Hydroalcoholic extract of Spartium junceum L. flowers inhibits growth and melanogenesis in B16-F10 cells by inducing senescence

Accepted Manuscript

Hydroalcoholic extract of Spartium junceum L. flowers inhibits growth and melanogenesis in B16-F10 cells by inducing senescence Valentina Nanni , Lorena Canuti , Angelo Gismondi , Antonella Canini PII: DOI: Reference:

S0944-7113(18)30191-0 10.1016/j.phymed.2018.06.008 PHYMED 52529

To appear in:

Phytomedicine

Received date: Revised date: Accepted date:

21 November 2017 21 March 2018 7 June 2018

Please cite this article as: Valentina Nanni , Lorena Canuti , Angelo Gismondi , Antonella Canini , Hydroalcoholic extract of Spartium junceum L. flowers inhibits growth and melanogenesis in B16-F10 cells by inducing senescence , Phytomedicine (2018), doi: 10.1016/j.phymed.2018.06.008

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ACCEPTED MANUSCRIPT

Hydroalcoholic extract of Spartium junceum L. flowers inhibits growth and melanogenesis in B16-F10 cells by inducing senescence

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Valentina Nannia, Lorena Canutia, Angelo Gismondia,1 and Antonella Caninia,*,1 Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica 1, 00133,

Rome, Italy

*Corresponding author

Ricerca Scientifica 1, 00133, Rome, Italy

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Prof. Antonella Canini, Department of Biology, University of Rome “Tor Vergata”, Via della

Tel. +39 06 7259 4333, Fax. +39 06 2023 500.

AG and AC are co-last authors

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E-mail address: [email protected].

Running title: Spanish broom exerts antineoplastic activity against B16-F10 melanoma cells

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ACCEPTED MANUSCRIPT ABSTRACT Background: Ultraviolet light exposure generates, in human tissues, radical species, which represent the main cause of photo-aging, DNA damage and skin cancer onset. On the other hand, Mediterranean plants, being continuously subjected to high solar radiation levels, are naturally adapted to take on this type of abiotic stress, thanks to the production of antioxidant secondary metabolites. For these reasons, several plant extracts were documented to be excellent

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antineoplastic drugs. Purpose: We investigated the potential antitumor activity of the flower extract obtained by Spartium junceum L., a Mediterranean shrub, correlating it with the plant metabolic profile. Study Design: After selecting the best extraction method to obtain as more secondary metabolites as possible from S. junceum flowers, we characterized the extract metabolic content. Then, by in vitro

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analyses, the antioxidant profile and the antineoplastic activity on B16-F10 murine melanoma cell of our extract were investigated.

Methods: Spectrophotometric assays, HPLC-DAD and GC-MS analyses provided us information about flower extract composition and antioxidant activity. MTT assay and Trypan Blue exclusion test were performed to assess the extract toxicity and the viability, after treatments, of B16-F10

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cancer cells and of C2C12 murine myoblasts. In vitro experiments (i.e. cytofluorimetry, protein analysis and qPCR) allowed us to analyze the effect of the plant extract on B16-F10 cell redox state,

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melanogenesis and cell cycle. Senescence induction was investigated by using a specific kit. Results: We observed that the hydroalcoholic extract of S. junceum flowers (HFE) strongly inhibited B16-F10 murine melanoma cell proliferation, while just a feeble effect was observed on

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C2C12 murine myoblasts. Moreover, we found that HFE exerted a pro-oxidant activity on melanoma cells, inhibited melanogenesis and caused cell cycle arrest in G2/M phase, inducing

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senescence. These anti-cancer properties of HFE could be related to the rich metabolic profile of the extract that we characterized by HPLC-DAD and GC-MS analyses.

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Conclusion: This evidence suggests that S. junceum phytocomplex can be used as a selective, nontoxic, economic and easily available anticancer drug.

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ACCEPTED MANUSCRIPT Keywords Senescence, Secondary metabolite, Cancer, Proliferation, Spanish broom, Antioxidant

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Abbreviations AAE, ascorbic acid equivalent; ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); AFE, aqueous flower extract; CNT, control sample; DAD, diode array detector; DER, drug extract ratio; DMEM, Dulbecco’s Modified Eagle’s Medium; DOX, doxorubicin; FRAP, ferric reducing ability of plasma; FMW, fresh material weight; GAE, gallic acid equivalents, HFE, hydroalcoholic flower extract; HHE, hydroalcoholic hip extract; HLE, hydroalcoholic leaf extract; HPLC, High performance liquid chromatography; GC-MS, gas chromatography-mass spectrometry; MTT, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; QE, quercetin equivalents; PBS, phosphate buffered saline; qPCR, quantitative polymerase chain reaction; ROS, reactive oxygen species; TIC, Total Ion Current.

Introduction

A lot of pharmaceutical products used to cure cancer, infections, chronic and age-related diseases, metabolic and immune disorders are natural substances or their derivatives (Newman and Cragg, 2016). Among them, the largest group is represented by phytochemicals, or rather secondary

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metabolites (i.e. phenolics, alkaloids, terpens) which are synthesized by plants to defend themselves from biotic and abiotic factors and promote their propagation (Gismondi et al., 2017). The

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application of these molecules in drug design and production is mainly linked to the great biological activity they show. Well-known examples are vancomycin (an antibiotic used against Gram-

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positive bacterial infections), staurosporine (a protein kinase inhibitor), rapamycin (an immunosuppressant) and taxol (an anti-cancer agent) (Clardy and Walsh, 2004; Paterson and

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Anderson, 2005).

