An Antioxidant Bioinspired Phenolic Polymer for Efficient Stabilization

Article pubs.acs.org/Biomac

An Antioxidant Bioinspired Phenolic Polymer for Efficient Stabilization of Polyethylene Veronica Ambrogi,†,¶ Lucia Panzella,‡,¶ Paola Persico,§ Pierfrancesco Cerruti,*,§ Carlo A. Lonz,† Cosimo Carfagna,§ Luisella Verotta,∥ Enrico Caneva,⊥ Alessandra Napolitano,‡ and Marco d’Ischia*,‡ †

Department of Materials and Production Engineering, University of Naples “Federico II”, P.le Tecchio 80, 80125 Napoli, Italy Department of Chemical Sciences, University of Naples “Federico II”, Via Cintia 4, 80126, Naples, Italy § Institute of Polymer Chemistry and Technology (ICTP-CNR), via Campi Flegrei 34, 80078 Pozzuoli (Na), Italy ∥ Department of Chemistry, University of Milan, via C. Golgi 19, 20133 Milano, Italy ⊥ Interdepartmental Center for Large Instrumentation (CIGA), via C. Golgi, 19, 20133 Milano ‡

S Supporting Information *

ABSTRACT: The synthesis, structural characterization and properties of a new bioinspired phenolic polymer (polyCAME) produced by oxidative polymerization of caffeic acid methyl ester (CAME) with horseradish peroxidase (HRP)-H2O2 is reported as a new sustainable stabilizer toward polyethylene (PE) thermal and photo-oxidative degradation. PolyCAME exhibits high stability toward decarboxylation and oxidative degradation during the thermal processes associated with PE film preparation. Characterization of PE films by thermal methods, photo-oxidative treatments combined with chemiluminescence, and FTIR spectroscopy and mechanical tests indicate a significant effect of polyCAME on PE durability. Data from antioxidant capacity tests suggest that the protective effects of polyCAME are due to the potent scavenging activity on aggressive OH radicals, the efficient H-atom donor properties inducing free radical quenching, and the ferric ion reducing ability. PolyCAME is thus proposed as a novel easily accessible, eco-friendly, and biocompatible biomaterial for a sustainable approach to the stabilization of PE films in packaging and other applications.

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INTRODUCTION In the last few years, there has been an increasing demand on the part of consumers and the market for natural or renewable resource materials that could be used in the place of synthetic counterparts as building blocks for thermoplastic and thermosetting resins.1−3 Several examples including the polymerization of natural monomers and their derivatives to obtain thermoplastic polymers with properties similar to those of commodity thermoplastics have been reported.4−6 Furthermore, health and environmental concerns posed by the use of several chemicals involved in the synthesis of thermosets underscored the need for the development of a sustainable supply of biobased resins.7−9 Interestingly, biobased phenolic functional polymers have been also described, acting as flame retardants or antioxidants when added in polymer matrices.10−13 It is known that hindered phenolics are widely used as primary antioxidants for polymers employed in food packaging.14−16 As a matter of fact, oxidative deterioration of packaged food may cause significant decreases in shelf life associated with off-flavors, off-odors, color changes, and nutritional loss and is therefore emerging as a major economic and health issue, especially with the ever increasing demand for long life foods. Although antioxidant additives, such as © 2013 American Chemical Society

butylated hydroxyanisole, butylated hydroxytoluene, tertbutylhydroquinone, and propyl gallate, have been added into food product formulations to inhibit oxidative reactions, issues regarding possible toxic, mutagenic, and carcinogenic effects of these additives have been raised, as previous observations indicated that small phenolic antioxidants may be released from polymer packaging films when in contact with water or beverages.17 Therefore, low and medium molecular weight natural phenolics, such as caffeic acid, natural rosemary extracts, ascorbic acid, α-tocopherol, curcumin, quercetin, and catechins, have been incorporated into different packaging materials and their stabilizing effects have been investigated.18−26 Little attention has been placed so far to the use of polymers of natural phenolics as bioavailable, biocompatible antioxidants for polymer stabilization in packaging and related technologies. Expected advantages with respect to the monomers would include lower volatility (with reduced adverse effects), greater chemical stability under processing conditions and lower tendency to be released from the polymer into the contact medium (food, water, and so forth). Received: October 18, 2013 Revised: November 26, 2013 Published: December 8, 2013 302

