Reactive & Functional Polymers 73 (2013) 1377–1383

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Reactive & Functional Polymers

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Laccase-initiated reaction between phenolic acids and chitosan

1381-5148/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.reactfunctpolym.2013.01.005

⇑ Corresponding author. Tel.: +386 2 220 7924; fax: +386 2 220 7990.E-mail address: mojca.bozic@um.si (M. Bozic).

Mojca Bozic a,⇑, Janez Štrancar b, Vanja Kokol a

a University of Maribor, Institute for Engineering Materials and Design, Smetanova ul. 17, SI-2000 Maribor, Sloveniab Jozef Štefan Institute, Laboratory of Biophysics, Jamova 39, SI-1000 Ljubljana, Slovenia

a r t i c l e i n f o a b s t r a c t

Article history:Available online 16 January 2013

Keywords:LaccaseChitosanPhenolic acidPolymerizationFunctionalization

Phenolic acids are known to possess antioxidant activities whilst chitosan is a biocompatible polymerwith antibacterial activity against a broad spectrum of bacteria. Merging both types of molecules couldtherefore provide several potential applications. In this work, antioxidant properties of phenolic acid–functionalized-chitosan were investigated after being prepared from structurally-different phenolic acids(caffeic and gallic acids) and chitosan using the laccase from Trametes versicolor as the reaction initiator. Alaccase-mediated oxidation kinetic of phenolic acids was monitored by UV–vis spectroscopy and cyclicvoltammetry, as well as spin-trapping electron paramagnetic resonance spectroscopy (ST-EPR). The pHwas shown to have a significant effect on the degree of phenolic acid self-polymerization, indicatingthe involvement of phenolate anions within the formations of coupled polyphenol products, and theirfunctionalities, i.e. antioxidant activity. All the phenolic acid-functionalized-chitosans displayed greatlyimproved ABTS radical cation scavenging capacities, compared with the untreated chitosan.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Laccase (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) areamongst the more popular oxidase enzymes used in various bio-technological applications (e.g. pulp delignification, bio-bleaching,treatment of industrial plant wastewater, fibers’ modifications,fruit juice processing, biosensor, and biofuel cell construction[1,2]). This is because they are able to catalyze the direct oxidationof an aromatic substrate in the presence of molecular oxygen,which is reduced to water during the reaction. This reaction com-prises the coupling of four single-electron oxidations of the reduc-ing substrate to the four electron-reductive cleavages of a di-oxygen bond, using four Cu atoms distributed amongst three sites[1]. Phenols are typical laccase substrates because their redoxpotentials (ranging from 0.5 to 1.0 V vs. NHE) are low enough to al-low electron abstraction by Cu1. They are oxidized into phenoxyradicals which, depending on the reaction conditions, can sponta-neously polymerize via radical coupling, or rearrange themselvesinto highly-reactive quinones through a dis-proportionate mecha-nism. The resulting quinones can spontaneously couple with e.g.chitosan primary amino groups via Schiff-base and Michael-addi-tion mechanisms in a subsequent non-enzymatic reaction [3,1,4].

Chitosan [5,6] has been proposed for use in food packaging,wound healing, biomedical devices, etc. because of its uniqueproperties like its biocompatibility, antimicrobial, and film-form-

ing capacities. However, in all the mentioned applications it isessential for this material to also possess antioxidant properties,within which chitosan is quite limited. In order to increasechitosan’s antioxidant capability, reactive functional groups ofchitosan (a primary amino group at the C2 position, as well asa secondary hydroxyl group at the C6 position) were shown tobe exploited for the grafting of laccase-mediated reactive pheno-lic acids, as reported in our previous work [3,4].

