Immobilization of caffeic acid on a polypropylene film: Synthesis and antioxidant properties

Article abstract of DOI:10.1021/jf101651y

The immobilization of caffeic acid (CA) on a polypropylene (PP) film was successfully performed through the covalent binding of the caffeoyl chloride on a modified polymeric surface of PP films grafted with hydroxyethyl methacrylate as monomer (PP-g-HEMA)

Full text of DOI:10.1021/jf101651y

9228 J. Agric. Food Chem. 2010, 58, 9228–9234
DOI:10.1021/jf101651y
Immobilization of Caffeic Acid on a Polypropylene Film:
Synthesis and Antioxidant Properties
‡
,
,†
*
DARIO
A
RRUA,† MIRIAM
C
RISTINA
S
TRUMIA
AND MO´ NICA
A
ZUCENA
N
AZARENO
†INQUINOA-CONICET, Facultad de Agronomı
del Estero, Avenida Belgrano (S) 1912 (CP 4200), Santiago del Estero, Argentina, and ‡Departamento de
Quımica Organica, Facultad de Ciencias Quımicas, Universidad Nacional de Cordoba,
Ciudad Universitaria (CP 5000) Cordoba, Argentina
´
a y Agroindustrias, Universidad Nacional de Santiago
´
´
´
´
´
The immobilization of caffeic acid (CA) on a polypropylene (PP) film was successfully performed
through the covalent binding of the caffeoyl chloride on a modified polymeric surface of PP films
grafted with hydroxyethyl methacrylate as monomer (PP-g-HEMA). The different reaction steps
were monitored by FT-IR spectroscopy. The synthesized films were characterized by Folin-
Ciocalteu method by measuring the available phenolic groups as caffeic acid equivalents linked
to the surface. The antioxidant efficiency of the modified polymers was evaluated by typical
spectrophometric methods, such as the bleaching of radicals DPPH• and ABTS•þ, and the inhibition
of the enzymatically induced coupled oxidation of linoleic acid and betacarotene. The available
phenolic groups on the modified film presented a good correlation with the antiradical activity toward
DPPH•. Moreover, the polymer synthesized in this work showed a good protective activity against
ascorbic acid oxidation in real samples of orange juice.
KEYWORDS: Antioxidant; active packaging; polyphenols; antiradical activity; grafting
INTRODUCTION
by the action of radical species formed in the product by endo-
genous as well as exogenous factors. Therefore, the presence of
antioxidants is very important to prevent these deleterious reac-
tions and extend the product shelf life (5). As the oxidation
reactions are a serious problem for the food industry, currently
synthetic antioxidants are used, butylated hydroxytoluene (BHT)
and butylated hydroxyanisole (BHA) being the most common.
However, the use of synthetic antioxidants is increasingly ques-
tionedbyconsumersbecauseoftheirhealthrisksandtoxicity(6,7).
Inthissense, the use ofantioxidants obtainedfromnaturalsources
has interesting advantages given their recognized safety when
compared to synthetic ones. Among natural phenolic compounds,
hydroxy and polyhydroxy derivates of cinnamic acids such as
caffeic, coumaric, and ferulic acids have been reported as good
free radical scavengers. Their reactivity against this unpaired-
electron species is dependent on both the number of phenolic
groups present in the molecule and the substitution pattern in the
aromatic ring (8,9). In a comparative study of the antioxidant and
antiradical capacities of a series of polyphenols, caffeic acid was
among the most active compounds (10).
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 case,
the development of polymeric materials containing the active
moiety covalently bound to their surface is an interesting alter-
native. Nowadays, the food industry has increasing interest in the
development of polymeric films with antioxidant properties.
Using this polymer modified on its surface, the antioxidant
groups are directly in contact with the medium where the free
radicals species are generated without contaminating the matrix.
Nowadays there is a great interest in the research and devel-
opment of “active packaging” in both the food and pharmaceu-
tical industries. An active packaging is able to interact directly
with the product and/or its surroundings to improve one or more
aspects of its quality or safety (1). The main aim of active
packaging is to have extra functions (related with the quality,
safety, and integrity of the product), which could be obtained by
incorporating active functional groups on the surface of packa-
ging systems.
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. 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. Photograft-
ing reactions are among these techniques and consist of the use of
energy for the formation of free radicals on the polymer surface
and the further polymerization reaction. Through this process, a
polymer with an active surface is obtained as a consequence of the
grafting of polymeric chains, where the functionality will depend
on the monomers used (2-4).