dx.doi.org/10.1021/bm4015478 | Biomacromolecules 2014, 15, 302−310

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units; nebulizer pressure, 50 psi; fragmentor voltage, 50 V, gain 1.0). ESI(-)MS spectra were obtained from samples dissolved in methanol. Synthesis of Caffeic Acid Methyl Ester (CAME). CAME was prepared as previously described.45 Briefly, caffeic acid (3 g) was dissolved in methanol (30 mL) and stirred under reflux following the addition of 96% sulfuric acid (3 mL). After 2 h, the mixture was allowed to cool and then was diluted with ethyl acetate (270 mL), washed twice with 5% aqueous bicarbonate and then with water until neutrality. Organic layers were desiccated and taken to dryness to give pure CAME (2.88 g, 89% yield).46 Preparation of PolyCAME. CAME (2 g) was dissolved in methanol (70 mL) and added to 0.1 M phosphate buffer (pH 6.8) (70 mL) to a final concentration of 75 mM. A solution (10 mL) of horseradish peroxidase (188 U/mL, final concentration 12.5 U/mL) and 30% hydrogen peroxide (2.4 mL, final concentration 150 mM) were added in two aliquots at 1 h time intervals. After 2 h under vigorous stirring, the mixture was centrifuged (3000 rpm, 15 min), the resulting precipitate was washed three times with water and lyophilized to give a yellowish-brown powder (1.59 g, 80% yield w/w). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Assay. The assay was carried out as described47 using a 200 μM solution of DPPH and a 0.33 mg/mL solution of polyCAME (both in methanol). Trolox was used as reference compound. PolyCAME (60 μL) or Trolox (20 μL) was added to DPPH (1.98 mL) and the absorbance at 515 nm was determined at 30 s intervals of time over 10 min. Ferric Reducing/Antioxidant Power (FRAP) Assay. The assay was carried out as described48 using a 20 mM solution of FeCl3 × 6H2O in water, a 10 mM solution of 4,6-tris(2-pyridyl)-s-triazine (TPTZ) in 40 mM HCl, and a 0.33 mg/mL solution of polyCAME prepared as above. A solution made up of acetate buffer (pH 3.6) (3 mL) plus Fe3+ solution (300 μL) and TPTZ solution (300 μL) was treated with 15, 60, 150, 300, or 450 μL of polyCAME in the order. After 10 min, the absorbance of all solutions was measured at 593 nm. Hydroxyl Radical Scavenging Assay. The assay49 was carried out using the following solutions: 20 mM FeCl2 × 4H2O in 40 mM HCl; 20 mM Na2EDTA in water; 50 mM H2O2; 10 mM salicylic acid in 0.2 M phosphate buffer (pH 7.4); 268 U/mL catalase in 0.2 M phosphate buffer (pH 7.4), and 0.33 mg/mL polyCAME (as above). The solution of polyCAME was further diluted in water to a final concentration of 0.4, 2, 5, 10, or 20 μg/mL. To 1.5 mL of 0.2 M phosphate buffer (pH 7.4), 500 μL of salicylic acid, 250 μL of EDTA, 250 μL of Fe2+, 2 mL of water or of polyCAME at the desired concentration, and 500 μL of the H2O2 solution were added. After 10 min, 500 μL of the catalase solution was added and the mixtures were analyzed by HPLC. Ascorbic acid or CAME was used as reference compound. HPLC analysis was carried out on an octadecylsilane column 250 × 4.60 mm (5 μm) using a gradient based on 10 mM phosphoric acid (pH 2.5)/acetonitrile as the eluant at a flow rate 0.7 of mL/min; detection wavelength was set at 280 nm. Percent inhibition was determined by quantitative analysis of dihydroxybenzoic acids in the presence and in the absence of the antioxidant (polyCAME, CAME, or ascorbic acid). Film Preparation. LLDPE films containing polyCAME were obtained by extrusion, using a Collin Teach-Line E20T single screw extruder equipped with a horizontal flat die, and a Collin CR72T chillroll and calendering unit. The temperature profile was as follows (from hopper to die): 150, 170, 180, 180, 170 °C. Prior to processing, polyCAME was dried under vacuum for 24 h at 60 °C and successively mechanically mixed with the polyethylene powder in a 1 wt % amount. The films containing polyCAME were transparent and amber-colored, presenting small solid additive inclusions dispersed throughout the sample. The average thickness of all LLDPE based films was 115 ± 10 μm. Film Characterization. Thermal oxidative behavior at high temperature was evaluated through differential scanning calorimetry (DSC) for oxidative induction times (OIT) measurements50 and thermogravimetric analysis (TGA) to determine the thermal stability. DSC measurements were carried out in duplicate according to ASTM 3895-80 standard test method using a Mettler Toledo DSC 30. Each sample was heated under nitrogen up to 200 °C at 20 °C min−1. After