Gallic and caffeic acids are well-known natural phenolic antiox-idants [7,8], the biological activity of which, however, depends onthe molecular structure, (primarily on the numbers and positionsof hydroxyl groups) of their monomers, as well as the poly-phenolsformed after the radical coupling reactions [9]. Thus, although, sev-eral low-molecular-weight poly-phenols have been shown to actas pro-oxidants and generate reactive oxygen species like hydro-gen peroxide [10], in contrast a relatively high-molecular-weightfraction of extracted plant poly-phenols was reported to haveexhibited enhanced antioxidant and anti-carcinogenic activities[11], being attributed to the increased numbers of AOH moieties,i.e. the increased number of electrons involved during theiroxidation.

From these perspectives, in this current paper we present ourstudies on the laccase-mediated polymerization capabilities ofcaffeic (CA) and gallic (GA) acids as di- and tri-hydroxyl phenolicacids, respectively, the antioxidant abilities of their polymeriza-tion products and designed phenolic acid–chitosan conjugates,with the goal of amplifying the antioxidant properties of thechitosan.

1378 M. Bozic et al. / Reactive & Functional Polymers 73 (2013) 1377–1383

2. Experimental

2.1. Materials and reagents

Medium molecular weight chitosan (Mw of 421.5 kDa) with adegree of de-acetylation �90% was purchased from Mahtani Chito-san PVT. Ltd., India. The gallic acid (GA), caffeic acid (CA), and thelaccase from Trametes versicolor, were obtained from Sigma–Al-drich, and used without further purification. All the reactions wereconducted in deionized water. The other reagents used were ofanalytical grade.

2.2. Kinetic of phenolic acids’ polymerization

GA and CA were dissolved at concentrations ranging from 1.67to 33.33 mM in 100 mM citrate and phosphate buffers pH 4.5 and6.5, respectively. The laccase dosages were 10 ll (0.055 g/10 mlappropriate buffer). Kinetic assays were carried out by monitoringthe enzymatic reactions, i.e. by measuring the product formation atselected wavelengths at room temperature. Specific wavelengthsfor the product formation from substrates were determinedaccording to the UV–vis absorption spectrum of the oxidationproducts from the particular substrates, by the laccase. The forma-tions of the UV–vis absorption spectra were monitored as a func-tion of enzyme reaction time. Kinetic parameters (Km and Vmax)were determined graphically from Lineweaver–Burk plots.

2.3. Cyclic voltammetry (CV) experiments

The oxidation and polymerization reactions were monitored bymeans of cyclic voltammetry (CV) using PGSTAT10. The Autolabpotentiostat/galvanostat was controlled by Autolab Nova softwareversion 1.7. All the experiments were carried out at a 50 mV s�1

scan rate in a 100 mL Metrohm cell with a triple-electrode config-uration. The working electrode was a glassy carbon (GCE) with asurface diameter of 3 mm (Metrohm). The counter and referenceelectrodes were platinum (Metrohm) and Ag/AgCl (Metrohm) elec-trodes, respectively. The renewal of the glassy carbon surface wasachieved by polishing with 1.0 and 0.3 lm alpha-alumina (Micro-polish, Buehler) on a micro cloth-polishing pad (Buehler), followedby washing in an ultrasonic Selecta bath for 2 min.

2.4. Spin-trapping electron paramagnetic resonance spectroscopy (ST-EPR) experiments

ST-EPR spectroscopy was done using an EPR Bruker ElexsysE500 spectrometer equipped with a high-sensitivity cavity andan Aqua-X sample holder. Spectra were obtained at 30 �C in pH6.5 buffer. The operational parameters were as follows: 331 mTcenter field, 10 mT sweep width, 9.8 GH microwave frequency,20 mW microwave power, modulation frequency of 100 kHz andamplitude of 0.1 mT, conversion time of 40.96 ms, and time-con-stant of 20.96 ms, 3 scans with a resolution of 1024 points. ADMPO standard spin-trap purchased from Sigma–Aldrich was usedto trap reactive oxygen species. Standard hypofine coupling wasused to identify radical species. The presence of hydroxyl radicalswas determined from the peak heights of the second low-field lineof the DMPO/�HO spin adduct.