Several scientific works reported that plant extracts inhibit the growth of a wide range of tumor cell

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lines (e.g. blood, skin, brain, colon, breast, prostate), both in vitro and in vivo, due to their antioxidant, anti-inflammatory, antiproliferative, differentiative or pro-apoptotic properties (Gullett et al., 2010; Lesgards et al., 2014). All this evidence suggests that plant molecules can represent non-toxic, economic and easily available chemopreventive and chemotherapeutic agents. Mediterranean flora, distributed in geographic areas with high solar radiation level, evolved specific adaptive strategies to sustain such type of environmental pressure, which would induce oxidative stress in plant tissues. In this context, the capacity to produce secondary metabolites with free radical scavenging activity is surely the most prominent (Ramakrishna and Ravishankar, 2011). These compounds, thanks to their chemical structure, essentially characterized by aromatic rings 3

ACCEPTED MANUSCRIPT and double bonds, can easily provide electrons to reactive species, disrupting the damaging oxidative chain reactions which occur in cell compartments (Gismondi et al., 2017). As regards human health, it is widely documented that ultraviolet (UV) light exposure generates radical species (e.g. ROS) in skin cells, causing oxidative stress, inflammation, photo-aging, DNA damage and, consequently, malignancy (Sekulic et al., 2008). Therefore, as skin cancer is the most common neoplasia in the World and melanoma is its deadliest form (The American Cancer Society, 2017), antiradical molecules extracted from Mediterranean plants could be used able to prevent

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onset, growth, progression and metastatization of such type of tumor (Działo et al., 2016; Jensen et al., 2010).

Spartium junceum L. (Spanish broom or weaver's broom) is a typical Mediterranean shrub species, belonging to Fabaceae family. Only two literature works demonstrated the anti-neoplastic activity of S. junceum extract (Abusamra et al., 2015; Cerchiara et al., 2012). In detail, Abusamra and

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colleagues (2015) tested flower hydromethanolic (80% methanol) extract on U-373 glioblastoma cells, observing a weak cytotoxicity effect, probably associated to a caspase-independent and nonapoptotic cell death. On the other hand, Cerchiara et al. (2012), using S. junceum flower aromatic water (obtained by vacuum distillation), performed a preliminary cytotoxicity screening on several

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cell lines (RPMI 7932, K562, MCF7-Bart, MCF7-ICLC, SW480 and NCTC 2544, as non-tumor control). In general, they observed a reduction of tumor cell growth, while no toxic effect was detected on NCTC 2544 cells.

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Taking into account these promising data and considering that no further work investigated the bioactivity of this Mediterranean plant, the present research aimed to increase the knowledge about

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the medicinal properties of this species. Therefore, metabolic profile and antitumor effect of S. junceum flower hydroalcoholic extract were studied in depth. In addition, the bioactivity of two

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evaluated.

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other Mediterranean plant species, Pittosporum tobira (Thunb.) W.T.Aiton and Rosa canina L. was

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ACCEPTED MANUSCRIPT Materials and methods Plant name and parts used for extract preparation Spartium junceum L. flowers were collected from plants grown in the Botanical Garden of the University of Rome “Tor Vergata”. Vegetal tissues were powdered with pestle, mortar and liquid nitrogen, resuspended with ddH2O (aqueous flower extract, AFE; 100 parts of distilled water) or 50% Ethyl Alcohol (hydroalcoholic flower extract, HFE; 50 parts of ethanol 96 % V/V and 50 parts of distilled water), at a concentration of 200 mg of plant powder per ml of solvent, and left in

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agitation for 24 h, at room temperature, in the dark. After centrifugation at 13.000 rpm for 20 min, the pellet was discarded, while the supernatant was sieved by 0.22 µm filter, in order to obtain liquid extracts from Spartium junceum flowers (100% genuine extract; DER genuine 1:1; 0% excipients). The quantity of genuine extract in the herbal medicinal product was 1 ml/ml. The preparations were completely dried out, using a vacuum concentrator (Concentrator Plus,

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Eppendorf), and finally stored at -20 °C. For the experiments, both AFE and HFE samples were resuspended in 1.5 ml of PBS 1X, to maintain the starting concentration (200 mg/ml). Determination of simple phenol and flavonoid content

Simple phenol content was assessed by Folin-Ciocalteu method, according to the modified protocol reported in Impei et al. (2015). In particular, results were expressed as micrograms of gallic acid

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equivalents per gram of fresh material weight (μg GAE/g FMW), using a gallic acid calibration curve (0-30 mg/l). Flavonoid quantitation was assessed by aluminum chloride colorimetric method,

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according to the protocol reported in Gismondi et al. (2017). In detail, data were reported as micrograms of quercetin equivalents per gram of fresh material weight (μg QE/g FMW), comparing

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them with a quercetin calibration curve (0-50 mg/l). In vitro antioxidant tests

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FRAP antiradical assay was performed according to the protocol reported in Gismondi et al. (2013). ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid; Sigma Aldrich) free radical

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scavenging assay was carried out according to the method described in Gismondi et al. (2014). Both in FRAP and ABTS spectrophotometric assay, ascorbic acid was used to obtain a calibration curve (0-30 mg/l). Relative results were expressed as milligrams of ascorbic acid equivalent per gram of fresh material weight (mg AAE/g FMW). High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD) analysis AFE and HFE samples were subjected to a qualitative chromatographic analysis by HPLC system equipped with SPD-M20A diode array detector (DAD, Shimadzu, Japan). The investigations were carried out using a Kinetex C18 (2.6 µm x 2.1 mm x 100 mm) column, formic acid 1% (phase A) and methanol (phase B) as solvents and an elution gradient, at flow rate of 0.35 ml/min, set as 5

ACCEPTED MANUSCRIPT reported: t0 min (A 85%, B 15%); t3.5 min (A 65%, B 35%); t9 min (A 25%, B 75%); t11 min (A 85%, B 15%); t20 min (A 85%, B 15%). UV-visible absorption spectra, at 257 nm and 360 nm, were obtained. Then, for the quantitative analysis of AFE and HFE, the same HPLC-DAD detection instrument and solvents were used, applying the following method: t0 min (A 85%, B 15%); t20 min (A 65%, B 35%); t55 min (A 10%, B 90%); t68 min (A 85%, B 15%); t70 min (A 85%, B 15%). In this last analysis, a Phenomenex Luna C18(2) (3 µm x 4.6 mm x 150 mm; 3µm) column and a flow rate of 0.95 ml/min were applied. Identification and quantitation of 16 different plant metabolites

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were carried out by direct comparison with different concentrations of relative pure standards (Sigma-Aldrich), on the basis of retention time, absorbance spectrum and chromatographic peak area. The concentration of each compound was expressed as micrograms per 100 milligrams of fresh material weight (µg/100 mg FMW). Gas Chromatography-Mass Spectrometry (GC-MS) analysis

