Novel acrylic polymers for food packaging: Synthesis and antioxidant properties

Food Packaging and Shelf Life 11 (2017) 84–90

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Novel acrylic polymers for food packaging: Synthesis and antioxidant properties Alessia Fazio* , Maria Cristina Caroleo, Erika Cione, Pierluigi Plastina Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, CS, Italy

A R T I C L E I N F O

Article history: Received 2 May 2016 Received in revised form 16 December 2016 Accepted 16 January 2017 Available online xxx Keywords: Tyrosyl acrylate Hydroxytyrosyl acrylate Candida antarctica lipase Polyacrylate Antioxidant polymer Cytotoxicity property

A B S T R A C T

In the present work, a strategy for the synthesis of novel polyacrylates covalently linked to natural antioxidants was accomplished in a two-step process. First, monomers were prepared via lipasecatalyzed transesterification of acrylic acid methyl ester with tyrosol (T) or hydroxytyrosol (HT). Then, tyrosyl acrylate (TA) and hydroxytyrosyl acrylate (HTA) were subjected to radical homopolymerization to give poly(tyrosyl)acrylate (PTA) and poly(hydroxytyrosyl)acrylate (PHTA), respectively. The monomers and the corresponding homopolymers were characterized with FT-IR and NMR techniques and by Folin-Ciocalteu method to give an estimation of the available phenolic groups, as T and HT equivalents, linked to the polymers. The results of DPPH radical scavenging assay indicate that the free radical scavenging activity of tyrosol and hydroxytyrosol was almost completely retained in the corresponding monomers and polymers. In addition, polyacrylate films did not exhibit any cytotoxic activities in vitro on RAT1 normal fibroblast cells, using MTT assay. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction The principal function of packaging is protection and preservation from external contamination (Robertson, 2006). This function involves retardation of deterioration, extension of shelf life, and maintenance of quality and safety of packaged food. Packaging protects food from environmental influences such as heat, light, the presence or absence of moisture, oxygen, pressure, enzymes, spurious odors, microorganisms, insects, dirt and gaseous emissions. Traditional food packages are passive barriers designed to delay the adverse effects of the environment on the food product. Otherwise, “active packaging” goes beyond the traditional role of packaging by imparting specific, intentional functionality to the packaging system. An active packaging presents on its surface active functional groups that interact with the food therein contained inhibiting its deterioration and preserving its organoleptic characteristics (Català & Gavara, 2001). Developments in active packaging have led to advances in many areas, including delayed oxidation and controlled respiration rate, microbial growth, and moisture migration. (Day, 2008; Kerry, O'Grady, & Hogan, 2006; Ozdemir & Floros, 2004). Antioxidant packaging includes antioxidant substances in

* Corresponding author. E-mail address: [email protected] (A. Fazio). http://dx.doi.org/10.1016/j.fpsl.2017.01.002 2214-2894/© 2017 Elsevier Ltd. All rights reserved.

food packaging systems to impart antioxidant activity. These agents can be applied into the packaging systems in different forms, mainly including independent sachet packages, adhesive-bonded labels, physical adsorption/coating on packaging material surface, being incorporated into packaging polymer matrix, multilayer films, and covalent immobilization onto the food contact packaging surface (Tian, Decker, & Goddard 2013). Both synthetic and natural antioxidant compounds are widely used in food and personal care products to increase stability, shelf life and preserve nutritional quality. There has been a growing interest in the substitution of synthetic food antioxidants like butylated hydroxyl anisole (BHA) and butylated hydroxyl toluene (BHT) by natural ones because they are suspected carcinogens and the US Food and Drug Administration (FDA) has therefore, restricted their use (Code of Federal Regulations, 2012; Kim & Lee, 2004; Mandlekar & Kong, 2000). Therefore, the use of natural antioxidants, such as hydroxy and polyhydroxy derivatives of cinnamic acids, is to be preferred to synthetic ones. Hydroxytyrosol (2-(3,4-dihydroxyphenyl)ethanol) and tyrosol (2-(4-hydroxyphenyl)ethanol) are phenolic compounds naturally present in olive trees (Olea europaea L.), fruits, olive oil and olive mill waste (‘alperujo’), both in their molecular form or as part of more complex molecules, mostly as esters of elenolic acid (Bendini et al., 2007; Silva, Gomes, Leitao, Coelho, & Boas, 2006). Several studies have reported various biological activities of hydroxytyrosol and tyrosol, such as antimicrobial (Bisignano et al., 1999), anti-

