Influence of harvest season on chemical composition and bioactivity of wild rue plant hydroalcoholic extracts

Accepted Manuscript Influence of harvest season on chemical composition and bioactivity of wild rue plant hydroalcoholic extracts Severina Pacifico, Simona Piccolella, Silvia Galasso, Antonio Fiorentino, Nadine Kretschmer, San-Po Pan, Rudolf Bauer, Pietro Monaco PII:

S0278-6915(16)30042-4

DOI:

10.1016/j.fct.2016.02.009

Reference:

FCT 8509

To appear in:

Food and Chemical Toxicology

Received Date: 15 December 2015 Revised Date:

3 February 2016

Accepted Date: 9 February 2016

Please cite this article as: Pacifico, S., Piccolella, S., Galasso, S., Fiorentino, A., Kretschmer, N., Pan, S.-P., Bauer, R., Monaco, P., Influence of harvest season on chemical composition and bioactivity of wild rue plant hydroalcoholic extracts, Food and Chemical Toxicology (2016), doi: 10.1016/ j.fct.2016.02.009. 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.

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Influence of harvest season on chemical composition and bioactivity of wild rue plant hydroalcoholic extracts

Severina Pacifico,1,* Simona Piccolella,1 Silvia Galasso,1 Antonio Fiorentino,1 Nadine Kretschmer,2

Department of Environmental Biological and Pharmaceutical Sciences and Technologies, Second

University of Naples, Via Vivaldi 43, I-81100 Caserta Italy 2

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San-Po Pan,2 Rudolf Bauer2 and Pietro Monaco1

Institute of Pharmaceutical Sciences, Department Pharmacognosy, Karl-Franzens University

Correspondence

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Graz, Universitaetsplatz 4/1, 8010 Graz, Austria

dr Severina Pacifico, Department of Environmental Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Via Vivaldi 43, I-81100 Caserta Italy; E-mail:

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[email protected] Phone +39 0823 274572

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Abstract

The rue (Ruta graveolens) copiousness in rural areas of the Campania Region based a thorough chemical and biological investigation aimed at exploring the seasonal variability of phenol constituents in rue leaves and its influence on their antioxidant, cytotoxic and anti-inflammatory

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capabilities. To this purpose, hydroalcoholic extracts were prepared from plant samples seasonally collected. LC-ESI-MS/MS techniques were employed to analyze qualitatively and quantitatively the seasonal rue phenol content, whereas different chemical antioxidant assays (by DPPH, ABTS, Fe3+

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RP, ORAC, and FCR methods) and XTT redox metabolic activity assay were performed to screen the seasonal phenol complex-related antioxidant and cytotoxic power. The ability of the rue leaf

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extracts to counteract cyclooxygenase-2 (COX-2) expression was also evaluated. Data obtained highlighted that the adopted extraction procedure markedly pauperized the furanocoumarin content in all the prepared rue extracts. Flavonol glycosides, along with the flavone acacetin and two sinapic acid derivatives were the main constituents of the spring harvest-derived extract, which exerted the highest antioxidant capability in cell-free systems and was capable to inhibit COX-2

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synthesis by 44% comparably to dexamethasone, used as positive control. Data provide new insights for developing a proper management of rue plants for new safe industrial purposes in

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herbal medicine field.

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Keywords: Ruta graveolens, LC-MS/MS phenol profile, antiradical capability, cytotoxicity, antiinflammatory activity

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1.

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Introduction

Healing plants have a long traditional history for their medicinal use (Petrovska, 2012; Ncube et al., 2012; Galasso et al., 2014; Pacifico et al., 2015a) and provide an unlimited source for drug discovery because of the unmatched availability of chemical diversity (Sasidharan et al., 2011). In

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fact, plants contain a broad array of bioactive compounds, mainly secondary metabolites, able to employ beneficial effects to humans, especially in the prevention of chronic degenerative diseases such as cancer, cardiovascular and neurodegenerative disorders. Indeed, the content in active

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ingredients of a medicinal plant is continuously affected by different endogenous, exogenous and/or biotic factors (Pacifico et al., 2015a; Pacifico et al., 2015b). Abiotic stresses deeply influence the

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secondary metabolites biosynthesis in spontaneous medicinal plants, which, even today, play a strategic role in the production of plant-based products. In fact, it is known that although the cultivation of medicinal species is in constant increase, it is still a marginal reality, and an amount between 75% and 90% of medicinal plants commercialized in the world still comes from wild harvest (ISMEA, 2013). Peculiarly, harsh abiotic stresses could reduce plant growth with huge

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impact on agriculture (Atkinson and Urwin, 2012). Drought, salinity, heavy metals, extreme temperatures, nutrient poor soils and other abiotic stresses account for major crops lost worldwide

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(Rodriguez et al., 2005). However, not all the effects are detrimental, as plants exhibit various defense mechanisms (Boscaiu et al., 2008), among which the synthesis of phenol compounds (Agati

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et al., 2012 and 2013), whose accumulation seems to counteract the overproduction of reactive oxygen species that triggers in plant. Thus, it is easy to image that depending on the habitat in which a plant grows and develops, it otherwise can modulate its ability to biosynthesize phenols, whose presence/absence makes the same plant an antioxidatively active/inactive source for human purposes (Nascimento and Fett-Neto, 2010; Ramakrishna and Ravishankar, 2011). Therefore, the variability of phenols biosynthesis as response to environmental and stress constraints overcoming (Hüsnü Can Baser, 2002; Edreva et al., 2008) cannot be neglected in the definition of 3

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pharmacological goodness of wild medicinal plants. It is feasible that the opportune knowledge of phenols stress-induced expression in wild medicinal plants could address the maintenance of their biodiversity safeguard and/or optimize their domestication in order to realize a production chain that exploits environmental conditions favorable to the production of plant characteristic phenols.

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Furthermore, the full knowledge of the phenolic constitution of medicinal and aromatic plant species, together with the determination of the health properties of their phenol plant complexes, could lead to their exhaustive and safe use in herbal medicine.

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In this context, Ruta graveolens L. wild plants, seasonally collected, were of interest.

