2.1 -2.1 1.7 -2.4 -1.3 -2.4 -1.4 2.4 2.2 2.7

C21H20O11 C28H46O4 C27H30O15

[M+H]+ [M+Na]+ [M+H]+

449.10784 469.32883 595.16578

449.10732 469.32851 595.16454

1.1 0.7 2.1

C55H74N4O5 C55H74N4O5 C55H74N4O5 C55H72O5N4M g C55H72O5N4M g

[M+H]+ [M+Na]+ [M+K]+ [M+H]+

871.57320 893.55514 909.52908 893.54259

871.57502 893.55332 909.53110 893.54426

-2.0 2.0 -2.2 -1.9

[M+Na]+

915.52453

915.52681

-2.5

ESI(-) FT-ICR Tentative Assignment Carbohydrates Hexose* Polyalcohol* Disaccharide* Organic Acids Acetic Acid* Lactic Acid Phosporic Acid Malonic acid Maleic Acid* Succinic Acid* Pyroglutamic acid Oxalacetic acid Malic Acid* Ascorbic Acid*

Formula

Adduct

Theor. m/z

Exp. m/z

Δppm

C6H12O6 C6H12O6 C6H14O6 C6H14O6 C12H22O11 C12H22O11

[M-H][M+Cl][M-H][M+Cl][M-H][M+Cl]-

179.05611 215.03279 181.07176 217.04844 341.10893 377.08561

179.05663 215.03232 181.07135 217.04889 341.10860 377.08536

-2.9 2.2 2.2 -2.1 1.0 0.6

C2H4O2 C3H6O3 H3PO4 C3H4O4 C4H4O4 C4H6O4 C5H7NO3 C4H4O5 C4H6O5 C4H6O5 C6H8O6

[M-H][M-H][M-H][M-H][M-H][M-H][M-H][M-H][M-H][M+Cl][M-H]-

59.01385 89.02441 96.96962 103.00368 115.00368 117.01933 128.03535 130.99860 133.01425 168.99092 175.02481

59.01378 89.02439 96.96974 103.00393 115.00337 117.01953 128.03507 130.99825 133.01426 168.99137 175.02497

1.2 0.2 -1.2 -2.4 2.7 -1.7 2.2 2.7 -0.08 -2.7 -0.9 41

Quinic Acid* Syringic Acid* Caffeoylquinic acid * Fatty Acids and derivatives Myristic Acid* Palmitic Acid* Sapienic Acid* Methyl palmitic acid Oleic Acid* Other compounds Hexenol/Hexanal N-α-Acetylcitrulline Deoxyribose-phosphate Trihydroxyflavanone-sulfate Luteolin-O-glucuronide Apiin* a Metabolites b Theor. c Exp. d The e

C7H12O6 C9H10O5 C16H18O9

[M-H][M-H][M-H]-

191.05611 197.04555 353.08781

191.05659 197.04518 353.08842

-2.5 1.9 -1.7

C14H28O2 C16H32O2 C16H30O2 C17H34O2 C18H34O2

[M-H][M-H][M-H][M-H][M-H]-

227.20165 255.23295 253.21730 269.24860 281.24860

227.20136 255.23345 253.21658 269.24896 281.24794

1.3 -2.0 2.8 -1.3 2.3

C6H12O C8H15N3O4 C5H11O7P C15H12O8S C21H18O12 C26H28O14

[M-H][M-H][M+Cl][M-H][M+Cl][M-H]-

99.08154 216.09898 248.99364 351.01801 497.04923 563.14063

99.08181 216.09839 248.99345 351.01830 497.04996 563.14122

-2.7 2.7 0.8 -0.8 -1.5 -1.0

identified also in petioles extracts are marked by an asterisk.

stands for calculated exact mass to charge ratio.

stands for experimental m/z value. error expressed in parts per million (ppm).

Compounds detected by ESI MS analyses of only organic extracts of celery blade leaves and

petioles.

Table 4. GC-MS analysis of blade leaves and petioles in organic extract. Blade leaves % Class Compound Abundance Monoterpene Limonene 2.9 Alcohol 2,4-Di-tert-butylphenol 0.7 Unknownc 12.8 Unknownc 6.5 Senkyunolide A 38.0 Phthalide Neocnidilide 9.1 Trans-ligustilide 3.6 Cis-ligustilide 0.6 Ether 1-methoxy-3-(2-hydroxyethyl)nonane 11.8 Phthalate compound 1.8 Phthalate Phthalate compound 0.8 Diisobutyl phthalate 3.9 7,10,13-hexadecatrienoic acid methyl ester 0.6 Methyl palmitate 2.6 Isopropyl palmitate 0.5 FAE Methyl linoleate 0.6 Methyl linolenate 1.6 Diterpene Phytol 0.9

KIa

KILb

1023 1509 1693 1717 1724 1730 1739 1800 1835 1840 1862 1868 1896 1921 2020 2092 2099 2109

1022 1518 1729 1735 1741 1797 1869 1926 2027 2106 2100 2109 42

FAE

Methyl stearate

0.7

2122

2128

12.5 22.3 47.5 5.0 1.6 11.1

1509 1868 1921 2092 2099 2122

1518 1869 1926 2106 2100 2128

Petioles Alcohol Phthalate FAE

2,4-Di-tert-butylphenol Diisobutyl phthalate Methyl palmitate Methyl linoleate Methyl linolenate Methyl stearate

aThe

Kovats Index (KI) values have been experimentally measured by using n-alkanes mixtures (C8-C24). bLiterature values (see the main text). cCompound related to senkyunolide (same EI spectrum and similar KI). FAE: fatty acid ester.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Highlights 

“Bianco di Sperlonga” PGI celery was investigated by a multi-methodological approach



A detailed phytochemical profile of celery aqueous and organic extracts was obtained



Biogenic amine and mycotoxin low contents confirmed celery quality and freshness

 Blade leave ethanolic extract showed antimutagenic and radical scavenger power

43