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carcinogenic (Owen et al., 2000) and anti-inflammatory (Haloui, Marzouk, Marzouk, Bouraoui, & Fenina, 2011), but especially as powerful antioxidant agents (Aeschbach et al., 1994; Visioli, Poli, & Galli, 2002; Perez-Bonilla, Salido, van Beek, & Altarejos, 2014). In fact, the antioxidant capacity of hydroxytyrosol is higher than that of other phenolic compounds with similar structures and other natural antioxidants such as vitamin C, vitamin E or resveratrol (Garcia-Garcia, Hernandez-Garcia, Sanchez-Ferrer, & Garcia-Carmona, 2013). In addition, a direct dissolution of the antioxidant in the food matrix is sometimes inconvenient because it would result in undesirable alteration in the product composition. In this perspective, the development of polymeric materials containing the active moiety covalently bound to their surface is an interesting alternative. Among the main advantages of synthetic polymers utilized in packaging industries such as polypropylene, poly(vinyl chloride), and polyethylene, their excellent physicochemical properties as well as the possibility of their being processed and their low cost can be mentioned. Poly(methyl methacrylate) (PMMA), owing to its excellent surface hardness, UV and abrasion resistance, and a myriad of coloring options from transparent to deep color, has been widely used in various sectors including transportation, architecture, electronics, and health (Unnikrishnan, Smita, & Nayak, 2014). However, these commodities present an inert surface. Therefore, one of the approaches to obtain active packages from synthetic polymers is carried out using surface modification techniques through chemical processes (Arrua, Strumia, & Nazareno, 2010). Recently, there has been an increasing interest in the immobilization of functional compounds onto the food contact surface of packaging by covalent linkages (Tian, Decker, & Goddard 2012). Covalent bonds can provide the most stable linkage between substrate film surface and active agents, a potential regulatory benefit. The active agents should not be labeled as food additives, as they are not likely to migrate from the package to the food (Tian, Decker, McClements, & Goddard 2014). Taking into account the above-mentioned precedents the main aim of this work was the synthesis and characterization of novel polyacrylates covalently linked to natural antioxidants, tyrosol (T) and hydroxytyrosol (HT). The active polymers could be used for food packaging applications as active monomaterial in direct contact with the food. In this perspective, the efficiency of the new antioxidant materials described herein in protecting a real food sample was also evaluated. Moreover, screening the polyacrylate derivatives for cytotoxicity properties on RAT1 fibroblast cell proliferation provides some level of assurance of their safety for food application. 2. Materials and methods 2.1. Materials Immobilized Candida antarctica lipase B (CALB, Novozym1435) was supplied by Novozymes A/S. Methyl acrylate (99%), FolinCiocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH
), tyrosol (98%) were purchased from Sigma Aldrich (Italy). All chemicals were used as received. The synthesis of 2-(3,4-dihydroxyphenyl)ethanol was performed according to literature method (Capasso, Evidente, Avolio, & Solla, 1999). Dimethylformamide (DMF) was freshly distilled.