This herbaceous perennial plant, native to the Mediterranean region, commonly known as rue, has

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been used for a long time as contraceptive, anti-inflammatory, antipyretic, anti-helminthic, to relieve symptoms of hangover, and applied externally as a poultice against rheumatic pain (Raghav et al., 2006; Saieed et al., 2006; Ratheesh and Helen, 2007; Asgarpanah et al., 2012; Malik et al., 2013). The rue copiousness in rural areas of the Campania Region based a thorough chemical and biological investigation aimed at exploring the seasonal variability of phenol constituents in rue

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2. Materials and Methods

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leaves and its influence on their antioxidant, cytotoxic and anti-inflammatory capabilities.

2.1 Plant collection and fractionation

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Ruta graveolens leaves were collected in the wild in Durazzano (Italy) on the tenth day of July and October 2012 and of January and April 2013. A voucher specimen has been deposited at the Herbarium of the Department of Environmental, Biological and Pharmaceutical Sciences and Technologies of the Second University of Naples. Durazzano is a small center in the Southern Italy (altitude 286 m above sea level) characterized by a mild Mediterranean climate, identified as Csa climate on the basis of Köppen and Geiger classification. Meteorological parameters, acquired from the Centro Agrometeorologico Regionale (C.A.R.) of the Campania Region, for the meteorological 4

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station located in Airola, are listed in Table 1. Three replicate samples (10.0 g each) of R. graveolens leaves for each collection time were ground in a porcelain mortar and pestle chilled with liquid N2. Frozen powdered samples were lyophilized using an FTS-System Flex-DryTM instrument (SP Scientific, Stone Ridge, NY, USA). Aliquots of dried leaves (1.0 g) underwent

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ultrasound-assisted extraction using an ultrasonic bath (Branson M3800, Carouge, Switzerland) at 40 kHz frequency. A hydro-alcoholic solution (H2O:MeOH; 1:1, v:v) was used as extracting solvent with a drug/solvent ratio equal to 1:5. Four sonication cycles were performed (30 min each) in order

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to achieve the maximum recovery of the leaf metabolic content. At the end of each sonication cycle, samples were centrifuged at 2,044 × g for 10 min in a Beckman GS-15R centrifuge (Beckman

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Coulter, Milano, Italy) fitted with rotor S4180. Obtained supernatants were dried under vacuum by a rotary evaporator (Heidolph Hei-VAP Advantage, Germany) to yield four crude extracts, RgSu (Ruta graveolens summer extract), RgAu (Ruta graveolens autumn extract), RgWi (Ruta graveolens winter extract) and RgSp (Ruta graveolens spring extract), which were stored at -20 °C

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until use.

2.2 RP-HPLC-ESI-MS/MS analyses

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Chromatographic analyses were carried out on a Dionex Ultimate 3000 HPLC system (ThermoScientific Vienna, Austria) equipped with Ultimate 3000 RS pump, Ultimate 3000 RS

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autosampler, Ultimate 3000 RS Column Compartment and Ultimate 3000 RS diode array detector (DAD). A Phenomenex® Synergy RP-80A column (4.0 µm particle size, 150 × 2 mm) was used for chromatographic separation. The mobile phase consisted of A: 0.1% formic acid in water and B: acetonitrile. Starting with 5% B, a linear gradient was followed to 15% B at 10 min, then increasing to 35% B at 30 min, 80% B at 40 min, and 100% B at 45 min, continuing for 5 min, before reequilibration to starting conditions. The flow rate was 0.3 mL/min and the injection volume was 5.0 µL. The DAD (Diode-Array Detector) acquisition range was 190–450 nm. 5

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The liquid chromatography (LC) system was coupled to a LTQ- XL mass spectrometer (Thermo Scientific Vienna, Austria) with an ESI (Electrospray Ionization) source and controlled by Thermo Tune Plus 2.7.0 software. ESI ion source operated in negative mode with the following parameters: dry gas flow (N2) 8.0 L/min with a capillary temperature set at 330 °C; source heater temperature

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set at 250 °C; sheath gas flow set at 50 arb; auxiliary gas flow set at 10 arb; source voltage set at 3.00 kV; capillary voltage set at -16 V. Mass spectra were recorded between m/z 50-2000. Collision-induced fragmentation experiments were performed in the ion trap using helium as a

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collision gas, with voltage cycles from 0.3 up to 2 V and collision energy set at 35 eV. To obtain further structural information, these ions were trapped and fragmented to yield the product ions

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patterns of the analytes. The constituents were identified by comparison of their specific chromatographic data, including UV spectra and MS/MS fragmentation patterns to literature data and reference compounds. Metabolites content was expressed relative to quercetin used as external standard. For this purpose quercetin calibration curves were prepared using ten different concentrations of the flavonol (3.06, 6.125, 12.5, 25.0, 50.0, 100.0, 400.0, 600.0, 800.0, and 1000.0

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µg/mL) injected in the same conditions of the samples.

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2.3 Determination of total phenols

Total phenol amount of investigated crude extracts was determined according to the Folin–

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Ciocalteau procedure, as reported by Pacifico et al. (2012). Analyzed samples (1.0 mg/mL in DMSO) were mixed with 0.500 mL of Folin–Ciocalteau reagent (FCR) and 4.0 mL of Na2CO3 (7.5% w/v). After stirring reaction mixture at room temperature for 3 h, the absorbance was read at 765 nm using a Shimadzu UV-1700 spectrophotometer (Shimadzu, Salerno, Italy). The content of total phenols of the samples was expressed as milligram gallic acid equivalents (GAEs) per 100 g of fresh material.

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2.4 Scavenging Capacity Assessment

Because multiple reaction characteristics and mechanisms as well as different phase localizations are usually involved, no single assay accurately reflect all of the radical sources or all antioxidants in a mixed or complex system. Thus, four different in vitro antiradical assays were employed

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(Pacifico et al., 2011; Di Maro et al., 2013). Each extract, previously dissolved in dimethyl sulfoxide (DMSO) as a stock solution of 125.0 mg/mL, was evaluated at different levels (DMSO final concentration was equal to 0.1% (v:v)). Tests were carried out performing three replicate

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measurements for three samples (n=3) of each extract (in total, 3×3 measurements). Recorded

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activities were compared to a blank arranged in parallel to the samples.