In this paper, we report a facile biocatalytic access to a novel bioinspired and biocompatible phenolic polymer of caffeic acid methyl ester (CAME), polyCAME, via a convenient horseradish peroxidase (HRP)/H2O2-catalyzed oxidation process27,28 and disclose the potent stabilizing effects of the new biopolymer on linear low density polyethylene (LLDPE), one of the most used polymers in food packaging. Caffeic acid and its derivatives are commonly found in cereals, fruits, and vegetables and are involved in the biosynthesis of lignin, the chief noncarbohydrate constituent of wood resulting from the oxidative polymerization and crosscoupling of 4-hydroxyphenylpropanoids.29 Caffeic acid derivatives are usually endowed with potent antioxidant properties and can act as scavengers of various oxidizing free radicals, including superoxide, peroxyl radicals, peroxynitrite, and nitric oxide,30−34 and exhibit also efficient metal chelating and antinitrosating properties.32,35−39 Polymers of caffeic acid (free acid) have been prepared by biocatalytic methods40 and have been investigated for biomedical, environmental, and technological applications,41 for example, antiviral and anticoagulation formulations42,43 and nanoscale surface patterning,44 but their potential as stabilizers of conventional polymers has remained apparently unexplored. Aims of this paper are (a) the optimization of the biocatalytic oxidation reaction for the preparation of polyCAME; (b) the structural characterization of polyCAME and a systematic assessment of its thermal stability and antioxidant properties; and (c) a detailed characterization of its effects on polyethylene (PE) in standardized tests of thermo- and photo-oxidative degradation. For the purposes of this study, CAME was preferred to the free acid for its less polar properties and a higher stability to decarboxylation and oxidative degradation during the thermal processes associated with PE film preparation.

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EXPERIMENTAL SECTION

Materials. All chemicals were purchased from Sigma-Aldrich and were used without any further purification. An unstabilized grade of a butene-copolymer linear low-density polyethylene (LLDPE), DJM1826, with a melt flow index (MFI) of 2.5 g 10 min−1 was supplied as a powder by Versalis (Italy). Methods. Solid-state 13C cross-polarization magic angle spinning (CP-MAS) spectra were collected at 125.77 MHz on a 500 MHz Bruker BioSpin NMR Spectrometer Avance 500, operating at a static field of 11.7 T and equipped with a 4 mm MAS probe, spinning the sample at the magic angle at speeds up to 15 kHz that with the addition of high power 1H decoupling capability allows to decrease or eliminate homo and heteronuclear anisotropies. All the samples were prepared by packing them in Zirconia (ZrO2) rotors, closed with Kel-F caps (50 μL internal volume) and the spinning speed (MAS) was optimized at 12 kHz after some experiments run in the range 4−12 kHz. Cross-polarization (CP) spectra under Hartmann−Hahn conditions were recorded with a variable spin-lock sequence (ramp CP-MAS) and a relaxation delay of 4s; a 1H π/2 pulse width of 3.0 μs was employed. Contact time was varied in the range 1−2.5 ms. In some experiments, high power proton decoupling was applied during acquisition without cross-polarization. The Cross-Polarization was also run with non-quaternary suppression experiment, rotor synchronized “NQS” refocused (CPNQS), to study the intensity of different heteronuclear dipolar interactions. In addition to CPNQS experiments, CP editing experiment by phase inversion (CPPI) was also used, as comparison, analyzing the evolution of dephasing 13C signal, depending on the heteronuclear dipolar interaction intensities. ESI spectra were collected on a LCQ Advantage Thermo Fisher Mass Spectrometer equipped with a ESI source (spray voltage, 4.5 kV; capillary temperature, 275.90 °C; sheath gas flow rate, 15 arbitrary 303