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reported in parts per million (d, ppm), downfield to tetramethylsilane (Me4Si) as an internal standard (d = 0.00 ppm), or referenced to residual solvent CHCl3 (1H NMR 7.27 ppm and 13C NMR 77.0 ppm); coupling constants J are given in Hertz. The FTIR spectra were performed using a Perkin-Elmer Paragon 1000 PC FTIR spectrometer. Quantitative analysis of ascorbic acid in natural juice samples was performed by high-performance liquid chromatography (HPLC). The determinations were carried out using a Shimadzu HPLC system, equipped with two SCL-10-AVP pumps, an SLC-10-AVP controller, and an SPD-20A UV–vis detector: the column used was a discovery HS C18 (Supelco) (250 mm  4.6 mm id, 5 mm particle size). 2.3. Synthesis of the monomers tyrosyl acrylate (TA) and hydroxytyrosyl acrylate (HTA) The reaction was an enzymatic transesterification where the primary hydroxyl group of tyrosol and hydroxytyrosol was regioselectively acylated by acrylic acid methyl ester via the acyl enzyme complex. In a typical reaction, antioxidant compound (1.6 mmol), acrylic acid methyl ester (16 mL, 176 mmol), Candida antarctica lipase B (200 mg, immobilized) were stirred for 24 h at 50  C. The enzyme was filtered off, the solvent was evaporated under reduced pressure and the product was purified by column chromatography (SiO2, hexane: acetone = 9:1; TA yield 96%, HTA yield 75%). TA and HTA were analyzed by GC–MS, FTIR, 1H and 13C NMR. 2.3.1. Characterization of tyrosyl acrylate (TA) MS m/e 192 (M+, 1), 121 (11), 120 (100), 107 (36), 77 (11), 55 (18); IR (neat) 3392 (br, s), 3021 (w), 2960 (w), 1704 (s), 1615 (m), 1516 (m), 1411 (m), 1302 (m), 1220 (s), 1064 (m), 983 (m), 812 (m) cm1; 1 H NMR (300 MHz, CDCl3) d (ppm) 7.12-7.04 (m, 2H, on phenyl ring), 6.82-6.75 (m, 2H, on phenyl ring), 6.40 (dd, J = 17.3, 1.4 Hz, 1H, CHH¼CH), 6.11 (dd, J = 17.3, 10.4 Hz, 1H, CHH¼CH), 5.83 (dd, J = 10.4, 1.4 Hz, 1H, CHH¼CH), 5.77 (s, 1H, OH), 4.33 (t, J = 7.1, 2H, CH2CH2OC¼O), 2.90 (t, J = 7.1, 2H, CH2CH2OC¼O). 13C NMR (75 MHz, CDCl3) d (ppm) 165.5, 154.5, 130.9, 130.1, 129.7, 128.5, 115.5, 65.4, 34.3. 2.3.2. Characterization of hydroxytyrosyl acrylate (HTA) MS m/e 208 (M+, 1), 137 (10), 136 (100), 123 (28), 77 (8), 55 (18); IR (neat) 3388 (br, s), 2958 (w), 2921 (w), 1699 (s), 1615 (m), 1520 (m), 1446 (m), 1286 (m), 1197 (s), 1065 (m), 983 (m), 812 (m) cm1; 1 H NMR (300 MHz, CDCl3) d (ppm) 6.82–6.71 (m, 2H, on phenyl ring), 6.66–6.58 (m, 1H, on phenyl ring), 6.40 (dd, J = 17.3, 1.4 Hz, 1H, CHH¼CH), 6.11 (dd, J = 17.3, 10.4 Hz, 1H, CHH¼CH), 5.83 (dd, J = 10.4, 1.4 Hz, 1H, CHH¼CH), 4.31 (t, J = 7.1, 2H, CH2CH2OC¼O), 2.84 (t, J = 7.1, 2H, CH2CH2OC¼O) 13C NMR (75 MHz, CDCl3) d (ppm) 165.9, 142.8, 141.5, 130.3, 129.3, 127.2, 120.2, 114.9, 114.4, 64.5, 33.3. 2.4. Polimerization of tyrosyl acrylate (TA) and hydroxytyrosyl acrylate (HTA) In a typical polymerization procedure, the monomer (0.25 mmol) was dissolved in 250 mL of dry DMF (1 M) followed by the addition of the initiator AIBN (0.018 mmol). Polymerization was conducted at 70  C for 48 h with continuous stirring. PTA yield was 84%, PHTA yield was 91%. PTA and PHTA were analyzed by FTIR, 1 H and 13C NMR.

2.2. Characterization All NMR spectra were recorded on a Bruker Avance 300 Ultrashield spectrometer equipped with a 5-mm probe. Proton (1H) and carbon (13C) measurements were performed in CDCl3 solutions at 300 and 75 MHz, respectively. All chemical shifts are

2.4.1. Characterization of poly(tyrosyl acrylate) (PTA) IR (KBr) 3412 (br, s), 3021 (w), 2962 (w), 1729 (s), 1616 (m), 1519 (m), 1453 (m), 1263 (m), 1166 (m), 1104 (m), 808 (m) cm-1; 1H NMR (300 MHz, CD3OD) d(ppm) 7.1–6.9 (m, 2H, on phenyl ring), 6.8–6.6 (m, 2H, on phenyl ring), 4.3–3.9 (m, 2H, CH2CH2OC¼O), 3.0–2.6 (m,