2.5. Phenolic acids’ functionalization of chitosan using laccase asreaction initiator

The chitosan film preparation and functionalization was per-formed at pH 4.5 (homogeneous method) and at pH 6.5 (heteroge-neous method) according to the reported methods [12] with slight

modifications. Briefly, the homogeneous method was performed byfirst preparing chitosan (1% w/v), phenolic acids (1 mM), and lac-case (9.6 U) solutions separately, and the pHs of each solution thenbeing adjusted to target values of pH 4.5. The chitosan solution washeated to 30 �C and an individual phenolic acid solution was addedunder constant stirring. Then the homogeneous distribution lac-case solution was slowly added whilst stirring, and allowed to re-act for 24 h at 30 �C. The laccase–chitosan conjugated i.e. the pelletwas removed from the solution by centrifugation at 5000 rpm for5 min. The supernatant solution was then cast onto 7 cm-diameterPetri dishes and oven-dried overnight at 45 �C. The dry films ob-tained were peeled off, and thoroughly washed with ethanol andwater. During the heterogeneous method, the pre-prepared chito-san films of 0.1 g were incubated in 50 mL of corresponding pheno-lic acid, and the laccase solution (1 mM GA or CA and 9.6 U oflaccase) at pH 6.5 for 24 h, under constant shaking at 30 �C. Thephenolic acid’s consumption was monitored by UV–vis spectros-copy analysis of the treating solutions, by measuring the GA andCA monomers’ concentrations at 260 and 285 nm, respectively.The control samples were treated in the same way, but withoutadding the enzyme. After the reaction, the films were rinsed outextensively with ethanol and water. Ethanol and water washingbaths were spectroscopically checked to determine the presenceof any phenolic acids within the washing baths.

2.6. ATR-FTIR spectroscopy analysis

The spectra of chitosan-control and phenolic acids-functional-ized-chitosan samples were recorded using a Perkin–Elmer Spec-trum One FTIR spectrometer with a Golden Gate ATR attachmentand a diamond crystal. The absorbance measures were carriedout within the range of 650–4000 cm�1, using 16 scans and a res-olution of 4 cm�1. The resolution of the spectra was improved bytheir de-convolution from a background scattering using a Gauss-ian function curve-fitting analysis. All the data analyses were per-formed using the Peakfit software version 4.12.

2.7. Antioxidant activity determination

The antioxidant activity was determined by mixing 1 mg of thetest sample and a 300 lL ABTS�+ free radical solution prepared bythe reaction between 7 mM ABTS in H2O and 2.45 mM potassiumpersulphate, stored in the dark at room temperature for 12 h. Be-fore usage, the ABTS�+ solution was diluted with phosphate buffersaline (PBS) in order to reach an absorbance of 0.700 ± 0.025 at734 nm. The inhibition of the ABTS�+ radical was monitored at734 nm and 25 �C, and the percentage inhibitions of this radicalwere calculated at the ends of 10 and 30 min using Eq. (1), whereAcontrol was the initial concentration of the ABTS�+ and Asample wasthe absorbance of the remaining concentration of ABTS�+ in thepresence of the sample. All the data were the averages of triplicateexperiments.

Inhibition or scavenging effect ð%Þ ¼ Acontrol � Asample

Acontrol� 100 ð1Þ

3. Results and discussion

3.1. UV–vis monitoring of poly-phenolic acid product’s formation bylaccase

The kinetics of the oxidation products formed by laccase fromthe di- and tri-hydroxyl-phenolic acids, CA and GA, were moni-tored by UV–vis spectroscopy. Based on the absorption spectra ofthe formed products, exact wavelengths were established for

Table 1Absorption maxima (nm) within the UV–vis absorption spectrum of the primaryproduct formation by laccase from the phenolic acids.

Wavelength (nm)

pH 4.5 pH 6.5

GA 395 395 (465, 600)*

CA 420 (poor solubility) 450

* Three products were detectable during GA laccase-catalyzed reaction at pH 6.5.The absorption maximums of the second and third primary products are shown inparentheses.

Table 2Kinetic data of the reaction of laccase with GA and CA at pH 4.5 and 6.5.