Several undesirable alterations that occur in foods, beverages,
and pharmaceuticals are due to the oxidation oforganic molecules
*Author to whom correspondence should be addressed (phone
þ54-385-4509500, int. 1617; fax þ54-385-4509525; e-mail nazareno@
unse.edu.ar).
pubs.acs.org/JAFC
Published on Web 07/28/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 16, 2010 9229
Figure 1. Representation of the sequential reactions steps for caffeic acid covalent immobilization on polypropylene films.
In this sense, Nerı
´
n et al. (11) have successfully designed anti-
were purchased from Sigma-Aldrich (Buenos Aires, Argentina). HEMA,
caffeic acid (97%), thionyl chloride, Folin-Ciocalteu reagent, linoleic acid,
β-carotene (95%), and lipoxidase preparation, type I-B, from soybean were
from Aldrich; benzophenone, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•),
and 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
(ABTS) were from Fluka. All chemicals were used as received. Tetrahydro-
furan (THF) was freshly distilled over benzophenone.
oxidant PP films by incorporating rosemary extracts containing
essential oils and phenolic compounds. This complex mixture of
antioxidants is fixed to the matrix, but the nature of the interac-
tions among the active compounds and the polymers has not been
discussed.
Another interesting approach concerning active polymers that
has recently been developed is the covalent binding of antioxidant
compounds to natural (12,13) and synthetic polymers (14,15) as
well as their use as comonomers (16). Even though these materials
could have interesting uses, they were not presented as a film
format.
Taking into account the precedents above-mentioned, the main
aim of this work was the binding of a natural antioxidant to a
polypropylene (PP) film surface. The functionalization of PP films
by introducing a hydroxyethyl methacrylate (HEMA) monomer
by UV-induced photografting reactions and the use of the intro-
duced functional groups in further reactions toward the covalent
linkage of caffeic acid were investigated. Characterization of the
modified film was carried out, and its antioxidant activity and
protective ability toward a real food sample were evaluated and
compared to the degree of caffeic acid incorporation.
Equipment. The FTIR spectra were performed on films in the transmis-
sion mode with a resolution of 8 cm-1 and 32 scans using a Nicolet 5 SXC
apparatus (Madison, WI). UV-vis absorption spectra were obtained with a
Unicam UV2 spectrophotometer (Unicam, Cambridge, U.K.). Quantita-
tive analysis of ascorbic acid in natural juice samples was performed by
high-performance liquid chromatography (HPLC). The determinations
were carried out using a Lab Alliance series III liquid chromatograph (Lab
Alliance, Alvarado, TX) coupled with a Shimadzu diode array detector
(Shimadzu Scientific Instruments, Columbia, MD). The column used was a
250 mm ꢀ 4.6 mm i.d., 5 μm, SS Wakosil RP-18, with a 4 mm ꢀ 4 mm i.d.
guard column of the same material (SGE Inc., Ringwood, Australia).
Preparation of Polypropylene Films Containing Caffeic Acid. The
covalent immobilization of caffeic acid on PP films was achieved through
two subsequent chemical reactions: first, the UV-initiated grafting polym-
erization of polypropylene films using HEMA as monomer, and, second,
the esterification reaction between the hydroxyl groups present in the
surface of polymeric films and the caffeoyl chloride. The mentioned
reactions are represented in Figure 1.
Grafting Copolymerization Reaction. The reactions were carried
out following the method used by Costamagna et al. (2) to obtain PP-g-
HEMA films. Briefly, the PP films were cut in 10 cm diameter circular
pieces and washed with distilled water and dried under vacuum at room
MATERIALS AND METHODS
Chemicals. PP films of 32 μm thickness were kindly provided by
´
Converflex-Arcor (Cordoba, Argentina). All commercially available chemicals

9230 J. Agric. Food Chem., Vol. 58, No. 16, 2010
Arrua et al.