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2H, CH2CH2OC¼O), 2.4–2.2 (m, 1H, CHCH2), 1.9–1.5 (m, 2H, CHCH2). 13 C NMR (75 MHz, CD3OD) d (ppm) 177.1, 157.9, 132.0, 130.7, 117.4, 67.8, 36.1, 31.6. 2.4.2. Characterization of poly(hydroxytyrosyl acrylate) (PHTA) IR (KBr) 3413 (br, s), 3021 (w), 2957 (w), 1725 (s), 1607 (m), 1522 (m), 1447 (m), 1364 (m), 1283 (m), 1190 (m), 1114 (m) cm1; 1H NMR (300 MHz, CD3OD) d(ppm) 6.9–6.4 (m, 3H, on phenyl ring), 4.3–3.9 (m, 2H, CH2CH2OC¼O), 3.0–2.6 (m, 2H, CH2CH2OC¼O), 2.4–2.9 (m, 1H, CHCH2), 2.0–1.5 (m, 2H, CHCH2). 13C NMR (75 MHz, CD3OD) d(ppm) 177.2, 147.0, 145.7, 131.5, 122.5, 118.1, 117.5, 67.8, 36.3, 31.6. 2.5. Evaluation of disposable phenolic groups by Folin-Ciocalteu procedure

25 mg of polyacrylates was added in a flask containing 5 mL of the orange juice, and the mixture was stirred at 35  C for 24 h during which ascorbic acid decomposition occurred. At the same time, an experiment with orange juice without polymers, as a control, was conducted under the same conditions in order to evaluate the natural decomposition of ascorbic acid. The antioxidant ability of PTA and PHTA was also compared with that of tyrosol and hydroxytyrosol, respectively. HPLC was used to monitor the disappearance of all prepared samples including the control. Analyses were performed at a constant flow rate of 1 mL/min using a pH 2.5 H2SO4 aqueous solution as mobile phase. The injection volume was 20 mL and the detection wavelength 254 nm. Quantitative analysis was carried out by constructing calibration curves of ascorbic acid within 10–500 mg/mL concentration range. 2.8. Thin film preparation

Amount of total phenolic equivalents was determined by the Folin-Ciocalteu colorimetric method (Fazio, Plastina, Meijerink, Witkamp, & Gabriele, 2013). Briefly, 100 mL of extract solution (1.5 mg mL1 MeOH) were mixed with 1 mL of diluited (1:10) FolinCiocalteu reagent, 800 mL of 10% Na2CO3 and distilled water to a volume of 5.0 mL. The mixture was left standing for 2 h at room temperature in the dark; the absorbance at 760 nm was measured by using a UV–vis spectrophotometer (model V-550, Jasco, Europe). All samples were assayed in triplicate, and data are expressed as means. Total phenolic content in each polymer was evaluated by constructing calibration curves of gallic acid within 0.001–0.5 mg/mL concentration range. The results were expressed as mg of gallic acid equivalents per gram of extract.

Spin coated films were prepared by using P6700 PI-KEM apparatus. PTA and PHTA were dissolved in acetone (10 mg/mL) and 500 mL of each solution were deposited onto a cleaned quartz slides and then rotated for 60 s at a spinning speed of 4200 rpm and an acceleration of 2500 rpm s1. All spin coated substrates were dried at 50  C in an oven for 6 h, cooled to room temperature and eventually stored at 4  C before using. The obtained films were analyzed by FTIR spectroscopy to verify whether the polymers had undergone some change during the operations of deposition and rotation of the polymeric solutions; the obtained spectra showed the same absorption bands of the corresponding polymers. Both spin coated polyacrylates were immediately used to evaluate the antioxidant ability of the films against ascorbic acid oxidation in natural orange juice.

2.6. Antioxidant activity (DPPH assay) of polyacrylic powders The free radical scavenging capacities of both monomers and polymers were determined by DPPH assay according to a known protocol (Fazio et al., 2013). 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) is a stable free radical, and when it reacts with a radical scavenger, its maximum absorbance at 517 nm decreases quickly; the antioxidant effect is proportional to the disappearance of DPPH in test samples. More specifically, standard MeOH solutions of monomers and polymers (1.5, 0.750, 0.150 and 0.015 mg mL1 MeOH) were prepared. 100 mL of each solution were mixed with 100 mL of the DPPH solution (3.9 mg in 10 mL of MeOH, corresponding to 1 mM), and the final volume adjusted to 3 mL by the addition of the necessary amount of MeOH, so as to obtain four different solutions with concentration of 50, 25, 5 and 0.5 mg mL1 for each compound. Then the mixtures were shaken vigorously and incubated for 30 min at room temperature in the dark. The colorimetric decrease in absorbance of each sample was then measured at 517 nm using a UV–vis spectrophotometer (model V-550, Jasco Europe). The control was a DPPH solution obtained by diluting 100 mL of the DPPH standard solution with MeOH to give a final volume of 3 mL. Experiments were carried out in triplicate, and the antiradical activity of the monomers and polymers was expressed as percent inhibition (I%) of the sample compared to the initial concentration of DPPH (control) according to the formula: I% = [(Absorbance517nmofcontrol-Absorbance517nmofsample)/Absorbance517nmofcontrol]  100. 2.7. Antioxidant capacity of polyacrylates in fresh orange juice PTA and PHTA were dipped in orange juice freshly squeezed and filtered, in order to determine the ability of the synthesized polymers to inhibit the oxidation of ascorbic acid. An amount of