2.4.1 Determination of DPPH radical scavenging capacity

In order to estimate the DPPH• (2,2-diphenyl-1-picrylhydrazyl) scavenging capability, investigated extracts (0.625, 1.25, 2.50, 5.00, 10.0, 25.0 and 50.0 µg/mL, final concentration levels) were dissolved in a DPPH• methanol solution (9.4 × 10-5 M; 1.0 mL) at room temperature. After 30 min,

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the absorption at 515 nm was measured by a Shimadzu UV-1700 spectrophotometer in reference to a blank. The results were expressed in terms of ID50 values on the basis of the percentage decrease

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of the initial DPPH• absorption by the test samples. TEAC (Trolox® Equivalent Antioxidant

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Capacity) values were also calculated.

2.4.2 Determination of ABTS radical cation scavenging capacity ABTS radical cation was generated by reacting ABTS ([2,2'-azinobis-(3-ethylbenzothiazolin-6sulfonic acid)]; 7.0 mM) and potassium persulfate (2.45 mM). The mixture was allowed to stand in the dark at room temperature for 12–16 h. Thus, the ABTS•+ solution was diluted with phosphate buffered saline (PBS) (pH 7.4) in order to reach an absorbance of 0.70 at 734 nm. Rue extracts (0.625, 1.25, 2.50, 5.00, 10.0, 25.0 and 50.0 µg/mL, final concentration levels) were dissolved in 7

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1.0 mL of diluted ABTS•+ solution. After 6 min of incubation, the absorption at 734 nm was measured by a Shimadzu UV-1700 spectrophotometer in reference to a blank. The results were expressed in terms of ID50 values on the basis of the percentage decrease of the initial ABTS•+

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absorption by the test samples. TEAC values were also calculated.

2.4.3 Determination of Fe(III) reducing power

R. graveolens extracts (0.625, 1.25, 2.50, 5.00, 10.0, 25.0 and 50.0 µg/mL, final concentration

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levels) were dissolved in potassium hexacyanoferrate (0.1 M, 2.5 mL) and NaH2PO4/Na2HPO4 buffer (0.2 M, pH 6.6, 2.5 mL). The mixture was incubated at 50 °C for 30 min. Then, an aqueous

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solution of trichloroacetic acid (100.0 mg/mL) was added. After 5 min, an aliquot of the reaction mixture (2.5 mL) was mixed with 2.5 mL of MilliQ water and 0.5 mL of ferric chloride (1.0 mg/mL). The absorbance was measured at 700 nm. The increase in absorbance with reference to the blank estimates the reducing power. The slopes of the inhibition percentage as a function of concentration for each sample and Trolox® were determined. The TEAC values were expressed as

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mg Trolox® equivalents per 1g of extract.

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2.4.4 Determination of oxygen radical absorbance capacity (ORAC) The ORAC assay was performed as follows: each extract (20 µL; 0.781, 1.56, 3.12, 6.25, 12.5 and

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25.0 µg/mL, final concentration levels) and fluorescein (FL; 120.0 µL; 70 nM, final concentration) were preincubated for 15 minutes at 37 °C in 75 mM phosphate buffer (pH 7.4). Then 2,2’-azobis(2-amidinopropane)-dihydrochloride (AAPH) solution (60.0 µL; 36 mM, final concentration) was rapidly added. In parallel with the test samples, a blank (FL + AAPH) and solutions of the reference antioxidant Trolox® (1-4 µM, final concentration levels) were properly prepared in PBS. The fluorescence decay (λex = 485 nm, λem = 525 nm) was recorded every minute for 120 min using a Tecan SpectraFluor fluorescence and absorbance reader. Antioxidant curves (fluorescence vs. time) 8

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were normalized to the curve of the blank. From the normalized curves, the area under the fluorescence decay curve (AUC) was calculated. Linear regression equations between net AUC (AUCantioxidant - AUCblank) and antioxidant concentration were calculated for all the samples. The antioxidant activity (ORAC value) was calculated by using the Trolox® calibration curve. The

2.4.5 Relative Antioxidant Capacity Index Determination (RACI)

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ORAC values were expressed as Trolox® equivalents (µM).

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RACI was calculated according to the method of Sun and Tanumihardjo (2007). The standard score was calculated as follows: (x – µ)/σ where x is the raw data, µ is the mean, and σ is the standard

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deviation. Standard scores have a mean of 0 and a standard deviation equal to 1.

2.5 Cytotoxicity assessment 2.5.1 Cell cultures

CCRF-CEM leukemia cells, and MDA-MB-231 breast cancer cells were grown in RPMI 1640

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medium (Gibco, Invitrogen, Vienna, Austria) supplemented with 2.0 mM glutamine (Sigma, MO, USA), 10.0% heat-inactivated fetal bovine serum (FBS, PAA laboratories, Austria) and 1.0%

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Pen/Strep (PAA laboratories, Austria) at 37 °C in a humidified atmosphere containing 5% CO2. U251 glioblastoma cells, and HCT-116 colon cancer cells were plated and grown under the same

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conditions, except that Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Invitrogen, Vienna, Austria) was used instead of RPMI. Human MRC-5 lung fibroblasts were grown in Minimum Essential Medium (MEM, Gibco, Invitrogen, Vienna, Austria) equally supplemented with 2.0 mM glutamine, 10.0% FBS, and 1.0% Pen/Strep.

2.5.2 XTT cell viability test

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Cytotoxicity was evaluated by the XTT test (Cell Proliferation Kit II, Roche Diagnostics, Mannheim, Germany), which allows the assessment of cell viability by determining the levels of activity of mitochondrial dehydrogenases towards 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2Htetrazolium-5-carboxanilide (XTT) (Galasso et al., 2014). Samples of each extract were prepared as

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a stock solution of 20.0 mg/mL in DMSO and further diluted in culture medium (DMSO final concentration was equal to 0.5% (v/v)). Tests were carried out performing three replicate (n=3) measurements for three samples of each extract (in total: 3×3 measurements). Recorded activities

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were compared to vehicle-treated (0.5% DMSO) control cells arranged in parallel to the samples. MDA-MB-231, U251, HCT-116 and MRC-5 cells were diluted to a final concentration of 5 ×

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104 cells/mL. One hundred micro-liters of the cells were seeded into the wells of 96-well culture plates (flat bottom) and grown for 24 h at 37 °C in a 5% CO2 atmosphere before extracts were added at final concentrations of 10.0, 25.0, 50.0, 100, and 250 µg/mL. Control cells were treated with 0.5% DMSO which did not affect the growth of the cells. In the case of CCRF-CEM cells, 100 µL of 1×104 cells/mL suspension were seeded in 96-well plates (flat bottom) and extracts were