temperature to a constant weight. Then, the piece of film was placed in a
glass Petri dish, and 0.5 mL of 0.2 M solution of the initiator benzophe-
none in the HEMA monomer and 0.5 mL of water were added. The dish
containing the reactive materials was placed in a photoreactor designed in
our laboratory and irradiated with UV light (medium-pressure UV lamp;
Engenlhard-Hanovia, Slough, U.K.) under a nitrogen atmosphere at
room temperature for 15 min. Once the grafting reaction finished, the
grafted films were extensively washed with a pH 8 NaOH solution before
analysis and finally with distilled water to remove traces of the unreacted
monomer and the free homopolymer chains formed. The samples were
dried under vacuum at room temperature to constant weight. The grafting
yield was determined by gravimetric measurements as the mean of five
single experiments using the equation
caffeic acid. Aliquots of 1 mL of each standard solution was added to
25 mL of DPPH• solution in methanol to reach final concentrations
between 5 and 25 μM. Absorbance changes were monitored at 515 nm for
30 min. Then, the percentage of the radical disappearance was determined
according to eq2, and a calibration curve was obtainedby plotting ARA%
versus caffeic acid concentration. A control reaction was performed using
PP-g-HEMA films.
ABTS•þ Bleaching Method. ABTS was dissolved in distilled water to
yield a 7 mM solution. Radical cation solution was prepared by incubating
1 mL of ABTS solution with 3.42 mL of 2.45 mM potassium persulfate
solution for 16 h in the dark at room temperature and subsequently diluted
with water to an absorbance of 1.00 ( 0.01 AU at 734 nm. To determine
the antiradical capacity of PP-g-HEMA-CA films, approximately 40 mg
of film was placed in a volumetric flask, containing 25 mL of ABTS•þ
solution, and the absorbance decrease at 734 nm was evaluated for 30 min.
All determinations were performed in triplicate. The ARA% for ABTS•þ
was calculated according to eq 2. The radical scavenging activity of PP-g-
HEMA-CA films was expressed in ARA%/100 mg of film as well as in
micromoles of caffeic acid per gram of film. For this purpose, a calibration
curve was prepared with a series of standard solutions of caffeic acid.
Aliquots of 1 mL of eachstandard solutionwas added to 25 mL of ABTS•þ
solution in methanol to reach to final concentrations between 0.1 and
1.5 μM. The absorbance of the system was monitored at 734 nm for 30 min.
Then, the percentage of the radical disappearance was calculated according
to eq 2, and a calibration curve was obtained plotting ARA% versus caffeic
acid concentration. A control reaction was performed using PP-g-HEMA
films. In the time scale of these experiments, the radical depletion
measured as a control assay in the absence of polymers or caffeic acid
addition was <1% and neglected.
Antioxidant Capacity of the Modified Films in the β-Carotene-
Linoleic Acid Cooxidation Induced by Lipoxygenase (LOX). The
experiments were carried out according to the procedure of Chaillou and
Nazareno (10) with minor modifications. First, linoleic acid solution was
prepared by mixing this compound with 219 mg of Tween 20 and diluting
with 25 mM borate buffer (pH 9.0) to a final concentration of
3.6 mg/mL. An aliquot of 1 mL of a saturated stock solution of β-carotene
in chloroform was mixedwith 1.1 g of Tween 20. Chloroform was removed
using a nitrogen stream for 20 min, and the final β-carotene aqueous
solution was prepared by adding pH 9.0 borate buffer. Once these
solutions were prepared in a volumetric flask containing 50 mg of
polymeric film, 0.55 mL of linoleic acid solution, 5.0 mL of β-carotene
solution, and 10 mL of borate buffer (pH 9.0) were added. The β-carotene
initial absorbance was adjusted to 1.00 ( 0.01 AU. Finally, an aliquot of
1 mL of 1.0 mg/mL LOX solution was added to initiate the reaction, which
was followed by monitoring the absorbance at 460 nm for 25 min. As a
control assay the same procedure was performed excluding the polymeric
film addition. Antioxidant activity (AOA) was calculated as suggested in
the literature (20) as the percentage inhibition of the β-carotene bleaching
by the polymeric film compared to that of the control.
Antioxidant Capacity of Polymeric Films in Orange Juice. The
protective ability of the films synthesized against ascorbic acid oxidation in
natural orange juice was tested by putting in contact the PP-g-HEMA-CA
films with juice samples. For this purpose, orange juice was freshly
squeezed and subsequently filtered. An amount of 60 mg of dry films
equivalent to 39 cm2 was added in a flask containing 25 mL of the orange
juice, and the mixture were stirred at 40 °C for 29 h to allow ascorbic acid
decomposition. As a control to determine the natural disappearance of
ascorbic acid under the same conditions, an experiment was performed
with orange juice without film. The antioxidant capacity of PP-g-HEMA-
CA films was also compared with that of a caffeic acid solution. In this
experiment, an aliquot of a caffeic acid solution containing 0.2930 μmol
was added to the 25 mL orange juice system. The depletion of ascorbic acid
concentration was monitored by HPLC. The ascorbic acid concentration
in the system was determined by taking 1 mL from the orange juice and
placing this aliquot in a vial containing 1 mL of a metaphosphoric
acid-acetic acid mixture. The sample was filtered and immediately
injected in the liquid chromatograph. The injection volume was 20 μL.