2.9. Antioxidant activity of polyacrylic films An amount of 5 mg of dry films equivalent to 1 cm2 were put in contact with fresh orange juice (3 mL) to test their antioxidant ability, similarly to that has be done for the polymer powders. Control with polyacrylate film without antioxidant pendants was also analyzed. The films were incubated at 35  C for 24 h. The ascorbic acid concentration in the orange juice in contact with the films was evaluated by HPLC analyses, in the same experimental conditions by which were analyzed powder polymer (the mobile phase used was pH2.5H2SO4 aqueous solution at 1 mL/min; injection volume was 20 mL and detection wavelength was 254 nm). 2.10. MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide] assay 2.10.1. Cell culture RAT-1 immortalized fibroblasts were purchased from Life Technologies. Cells were grown in Minimum Essential Medium (MEM) (Gibco) without antibiotics supplemented with 10% fetal calf serum (FCS) (Invitrogen), 2 mM glutamine and maintained in log phase by seeding twice a week at density of 3  105 cells/mL in a humidified 5% CO2 incubator at 37  C as recommended by ATCC. Cell number has been estimated with a Burker camera. 2.10.2. MTT proliferation assay Cell viability was determined by the MTT assay (Wang et al., 2000; Cione et al., 2013) measuring the reduction of 3-(4, 5dimethylthiasol-2-yl)-2,4,-diphenyltetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where is reduced to an insoluble, colored, formazan product. The amount of color produced is directly proportional to the number of viable cells. RAT1 cells were

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incubated with a thin film prepared from solution in a range of concentration of 0.25–1 mg/mL of PA, PTA and PHTA and disposed a in 6-well plates. Cells were treated at 12, 24, 48 and 72 h. At time of assay, 10 mL of MTT (5 mg/mL in PBS) was added to each well and incubated for 3 h at 37  C. The medium was then carefully aspirated, and 100 mL of dimethyl sulfoxide (DMSO) was added to solubilize the colored formazan product, agitating the plates for 5 min on a shaker. The absorbance of each well was measured with a spectrophotometer at a test wavelength of 570 nm with a reference wavelength of 690 nm. The optical density (OD) was calculated as the difference between the absorbance at the reference wavelength and the absorbance at the test wavelength. The results were expressed as the percentage of cell viability (OD of drug treated sample/OD of control)  100. The experiments were carried out in duplicate. 3. Results and discussion 3.1. Monomer synthesis An enzymatic strategy was developed to use mild and highly selective methods to covalently couple the primary hydroxyl group of the antioxidant compounds, tyrosol and hydroxytyrosol, with methyl acrylate via the acyl enzyme complex. C. antarctica lipase B (CALB)-catalyzed transesterification requires mild reaction conditions (T = 50  C) and the methyl acrylate was used as the reagent and as well as solvent. The mentioned reactions are represented in Fig. 1. The optimal reaction conditions were obtained after having carried out a screening on the amount of immobilized enzyme and on the time, as shown in Table 1. The results showed that higher yields for the monomers are obtained with the minimum possible amount of CALB and conducting the reaction for 24 h (TA yield = 96%; HTA yield = 75%). The products were characterized by FT-IR spectroscopy and compared with the FT-IR spectrum of the pure methyl acrylate known in the literature (Lide, 1994). The IR spectrum of the monomer TA confirms the formation of the product due to the presence of the peak at 1702 cm1 relative to the ester bond between the acrylic group and the tyrosol. Moreover, the presence of tyrosol in the structure is highlighted by the signals related to the stretching of the aromatic OH (3391 cm1), of the aromatic CH (3021 cm1) and the carbon–carbon bond of the benzene ring (1516 cm1). The peak at 812 cm1 confirms the presence of a 1,4disubstituted benzene, while the band of medium intensity at 1615 cm1 is attributed to the asymmetrical stretching of the vinyl group (Fig. S1). The IR spectrum of the HTA confirms the formation of the product due to the presence of the peak at 1699 cm1 relative to the ester bond between methyl acrylate and hydroxytyrosol. The peak at 3387 cm1 that corresponds to hydroxylic groups linked to the aromatic ring of the 1-(2-hydroxy) ethyl-3,4-dihydroxybenzene highlights the presence of hydroxytyrosol in the structure of the monomer. Stretching of the carbon–carbon bond in the aromatic ring generates the peak at 1520 cm1 (Fig. S2).