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immediately added. All cells were incubated with investigated extracts for 24, 48 and 72 h before a freshly prepared XTT solution was added as specified by the manufacturer: XTT-labeling reagent

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and electron-coupling reagent were mixed in a ratio of 50:1 and 50 µL of this mixture were added to each well of the 96-well plate. The plates were then incubated for 1.5 h (MDA-MB-231, U251,

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HCT-116 and MRC-5 cells) or 4.0 h (CCRF-CEM cells) at 37 °C in a 5% CO2 humidified atmosphere and read out after incubation using a Tecan Rainbow microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Vinblastine (0.01 µM) served as positive control. The cytotoxic effect of the treatment was determined as percentage of viability compared to untreated cells using the following formula: (absorbance of treated cells/absorbance of untreated cells)×100 (Pacifico et al., 2014).

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2.6 Cyclooxygenase-2 (COX-2) gene expression assay 2.6.1 Cell culture and reagents

THP-1 human leucemic monocytic cell line (European Collection of Cell Culture; Item No. 88081201) was maintained in RPMI 1640 (Gibco®, NY, USA) supplemented with 2 mM L-

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glutamine, 10 % heat-inactivated fetal bovine serum (FBS, Gibco®, NY, USA), 10 mM HEPES (N2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, Gibco®, NY, USA), 100 U/mL penicilin and 100 µg/mL streptomycine (Pen/strep, Gibco®, NY, USA) at 5% CO2 and 37 °C humidified

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atmosphere. For the initiation of monocyte-macrophage differentiation, 1×106 cells/24 wells were seeded in RPMI 1640 medium containing 12 nM PMA (Phorbol 12-myristate 13-acetate, Sigma,

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MO, USA) for 48 hours. After differentiation, cells were treated with plant extracts (50 µg/mL) for 1 h and stimulated with 7.5 ng/mL final concentration LPS (lipopolysaccharide, Sigma, MO, USA) for additional 3 hours. Cells treated with DMSO (≤ 0.1%) were used as calibrator sample. Before RNA extraction, cells were washed 3 times with cold PBS to remove non-attached cells.

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2.6.2 RNA extraction and reverse transcription-polymerase chain reaction Total RNA was extracted using GenEluteTM Mammalian Total RNA Miniprep Kit (Sigma, MO,

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USA) and reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, NY, USA) according to manufacturer´s manual. The thermal cycling

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conditions were set to 25 °C for 25 min, 37 °C for 120 min and 85 °C for 5 seconds.

2.6.3 Real-time Polymerase Chain Reaction (PCR) COX-2 gene expression analysis was performed on ABI-7300 Real-Time PCR System (Applied Biosystems, NY, USA) using Pre-developed TaqMan® Assay (Applied Biosystems, NY, USA). Target cDNA was amplified using COX-2 primers: forward 5´- GAA-TCA-TTC-ACC-AGG-CAAATT-G – 3´, reverse 5´- TCT-GTA-CTG-CGG-GTG-GAA-CA-3´ and COX-2 probe: FAM- 5´11

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TCC-TAC-CAC-CAG-CAA-CCC-TGC-CA-3´-TAMRA (Applied Biosystems, NY, USA) under following cycling conditions: 50 °C for 2 min, 95 °C for 10 min, followed by 40 PCR cycles of 95 °C for 15 sec and 60 °C for 1 min. Target genes were normalized to glyceraldehyde-3-phosphate

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dehydrogenase (GAPDH) and relative quantified using the 2-(∆∆CT) method (Galasso et al., 2014).

2.7. Statistical analysis

All data were expressed as mean values ± SD. Student's t-test was applied in order to determine

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statistical significance (significance level was set at P < 0.05). All analyses were performed with

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XLSTAT 2014.5.03 software package.

3. Results and Discussion

The metabolic profile of the hydroalcoholic extracts from Ruta graveolens leaves (Fig. 1) showed that they were relatively poor in phenolic constituents. In fact, only thirteen metabolites were identified in the most complex extract (Table 2); their structures were depicted in Fig. 2. Compound

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1 was identified as gentiobiose, a disaccharide composed of two units of D-glucose joined with a β(1→6) linkage. In fact, it showed the [M-H]‒ ion at m/z 341, and disaccharide characteristic

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fragment ions at m/z 179 and 161. The presence of product ions at m/z 281 and 251, fragments from the sugar rings which are diagnostic of the linkage position, allowed us to characterize the

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disaccharide as gentiobiose (Garozzo et al., 1991; Zhu and Cole, 2011). The MS spectrum of compound 2 showed the [M-H]− ion at m/z 337 and fragment ions in accordance with the presence of a 4-O-p-coumaroyl quinic acid. In fact, the [M-H]− ion provided in the MS2 spectrum, beyond the fragment ion at m/z 191, the ion at m/z 173 [quinic acid-H-H2O]− as base peak (Clifford et al., 2003; Parejo et al., 2004; Plazonić et al., 2009). MSn spectra of compound 3 were in accordance with the presence of a bis-C-glycosylated flavone. The MS spectrum exhibited the deprotonated molecule at m/z 593, which produced MS2 ions at m/z 12

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473 [M-H-120]− (base peak), m/z 551 and 431. The neutral loss of 120 Da allowed us to hypothesize the presence of a hexose residue linked by C-C bond to apigenin (m/z 341 = [aglycone+71]−; m/z 311 = [aglycone+41]−). Further collision of the ion at m/z 473 yielded the ion at m/z 353 (loss of 120 Da) indicating the presence of a second C-hexosyl moiety. The fragment ion

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at m/z 383 ([aglycone+113]-) allowed us to confirm the aglycone identity (Ferreres et al., 2003). As C-linked saccharide residues were found only at the C-6 and/or C-8 positions of flavone skeletons, compound 3 was assigned as apigenin-6,8-di-C-hexoside.