The mobile phase used was pH 2.5 H2SO4 aqueous solution at 1 mL/min at
24 ( 1 °C. The detection wavelength was 254 nm. A calibration curve was
performed with authentic samples of ascorbic acid in a concentration
range between 15 and 450 μg/mL.
grafting ð%Þ ¼ ½ðWgf - Wngf Þ=Wngf ꢁ ꢀ 100
ð1Þ
where Wgf and Wngf are the dry weights of grafted and nongrafted films,
respectively.
Immobilization of Caffeic Acid on PP-g-HEMA Films. These
reactions were performed under anhydrous conditions with a nitrogen
flow. First, 0.4 g of caffeic acid and 0.84 mL of thionyl chloride were
dissolved in 30 mL of anhydrous THF. Then, the reaction was allowed to
proceed at reflux for 6 h. After that, the THF and thionyl chloride were
evaporated under vacuum, and a brown solid was obtained, which
correspond to the caffeoyl chloride. Then, 0.300 g of PP-g-HEMA, 66 μL
of triethylamine, and 30 mL of anhydrous THF were added to the reaction
flask containing this acyl chloride. The coupling reaction was carried out at
reflux for 10 h. Once the reaction had finished, PP-g-HEMA-CA films were
washed twice with 50 mL of THF and three times with 50 mL of methanol to
eliminate the unreacted agents. Afterward, the modified film was dried
under vacuum to constant weight.
Determination of Available Phenolic Groups on the Modified
Film. This was determined using the Folin-Ciocalteu method (17) with
some modifications. In a volumetric flask 100 mg of PP-g-HEMA-CA
film, 1 mL of Folin-Ciocalteu reagent, and 10 mL of distilled water were
placed and stirred. After 3 min, 4 mL of 2% Na2CO3 and 10 mL of water
were added to a final volume of 25 mL. The reaction was kept at room
temperature for 48 h. After that, the absorbance was determined at 760 nm
against a control sample of PP-g-HEMA film prepared under the same
reaction conditions. An experiment in the absence of film was also
performed and used as blank for these measurements. To determine the
total phenolic content of PP-g-HEMA-CA films, a calibration curve was
prepared using caffeic acid standard solutions between 5 and 25 μM. The
results were expressed as micromoles of caffeic acid per gram of dry
polymer. All determinations were performed in triplicate.
Determination of Antiradical Activity of the Synthesized Film.
Theradical scavenging propertiesofPP-g-HEMA-CAfilms were evaluated
against two different radicals using their well-known bleaching methods:
2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) (18) and 2,20-azinobis-
(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS•þ) (19). All
determinations were performed in triplicate.
DPPH• Bleaching Method. Approximately 40 mg equivalent to
26 cm2 of PP-g-HEMA-CA films was placed in a volumetric flask,
containing 25 mL of DPPH• solution in methanol (initial absorbance at
515 nm equal to 1.00 ( 0.01 AU). The remaining DPPH• was determined
by monitoring absorbance changes at 515 nm for 30 min. The antiradical
activity (ARA%) of the polymeric films was calculated according to the
equation
ARA% ¼ 100 ꢀ ð1 - Ass=A0Þ
ð2Þ
where ASS is the absorbance of the solution in the steady state and A0 is the
absorbance of DPPH• solution before the addition of the active film. The
absorbance of the system in the steady state was estimated by mathema-
tical fitting of kinetic curves performed with Origin 7.0 software. In the
time scale of these experiments, the radical depletion measured as a control
assay in the absence of polymers or caffeic acid addition was <1%. These
radical consumption values were negligible and had no effect in the
calculations of activity according to eq 2. The antioxidant capacity of
PP-g-HEMA-CA films was expressed in ARA%/100 mg of film as well as
in micromoles of caffeic acid per gram of film. For this purpose, a
calibration curve was prepared with a series of standard solutions of

Article
J. Agric. Food Chem., Vol. 58, No. 16, 2010 9231
Figure 2. FTIR spectroscopic changes after polypropylene (PP) film modification (grafting with HEMA monomer) and caffeic acid binding on the film surface:
(A) PP; (B) PP-g-HEMA; (C) PP-g-HEMA-CA.