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Table 1 Enzymatic Transesterification reaction of the monomers.a Monomer

Entry

Enzyme amount (mg)

Time (hours)

Yield (%)

TA

1 2 3

400 200 200

24 48 24

90 89 96

HTA

1 2 3

400 200 200

24 48 24

70 70 75

In the 1H NMR spectra of the monomers TA and HTA (Figs. S3 and S4), methylene proton appeared at d 4.33 and 4.31 respectively, which otherwise appeared at d 3.86 in tyrosol and hydroxytyrosol. This downfield shift indicated the formation of an ester involving the primary hydroxyl of T and HT. In the 13C NMR spectra of the monomers TA and HTA (Figs. S5 and S6) methylene carbon appeared at d 65.4 and 64.5, respectively, which otherwise appeared at d 63.9. Furthemore, the study of integral values of 1 H NMR signals, as well as peak position in proton and carbon NMR, confirmed that the expected products were formed. 3.2. Synthesis of Tyrosol and HydroTyrosol functionalized poly (acrylate) (PTA and PHTA) The polymerization reactions were of radical type; 2,2-azobis (2-metilpropionitrile (AIBN) was used as the radical initiator (Fig. 2). At first exploratory polymerization tests were carried out in the presence of equimolar amounts of catechol in order to verify if the presence of the phenolic groups in the monomers synthesized could inhibit the initiation. The formation of the polymer during the initial tests confirmed the possibility of conducting the polymerization by radical route. Then exploratory tests have been used to establish the optimal reaction conditions for the polymerization of both monomers (time, temperature, solvent, [monomer], [AIBN]). PTA and PHTA, similarly to the monomers were analyzed by FTIR, 1H and 13C NMR. The FT-IR spectra of the polymers (Figs. S7 and S8) shows the characteristic bands of aromatic –OH (3411–13 cm1) and ester bond (1725–28 cm1), but the comparison with the characteristic IR bands of the monomers was enough to confirm that the polymerization occurred: the disappearance of the stretching band of the vinyl group of the monomer, as a result of the polymerization, is not detectable because the bands related to the stretching of the unsaturations of the aromatic rings along the polymer chain and vinyl double bond fall in the same range of the spectrum. In the 1H NMR spectrum of the polyacrylates PTA and PHTA (Figs. S9 and S10) two protons corresponding to the vinyl group disappeared, indicating the polymerization of vinyl group. In the 13 C NMR spectra (Figs. S11 and S12), peaks at d 128.6 and 130.3 for vinyl carbons disappeared and new peaks for the polymerized backbone appeared in the aliphatic region. These results indicated

Fig. 1. Enzymatic synthesis of tyrosyl acrylate (TA) and hydroxytyrosyl acrylate (TA).

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A. Fazio et al. / Food Packaging and Shelf Life 11 (2017) 84–90

Fig. 2. Polimerization of tyrosyl acrylate (TA) and hydroxytyrosyl acrylate (HTA).

the successful coupling of tyrosol and hydroxytyrosol through the primary hydroxyl to the polyacrylate backbone. 3.3. Evaluation of disposable phenolic groups by the Folin-Ciocalteu procedure Folin-Ciocalteu method gave an estimation of the available phenolic groups, as T and HT equivalents, linked to the polymers, and indirectly provided information about the polymerization degree. Phenolic compounds undergo a complex redox reaction with phosphotungstic and phosphomolybdic acids present in the Folin-Ciocalteu reactant. The color development is due to the transfer of electrons at basic pH to reduce the phosphomolybdic/ phosphotungstic acid complexes to form chromogens in which the metals have lower valence (Pan et al., 2007). For each biopolymer, disposable phenolic groups were expressed as milligram equivalents of gallic acid. These values were 164 and 406 mg GAE/g of dry PTA and PHTA, respectively. From the results obtained it is deduced as the number of available phenol groups on the surface of the polymer appears higher in the case of poly (acrylic acid 2-(3,4dihydroxyphenyl)-ethyl ester) (406 mg GAE/g polymer) compared to the poly (acid acrylic 2-(4-hydroxyphenyl)-ethyl ester) (164 mg GAE/g polymer). 3.4. Scavenging effect on DPPH radicals The DPPH radical is a stable organic free radical with an absorption maximum band around 515–528 nm and, thus, it is a useful reagent for evaluation of antioxidant activity of compounds. The DPPH test, based on the disappearance of the colored synthetic radical DPPH
, measures the ability of antioxidant compounds for trapping free radicals by donating hydrogen atoms or electrons, producing in consequence the bleaching of the colored radical solutions. The results showed that the scavenging effects of TA on DPPH radicals increased with the concentration: it varied from 0.08% at 0.5 mg/mL to 22.3% at 50 mg/mL. In contrast, HTA exhibited a high value of antioxidant ability even at lower concentration (94.2% at 5 mg/mL) (Table 2). Furthermore, the data reported in table show that the antioxidant activity of the hydroxytyrosyl acrylate (HTA) is much higher, at the same concentration, than that of the tyrosyl acrylate (TA). In fact, at the highest concentration (50 mg/mL) the