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Compound 4 was identified as a feruloyl quinic acid isomer. The fragmentation of the [M-H]‒ ion at m/z 367 provided a fragment ion at m/z 193, likely a feruloyl moiety, and, as base peak, the

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fragment ion at m/z 173 [M-H-feruloyl acid]‒ allowing us to localize the linkage of the feruloyl moiety at the hydroxyl function on C-4 carbon of quinic acid (Clifford et al., 2003). Compound 5 was cnidioside A, a benzofuran glycoside previously isolated from the ethanol extract of the dried aerial parts of Ruta graveolens (Chen et al., 2001). The dissociation of the [M-H]− ion at m/z 367

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gave rise to ions at m/z 323 and 205, arising from losses of a CO2 unit and a hexosyl moiety, respectively. The fragmentation pattern relative to the deprotonated molecular ion at m/z 423 of the metabolite 6, identified only in RgWi sample, allowed us to tentatively identify it as isorutarin, a

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molecule previously isolated from Ruta graveolens (Dhale et al., 2010), belonging to the furanocoumarin class. In the MS2 spectrum the fragment ion at m/z 261 was in accordance with the

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loss of a hexose moiety, whereas the fragment ion at m/z 245 was probably due to the prenylated precursor. The fragment ion at m/z 179 probably consisted in the caffeoyl anion. Metabolite 7 was putatively identified as ranupenin-3-O-rutinoside (also known as gossypetin 7-methyl ether-3-Orutinoside), a yellow flower pigment already isolated from Ruta graveolens and from five other Ruta species (Harborne and Boardley, 1983). The mass spectrum showed the [M-H]− ion at m/z 639 which provided the ions at m/z 624 (-15 Da), and 331. This latter, probably due to the neutral loss of a deoxyhexosylhexose residue (-308 Da, e.g. rutinose), was identified in the aglycone ranupetin. 13

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The presence of the aglycone radical at m/z 330 was also observed. Further collision of the ion at m/z 331 yielded the ions at m/z 316 (-15 Da), due to the loss of a methyl radical, and at m/z 181 and 153, both attributable to retrocyclization cleavages involving the flavonol A ring. A fragment at m/z 110 seemed to confirm the presence of a dihydroxylated B ring.

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Metabolite 8 was rutin. The identification of the compound was addressed by the comparison with mass spectra and retention time of the relative reference standard. MS spectrum showed the deprotonated molecular ion at m/z 609, whose dissociation generated the fragment ion at m/z 301 as base peak (-308 Da). Compound 9, identified only in RgWi sample, was putatively identified as

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7,3’-O-dimethyl-gossypetin-3-O-rutinoside. When the [M-H]− ion at m/z 653 dissociated, the

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fragment ion at m/z 345 [M-H-308]− was provided, together with the ions at m/z 331, 330, and 287. The first fragment suggested the loss of a deoxyhexosylhexose moiety, whereas the fragment ions at m/z 330 and 315 were attributable to the loss of one and two methyl radicals. As methylation on gossypetin skeleton was found at C-7 or C-3’ carbon, methyl functions were localized therein (Harborne and Boardley, 1983). The mass spectrum of metabolite 10 showed the [M-H]− ion at m/z

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623, which provided the fragment ion at m/z 315 by the neutral loss of 308 Da due to a deoxyhexosylhexosyl moiety. The collisionally activated dissociation of the fragment ion at m/z 315

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provided the ion at m/z 300 [M-H-CH3·]‒, allowing us to identify the aglycone as 3’-Omethylquercetin (isorhamnetin). MS4 spectrum, relative to the fragment ion at m/z 300, showed the

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ions at m/z 271 and 255 confirming the presence of isorhamnetin (Cuyckens and Claeys, 2004). The metabolites 11 and 12 putatively corresponded to 1,2-di-O-sinapoyl gentiobiose and 1-O-sinapoyl2-O-feruloyl gentiobiose. Similar compounds were previously isolated from Ruta graveolens (Chen et al., 2001). The MS spectra of both compounds showed a common loss of 224 Da from the deprotonated molecular ion, probably due to sinapic acid. In particular, the [M-H]‒ ion at m/z 753 for compound 11 provided in MS2 spectrum the fragment ions at m/z 547, 529 and 265. The first one was due to the loss of 206 Da, according to the presence of a sinapoyl moiety, which was 14

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confirmed by the neutral loss of 224 Da leading to the ion at m/z 529. The further dissociation of the ion at m/z 547 gave the ions at m/z 385 (-162 Da) and 367 (-180 Da), which allowed us to suppose the presence of a hexose. Other fragment ions in the MS3 spectrum were detected at m/z 325, 295 and 265, due to typical neutral losses of disaccharidic moiety (-222, -252 and -282 Da). On the basis

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of MS/MS data, compound 11 likely corresponded to 1,2-disinapoylgentiobiose. Compound 12 was putatively identified as 1-sinapoyl-2-feruloylgentiobiose (Chen et al., 2001). In fact, its [M-H]‒ ion at m/z 723 dissociated providing, beyond others, the ions at m/z 547 and 517, which were due to neutral losses of 176 Da and 206 Da, respectively. These losses were attributable to the presence of

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a feruloyl (-176 Da) and a sinapoyl moiety. The ion at m/z 499, also displayed as MS2 fragment ion,

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confirmed the presence of sinapic acid. Both the hydroxycinnamoyl acids were supposed to be linked to the same hexose residue of a gentiobiose unit. Confirming this hypothesis, the fragment ions at m/z 295 (-222 Da), 265 (-252 Da) and 235 (-282 Da) were detected (Zhu and Cole, 2011). Both the hydroxycinnamic acid esters were reported as constituents of broccoli inflorescences

al., 1997).