RESULTS AND DISCUSSION
Immobilization of Caffeic Acid on PP-g-HEMA Films. There
are numerous reports about esterification reactions of polyphe-
nolic acids that occurred using carbodiimides as coupling
agent (9, 15) and through the previous formation of the acyl
chloride of the polyphenolic compound, either with protection of
the phenolic group (23, 24) or without it (25-27). Among the
different synthetic methods, two different esterification reactions
to immobilize caffeic acid were tried. First, a synthetic method
described by Chen et al. (9) was performed as a direct reaction
between PP-g-HEMA films and caffeic acid using N,N0-dicyclo-
hexyl carbodiimide (DCC) as coupling agent. However, no
covalent immobilization of caffeic acid was obtained in this
experiment. Second, the esterification reaction of PP-g-HEMA
film and caffeoyl chloride, previously synthesized from the caffeic
acid, was carried out in anhydrous THF, obtaining better final
results. The acyl chloride formation from the polyphenolic acid
was monitored by FTIR determinations, looking for the band at
approximately 1780 cm-1, which corresponds to the stretching
vibration of the carbonyl group of the caffeoyl chloride (data not
shown). No further changes were observed in this signal after 6 h,
indicating that the reaction had been completed. A subsequent
reaction to make the covalent linkage of the polyphenolic com-
pound on PP-g-HEMA films was carried out in THF as solvent.
Figure 2 shows the FTIR spectra of PP-g-HEMA (Figure 2B) and
PP-g-HEMA-CA films (Figure 2C), which confirmed the caffeic
acid linkage on the PP-g-HEMA matrix. By comparison of the
two FTIR spectra, in the spectrum of PP-g-HEMA-CA new
bands at 1600 and 1520 cm-1, corresponding to the aromatic
carbon-carbon stretching vibrations, were found. Therefore, to
the best of our knowledge, this is the first approach toward the
caffeic acid covalent immobilization on polyolefin films.
Grafting Copolymerization Reaction. Surface grafting polymer-
ization enables the introduction ofspecific properties derived from
the grafted layer while also preserving the bulk and structural
properties of the underlying material. The introduction of func-
tional groups on the polymeric films via grafting polymerization is
a versatile approach for the preparation of materials with the
controlled incorporation of functional groups (21). In this work,
the chemical modifications of the PP surfaces were carried out by
radical grafting polymerization initiatedby UV lightusing HEMA
as the grafting monomer according to the conditions proposed by
Costamagna et al. (4). The yield of chemical modification on the
surface was evaluated by gravimetric measurements, obtaining
11.9% (standard deviation = 0.7%) of grafting yield. For surface
modification applications, thick grafting layers are unnecessary
and even undesirable because they may change the physical
properties of the bulk polypropylene film. The presence of water
in the reaction medium promotes the formation of thin grafted
layers on the surface because it prevents the diffusion of the
monomer inside the polymeric film. Moreover, the grafting agent
and the grafted chains are soluble in water, and this fact facilitates
the radical reaction propagation on the polymer surface (4). In
addition, water is inert to the triplet excited state of the benzo-
phenone photoinitiator, so the grafting reaction is not inhib-
ited (22). The remaining homopolymer formed as side product
was removed by exhaustive washing of the grafted films with a
very slightly basic solution (pH 8) and then with distilled water.
After these washing processes, the typical signal at 1630 cm-1 of
the CdC groups from the HEMA monomers was not observed in
the IR spectrum of the grafted films, demonstrating the absence of
residual monomer. The IR spectra of the PP and PP-g-HEMA
films are shown in Figure 2, spectra A and B, respectively.