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AFE and HFE dried pellets were resuspended in 1.5 ml of methanol. Two µL of each sample were injected, in splitless modality, in a GC-MS QP2010 system (Shimadzu, Japan). The run was performed with an injection temperature of 250 °C, a DB-5 (30 m x 0.25 mm x 0.25 μL; Agilent Technologies, Santa Clara, USA) column and Helium as carrier gas at constant flow rate of 1

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ml/min. Temperature gradient applied to GC oven, during the analysis, was: at 70 °C for 2 min, then 200 °C at a rate of 25 °C/min, stay for 5 min, followed by a temperature of 300 °C at a rate of 3 °C/min, stay for 10 min. MS analysis was carried out in TIC (Total Ion Current) modality,

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applying an electron impact (EI) at 70 eV and scanning range from 100 to 900 m/z. Other parameters were: ion source temperature of 230 °C, interface temperature 250 °C and solvent cut

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time of 2 min. Compounds were identified by comparing their mass spectra with those of pure standards registered in the NIST v.14 library associated to the detection software (LabSolution

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v2.53 SU3, Shimadzu, Japan). Similarity values were considered acceptable only if higher than

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Cell culture and treatments Murine melanoma (B16-F10) and murine myoblast (C2C12) cell lines were maintained and propagated in Dulbecco’s Modified Eagle’s Medium (DMEM), under standard conditions (Gismondi et al., 2010). With respect to the other assays, for cell experiments, HFE dried pellet was resuspended in 250 µL of PBS 1X, reaching the final concentration of 1.2 g of plant material per ml of solvent. Cell treatments were performed incubating cells, for 24 and 48 h, with different concentrations of HFE: the equivalent of 0.1, 0.2, 0.5, 1, 2, 4, 6, 8 and 10 mg of raw plant material per ml of cell culture medium (HFE mg/ml). Control samples (CNT) were represented by cells

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ACCEPTED MANUSCRIPT treated with PBS 1X, for the same time and volume of the highest treatments. Doxorubicin (DOX; Sigma-Aldrich Co.), a well-known senescence inducer, was used at 0.3 μM for 48 h. Cell growth study, Selectivity Index (SI) calculation and Trypan Blue exclusion test Cell growth was measured by a 3-(4,5-dimethyl-thiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) based kit (Sigma-Aldrich Co., Italy), according to manufacturer’s instruction. Results were expressed as percentage variation of cell proliferation with respect to the control (PBS), taken as unit (100%). The degree of anticancer selectivity of the plant extract was expressed as following:

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Selectivity Index (SI) = IC50 C2C12 normal cell line⁄IC50 B16-F10 cancer cell line where IC50 (Inhibitory Concentration) is the concentration at which 50% of cell proliferation is inhibited.

Trypan Blue exclusion test was performed to measure HFE cytotoxicity by counting alive and dead cells with a Neubauer modified chamber, after staining with Trypan Blue dye (1%, w/v). diacetate (DCFH-DA)

carbonylated protein content evaluation

test,

cell

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2′,7′-dichlorodihydrofluorescein

cycle analysis

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DCFH-DA assay was performed as widely reported in Gismondi et al. (2014). In particular, negative and positive controls were always performed, incubating cells, respectively, with PBS 1X

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and 5 mM of H2O2 for 4 h at 37 °C before the exposure with DCFH-DA. Cell cycle was studied as described by Gismondi et al. (2013). In both studies, 10.000 cell events per sample were analyzed

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by FACSCalibur flow cytofluorimeter (Becton Dickinson, Milan, Italy) associated with CellQuest software. Carbonylated proteins were detected using the OxyBlot Protein Oxidation Detection Kit (Millipore, Vimodrone, Italy), according to manufacturer’s guidelines. For quantitation, the signal

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of carbonylated proteins was normalized to β-Actin, used as loading control, as reported in Gismondi et al. (2014).

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Cellular senescence assay

Senescence was investigated using a specific kit (Sigma-Aldrich Co., Italy) able to detect β-

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galactosidase activity, a specific marker for this biological process, staining senescent cells in blue. Stained cells were observed by light microscopy (20X, Nikon ECLIPSE E100). Images from random microscopic fields (5 per well) were captured by a camera (Progress capture pro 28.0.1) and colored cells were counted using ImageJ Software (version 1.48v; USA). Real-time PCR (qPCR) analysis Total RNA was extracted from cells by Pure Link RNA Mini Kit (Ambion, Life Technologies). cDNA synthesis and qPCR analysis were performed as widely reported in Mastropasqua et al. (2017). Primers used in this work were reported in Supplementary Material 1. The amount of mRNA for each gene was quantified using the 2-ΔΔCt formula, where the threshold cycle (Ct) of the 7

ACCEPTED MANUSCRIPT target gene detected in the treated sample is normalized for the internal reference gene (Actb, ΔCt) and for the respective value observed in control cells (ΔΔCt). Protein analysis Total protein extraction, separation on 12% SDS-polyacrylamide gel and Western Blotting technique were performed according to Gismondi et al. (2013). Used antibodies were: mouse monoclonal anti-Actin (Cell Signaling) and goat polyclonal anti-MITF (Microphthalmia-associated

and anti-goat secondary antibodies (Santa Cruz). Statistical analysis

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transcription factor, Santa Cruz) primary antibodies; horseradish peroxidase-conjugated anti-mouse

All results were expressed as means ± standard deviation (s.d.) of different measurements obtained by independent experiments (n≥3). Significance of the data was estimated with one-way ANOVA

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test. p value  < 0.05 was considered statistically significant. (p values: * < 0.05; ** < 0.01; *** <

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0.001).