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(Vallejo et al., 2003), and were found to be highly effective at preventing lipid damage (Plumb et

The deprotonated molecular ion at m/z 283 of compound 13, which provided an abundant fragment

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ion at m/z 268, was in accordance with acacetin (4’-O-methylapigenin; Galasso et al., 2014). Among the identified metabolites, only compounds 1, 8, 10 and 11 were found in all the

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hydroalcoholic extracts from Ruta graveolens (Table 2). RgWi and RgSp were richer in phenolic compounds than RgSu, whereas the extract from RgAu contained only the four metabolites mentioned above. The metabolite broadly present in all the extracts was rutin (8), whose relative abundance seemed to be strongly collection time dependent. In fact, RgSp possessed a rutin content 3.85, 10.97 and 1.56-fold those of RgSu, RgAu and RgWi, respectively. The same sample resulted particularly rich in isorhamnetin rutinoside (10) and in sinapoyl derivatives (11 and 12). This latter was absent in the RgAu sample. Furthermore RgSp contained appreciable amounts of ranupenin-315

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O-rutinoside (7) and acacetin (13). Data from MS quantitative analysis emphasized that rue responds to abiotic stress conditions as drought and low precipitation rate increasing flavonoid glycosides production. Rue plants were collected in Durazzano, a small center in the Southern Italy (altitude 286 m above sea level) characterized by a mild Mediterranean climate, identified as Csa

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climate on the basis of Köppen and Geiger classification. Four collection times, between 2012 and 2013 years, were considered, each of them represented the first month of season. Data from ISPRA (Istituto Superiore per la Protezione e la Ricerca Ambientale) reports show that 2012, as well as

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2013, was a year warmer than the climatological mean, both globally and in Italy (Desiato et al., 2013; 2014). The summer of 2012 was characterized by a strong lack of rainfull and strong positive

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thermal anomalies that have made it one of the hottest summers of the last fifty years. In the first July decade the average temperature in Durazzano was equal to 25.5 °C with an average relative humidity of 59.4%. On 10th July the maximum temperature was 34.8 °C. The drought condition and the absence of precipitations characterizing all the summer of 2012 and ended on September. On October 2012, typically autumnal conditions were present and rue appears to lack a good portion of

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flavonoid glycosides component. Peculiarly, rutin (8), the main rue constituent, underwent a deep decrease in this season. Flavonoid glycosides were again present in RgWi sample and underwent a

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further increase in RgSp sample.

The antioxidant capacity evaluation of hydroalcoholic extracts from rue leaves was conducted by

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the application of different tests. Since the antioxidant activity of plant extracts is often related to the amount of polyphenols contained therein, one of the adopted methods made use of the FolinCiocalteu reagent (FCR, Folin Ciocalteu Reagent). On the basis of the gallic acid calibration curve, total polyphenol content (TPC, Total Polyphenol Content) was determined to be highest for the extract RgSp (90.08 GAE mg/g), comparable for the extracts RgSu (56.76 GAE mg/g) and RgWi (50.30 GAE mg/g), while it showed its lowest level for the extract RgAu (35.47 GAE mg/g).

16

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As reported in Table 3, the extracts under study showed significant differences as scavengers towards the radical species, ABTS•+ and DPPH•, used as probe in radical scavenging activity tests. The ID50 values calculated showed that the extract RgSp exerted a scavenging activity higher than the other extracts. In particular, it was 1.7, 2.4 and 3.9-fold more effective compared to RgWi,

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RgSu and RgAu, respectively. These data well correlated with the TPC values obtained by FCR method and with polyphenolic profile analysis data, which emphasized that the extract RgSp was richer in phenolic constituents with the metabolite rutin was similarly present in all the extracts. It

various antioxidant systems in vitro (Yang et al., 2008).

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was demonstrated that rutin exerts a powerful antioxidant capacity, concentration-dependent, in

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Many structural factors may contribute in the activity exertion: the o-dihydroxyphenolic structure (catechol) of the B ring, the double bond between the C-2 and C-3 carbons together with a 4-oxo carbonyl function in the C ring and the hydroxyphenolic function at C-5 position. The presence of alcoholic oxygen engaged in acetal bond with the glucose residue of rutinose makes the antiradical activity of rutin lower than that of its aglycone quercetin, when tested in pure form. Scientific

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evidence point out that rutin is able to inhibit the DPPH radical to a lesser extent when compared to the flavone luteolin, which, lacking the hydroxyl function at C-3 position, shares with rutin

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potentially active sites for the antioxidant activity exertion. The lower capacity could be explained by the higher folding of B ring, with respect to the A and C rings, caused by glycosylation with

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torsional angles of about 30° (Van Acker et al., 1996). The evaluation of the Fe3+ reducing power confirmed that RgSp (78.23 µg of Trolox® equivalents/g of extract; Table 3) was the extract with greater effectiveness. Significant differences were found between the results obtained from the tests described above and those obtained by the ORAC method. In fact, this latter promoted as the higher radical scavenging extract the one obtained from the autumn collection of plant leaves (RgAu). The results can be explained in the light of the extract metabolic profile and of evidences reported in literature that molecules, such as rutin and 17

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hydroxycinnamic acids, are responsible for high ORAC values (Wolfe and Liu, 2007). Although RgAu is characterized by only four constituents, it is rich in rutin and disinapoyl gentiobioside (11), which represent 54.4 and 43.4% of the extract. The extracts RgWi and RgSp, which appeared to be less active in the ORAC test, are rich in rutin (73.0 and 64.3%), but have a low content of the

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disinapoyl gentiobioside, which represents in both extracts only 10% of all the identified metabolites.

On the basis of data obtained from individual tests and taking into account the relative correlation

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indices, for each extract the RACI (Relative Antioxidant Capacity Index) value was calculated. Data obtained by applying the ABTS method positively correlated with those obtained by the DPPH

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method (R = 0.983) and with the content of phenols (R = 0.946) (Table 4A). Similarly, a particularly positive correlation was observed between data from DPPH method and those from the TPC (R = 0.874). However, a strong negative correlation was found between the ORAC data with those calculated for the other methods. The RACI index allows the comparison of the antioxidant activity of plant complexes estimated through different methods and gives a more comprehensive

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determination of the whole antioxidant capacity of a system (Table 4B). The results confirmed that the most active sample is RgSp (RACI = 0.866) and highlighted as the least active the extract

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collected in the summer season, RgSu (RACI = -0.356). The cytotoxicity evaluation was carried out by the XTT test that allows to estimate the number of

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live cells present in culture, and then to evaluate the effect of the treatment with an added exogenous agent on the vitality of the cell population (Roehm et al., 1991). The test is based on the capacity of the tetrazolium compound XTT (yellow) to be metabolized by mitochondrial dehydrogenases, active in living cells, in a water-soluble derivative of formazan (bright orange). The inhibition of redox mitochondrial activity was evaluated on CCRF-CEM, HCT-116, MDAMB-231, U-251cancer cell lines and on normal-like MRC-5 lung fibroblasts at 24 h, 48 h and 72 h exposure times. The results showed that redox mitochondrial activity of the tested cells was 18