Determination of Total Phenolic Content. To quantify the
available phenolic groups present on the polymeric film surface,
the Folin-Ciocalteu method was used. This method has been
extensively used in the quantification of phenolic compound
content in wines, vegetable extracts (28, 29), foods, and also
polymeric materials (16). It is based on a colorimetric oxida-
tion-reduction reaction by which the Folin-Ciocalteu reagent is
reduced by the phenolic compounds at basic pH. Total phenolic
contents determined in the modified films were compared with
their free radical scavenging properties. Table 1 shows that the
The characteristic PP bands were observed at 2842 and 1458
cm-1, corresponding to the -CH2 and -CH3 bending vibrations,
respectively. The PP-g-HEMA film spectrum presented typical
bands at 3400 and 1727 cm-1, which correspond to the -OH and
-CdO stretching vibrations, respectively. Therefore, the surface
activation of PP films was successfully performed through UV-
initiated grafting polymerization reactions. The free alcohol
groups were further used in the subsequent chemical reactions
to make covalent linkages with caffeic acid.

9232 J. Agric. Food Chem., Vol. 58, No. 16, 2010
Arrua et al.
available phenolic content in PP-g-HEMA-CA films was equiva-
lent to 6.7 μmol of caffeic acid/g of dry film or 0.011 μmol of
caffeic acid/cm2 of dry film.
scavenging reaction by the polymeric films. These results showed
that the radical-scavenging properties presented for the films
prepared in this work can be attributed to the caffeic acid
molecule linked to the polymeric surface.
Determination of Antiradical Activity of the Modified Films.
Numerous methods have been developed to evaluate the anti-
oxidant power of natural and synthetic substances and also more
complex systems such as food and beverage extracts (30, 31).
These methods, based on the disappearance of colored synthetic
radicals such as DPPH• or ABTS•þ, were chosen because of their
simplicity and versatility. They measure 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 free radical scavenging abilities
of PP-g-HEMA-CA and PP-g-HEMA films were determined by
both DPPH• and ABTS•þ bleaching methods and expressed in
ARA%/100 mg of film. Table 1 shows the values obtained by
both methods.
The antiradical capacity of the PP-g-HEMA-CA film deter-
mined in the DPPH• assay obtained from the caffeic acid
calibration curve was equivalent to 6.6 ( 0.4 μmol of caffeic
acid/g of dry film, indicating a good ability to scavenge the radical
in methanolic medium. Because this value is very similar to that
obtained from the total phenolic content, this activity of the
modified films can be ascribed to the presence of active phenolic
groups over the polymeric surface.
Compared with other antioxidant polymers previously re-
ported, the activity value reached for PP-g-HEMA-CA obtained
in this work was of the same order as the obtained for water-
soluble antioxidant polymers based on ferulic acid and
methacrylic acid as comonomers (16). Therefore, the radical
scavenging behavior that presents the polyphenolic compound
covalently bound to a polymeric film was similar that obtained
for soluble polymers in which the antioxidant moiety forms part
of the polymeric chain. When the DPPH• assay was performed
with the control polymer PP-g-HEMA, a negligible decrease in
absorbance at 515 nm was found and ARA% was only of 2.2%/
100 mg of film, whereas the inhibition obtained for PP-g-HEMA-
CA films was 89%/100 mg of film. This difference is shown in
Figure 3A, which represents the kinetic behaviors of the radical
The reuse of the antioxidant film was assayed after the DPPH•
assay; therefore, it was washed with water and rinsed with
methanol. However, subsequent assays indicated the complete
loss of activity. Moreover, several attempts were carried out to
recover it by regenerating the active groups. One of them
consisted of soaking the already used film in ascorbic acid
solutions; however, no positive results were found.
With regard to the ABTS•þ assay, the radical scavenging
capacity of the PP-g-HEMA-CA film was 18%/100 mg of film
and equivalent to 0.19μmol of caffeic acid/g of dry film, this value
being markedly lower than that obtained for the DPPH• test. This
difference could be attributed to the different media in both
assays. In contrast to the DPPH• tests, which used methanol as
solvent, the aqueous medium used in ABTS•þ assays seems to
be less compatible with the hydrophobic surface presented on the
polymeric films after the esterification reactions. Therefore,
the antioxidant molecules immobilized on the polymeric surface
are less available to scavenge the radical species presented in the
aqueous solution. With respect to the results obtained for the
control polymer PP-g-HEMA, the value measured of ARA%/
100 mg of film was 2.1%, markedly lower than the 18% obtained
for PP-g-HEMA-CA film. As in the DPPH• assay, these values
indicate that the radical scavenger properties shown for the
modified polyolefins can be ascribed to the antioxidant molecule
immobilized on the polymeric surface. The kinetic behaviors of
ABTS•þ bleaching by PP-g-HEMA-CA and PP-g-HEMA films
are shown in Figure 3B.