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ACCEPTED MANUSCRIPT Results Hydroalcoholic extraction of S. junceum flowers is preferable than aqueous one Plant material was processed and subjected to aqueous (AFE) or hydroalcoholic (HFE) extraction, as widely described in Materials and Methods section. In order to understand which solvent was more suitable for isolation of secondary metabolites from S. junceum flowers, both extracts were analyzed by spectrophotometric and chromatographic approaches. Content of phenolic compounds and antiradical power of the extracts were estimated. HFE showed comparable levels of simple

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phenols (112.25 ± 12.55 μg GAE/g FMW) but higher doses of flavonoids (96.10 ± 1.50 μg QE/g FMW), with respect to AFE (96.60 ± 5.80 μg GAE/g FMW and 20.00 ± 1.85 μg QE/g FMW, respectively). Similarly, FRAP and ABTS assays, performed to determine in vitro antioxidant activity of the samples, corroborated previous data. Indeed, in FRAP test, HFE showed a stronger

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free radical scavenging effect (0.90 ± 0.01 mg AAE/g FMW) compared to AFE (0.73 ± 0.03 mg AAE/g FMW). On the other hand, in ABTS analysis, both extracts evidenced similar antiradical property (6.79 ± 0.08 mg AAE/g FMW and 6.89 ± 0.03 mg AAE/g FMW). As second step, a preliminary HPLC-DAD analysis was carried out, to obtain qualitative information about HFE and AFE

metabolic

profiles.

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Material

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spectrophotometric data, HFE presented UV-visible absorption profiles, at 257 and 360 nm (main wavelengths at which flavonoids and simple phenols absorb light, respectively), slightly richer than

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AFE ones. In particular, compared to AFE, HFE chromatographic profiles showed more peaks at high retention times (>4 min of elution).

AFE and HFE share only a portion of S. junceum phytocomplex

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In-depth chromatographic analyses were performed on AFE and HFE. HPLC-DAD detected and quantified 16 ubiquitous plant molecules in both extracts, by direct comparison with relative pure

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standards (Table 1). Kaempferol-3-o-glucoside and chlorogenic acid were the most abundant compounds in AFE (17.3 µg and 12.6 µg per 100 mg FMW, respectively). On the contrary, 4-

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hydroxybenzoic acid (36 µg/100 mg FMW) and rutin (10.21 µg/100 mg FMW) appeared as predominant molecules in HFE. A further characterization of AFE and HFE was performed by GCMS (Tables 2 and 3), in order to identify less polar metabolites. In this context, aqueous and hydroalcoholic extracts showed a total of 20 and 38 different metabolites, respectively. The most abundant molecules were o-Cymene (54.18%) and coumaran (14.92%) in AFE and coumaran (37.12%) and 5-methyl-1-phenylbicyclo[3,2,0]heptane (8.45%) in HFE. HFE extract inhibits B16-F10 growth without inducing cell death According to all previous evidence, our investigations continued by studying HFE bioactivity on B16-F10 murine melanoma cells, a highly aggressive and drug-resistant tumor cell line. 9

ACCEPTED MANUSCRIPT First of all, MTT assay was performed to evaluate the possible effect of S. junceum hydroalcoholic extract on tumor cell growth. However, C2C12 non-tumor murine myoblasts were also subjected to the same treatment, to verify that HFE was not toxic for normal cells. Screening experiments were carried out treating cells, for 24 and 48 h, at different concentrations (from 0.1 to 10 HFE mg/ml), in order to individuate doses able to affect tumor cell growth without inducing high toxicity on nontumor cells. PBS was used as appropriate control. Lower concentration of HFE did not exert any relevant antiproliferative effect on B16-F10 and C2C12 cells (Supplementary Material 3). On the

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contrary, as shown in Fig. 1A, after 24 h of incubation, S. junceum flower hydroalcoholic extract slightly decreased cell proliferation of 14.8%, 19.55%, 22.65%, 31.83%, respectively at 4, 6, 8, 10 HFE mg/ml, compared to CNT sample. The same extract did not noticeably influence C2C12 cell growth, at any concentration, with respect to the control (in detail, 0.05%, 6.06%, 11.52%, 17.34%, 22.79% of reduction, respectively at increasing doses) (Fig. 1B). The best results were obtained

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after 48 h of treatment. Indeed, as shown in Fig. 1C, we observed a significant reduction of B16F10 cell amount, especially at higher treatment concentrations (10.45%, 23.75%, 37.25%, 41.15%, 56.43% after exposure to 2, 4, 6, 8, 10 HFE mg/ml, in that order), while C2C12 proliferation was less impaired (23.57%, 28.10%, 26.76%, 24.15%, 26.15% of decrease at 2-10 HFE mg/ml,

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respectively) (Fig. 1D).

In addition, IC50 value and selectivity index (SI) of HFE treatment, for normal C2C12 myoblasts and cancerous B16-F10 cells, were calculated. IC50 value for C2C12 cells was 19.58 mg/ml, after

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24 and 48 h of incubation, whereas these values were 15.55 and 9.20 mg/ml, respectively, for B16F10 cells. The corresponding selectivity indexes of C2C12/B16-F10 were 1.26 and 2.13, in that

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order, at 24 and 48 h.

Finally, cytotoxicity was evaluated performing the Trypan Blue exclusion test. In particular, by

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counting the amount of alive cells, we generated B16-F10 and C2C12 cell growth curves, confirming MTT results (Fig. 1E and 1F). Moreover, cell death levels were measured by detecting

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the percentage of dead cells. As reported in Fig. 1G, surprisingly, we observed that no HFE treatment exerted a strong toxic effect on B16-F10 cells, although a significant cell proliferation decrease was highlighted by MTT assay and proliferation curves. As regards C2C12, detected citotoxicity was not relevant, with respect to the control, except that after 48 h of treatment with 8 and 10 HFE mg/ml (21.8% and 26.8% of dead cells, respectively). HFE exerts time- and dose-dependent pro-oxidant activity Considering the great antiproliferative effect of HFE on B16-F10 cells at 4, 8 and 10 mg/ml doses, not associated to cytotoxicity, only these concentrations were selected for further analyses.