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Figure 1. ESI(-)MS spectrum of polyCAME. the test temperature was attained, the purging gas was turned to oxygen. The heat flow was recorded in isothermal conditions as a function of time. The beginning of material oxidation was apparent by a sudden increase in the slope of the exothermal heat flow curve. OIT was taken as the time elapsed after gas switching to the beginning of the slope increase, determined as the intersection point between the baseline and the tangent line to the curve at the inflection point. TGA was performed through a TA Instruments Q5000 thermobalance. For each measurement, about 5 mg of sample was heated from room temperature up to 700 °C at a rate of 10 °C min−1 in air atmosphere. Tests were carried out in duplicate. In order to evaluate the photo-oxidative stability at low temperature, the prepared films were aged at 40 °C up to 1800 h under dry conditions in an Angelantoni Sunrise environmental chamber equipped with a mercury lamp simulating solar irradiation (200 < λ < 700 nm, average incident light intensity 100 μW/cm2) and periodically examined by chemiluminescence (CL), FTIR ,and tensile tests. CL experiments were performed by means of a Lumipol 3 luminometer manufactured by the Polymer Institute of Slovak Academy of Sciences, Bratislava, Slovakia. The measurements were carried out in nitrogen flow. The samples were circular cuts of polymer film weighing about 6 mg. Each sample was heated from 30 to 230 °C at 5 °C min−1 and then cooled down to room temperature. FTIR spectroscopy on films subjected to accelerated aging was carried out by means of a Perkin-Elmer Spectrum 100 spectrometer. Spectra were recorded in transmission mode as an average of 32 scans in the range 4000−400 cm−1 with a resolution of 4 cm−1. Each sample was analyzed periodically and then returned to the oven for continued aging. The build-up of hydroxyl, carbonyl, and vinyl groups during photo-oxidation was calculated by subtracting the relative peak area at time zero from that of the aged samples in the respective absorption ranges. The broad carbonyl stretching region (1900−1600 cm−1) was deconvoluted using the OriginPro 8.5 software program, assuming Lorentzian bandshapes.51

Tensile tests were performed at the selected aging times on eight specimens for each formulation, using an Instron model 5564 dynamometer equipped with a 1 kN load cell at 23 ± 2 °C and 45 ± 5% RH with a 10 mm min−1 clamp separation rate.

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RESULTS AND DISCUSSION Synthesis and Structural Characterization of polyCAME. PolyCAME was prepared by HRP/H2O2 oxidation of 75 mM CAME in phosphate buffer, pH 6.8. The polymer was obtained in ca. 80% yield on a weight basis as a brown precipitate absorbing broadly in the UV−vis spectrum. ESI(-)MS analysis (Figure 1) showed well-detectable clusters of peaks in the mass range up to 2000 m/z, suggesting the presence of significant levels of oligomers up to at least the decamer stage, peaking at the tetramer. It is clear that the analytical technique used does not allow to draw definitive conclusions about the degree of polymerization of the material and the relative composition in terms of oligomers abundance. Analysis of the clusters of the pseudomolecular ion peaks [MH]− revealed a repetitive unit of 192 Da that by comparison with the molecular weight of CAME of 194 suggested the involvement of the propenoic chain in the coupling to give a benzodihydrofuran moiety in a fashion analogous to that appearing in the dimers obtained from CAME by peroxidase oxidation (Scheme 1).52 Other series of peaks well apparent in the spectrum and regularly separated one from the other by 192 Da can be attributed to the addition of the nucleophiles available in the medium during the polymerization reaction, namely hydrogen peroxide/water or methanol onto the terminal catechol moiety in the oxidized o-quinone form. Further oxidation of the benzodihydrofuran unit by the 304


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Scheme 1. Proposed Representative Structures and Mechanism of Formation of CAME Oligomers Responsible for Main Peaks of the ESI(-)MS Spectruma

Figure 2. 13C CP-MAS NMR spectrum of polyCAME.