3.5. Ability of polymers to inhibit vitamin C oxidation in orange juice samples Postharvest processing plays a key role to preserve fruit quality including ascorbic acid content during storage and marketing (Barrett & Lloyd, 2012). For this reason, a control experiment, during which a sample of orange juice was left standing for 24 h at 35  C, was carried out to allow the natural decomposition of ascorbic acid. In this way, the antioxidant abilities of the synthesized polyacrylates in a real food system was determined; HPLC analyses have provided the values about the ascorbic acid content during 24 h in orange juice (control), orange juice added with polymers and juice in contact with antioxidant compounds. Table 3 showed all ascorbic acid retention values. The results highlighted the time-dependence of the ability of polymers to inhibit the vitamin C oxidation in orange juice samples. Ascorbic acid retention values after 5 h for both PTA and PHTA are high (60.76% and 80.93%, respectively), but the retention of ascorbic acid after 24 h was significantly higher for the juice sample in contact with PHTA (71.74%) than for the juice added with PTA (33.21%). Moreover, the polymer PHTA synthesized in this work showed excellent abilities to inhibit the oxidation of vitamin C and, therefore, promising applications as food packaging in different fields. 3.6. Antioxidant activity of polyacrylic films

Table 2 I%DPPH of antioxidant compounds, monomers and polymers.a Radical scavenging activity 0.5 mg/mL

5 mg/mL

25 mg/mL

50 mg/mL

T

0.1  0.02

6.3  0.2

11.3  1.1

24.3  1.7

HT

30.3  1.1

95.5  1.4

97.1  3.1

98.5  2.2

Monomers

TA HTA

0.08  0.01 21.3  1.1

6.5  0.3 94.2  2.6

10.3  0.7 95.3  1.4

22.3  0.9 97.0  2.1

Polymers

PTA PHTA

0.08  0.01 19.0  1.3

4.2  0.4 86.2  1.6

7.0  0.5 92.3  1.8

17.1  0.7 94.0  1.1

Antioxidant compounds

IDPPH% of HTA is equal to 97, while that of TA is much lower (22.3). The radical scavenging behavior of polyacrylates PTA and PHTA was similar to that obtained for the corresponding monomers. These results demonstrated that the formation of polymers preserved the antioxidant activity of the monomers. The antioxidant capacity of PHTA at 50 mg/mL (94%), in agreement with I%DPPH of the corresponding monomer at the same concentration (97%), is higher than that of PTA (17.1%). Table 2 shows the values of I%DPPH of polyacrylates and monomers on DPPH; these results were compared with that of tyrosol (T) and hydroxytyrosol (HT). It is clear that the free radical scavenging activity of the antioxidant compounds T and HT was almost completely retained in the corresponding monomers and polymers. The available phenolic groups on polyacrylates presented a good correlation with the antiradical activity toward DPPH
. The poly(hydroxytyrosyl)acrylate (PHTA), which exhibited the highest antioxidant activity, also had the highest total phenolic content.