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ACCEPTED MANUSCRIPT First of all, in order to clarify if HFE bioactivity might be exerted by regulating cell oxidative status, the amount of intracellular reactive oxygen species (ROS) was monitored, using DCFH-DA assay. B16-F10 cells were incubated with 4, 8 and 10 mg/ml HFE, for 4, 24 and 48 h, or with hydrogen peroxide (5 mM, 4 h) as positive control. As shown in Fig. 2A, after 4 h of treatment with 4 HFE mg/ml, cells showed a slight decrease of ROS level (16.84%), compared to the control (PBS). On the contrary, using 8 and 10 mg/ml of HFE, they increased ROS amount of 43% and 61.1%, respectively. However, in all cases, during the time course, ROS levels slightly decreased.

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Indeed, respectively at 24 and 48 h, compared to the control, intracellular fluorescence was equal to -5.54% and -6.03% at 4 HFE mg/ml, +19.1% and +11.8% at 8 HFE mg/ml and +24.4% and +21.4% at 10 HFE mg/ml.

HFE pro-oxidant activity on B16-F10 cells was confirmed by studying the level of carbonylated proteins. After incubation, for 4, 24 and 48 h, with 4, 8 and 10 mg/ml of HFE, cell proteins were

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extracted. Oxidized proteins were labeled, using a specific kit and analyzed by Western Blotting. As indicated in Fig. 2B, 4 HFE mg/ml treatment did not induce substantial oxidative post-translational modifications, whereas higher doses increased carbonylation phenomena, in a dose- and timedependent way. In detail, 8 HFE mg/ml determined an increase of protein carbonylation, after 4, 24

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and 48 h, respectively, of 150.3%, 99% and 22.5%. Similarly, 10 HFE mg/ml, as shown in the representative Western Blotting analysis reported in Fig. 2C, increased oxidized proteins of 191.1%,

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91.9% and 68.3%.

HFE extract decreases melanogenesis-related gene expression and protein levels As Microphthalmia-associated transcription factor (Mitf) pathway is fundamental and peculiar in

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melanoma cells (Kawakami and Fisher, 2017), the expression level of Mitf, Tyrosinase (Tyr) and Tyrosine-related protein 1 (Tyrp1) genes, in B16-F10 cells, after 48 h of treatment with HFE, was

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investigated. Four mg/ml HFE caused the reduction of Mitf and Tyrp1 transcripts of 12.9% and 22.8%, in that order, while a slight increase of Tyr mRNA level (5.5%) was detected, compared to

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the control. In the case of 8 mg/ml HFE treatment, instead, all mRNA levels decreased. However, the lowest values were obtained at 10 mg/ml of HFE, which reduced Mitf, Tyr and Tyrp transcripts of 64.7%, 25.4% and 54.9%, respectively, in comparison with the control (Fig. 3A). To validate previous qPCR data and quantify Mitf protein level, a Western Blot analysis was performed (Fig. 3B). With respect to the control (considered as unit, 100%), after 48 h of incubation with 4, 8 and 10 mg/ml of HFE, a similar decrease of Mitf was observed in all samples (73.4%, 70.7% and 68.2%, respectively), as shown by densitometric quantitation performed using Actin as internal loading control (Fig. 3C).

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ACCEPTED MANUSCRIPT HFE arrests cell cycle and induces senescence in B16-F10 melanoma cells Finally, to better clarify HFE activity on melanoma cells, we analyzed B16-F10 cell cycle after treatment with HFE, for 24 and 48 h. In both cases, we observed an increase of cells in G2/M phase, with respect to the control, which was directly proportional to the treatment dose. In Fig. 3D, we reported the results obtained after 48 h of treatment. As shown, 4, 8 and 10 mg/ml of HFE treatments caused an increase of 6.3%, 7.2% and 9.4% of cells in G2/M phase, respectively. Moreover, flow cytometric assay confirmed the absence of a pronounced cell death in B16-F10 cell

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line after HFE treatments, as previously demonstrated by Trypan Blue exclusion test. Indeed, the amount of cells in sub-G0 phase was 2.48%, 6.33%, 8.19% and 11.59%, in that order, in CNT, 4, 8 and 10 mg/ml of HFE.

This experiment suggested that HFE could induce senescence in murine melanoma cells. To confirm it, a specific kit able to stain (in blue) senescent cells was used. Images of each sample,

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obtained by optical microscopy, were reported in Fig. 4. We compared negative (PBS; Fig. 4A) and positive (doxorubicin-treated cells; Fig. 4B) controls (which showed 0.3% and 89.1% of senescent cells, in that order) with HFE treated cells. After 24 and 48 h of treatments with 4 mg/ml of HFE, we detected only 1.2% and 1.4% of blue cells, respectively (Fig. 4C and 4D). Using 8 mg/ml HFE,

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for 24 and 48 h (Fig. 4E and 4F), the percentage of stained cells slightly increased at 4.7% and 4.8%, respectively. The best results were obtained with 10 mg/ml HFE treatments (Fig. 4G and 4H): the extract induced a pronounced senescence phenotype (65.6% and 71.4% of blue cells,

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respectively, at 24 and 48 h).

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ACCEPTED MANUSCRIPT Discussion In order to face biotic and abiotic stresses typical of Mediterranean areas, such as exposure to high levels of UV light, plants produce huge amounts of secondary metabolites. These molecules, showing an elevated bioactivity even on mammalian systems as pro- or antioxidant agents, are considered of great scientific, medical and pharmaceutical interest. For this reason, in the present work, we investigated the effect of extracts obtained from three Mediterranean plants (P. tobira (Thunb.) W.T.Aiton, R. canina L. and S. junceum L.) on neoplastic and non-tumor murine cell

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lines. The capacity of these plant extracts to regulate the growth rate of B16-F10 murine melanoma cells and C2C12 murine myoblasts was investigated. P. tobira leaf extract was toxic for C2C12 cells and unable to exert any relevant effect on melanoma cell proliferation, whereas R. canina hip extract seemed to have pro-tumor activity on B16-F10 cells (Supplementary Material 4). This evidence induced us to exclude P. tobira and R. canina extracts from further analyses, focusing our

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attention on S. junceum.