145, 128, and 115 ppm suggesting that (1) the ester group is not lost in the polymerization reaction and (2) a significant proportion of the carbon positions of the catechol moiety has not undergone substantial changes. That these latter resonances were mainly due to CH carbons was confirmed by the substantial suppression of these signals in the CPNQS spectrum, which showed only the signals at 165 and 128 ppm. In line with this interpretation was also the CP with polarization inversion (CPPI) spectrum (Figure S2 in Supporting Information). A major difference of the CP-MAS spectrum of polyCAME with respect to that of the starting material CAME is the presence in the aliphatic region of the weak but well discernible pattern of carbon resonances in the range 70−90 ppm, suggesting the presence of O-substituted benzylic positions as those appearing in the dihydrobenzofuran units of the structures in Scheme 1 and in good agreement with the reported chemical shifts values of the benzodihydrofuran alpha carbons.52 Support to the formation of the dihydrobenzofuran ring at the expenses of the original double bond of the CAME units was also provided by (a) the significant increase of the signal around 51 ppm, possibly due to the contribution of the original C-2 carbons, and accordingly (b) the appreciable decrease of the broad signal between 135 and 150 ppm with respect to the other signals present also in the 13 C spectrum of CAME. Antioxidant Properties of polyCAME. The antioxidant properties of polyCAME were investigated with respect to standard or reference compounds using commonly used assays such as (a) the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, which determines the H-donor capacity of the antioxidant as quencher of the stable DPPH free radical;47 (b) the ferric reducing/antioxidant power (FRAP) assay, which measures the ability of the antioxidant to reduce a Fe3+-tripyridyltriazine complex to the Fe2+ form;48 (c) the ferrous oxidation-xylenol orange (FOX) assay, which determines the efficiency of the compound to inhibit the effect of hydrogen peroxide on oxidation of Fe2+ to Fe3+;53 (d) the 2,2′-bipyridyl assay, which determines the iron-chelating properties of a substance;54 and (e) the salicylate assay, which determines the ability of the compound to act as OH radical scavenger by inhibiting hydroxylation of salicylic acid by the Fenton reagent to give 2,3and 2,5-dihydroxybenzoic acids (HPLC quantitation).49 In the DDPH assay, polyCAME was found to exert a modest (22 ± 3%) but significant inhibitory effect as compared to trolox (83 ± 1%). At a final concentration range of 1.5−40 μg/mL,

a

The order of the structural units is arbitrary and the coupling mode is based on literature data on caffeic acid oligomers.42,43,52.

peroxidase/H2O2 system may have occurred on a limited number of units. Other valuable structural information were obtained by solid state 13C NMR run in the CP-MAS modality. Preliminarily, the spectrum of CAME was run under the same modality (Figure S1 in Supporting Information). In addition to the signals around 165 and 51 ppm due to the carbomethoxy group, intense signals around 145, 128, and 125 ppm, as well as in the range 113−115 ppm, were well apparent in close analogy to the 13 C spectrum run in solution.46 In the CP nonquaternary suppression (CP-NQS) spectrum, the signal at 145 ppm was significantly reduced, those at 113− 115 ppm were completely suppressed, while a single signal remained at 128 ppm (Supporting Information Figure S1). On this basis, it was possible to identify as a CH carbon that at 145 ppm, likely due to the C-2 carbon of the propenoic chain. Those at 125 and 113−115 ppm (3 signals) were attributable to the C-3 carbon of the propenoic chain, and to the C-2′, C-5′, and C-6′ carbons of the benzene moiety (see Supporting Information Figure S1 for numbering and structural assignment). On this basis, the CP-MAS spectrum of polyCAME (Figure 2) was then analyzed. In addition to intense signals corresponding to the carbomethoxy functionality, this showed broad signals around 305