Substrate

GA CA

pH 4.5a pH 6.5b pH 4.5 pH 6.5c

Detection absorbance 395 nm 395 nm 450 nmKm (mM) 1.23 ± 0.12 15.15 ± 0.81 – 3.52 ± 0.8Vmax (lM/min) 6.16 ± 0.46 1.97 ± 0.2 – 0.15 ± 0.032Vmax/Km (�10�3 min�1) 5.01 0.13 – 0.043

The Km and Vmax values for CA could not be determined due to the incompletedsolubility of the substrate at pH 4.5.

a Linear regression coefficient (R2) of the Lineweaver–Burk plots: 0.998.b Linear regression coefficient (R2) of the Lineweaver–Burk plots: 0.94.c Linear regression coefficient (R2) of the Lineweaver–Burk plots: 0.81.

M. Bozic et al. / Reactive & Functional Polymers 73 (2013) 1377–1383 1379

determining the kinetic constants Km and Vmax on the phenolicsubstrates.

When GA and CA were used as substrates at different pH medi-ums, the UV–vis absorption spectrums obtained by laccase-cata-lyzed reactions were quite different. The laccase-catalyzedoxidation reaction at pH 4.5 led to absorption maximums at 395and 420 nm for CA and GA, respectively, representing one newly-

Fig. 1. Cyclic voltammetry kinetics of products’ formations with laccase’ using 1 mM Gsample).

formed product, whereas three maximum absorptions at 395,465, and 600 nm appeared for GA at pH 6.5 (Table 1). At pH 6.5,the GA anions derived from proton dissociation dominated (occur-rence >99.5%) and the carboxyl, as the withdrawing group, becamedeprotonated and with that the electron-donating group, which fa-vored H-atom transfers- and electron-donation-radical based cou-pling reactions [13]. Laccase oxidation of GA at the pH 6.5 systemled to the formation of a primary product at 395 nm and two addi-tional ones with extended conjugation having visible regionaldetection. On the other hand, CA oxidation at pH 6.5 did not revealany formation of new products. This can be explained by the re-duced CA carboxyl electron-donating effect (because of vinyl dou-ble-bond separation) and one missing hydroxyl group on thebenzene ring.

The pH dependence of Km and Vmax for the laccase-catalyzed GAand CA is presented in Table 2. The GA–laccase reaction system atpH 6.5 displayed a 12.3-fold higher apparent Km than at pH 4.5,suggesting that the affinity of the laccase was significantly influ-enced, i.e. decreased by the increased pH medium. In addition,the rate of the reaction was significantly higher at pH 4.5, as indi-cated by Vmax and by the catalytic efficiency (Vmax/Km) of the reac-tion, which was in accordance with the decreased laccase activityat pH 6.5 [4]. The Km value for CA at pH 4.5 could not be deter-mined due to the insolubility of the substrate at concentrationsof Vmax. When comparing the substrate-dependent kinetics at pH6.5, the Km value of GA was 4.3-fold higher than that for CA, indi-cating a lower affinity of laccases towards GA. On the other hand,the maximum reaction rate Vmax and the catalytic efficiency Vmax/Km were higher for GA. It can be assumed that the number of hy-droxyl groups on the benzene ring and their polarity and aciditycould cause different orientations of the substrate regarding theactive site of the laccase. The CA with catechol (two ortho posi-tioned hydroxyl groups) structure seemed to be preferential whencompared to GA with a triple hydroxyl groups on benzoicstructure.

A at pH 4.5 and 6.5 (insets are the corresponding cyclic-voltagrams of the control

Fig. 2. Cyclic voltammetry kinetics of products’ formations with laccase, using 1 mM CA at pH 4.5 and 6.5 (insets are the corresponding cyclic-voltagrams of the controlsample).

0

20

40

60

0.1 10 1000

OH

.[r

adic

als]

Time [min]

1 mM GA, pH 6.5

0

20

40

60

0.1 10 1000

OH

.[r

adic

als]

Time [min]

1 mM CA, pH 6.5

Fig. 3. Kinetic of hydroxyl radicals formed during the reaction within different aqueous systems.