Two solvents, with different polarity (water and 50% ethyl alcohol), were employed to extract secondary metabolites from Spanish broom flowers. With respect to the aqueous solution (AFE), the hydroalcoholic extract (HFE) presented a higher content of plant metabolites, as demonstrated

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by spectrophotometric, HPLC-DAD and GC-MS analyses. In particular, by HPLC-DAD technique, we detected and quantified different ubiquitous plant chemical compounds in both samples (Table 1). Significant differences were observed; for instance, the most abundant compounds (4-

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hydroxybenzoic acid, rutin) of HFE were totally absent in the aqueous extract. On the other hand,

HFE profile.

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AFE showed the presence of chlorogenic acid, kaempferol and apigenin, which were not present in

UV-visible absorption chromatographic profiles of HFE revealed the presence of additional peaks at

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high retention times, corresponding to molecules with lower polarity, compared to AFE (Supplementary Material 2). For this reason, we also compared AFE and HFE metabolic profiles by

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GC-MS technique, in order to identify less polar compounds. These analyses (Table 2 and 3) allowed us to identify different and peculiar molecules in both extracts. The common one was coumaran, even if it was present in different relative abundances. Moreover, in HFE, we identified specific metabolites, such as 3-methylcinnoline, methylquinol and thymol, whose presence in Spanish broom flowers has not been documented yet. Indeed, since only rare scientific papers report the chemoprofile of S. junceum flowers (Bilia et al., 1993; Cerchiara et al., 2012; De Rosa and De Stefano, 1982; Mancini et al., 2010; Yeşilada and Takaishi, 1999; Yeşilada et al., 2000), the present research provides new and important information about Spanish broom phytocomplex.

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ACCEPTED MANUSCRIPT HFE, probably due to its chemical composition, showed a greater antiradical power with respect to AFE, in FRAP and ABTS in vitro tests. All these data indicated that the hydroalcoholic extraction was better than the aqueous method to isolate bioactive compounds from S. junceum flowers. Consequently, next investigations were carried out only on HFE. The second purpose of our work was the study of HFE bioactivity on B16-F10 cells. The underlying hypothesis was the possibility that S. junceum antioxidant molecules could modulate the oxidative state typically dysregulated in animal tumor cells (Benhar et al., 2002), restoring their

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redox equilibrium.

To achieve it, B16-F10 cells were treated, for 24 and 48 h, with different concentrations of HFE (from 0.1 to 10 mg/ml) and subjected to MTT assay (Supplementary Material 3; Fig. 1A, 1C). We individuated 4, 8 and 10 mg/ml as the most effective doses on murine melanoma cells. Our results showed that S. junceum extract was extraordinarily able to reduce B16-F10 growth, in a dose and

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time dependent manner, reaching more than 50% of inhibition in cell proliferation.

Since the main goal would have been to demonstrate the antineoplastic activity of our extract and its concomitant safety on healthy tissues, we also tested HFE on C2C12 murine myoblasts, a nontumor proliferating cell model system. Surprisingly, we discovered that HFE did not strongly

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reduce C2C12 cell growth (Fig. 1B and 1D). To validate it, we proceeded calculating IC50 values and selectivity index for HFE treatments on C2C12 and B16-F10 cells. HFE was found to exert a time-dependent and selective cytotoxicity on B16-F10 cell line. Indeed, as known in literature, a SI

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value greater than, or equal to, 2.0 is an interesting selectivity index (Badisa et al., 2009). MTT data were further confirmed by generating cell proliferation curves, after staining with Trypan

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Blue solution (Fig. 1E and 1F). Detecting dead cells, this specific dye allowed us also to obtain cytotoxicity value of HFE treatments. In particular, we observed that the extract did not determine

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high level of cell death in both cell lines (Fig. 1G). The only significant toxic effects (>20%) were observed on C2C12 cells treated for 48 h with 8 and 10 HFE mg/ml. This evidence indicated that

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the reduction of C2C12 cell growth in presence of HFE might be associated to Spanish broom toxic activity on myoblasts, a phenomenon which did not occur on B16-F10 (where dead cells were always < 12%). The inability of HFE to induce B16-F10 cell death was also confirmed by propidium iodide flow cytometric assay, measuring sub-G0 events. All these findings supported the hypothesis that S. junceum flower extract, being non-toxic for melanoma cells, reduced B16-F10 proliferation according to a different molecular pathway with respect to cell death. These interesting results prompted us to investigate which mechanism was activated by HFE in B16-F10 cells. Since we demonstrated HFE was rich in antioxidant metabolites, we firstly 14

ACCEPTED MANUSCRIPT evaluated its possible effect on B16-F10 cell redox state. We observed that, at high doses, our extract exerted a pro-oxidant activity (Fig. 2). Indeed, despite polyphenolic compounds are considered natural free radical scavengers, several studies demonstrated that they can act as prooxidants, under certain conditions (e.g. high doses, presence of metal ions) (Bouayed and Bohn, 2010; Gismondi et al., 2015). In light of these considerations, HFE antiproliferative effect on B16F10 cells was supposed to be associated to radical properties of its chemical components. Indeed, as suggested by Kim and colleagues (2014) and Liu-Smith and Meyskens (2016), both high levels of

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ROS and specific secondary metabolites seem to possess an antimelanogenic activity in melanoma and melanocytic cells. More in detail, they negatively target Mitf pathway, usually linked to cell life, differentiation and proliferation, which includes Tyrosinase (Tyr) and Tyrosine-related protein 1 (Tyrp-1) enzymes. To validate this theory, in treated B16-F10 cells, we measured the mRNA levels of Mitf, Tyr and Tyrp1 by qPCR (Fig. 3A) and the amount of Mitf protein by Western

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Blotting (Fig. 3B and 3C). Results clearly demonstrated that HFE exerted an antimelanogenic effect, turning off Mitf signaling.

Since low levels of Mitf (Goding, 2011; Schwahn et al., 2005) and high dose of ROS (Petrova et al., 2016; Ziegler et al., 2015) were also associated with senescence, we wondered if the

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antiproliferative effect of HFE on B16-F10 could be due to the induction of this process. Senescence is an irreversible phenomenon, which leads to the arrest of cell metabolism and division, usually at G0/G1 or G2/M phases (Gire and Dulic, 2015). For this reason, we monitored

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the cell cycle of HFE-treated cells (Fig. 3D). We found that Spanish broom extract determined a dose-dependent increase of cells in G2/M phase. However, the crucial confirmation that HFE

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triggered senescence in B16-F10 cells was obtained using a kit for the detection of β-galactosidase activity. Indeed, this enzyme is present at pH 6 only in senescent cells and not in quiescent and

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tumor ones (Bernardes de Jesus and Blasco, 2012). In our case, high concentrations of HFE

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significantly increased the amount of cells with aging phenotype (Fig. 4).