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(Tonset) was determined as 260 ± 2 °C, and the temperature related to 10 wt % loss (T10%) as 236 ± 5 °C. The stability of polyCAME to photo-oxidation by simulated solar light irradiation at 40 °C was investigated using FTIR spectroscopy. The IR spectrum of the unaged polymer (Figure S3 in Supporting Information) shows a broad absorption at 3430 cm−1 (O−H) and sharp peaks at 2954 cm−1 (C−H), 1697 cm−1 (conjugated CO), 1630 cm−1 (aromatic CC), and 1172 cm−1 (C−O). The spectra of irradiated polyCAME show that the polymer did not suffer detectable structural modification even after 1000 h photo-oxidation at 40 °C, suggesting sufficient intrinsic stability for the PE stabilization experiments. Stabilizing Activity of polyCAME on Polyethylene Films. LLDPE films containing polyCAME at 1 wt % amount were obtained by extrusion. The films were transparent and amber-colored with an average thickness of 115 ± 10 μm. The effectiveness of polyCAME as a PE stabilizer was determined by means of TGA and oxidative induction time (OIT) measurements through differential scanning calorimetry (DSC), which are considered fast and reliable tools to evaluate polymer formulation stability at high temperatures. It is worth noting, however, that the correlation between these methods and longterm stability in polymer/antioxidant systems could not be straightforward, possibly leading to inconsistent extrapolation of data to lower temperature conditions.55,56 Therefore, on the present study high-temperature characterization was combined with methods based on aging treatments at low temperatures, which can provide information on polymer durability under service conditions. Accordingly, LLDPE based films were first characterized by DSC and TGA and then subjected to a photooxidative treatment at low temperature. The progress of oxidation was investigated by CL and FTIR spectroscopy, complemented by data from mechanical tests. High Temperature Stability: DSC and TGA Characterization. The oxidation of polymers occurs through an overall exothermic reaction that causes an abrupt increase in the heat flow measured by DSC. The time needed (OIT) for the DSC signal to increase must be considered for the evaluation of the antioxidant efficiency. This method represents one of the most commonly used tool in academic and industrial environment to assess the oxidative stability of polyolefins50 because it is easy and rapid to carry out and provides prompt data evaluation. More specifically, the longer the OIT, the more stable is the material. Figure 5a displays the averaged curves of heat flow versus time upon oxidation of the samples in oxygen at 200 °C, along with the OIT values, calculated by subtracting from the overall experimental time the interval related to the heating and isothermal steps performed before switching to oxygen (570 s). From the figure, it can be inferred that PE exhibited shorter induction time (OIT = 30 s) than the doped film (OIT = 780 s), indicating that oxidation was significantly retarded. These results revealed the effective antioxidant activity ascribed to polyCAME that even at a loading as low as 1 wt % was able to increase OIT values by 25 times in comparison with the pure polymer. This result compares well with those described in literature for polyolefins stabilized with different natural or naturally based antioxidants. For example, when LLDPE was stabilized with polyphenols such as gallates,57 quercetin,26 and curcumin22 in amounts ranging from 0.1 to 0.5 wt.%, increases in OIT values comparable to that of the present work were reported. Similar outcomes were also described for polypropylene-based formulations. In particular, Samper et al.25

polyCAME exhibited a reducing capacity as 0.15 trolox equivalents toward 1.7 mM FeCl3 in 0.3 M acetate buffer, pH 3.6. Figure 3 reports the effects of polyCAME in the OH radical scavenging salicylate assay.

Figure 3. Inhibitory effect of polyCAME (open bars) versus ascorbic acid (full bars) on salicylate hydroxylation induced by the Fenton reagent. Data are average values for three separate experiments (SD < 3%).

PolyCAME proved to be as efficient as ascorbic acid in inhibiting salicylate hydroxylation. For comparison, CAME was also tested at 3 μg/mL showing a ca. 75% inhibition. An IC50 of 0.85 μg/mL could be determined for polyCAME and of 0.81 μg/mL for ascorbic acid. No detectable effect of polyCAME was observed in the FOX and iron chelation assays. From the above set of antioxidant capacity data, it could be concluded that polyCAME is a highly effective scavenger of OH radicals generated by Fenton-type systems, a good free radical quencher by H-atom transfer toward DPPH, a modest ferric ion reductant, but a poor metal chelating agent and hydrogen peroxide scavenger. Thermal and Photo-Oxidative Stability of polyCAME. The thermal stability of polyCAME was assessed by TGA. As apparent from Figure 4, polyCAME underwent a rapid weight loss of about 5% in the range from 50 to 100 °C due to the volatilization of the moisture contained in the sample. After a plateau at constant weight, a smooth slope change occurred around 250 °C, associated to a weight loss of about 60%. Above 400 °C, an abrupt weight decrease was noticed, leading to complete polyCAME thermal degradation with no residual char. The temperature related to weight loss onset

Figure 4. Integral and derivative thermogravimetric curves of polyCAME heated at 10 °C min−1 under air. 306