Antioxidant Capacity toward β-Carotene-Linoleic Acid Coox-
idation Induced by LOX. The PP-g-HEMA and PP-g-HEMA-CA
polymeric films showed extremely low but similar inhibitions of
the β-carotene-linoleic acid cooxidation induced by LOX; the
antioxidant activities determined corresponded to 0.01 and
1.54%, respectively. This clearly indicates that the linkage of
caffeic acid to the film decreases its protective ability in this
system. The antioxidant action against the β-carotene bleaching
in the cooxidation process can take place by different mechanisms
such as the inhibition of the prooxidant enzyme, which initiates
the oxidation as well as the scavenging of peroxyl radicals
involved in the propagation step. According to their polarity,
such intermediates are located in the micelle interface of this
system. In this microheterogeneous medium, where the reagents
are distributed in different phases, their reactivity is highly
conditioned or limited by their partition in the appropriate phase.
In contrast to free caffeic acid in the solution bulk, the caffeic acid
Table 1. Antiradical Capacities against DPPH• and ABTS•þ and Total
Phenolic Content by Folin-Ciocalteu Method of PP-g-HEMA-CA Films
phenolic content
DPPH•
ABTS•þ
(μmol of caffeic acid/g
of dry film)
sample
(ARA%/100 mg) (ARA%/100 mg)
PP-g-HEMA-CA
PP-g-HEMA
89 ( 6
2.2 ( 0.4
18 ( 2
2.1 ( 0.3
6.7 ( 0.5
2.3 ( 0.2
Figure 3. Kinetic behaviors of DPPH• (A) and ABTS•þ (B) solutions after addition of PP-g-HEMA (b) and PP-g-HEMA-CA (9) polymeric films.

Article
J. Agric. Food Chem., Vol. 58, No. 16, 2010 9233
Table 2. Ascorbic Acid Depletion in Orange Juice Samples after 29 h in
Aerobic Conditions at 40 °C
antioxidant addition
untreated juice. However, the highest ascorbic acid protection
was obtained for the experiments with caffeic acid added to the
juice, although at a lower concentration than the film. This
confirms that the synthesized film has protective activity against
the oxidation of the vitamin in a natural matrix, but it is less
reactive in aqueous media than in alcoholic systems.
In summary, the immobilization of caffeic acid on a grafted PP
film was successfully performed through the covalent binding of
the caffeoyl chloride on a PP-g-HEMA polymer surface. The
available phenolic groups on the modified film presented a good
correlation with the antiradical activity toward DPPH•, although
a lower reactivity as ABTS•þ scavenger in aqueous medium was
observed. Moreover, the polymer synthesized in this work
showed protective properties against the oxidation of a juice
constituent and, therefore, promising applications as food packa-
ging in different fields. Further studies are in progress searching
for higher hydrophilicity of the grafted layer as well as the
development of new synthetic pathways toward the immobiliza-
tion of natural antioxidants.
(μmol of caffeic acid
equiv/L of juice)
ascorbic acid
retention (%)
sample
orange juice (control)
orange juice þ PP-g-HEMA-CA film
orange juice þ CA
no addition
16.0 μmol
11.7 μmol
37 ( 1
74 ( 1
83 ( 1
ABBREVIATIONS USED
LOX, lipoxygenase; FC, Folin-Ciocalteu; DPPH•, 2,2-diphenyl-
1-picrylhydrazyl radical; GA, gallic acid; LA, linoleic acid; ABTS•þ
,
2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid); AOA,
antioxidant activity; ARA, antiradical activity; CA, caffeic acid;
HEMA, hydroxyethyl methacrylate; PP, polypropylene; FTIR,
Fourier transform infrared; HPLC, high-performance liquid chro-
matography; THF, tetrahydrofuran; AU, absorbance unit; BHT,
butylated hydroxytoluene; BHA, butylated hydroxyanisole; DCC,
N,N0-dicyclohexylcarbodiimide.
Figure 4. Time dependence of the ascorbic acid retention (%) in 25 mL
of orange juice at 40 °C in contact with a caffeic acid containing film
(16.0 μmol/L) (9), taking as a reference caffeic acid addition (11.7 μmol/L)
(b) and using as a control the juice without additions (2).
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Received for review April 29, 2010. Revised manuscript received July 17,
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