Conclusion

In conclusion, we proved that S. junceum hydroalcoholic flower extract, probably thanks to its rich biochemical profile and pro-oxidant activity, is able to inhibit melanogenesis and induce senescence in B16-F10 murine melanoma cells, preventing hyper-proliferating events. All these data led us to believe that the Spanish broom phytocomplex can represent a promising candidate as antimelanoma agent. Indeed, it would represent an alternative agent to actual toxic chemotherapeutics (e.g. cisplatinum; Wehler et al., 2017), being slightly damaging for non-tumor cells and highly selective and efficient against cancer cells. 15

ACCEPTED MANUSCRIPT Conflict of interest The authors state no conflict of interest. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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34. The American Cancer Society. What are the key statistics about melanoma skin cancer? Last Revised: 2017. http://www.cancer.org/cancer/skincancermelanoma/detailedguide/melanoma-skin-cancer-key-statistics. 35. Wehler, E., Zhao, Z., Pinar Bilir, S., Munakata, J., Barber B., 2017. Economic burden of toxicities associated with treating metastatic melanoma in eight countries. Eur J Health Econ. 18, 49-58. 36. Yeşilada, E., Takaishi, Y., 1999. A saponin with anti-ulcerogenic effect from the flowers of Spartium junceum. Phytochemistry 51, 903-8. 37. Yeşilada, E., Tsuchiya, K., Takaishi, Y., Kawazoe, K., 2000. Isolation and characterization of free radical scavenging flavonoid glycosides from the flowers of Spartium junceum by activity-guided fractionation. J Ethnopharmacol. 73, 471-8. 38. Ziegler, D.V., Wiley, C.D., Velarde, M.C., 2015. Mitochondrial effectors of cellular senescence: beyond the free radical theory of aging. Aging Cell 14, 1-7.

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ACCEPTED MANUSCRIPT Table legends Table 1. HPLC-DAD chemical profiles of AFE and HFE. List and concentration of metabolites detected in AFE and HFE by HPLC-DAD analysis. Results were expressed as µg of molecule per 100 mg of fresh material weight (µg/100 mg FMW) and represent the mean ± s.d. of six

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HPLC-DAD detected compound μg/100 mg FMW ± s.d. AFE HFE 4-Hydroxybenzoic n.d. 36.00 ± 0.04 acid Rutin n.d. 10.21 ± 0.01 Ellagic acid 3.00 ± 0.03 2.87 ± 0.01 Kaempferol-3-o17.37 ± 0.05 2.16 ± 0.02 glucoside Caffeic acid 1.09 ± 0.01 1.47 ± 0.04 Caffeic acid 1,10.26 ± 0.01 1.07 ± 0.03 dimethylallyl ester Caffeic acid phenolic 1.90 ± 0.02 0.94 ± 0.02 ester Quercetin-3-o3.10 ± 0.05 0.94 ± 0.03 arabonoside Gallic acid n.d. 0.82 ± 0.04 Kaempferol 0.33 ± 0.01 0.63 ± 0.03 p-Coumaric acid 6.70 ± 0.04 0.67 ± 0.02 Genistein n.d. 0.48 ± 0.02 Vanillic acid n.d 0.11 ± 0.01 Quercetin 1.17 ± 0.03 0.04 ± 0.01 Chlorogenic acid 12.57 ± 0.04 n.d. Apigenin 0.19 ± 0.02 n.d.

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independent replicates.

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Table 2. GC-MS chemical profile of AFE. List of secondary metabolites detected in AFE by GCMS analysis. The relative abundance of each compound was reported as percentage value with

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respect to the total mixture (100%). The values correspond to the mean of three independent

peak area.

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replicates. The maximum s.d. for each measurement was lower than 5% of the respective molecule

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GC-MS detected AFE compound o-Cymene Coumaran 2,6-Cyclooctadiene-1-carboxylic acid, 4,5dimethyl-, methyl ester 2,6-Dioxaadamantane, 8-iodo-4-acetoxy2,4-Diphenyl-4-methyl-2(E)-pentene Carbamic acid, N-(2,3-dimethylphenyl)-, oxiranylmethyl ester 1,2,4-Thiadiazole, 5-amino-3-propylHeptanal, diethyl acetal N-Benzyl-N-ethyl-p-isopropylbenzamide 1-(4-Hydroxy-6-methyl-2-pyrimidinyl)-2aziridinone 2(1H)-Quinolinone, 3,4-dihydroThiocyanic acid 3-pyridyl ester 2-Butenoic acid, 2-methoxy-3-methyl-, methyl ester

% 54.18 14.92 6.44 6.01 4.35 4.21 3.85 3.33 0.54 0.37 0.36 0.21 0.21

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ACCEPTED MANUSCRIPT Propenamide, 3-(2-hydroxyphenyl)-N,NdimethylNaphthalen-4a,8a-imine, 1,4,5,8tetrahydroSuccinic acid, 8-chloroctyl ethyl ester 2,4-Nonadien-1-ol 3-Tosyl sedoheptulose Malonic acid, 2-(2,3dihydrobenzo[b]furan-5-yl)-2-hydroxy-, dimethyl ester 5-Methyl-1,3-oxathiane

0.20 0.19 0.16 0.16 0.14 0.12

0.05

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Table 3. GC-MS chemical profile of HFE. List of secondary metabolites detected in HFE by GCMS analysis. The relative abundance of each compound was reported as percentage value with respect to the total mixture (100%). The values correspond to the mean of three independent replicates. The maximum s.d. for each measurement was lower than 5% of the respective molecule

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% 37.12 8.45 6.88 6.87 6.19 4.46 4.36 3.89 3.58 3.56 2.83 2.58 2.47 1.94 0.80 0.59 0.44 0.42