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weight loss is reported in the inset of Figure 5b. The two films showed almost constant weight values until 200 °C, indicating that moisture absorption was negligible. For temperatures ranging from 200 to approximately 245 °C, a weight increase followed by a rapid drop was observed. The weight increase can be attributed to the oxygen uptake by the polymeric matrix, leading to the formation of unstable peroxidized polymer species.58,59 These structures decompose involving cleavage of the polymer chain bonds with the release of volatile compounds and rapid weight loss. The extrapolated onset temperature at which the test specimen begins to gain weight could be taken as the OOT value.60,61 Differences in OOT values may be used to sort the samples in ranking order according to their stability. The relative values are summarized in Table 1: neat LLDPE exhibited the lower onset temperature, while the presence of polyCAME increased the OOT value by about 40 °C, likely due to the outstanding radical scavenging property ascribed to the polyphenolic structure. The higher thermal stability of PE/ polyCAME was also confirmed by Tonset and T10% temperatures, which showed values about 10 °C higher than those of the plain LLDPE. Long-Term Stability: Chemiluminescence, FTIR, and Mechanical Tests. The prepared films were subjected to a photooxidative treatment at 40 °C under UV−vis irradiation up to 1800 h and periodically analyzed. It is well-known that photo-oxidation of PE occurs through a mechanism involving a complex series of steps, as detailed in Supporting Information. According to the reported mechanism,62 in the termination stages coupling of two alkyl peroxy radicals produces alcohol, singlet oxygen, and excited carbonyl species, which can decay through a photon emitting reaction generating CL. Therefore, CL analysis represents an effective tool to identify the presence of peroxide species since the early stages of LLDPE oxidation. The amount of peroxy radicals is connected to the hydroperoxide concentration, the latter being related to the CL intensity.63 Figure 6 shows the nonisothermal CL runs under nitrogen of PE after different periods of photoaging at 40 °C in air (a) and the evolution of the area under CL curves as a function of the photo-oxidation time (b), respectively. From Figure 6a, it is apparent that up to 212 h of aging time CL intensity increases constantly and the induction period is shifted toward lower temperatures. The areas under the CL curves, corresponding to the relative concentration of peroxides accumulated at different irradiation times64 were calculated and reported in Figure 6b as a function of aging. It can be evidenced that a certain amount of peroxide is already present in the pure polymer prior to irradiation. It is likely that high-temperature processing during film preparation induced formation of such peroxides in the polymer backbone. On the other hand, polyCAME efficiently protected PE from degradation due to thermal processing. Upon irradiation, the CL area rapidly increased for PE, indicating that these samples were not stabilized against photooxidation. The kinetics of peroxide accumulation were significantly slowed in the presence of polyCAME, as an induction time of about 200 h was measured before the CL signal increased significantly. The protective effects of polyCAME were not attributable to a simple UV screening effect as deduced from by the negligible absorption of the additive in the doped LLDPE films. In order to better elucidate the mechanism of oxidation of polyethylene in the presence of polyCAME, the irradiated samples were also analyzed by FTIR spectroscopy throughout

Figure 5. (a) Heat flow curves versus time during LLDPE-based films oxidation at 200 °C. (b) Thermogravimetric curves of LLDPE-based films heated at 10 °C min−1 under air.

found that quercetin at 0.5 wt % was able to increase OIT values by 1 order of magnitude; a similar result was reported by del Mar Castro Lòpez24 by use of 2 wt % catechin and green tea extracts. TGA under air provided an insight on the influence of polyCAME on polymer degradation behavior at high temperatures. The thermogravimetric traces of all the samples heated at 10 °C/min up to 650 °C are shown in Figure 5b. Oxidative onset temperature (OOT) and temperatures related to weight loss onset (Tonset) and 10 wt % loss (T10%) are summarized in Table 1. From the figure, two consecutive weight loss steps could be observed for both samples: the first, starting between 250 and 260 °C, corresponding to the thermal decomposition, was followed by a second stage (at around 450 °C), where combustion and complete volatilization of the char formed during the first step took place. A particular of the early stage of Table 1. Oxidative Onset Temperature (OOT) and Temperatures Related to Weight Loss Onset (Tonset) and 10 wt % loss (T10%) for LLDPE-Based Films sample

OOT [°C]

Tonset [°C]

T10% [°C]