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inhibited in a time-dependent manner. In particular, at 24 h exposure time, all the extracts, at the highest tested doses, weakly inhibited CCRF-CEM leukemic cells redox mitochondrial activity; the summer extract 100 µg/mL dose also provided a weak inhibition towards HTC-116 cells, whereas U-251 cells underwent a mild inhibition when treated whit the highest dose of autumnal and winter

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extracts. At 48 h and 72 h exposure times, the slight inhibitory effect (< 25%) was detected for all the tested cancer cell lines. MDA-MB-231 breast cancer cell line seemed to be peculiarly responsive to winter extract, whose highest dose was able to define, after 48 h, an activity decrease

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around 50%. Analogously, the RgWi 50 and 100 µg/mL doses reduced by almost 50% MDA-MB231 breast cancer cell line redox mitochondrial activity (Table 5). Our findings are in line with

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those of Varamini et al. (2009), who, investigating the cytotoxic effects of the ethanolic crude rue extract towards different cancer cell lines, found it had roughly no cytotoxicity against solid tumor cells (e.g. HeLa and MCF-7 cell lines), whereas it showed an inhibitory effect against haematopoietic cell lines. Furthermore, the researchers performed cell cycle analysis, which evidenced that R. graveolens extract caused profound increase in the number of cells with the sub-

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G1 DNA content allowing them to hypothesize that cytotoxic activity of this plant has been due to its apoptosis inducing properties.

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The harvest-time dependent anti-inflammatory properties of the prepared rue extracts were also evaluated through the investigation of their ability to modulate the expression of COX-2 genes. A

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number of inflammatory processes are associated with the synthesis of prostaglandins, which are responsible for the perception of pain. The main enzyme responsible for the synthesis of prostaglandins is a cyclooxygenase membrane-associated, which exists in two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed, whereas COX-2 is the inducible isoform and its expression is strongly related to the development of chronic degenerative diseases. The synthesis of COX-2 can be inhibited by intra- or extra-cellular agents that inhibit nuclear factor kappa-lightchain-enhancer of activated B cells (NF-κB). In particular, the NF-κB activation can be inhibited 19

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directly or through an up-regulation of the inhibitory protein IκB. Synthetic drugs or natural agents, that are able to act as receptor ligands and modulate the expression of COX-2 genes, may be considered as anti-inflammatory agents. Data obtained showed that the extract RgSp was the most active; its anti-inflammatory efficacy was fully comparable to that of dexamethasone (dex), a well-

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known anti-inflammatory agent used as a positive control (Fig. 3). Moreover, both autumnal and winter extracts slightly induced COX-2, whereas a mild pro-inflammatory effect was detected for the summer extract. Considering quali-quantitative MS profile data, the strong activity of the spring

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extract seems not to be related to its rutin content. In fact, although the molecule was highly produced in the RgSp extract, calculating its abundance percentage in each extract, it was observed

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that the extract richer in rutin is RgWi, in which the molecule is 73.0% of the identified metabolites. The extract RgSp differs from the winter one for its higher content of the rutinoside of the methyl derivative of gossypetin (7; 15% of total metabolites in RgSp and only 1.3% for RgWi) and of the sinapoyl feruloyl gentiobioside (12). The extract RgSp was also the only one among the hydroalcoholic extracts from Ruta graveolens to contain acacetin (13).

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Although the anti-inflammatory properties of rutin have been investigated for a long time in vitro and in vivo (Selloum et al., 2003), the presence of other molecules in the extract makes it active.

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This finding is in line with Raghav et al. (2006), who, investigating the effect of plant extract of Ruta graveolens on murine macrophage cells (J-774) challenged with lipopolysaccharide (LPS),

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stated that rue plant extract at the lowest concentration (100 µg/mL) having only 6 µM rutin was more effective in inhibiting nitric oxide production as compared than pure rutin with a concentration 13-fold higher (80 µM). The authors evidenced that the inhibition of nitric oxide production by the plant extract was due to the combinatorial effect of the whole extract on the transcription of inos gene. Acacetin was shown to exert nociceptive/inflammatory properties (Carballo-Villalobos et al., 2014) and that gossypetin was found to have anti-inflammatory activity in the carrageenan edema test (Ferrándiz and Alcaraz, 1991;Chirumbolo, 2010). 20

ACCEPTED MANUSCRIPT 4. Conclusions Rue plants are broadly distributed in the wild in the Campania Region (Italy). The employment of ultrasound-assisted maceration in hydroalcoholic solution allowed us to obtain extracts poor in

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furanocoumarin constituents, whereas LC-MSn based metabolic profiling analyses aimed to the establishment of the variation of rue phenol composition during the year. RgSp extract, which was characterized by the highest TPC (90.08 GAE mg/g), also proved to be the most effective scavenger

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towards ABTS•+ and DPPH•, with an ID50 value 1.7, 2.4 and 3.9-fold lower than RgWi, RgSu and RgAu, respectively. Furthermore, its effect on the expression of COX-2 genes in THP-1 cells

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established an anti-inflammatory efficacy (COX-2 inhibition equal to 44.2±3.3%) fully comparable to dexamethasone (44.3±5.8%). The high presence of rutin and its synergy with other flavonoid and hydroxycinnamoyl substances, characterizing the spring extract, suggest its possible use to prevent and/or slow down the deleterious effects of oxidative stress and inflammation. Data provide new

Conflict of Interest

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herbal medicine field.

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insights for developing a proper management of rue plants for new safe industrial purposes in

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The authors report no conflict of interest.

21

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Figures legend

Fig. 1. TICs (Total Ion Current chromatograms) from RgSu, RgAu, RgWi, RgSp extracts. Fig. 2. Structures of the identified metabolites in rue extracts (rutinose = 6-O-α-L-rhamnosyl-Dglucose). Fig. 3. Effects of Ruta graveolens extracts on the expression of COX-2 genes in human leukemic

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THP-1 cells. dex = dexamethasone.

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Tables legend

Table 1 Agrometeorological mean parameters for harvest months, acquired from Campania Region C.A.R. Table 2 LC-MS/MS data of the compounds identified in the different Ruta graveolens extracts (RgSu, RgAu, RgWi, RgSp). Quantitative data are expressed as quercetin equivalents (µg/mL). n.d. = not detected.