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GC-MS detected HFE compound Coumaran 5-Methyl-1-phenylbicyclo[3,2,0]heptane 3-Methylcinnoline l-(+)-Ascorbic acid 2,6-dihexadecanoate Neronine Stearic acid Lepidine, 2-chloro-6-methoxyThymol Coumarin, 3,4-dihydro-4,4,5,7,8pentamethyl-6-fluoroPalmitic acid, ethyl ester Linolenic acid Methylquinol Quinoline derivative Linolenic acid, ethyl ester 2-Propylquinoline 9-12-Octadecadienoic acid, methyl ester (-)-Isolongifolol, methyl ether 3,11-Diazatricyclo[7,3,1,0(3,8)]trideca-57-dien-4-one,11-(2-hydroxyethyl)Isoquinoline,1,2,3,4-tetrahydro-7methoxy-2-methyl-8-(phenylmethoxy)derivative beta-Vatirenene 3,4,5,6-Tetradehydrospartein-2-one Retinal 2-Nitro-2'-methoxy-stilbene 6-Methoxy-3-methyl-8-nitro-5-[[4phenoxy]phenoxy]quinoline Ergocalciferol Isoflavone C(14a)-Homo-27-nor-14,betagammaceran-3-alpha-ol Methyl (19trans)-16[(acetyloxy)methyl]sarpagan-17-oate Isolongifolene, 4,5,9,10-dehydroSolasodine Quebrachamine

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peak area.

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0.31 0.28 0.24 0.13 0.13 0.12 0.12 0.11 0.09 0.09 0.09 0.08

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ACCEPTED MANUSCRIPT Lup-20(29)-en-28-oic acid, 3-betahydroxy-methyl ester Obscurinervinediol Caryophyllene oxide Lupeol Nerolidyl propionate Benzene, 1,1'-(3,3-dimethyl-1butenylidene)bisBacchotricuneatin c

0.07 0.06 0.06 0.05 0.05 0.04 0.03

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Figure legends

Fig. 1. MTT assay, proliferation curves and cytotoxicity analysis. B16-F10 (A and C) and C2C12 (B and D) cell growth was measured after 24 (A and D) and 48 (C and D) h of treatments with PBS and different concentrations (2-10 mg/ml) of HFE. Results, expressed as percentage with respect to the PBS control (considered as unit, 100%), represented the mean ± s.d. of four

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independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001 vs control). Proliferation curves of B16-F10 (E) and C2C12 (F) cells were generated counting, with a Neubauer modified chamber, the amount of alive cells, after staining with Trypan Blue, at 0, 24 and 48 h of treatments with HFE. Results were indicated as mean ± s.d. of four independent experiments (p < 0.05 vs control). (G)

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The percentage of C2C12 and B16-F10 dead cells, obtained by Trypan Blue exclusion test, after 24 and 48 h of HFE treatments (2-10 mg/ml), was reported. All results were indicated as mean ± s.d. of

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four independent experiments (p < 0.05 vs control).

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Fig. 2. Cell redox state analysis. (A) The amount of intracellular ROS was quantified in B16-F10 cells, treated with 4, 8 and 10 mg/ml of HFE for 4, 24 and 48 h, by DCFH-DA fluorescent assay. ROS concentration was reported as percentage compared to the control (PBS). Hydrogen peroxide

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treatment was performed as positive control. Data were expressed as mean ± s.d. of three independent measurements (n.s. = not significant; *p < 0.05; **p < 0.01; ***p < 0.001 vs control).

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(B) Carbonylated protein levels were measured, after treatments with HFE 4, 8 and 10 mg/ml for 4, 24 and 48 h, by using the OxyBlot Protein Oxidation Detection Kit and by Western Blotting

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analysis. Results were reported as percentage amount of carbonylated proteins, with respect to the control (PBS, 100%). Actin signal was used as normalizer, representing a loading control. Data represented the mean ± s.d. of three independent experiments (*p < 0.05; **p < 0.01 vs control). (C) A representative Western Blot analysis of oxidized protein levels, after exposure to HFE 10 mg/ml for 4, 24 and 48 h, was shown.

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Fig. 3. qPCR and cell cycle analyses. (A) Mitf, Tyr and Tyrp1 mRNA levels, measured by qPCR in B16-F10 cells treated for 48 h with 4, 8 and 10 mg/ml of HFE, were reported. Gene expression,

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calculated as transcript amount after normalization for Actin mRNA, was reported as percentage with respect to the PBS CNT (considered as unit, 100%). Data represented the mean ± s.d. of three

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independent measurements (*p < 0.05; **p < 0.01 vs control). (B) A representative Western Blot analysis of Mitf and Actin protein levels was shown. The double band visualized by anti-Mitf

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antibody is due to the existence of two Mitf isorforms (UniProt Database; isoform M ID: Q08874-8, isoform M1 ID: Q08874-9). (C) Quantitation of Mitf protein, in B16-F10 cells treated for 48 h with 4, 8 and 10 mg/ml HFE, was reported. Results, obtained from the ratio between Mitf and Actin (used as loading control), were indicated as percentage values with respect to the control (PBS, 100%). Data indicated the mean ± s.d. of three independent experiments (*p < 0.05; **p < 0.01 vs control). (D) Cell cycle analysis, of B16-F10 cells treated with 4, 8 and 10 mg/ml HFE for 48 h, was shown. For each sample, the percentage amount of cells in every cycle phase (G0/G1, S and G2/M) was measured by cytofluorimetric analysis. Results were expressed as mean ± s.d. of three independent experiments (*p < 0.05; ***p < 0.001 vs control). 23

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Fig. 4. Senescent cells detection. B16-F10 cells were treated with PBS (negative control; A panel), doxorubicin (positive control; B panel), 4 mg/ml (C and D panels), 8 mg/ml (E and F panels) and 10 mg/ml (G and H panels) HFE, for 24 (C, E and G panels) and 48 h (A, B, D, F and H panels),

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and then subjected to a specific kit able to stain in blue senescent cells. Images were captured by a camera associated with a light microscope. The arrows indicate single cells showing the senescent

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phenotype. The black bars indicate 60 μm.

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Graphical abstract 25

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