1380 M. Bozic et al. / Reactive & Functional Polymers 73 (2013) 1377–1383

3.2. CV kinetic of poly-phenolic acid products’ formation by laccase

Cyclic voltammetry was used to follow the formation of electro-active GA and CA products by laccase catalyzes. As presented inFig. 1, the electrochemical oxidation mechanism of GA at GCE in-volved two oxidation processes (anodic peaks, Ia), firstly at around350 mV and secondly at around 650 mV. The charge-transfer pro-cess at the first peak corresponded to the oxidation of the first phe-nolic hydroxyl group of GA, and a second oxidation peak to theelectron transfer of the second hydroxyl group, their reductioncounter-parts were lacking. The oxidation peaks’ positions weremoved to lower values at an increased pH. The GA had four poten-tial acidic protons, having pKa values of 4.0 (carboxylic acid), 8.7,11.4, and >13 (phenolic OHs). The known pKa values of the gallateradical were �4 for the carboxyl group and 5.0 for phenolic hydro-gen, so that it was deprotonated at neutral pH [14]. The resultingquinone Q�� was then easily oxidized because the Q��/Q standard

potential was more negative than the QH�/QH+ standard potential.In addition, the possibility of dual intra-molecular hydrogen-bond-ing stabilization of the electro-chemically generated GA-quinoneradical anions could provoke a shift of the reduction potentials to-ward less negative potentials. During the laccase treatment bothanodic peaks decreased, but had not completely disappeared after5 h, suggesting that a fraction of GA still remained within the solu-tions, especially at pH 6.5, where the reaction-rate was highlydiminished (Fig. 1 – left graphs). No electrochemical detection ofnewly-formed products, even after 24 h of incubation, indicatedno formation or by CV undetectable electro-active GA products ata 1 mM starting concentration. However, when a concentrationof 10 mM GA and 153 U of laccase was used, two decreasedirreversible electrodic processes were detected, a broad one at apotential of 350–450 mV, and another at 700 mV, by CVmeasures as reported in our previous work [12]. Effective CVdetection of a newly-formed phenolic product was clearly

Fig. 4. Fourier self-de-convolution and the curve fitting of an FTIR absorbancespectra taken in attenuated total reflectance (ATR) mode within the range of 1850–850 cm�1.

M. Bozic et al. / Reactive & Functional Polymers 73 (2013) 1377–1383 1381

concentration-dependent, since the existence of newly-formedproducts was confirmed by UV–vis spectroscopy. Besides concen-tration detection, CV was applied to show GA’s and CA’s effective-ness as antioxidants by the positions of their oxidation peaks(Figs. 1 and 2 – right graphs). GA’s first peak during laccase incuba-tion at both pH’s moved to the lower potentials, showing laccase-induced substrate’s readiness to oxidize, and thus more reactivethan antioxidants [15]. The second peak’s potential remained moreor less constant at both pHs throughout the whole process.

The reaction on GCE with CA involved an ortho-diphenol moi-ety reaction to an ortho-quinone, a feature typical of antioxi-dants that react within the 200–400 mV anodic regions(depending on the concentration and scan rate) [16]. CA-gener-ated cation was radically stable enough so that the cyclic vol-tammogram appeared as a single-electron chemically-reversibleoxidation process. The degree of reversibility was increased byincreased pH (Ic/Ia = 0.8 at pH 4.5 and Ic/Ia = 0.9 at pH 6.5). Onthe other hand the ease of reversibility, as indicated by the peakpotential separation (DE), was higher at pH 4.5 (DE = 78 mV)than at pH 6.5 (DE = 164 mV). The presence of laccase in the1 mM CA solution of pH 4.5 led to the appearance of a singlenew timely-decreased anodic peak (Ia) seen at 757 mV and anew cathodic peak (Ic) at 24 mV, after 5 h of incubation, indicat-ing the redox reversibility of the newly-synthesized oligomeric/polymeric compounds (Fig. 2 – left graphs). At pH 6.5 no detec-tion of new electro-active compound was observed but, as in thecase of GA, when using a 10 mM concentration (as in our previ-ous study [12]), one new reversible peak was detected. The oxi-dation peaks’ positions were more or less constant over time,thus indicating no obtained new compound with a higher anti-oxidant status (Fig. 2 – right graphs).