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Table 3 Antioxidant activity of hydroalcoholic extracts from Ruta graveolens expressed as ID50 (µg/mL) vs ABTS●+ and DPPH● and TEAC values (Trolox® Equivalents Antioxidant Capacity, ID50Trolox/ID50campione×100). TEAC value from Fe3+ RP (Reducing Power) and ORAC data are also shown.

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Table 4 A) Correlation coefficients between the different antioxidant methods applied; B) Standard scores of the antioxidant capability of each extract for each applied method and relative RACI

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values.

Table 5 Effects of extracts RgSu, RgAu, RgWi and RgSp on redox mitochondrial activity of HCT116, MDA-MB-231, U-251, MRC-5 and CCRF-CEM cell lines after 24, 48 and 72 h exposure times. vnb = Vinblastine. Values are the mean ± SD of measurements were carried out on 3 samples

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(n = 3) analyzed twelve times.

28

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RgSp

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RgWi

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RgAu

RgSu

Fig. 1.

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29

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Fig. 2.

30

Fig. 3.

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31

Table 1 Jul 2012a

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Jan 2013

Apr 2013

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32.6 23.3 12.1 21.2 maximum temperature 24 h °C 16.7 10.5 3.8 8.84 minimum temperature 24 h °C 24.8 16.7 7.65 15.0 average temperature 24 h °C 86.4 92.2 89.2 87.1 maximum humidity 24 h %Sat 30.4 49.0 58.2 39.2 minimum humidity 24 h %Sat 59.2 76.2 77.3 65.7 average humidity 24 h %Sat 2.06 1.80 2.53 2.52 wind speed 24h m/s 1.80 5.48 6.43 2.20 precipitation 24 h mm a RgSu sampling time; bRgAu sampling time; cRgWi sampling time; dRgSp sampling time

32

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Table 2

1

RT (min) 1.5

2 3

11.7 12.5

337 593

4

13.2

367

5 6 7

14.5 15.3 16.4

367 423 639

8

16.9

9

Peak

[M-H]341

MSn

tentative assignment

281;251;221;179 (→161; 143; 131; 119; 113; 89); 161 191;173 (→153; 111; 93) 551 (→503;473;417;397;235); 473 (→383;353;325;297;283); 431(→341;311) 193(→149;134) 173 (→155;111;93;71)

Gentiobiose

RgSu

RgAu RgWi

RgSp

36.75

0.13

1.04

0.24

n.d.

n.d.

0.57

n.d.

n.d.

n.d.

n.d.

0.50

4-O-feruloylquinic acid

n.d.

n.d.

1.03

n.d.

cnidioside A furanocoumarin (e.g. isorutarin) ranupenin-3-O-rutinoside

trace n.d.

n.d n.d.

trace 0.40

0.61 n.d.

n.d.

n.d.

1.07

9.46

609

323; 205;161 261;245;179 624;331 [→316 (→ 271/270; 244;194;166;140;110) ; 181;153;110] ;330 301 (→179;151)

Rutin

24.30

8.61

60.02

93.64

18.6

653

345; 331; 330 (→315;302;287); 287

n.d.

n.d.

0.22

n.d.

10

19.1

623

315[ (→300(→271;255)];300

7,3’-O-dimethyl-gossypetin-3-Orutinoside isorhamnetin-3-O-rutinoside

2.79

0.23

8.03

15.25

11

21.3

753

12.52

6.87

8.98

15.93

12

21.7

723

547 [→385; 367; 325; 295; 265; 223 (→208; 193;179; 164) ;205; 193;190;175]; 529;265 547;517(→355;337;295;265; 235;207;205;193;175;161); 499;223 268

0.15

n.d.

0.81

6.96

n.d.

n.d.

n.d.

3.11

RI PT

SC

M AN U

TE D

283

EP

25.6

disinapoyldihexoside (e.g. 1,2disinapoylgentiobioside) sinapoylferuloyl dihexoside (e.g. 1-sinapoyl-2-feruloyl gentiobioside) acacetin

AC C

13

4-O-p-cumaroylquinic acid 6,8-C-dihexosyl-apigenin

33

ACCEPTED MANUSCRIPT

sample

ID50 ●+

ABTS 115.19 186.27 82.30 48.23 1.71

DPPH

●

67.40 117.21 32.19 21.12 1.22

TEAC ●+ ABTS

TEAC ● DPPH

TEAC Fe(III)RP

ORAC Values

1.48 0.91 2.07 3.54

1.81 1.04 3.78 5.77

23.41 68.43 54.55 78.23

1847.76 1981.17 880.13 1144.01

AC C

EP

TE D

M AN U

SC

RgSu RgAu RgWi RgSp Trolox®

ID50

RI PT

Table 3

34

Table 4

ACCEPTED MANUSCRIPT ABTS 1

ABTS DPPH Fe3+ RP ORAC TPC

Fe3+ RP 0.463077 0.491947 1

DPPH 0.983399 1

ORAC -0.71496 -0.8262 -0.32201 1

B) DPPH -0.608 -0.971 0.321 1.259

Fe3+ -1.370 0.514 -0.067 0.924

ORAC 0.719 0.968 -1.090 -0.597

TPC -0.060 -0.983 -0.340 1.384

RACI -0.356 -0.287 -0.223 0.866

AC C

EP

TE D

M AN U

SC

RgSu RgAu RgWi RgSp

ABTS -0.460 -0.964 0.062 1.362

TPC 0.946297 0.874591 0.292833 -0.48347 1

RI PT

A)

35

Table 5

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

L = low dose = 25 µg/mL; M = medium dose = 50 µg/mL; H = high dose = 100 µg/mL = no cytotoxicity; = RAI < 25%; = RAI < 50%; = RAI > 50%.

36

ACCEPTED MANUSCRIPT Highlights 1. Ruta graveolens is copious in rural areas of the Campania Region 2. Rue leaf seasonal extracts were prepared by ultrasound assisted maceration 3. LC-ESI-MS/MS analyses highlighted the seasonal rue phenol variability

RI PT

4. Flavonol and sinapic acid derivatives were abundant in the spring extract

AC C

EP

TE D

M AN U

SC

5. Rue spring extract showed marked antioxidant and anti-inflammatory capabilities