PE PE/polyCAME

202 ± 2 243 ± 2

247 ± 3 256 ± 4

327 ± 2 338 ± 3 307


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aging. The films subjected to photo-oxidative degradation at 40 °C showed an increase of absorption intensity over time, but after 1007 h the absorption intensity in the presence of polyCAME was significantly lower. A detailed analysis of the kinetic evolution of the functional groups formed during photo-oxidation of LLDPE and of the stabilization pathway by polyCAME is described in Supporting Information (Figures S4−S7 and Scheme S1). The increase in carbonyl groups observed by spectroscopic analysis of the aged films was correlated to the changes in tensile properties from 0 to 1800 h. Table 2 reports elastic modulus (E), stress at break (σb) and deformation at break (εb) of the unaged films (t = 0). Table 2. Elastic Moduli (E), Stress at Break (σb), and Deformation at Break (εb) of the Unaged LLDPE-Based Films sample

E (MPa)

σb (MPa)

εb (%)

PE PE/polyCAME

151.0 ± 6.4 151.8 ± 7.8

28.2 ± 0.7 22.9 ± 2.6

816.2 ± 32.1 756.2 ± 61.0

Stress at break value was significantly lower for the samples containing polyCAME, while the elastic modulus and deformation at break value were not affected. It is also worth noting that larger standard deviations were calculated for these samples, suggesting that the additive solid inclusions are defects acting as initiation sites for crack propagation. Figure 8 reports the percent relative changes of deformation (a) and stress (b) at break, as well as elastic modulus (c) for LLDPE-based films as a function of the irradiation time. Whereas PE did not withstand a prolonged photo-oxidative treatment, showing a drop in stress and deformation at break soon after 46 h, in the presence of polyCAME the polymer retained about 75% deformation at break after 550 h, under which conditions the undoped samples failed dramatically. The loss of mechanical properties of PE can be correlated with the decrease in the average molecular weight upon aging. Finally, the photooxidative treatment brought about a significant increase of elastic modulus for PE starting from 400 h. This can be attributed to the massive reduction of molecular weight, which led to chains rearrangements and to increased polymer crystallinity.66,67

Figure 6. (a) CL emission on heating of PE photooxidized for different time periods at 40 °C (the 1007 h trace includes also the cooling step). (b) Evolution of the area under CL curves as a function the photo-oxidation time.

the aging process. The evaluation of different oxidation products was performed by monitoring the absorption intensity changes of vinyl groups (900−916 cm−1), carbonyls (1680− 1780 cm−1), and hydrogen-bonded hydroxyl species (−OH and −OOH) (3280−3520 cm−1) in the IR-spectra.65 Figure 7 shows the FTIR spectra of PE and PE/polyCAME in the hydroxyl, carbonyl, and vinyl range before and after 1007 h

Figure 7. FTIR spectra of PE and PE/polyCAME in the hydroxyl, carbonyl, and vinyl regions at time 0 and after 1007 h of aging. 308


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evidence suggests that polyCAME acts primarily by scavenging high reactive oxygen radicals and repairing free radical damage to LLDPE by efficient H-atom transfer. On the basis of these results, polyCAME is proposed as a new efficient bioinspired and biocompatible additive for a sustainable approach to PE stabilization with considerable potential advantages with respect to monomer CAME and related simple phenols in terms of reduced extent of leakage, stability to thermal and oxidative injury, and lower toxicity. With respect to caffeic acid polymers reported in the literature, it displays ester functions imparting reduced susceptibility to decarboxylation and a less polar character favoring interaction with PE chains.

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ASSOCIATED CONTENT

S Supporting Information *

NMR spectra of CAME and polyCAME, thermal and photooxidative stability of polyCAME, analysis of photo-oxidation of LLDPE-based samples through FTIR, and antioxidant assays. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(M.d.I.) E-mail: [email protected]. Fax: +39 081674393. *(P.C.) E-mail: [email protected]. Fax: +39 0818675230. Author Contributions ¶

V.A. and L.P. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from Italian MIUR (PRIN 2010-2011- 2010PFLRJR project and PRIN 2010-11-PROxi project).

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REFERENCES

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Figure 8. Percent relative changes of (a) deformation and (b) stress at break, and (c) elastic moduli for LLDPE-based films as a function of the irradiation time.

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CONCLUSIONS A novel phenolic biopolymer (polyCAME) has been synthesized by a mild biocatalytic procedure from caffeic acid methyl ester (CAME) and was extensively characterized for its antioxidant capacity and stability properties. When assayed in widely used tests for thermo-oxidative and photo-oxidative degradation of LLDPE, polyCAME exerted potent inhibitory effects both in the short term and in the long term. Available 309


Biomacromolecules

Article

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