In separation, control experiments were carried out in the ab-sence of laccase and in the presence of phenols’ components. Thecontrol samples did not show any new UV–vis absorption bandsor CV current peaks decreasing (data not shown) within 5 h, indi-cating the stabilities of GA and CA at pH 4.5 and 6.5.

3.3. ST-EPR results

The ST-EPR experiments revealed some additional resultsabout oxidation kinetic, as well as about the diffusion and cou-pling of radical species in chitosan solution. It should be ad-dressed that the ST-EPR experiments trapped the radicals intosemi-stable spin adducts, where the spin-traps acted as integra-tors. However, since the spin-traps needed to react with radicals,it was designated as an unstable molecule, and the adduct (spin-trap with caught radical) also as unstable for the same reason.Therefore, if there were some of the oxygenation or reductionpresent, adduct would oxidize or reduce into nitrones or hydrox-yanions, respectively.

In order to examine the efficiency of the laccase-mediated func-tionalization of chitosan by homogeneous method, the EPR signalintensities of DMPO/�HO spun adducts for both phenolic acids,were compared without and in the presence of chitosan (Fig. 3).

A small number of hydroxyl radicals, which are always presentin a water medium, were trapped using a spin-trap into a spin-ad-duct, and shuttled for about 100 min. However, when laccase waspresent the adducts (nitoxides) became oxygenized into EPR-immiscible nitrones in less than 10 min. With the addition of GAor CA into the laccase solution the spin-trap adduct oxidation slo-wed down by a factor of two, meaning that the phenolic acids tookover approximately half of the oxidation pathways. In the presenceof chitosan the amount of radicals produced by the laccase signif-icantly increased and became localized, indicating that the oxida-tion of the spun adducts had been postponed for 10 min. It waslikely that the radicals trapped on the phenolic acids had reacted

with the chitosan due to the restricted diffusion, instead of reacting(oxidizing) with the spun-adduct floating around. The dynamicsregulated by the restricted diffusion was the same for both theGA and CA acids.

Fig. 5. Time-dependent antioxidant activities of phenolic acids, laccase polymerized phenolic acids, non-functionalized and by phenolic acid functionalized chitosan at pH 4.5and 6.5, measured as reductions of the ABTS cation radicals.

1382 M. Bozic et al. / Reactive & Functional Polymers 73 (2013) 1377–1383

3.4. ATR-FTIR characterization of phenolic acid–functionalized-chitosan

Fig. 4 shows the ATR-FTIR spectra of chitosan references andphenolic acids’ functionalized chitosan films produced by hetero-geneous (at pH 6.5) and homogeneous (at pH 4.5) methods.

By de-convolution of the spectral region between 1750 and1450 cm�1, two bands for chitosan were observed: the bands at1624, and 1531 cm�1 corresponding to amide I and amide II struc-tures, respectively. For films obtained by phenolic acids’ function-alization at pH 4.5 three de-convoluted spectra were observed: inthe case of GA the bands at 1608 cm�1, 1516 cm�1 and 1711 cm�1

corresponding to amide I, amide II, and a new band for ester struc-tures, respectively, and in the case of CA the bands at 1602 cm�1,1511 cm�1 and 1712 cm�1. New bands at around 1715 cm�1 wereassigned to the ester bonds between the chitosan and dissociatedGA, whereas for CA functionalization these ester bands were attrib-uted to the CA polymerized products [12]. Functionalization at pH6.5 also revealed three de-convoluted spectra: GA at 1600, 1529,and 1651 cm�1 corresponding to amide I, amide II, and a new bandfor imine structures, respectively, and the same structures for CA at1600, 1524, and 1645 cm�1. The new bands at around 1650 cm�1