The polyacrylate films were assayed with juice samples in the same conditions of the powder polymers. Ascorbic acid retention values for the experiments are presented in Table 3. The results showed that the content of vitamin C decreased with the time for both films, similarly to what happened for the polymer powders. The abilities of powder polymers and the corresponding films to inhibit the ascorbic acid oxidation were comparable on equal experimental conditions (time and temperature). The retention percentages for PTA and PHTA films after 5 h were 54.73 and 78.43 respectively, but they were lower after 24 h (31.69 for PTA and 70.55 for PHTA). The obtained values show that PHTA film exerts a

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Table 3 Ascorbic acid decomposition in orange juice samples at 35  C.a Sample Orange Orange Orange Orange Orange Orange Orange

juice (control) juice + 25 mg T juice + 25 mg PTA juice + 25 mg HT juice + 25 mg PHTA juice + 5 mg PTA film juice + 5 mg PHTA film

ascorbic acid retention (%) (t = 5 h)

ascorbic acid retention (%) (t = 24 h)

43.93  0.9 64.80  1.5 60.76  2.1 88.64  3.5 80.93  2.9 54.73  1.5 78.43  1.7

19.42  0.6 36.21  1.1 33.79  1.9 75.12  1.3 71.74  3.1 31.69  2.5 70.55  1.1

Fig. 3. Cytotoxicity test on RAT1 cell line at different times.

greater ability to reduce the semiascorbyl radical (generated by oxidation of ascorbate). 3.7. Effect of functionalized polyacrylates on cell viability in RAT1 fibroblast cells Cytotoxicity screening of polyacrylate derivatives on RAT1 fibroblast cell proliferation was determined using the MTT assay after 12, 24,48 and 72 h of incubation. The viability of the control samples was set as 100%. All polyacrylate films were assayed for cytotoxic activities, in order to validate the safety of these new compounds. The cytotoxicity test was performed on RAT1, a

Fig. 4. Concentration dependent action of poly(acrylate) (PA), poly(tyrosyl acrylate) (PTA) and poly(hydroxytyrosyl acrylate) (PHTA) on RAT1 cell line cytotoxicity test at 48 h (A) and 72 h (B).

normal cell line. One important criterion for a food antioxidant is that it must have minimal or no side-effects on normal human cells to be safe, also for edible purpose. Films for food packaging need to be assessed for beneficial effects to support claims, but also for toxic effects aiming at defining sub-toxic concentration. Based on the Food and Drug Administration (FDA) in vitro toxicity protocol, ISO 10993-5, percentage of cell viability above 80% is considered as non-cytotoxic, 60–80% weak, 40–60% moderate and below 40% strongly cytotoxic (Jorge, Marian, Petr, & Petr, 2014). The effectiveness of all the compounds tested as thin film in reducing or incrementing cell viability was not evident neither at 12, 24, 48 and 72 h incubation (Fig. 3). Therefore, testing directly the films, on cell viability, prepared from solution in a range of concentration of 0.25–1 mg/mL of PA, PTA and PHTA, and disposed in a 6-well plates for 48 h (Fig. 4A) and 72 h (Fig. 4B) showed no toxicity for all the tested samples. The in vitro test demonstrated that these substances were avoided of toxicity. 4. Conclusions In the present study, polyacrylates with antioxidant activity were successfully synthesized via a two-step process. The first step was a mild enzymatic transesterification where the primary hydroxyl group of tyrosol and hydroxytyrosol was regioselectively acylated by acrylic acid methyl ester. The resulting monomers were subjected to a radicalic polymerization in the second step. The polymers were characterized by FT-IR, 1H NMR and 13C NMR. NMR spectroscopy has proved indispensable to confirm the formation of the polymer. The antioxidant efficiency of the polyacrylates, evaluated by DPPH test, was similar to that obtained for the corresponding monomers, which demonstrated that the formation of the polymers preserved the activity of the pendant antioxidant compounds. These results were compared with those of tyrosol (T) and hydroxytyrosol (HT); it is clear that the synthesized monomers and polymers retain the free radical scavenging activity of the antioxidant compounds T and HT. Polymerized hydroxytyrosyl acrylate (PHTA), when used at a concentration up to 50 mg/mL, fully scavenged DPPH free radicals. A good correlation between the available phenolic groups on the polyacrylates and the antiradical activity towards against DPPH
was also observed. The poly(hydroxytyrosyl)acrylate (PHTA), which exhibited the highest antioxidant activity, also had the highest total phenolic content. All polyacrylate films did not exhibit any cytotoxic activities in vitro. The test was performed on RAT1 normal fibroblast cells. Although, cytotoxicity test is not enough for food contact materials, we performed it as a preliminary approach to rule out any possibility of toxicological properties of our films. However, further studies on the safety assessment for food industry application and/ or future food packaging product based on polyacrylates derivatives are still needed. The general problem arising from the use of food contact materials derives from their content of substances

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