were attributed to the C@N stretching mode of the imines, sup-porting the occurrence of a Schiff-base reaction, whereas the Mi-chael addition-type reaction i.e. amine bonding confirmed byamide II band broadening [12]. The shift of the amide II and I bandsto lower wavenumbers in all the functionalized chitosan films, be-sides amine bonding, additionally indicated the formation of amidebonding, respectively. It can be concluded from these results thatthe phenolic acids were successfully coupled onto the chitosanpolymer via a combination of ester, amino, imino or amide bond-ing, depending on the phenolic acid type and pH condition usedduring treatment.

3.5. Antioxidant activity

The antioxidant activity of synthesized the poly-phenolic wasreduced compared to the monomer’s solution, which was expectedaccording to the reduced polymer concentrations within the com-parable solutions (Fig. 5 – upper-graphs). Nevertheless, the pH ef-fect on the synthesized poly-phenolic functionalities can be clearlyseen. GA synthesized polymers at pH 6.5 reached about 35% higherantioxidant capacity than GA polymers synthesized at pH 4.5, andeven at reduced concentrations reached comparable antioxidantactivities to GA monomer’s solution. The results were in correla-tion with UV–vis spectroscopic analysis when three new synthe-sized compounds were indentified (Table 1). In the case of CA,the profile of antioxidant activity was vice versa, where synthe-sized polymers at pH 4.5 reached approximately 32% higher anti-oxidant capacity than CA polymers synthesized at pH 6.5,comparable to the antioxidant activity of the CA monomers’ solu-tions. A new CA compound synthesized at pH 4.5 showed elec-tro-activity in addition, confirmed by CV measures (Fig. 2).

The antioxidant profiles of the control and phenolic acid-func-tionalized-chitosan films showed that the chitosan film itself pos-sessed some antioxidant activity (Fig. 5 – lower graphs), which canbe explained by several mechanisms. One is a free-radical scaveng-ing activity, in which the chitosan may eliminate various free-rad-icals by the action of nitrogen on the C-2 position. The scavengingactivities of chitosan derivatives against �OH may be derived fromsome or all the following speculations: (i) the hydroxyl groups inthe polysaccharide unit can react with �OH following a typical H-abstraction reaction; (ii) �OH can react with the residual free aminogroups NH2 to form stable macromolecular radicals; (iii) the NH2

groups can form ammonium groups NH�3 by absorbing H+ fromthe solution, then reacting with �OH through additional reactions[17]. The antioxidant abilities of the GA and CA functionalized

M. Bozic et al. / Reactive & Functional Polymers 73 (2013) 1377–1383 1383

chitosans using both methods (i.e. homogeneous at pH 4.5 and het-erogeneous at pH 6.5) were greatly increased, when compared tothe non-functionalized one. At pH 4.5 phenolic acid functionaliza-tion was carried out within the chitosan solution, where laccasepolymerization ability was indentified besides electrostatic inter-actions and additional ester-bond formation between the hydroxylgroup on C-6 chitosan and carbonyl group on GA [12]. ABTS’s scav-enging ability profile was similar to the scavenging abilities of cor-responding polymerized solutions i.e. CA functionalized chitosanpossessed higher activity at pH 4.5, whilst for GA functionalizedchitosan it was higher at pH 6.5.

4. Conclusions

The laccase-catalyzed oxidation process for GA and CA – pro-duced different numbers of new structures with good antioxidantactivities, whereas in the case of GA the process at pH 6.5 producedstructures with higher antioxidant activity, and in the case of CAhigher antioxidant activities were obtained for those synthesizedstructures at pH 4.5. The GA and CA functionalized chitosan atpH 4.5 and 6.5 showed great enhancement of the antioxidant abil-ity, the antioxidant profile of which was similar for newly-synthe-sized GA and CA polymers, depending on the pH medium.

Acknowledgment

This work was supported by the Slovenian Research Agency(Grant no. L4-3641).

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