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Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Development of an active food packaging system with antioxidant properties based on green tea extract a
b
b
a
Daniel Carrizo , Giuseppe Gullo , Osvaldo Bosetti & Cristina Nerín a
Aragon Institute of Engineering Research (I3A), EINA, GUIA group, Department of Analytical Chemistry, University of Zaragoza, Zaragoza, Spain b
Goglio S.p.A., R&D Chemical Laboratory, Packaging Division, Daverio, Varese, Italy Accepted author version posted online: 25 Nov 2013.Published online: 05 Feb 2014.
To cite this article: Daniel Carrizo, Giuseppe Gullo, Osvaldo Bosetti & Cristina Nerín (2014) Development of an active food packaging system with antioxidant properties based on green tea extract, Food Additives & Contaminants: Part A, 31:3, 364-373, DOI: 10.1080/19440049.2013.869361 To link to this article: http://dx.doi.org/10.1080/19440049.2013.869361
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Food Additives & Contaminants: Part A, 2014 Vol. 31, No. 3, 364–373, http://dx.doi.org/10.1080/19440049.2013.869361
Development of an active food packaging system with antioxidant properties based on green tea extract Daniel Carrizoa, Giuseppe Gullob, Osvaldo Bosettib and Cristina Nerína* a
Aragon Institute of Engineering Research (I3A), EINA, GUIA group, Department of Analytical Chemistry, University of Zaragoza, Zaragoza, Spain; bGoglio S.p.A., R&D Chemical Laboratory, Packaging Division, Daverio, Varese, Italy
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(Received 20 December 2012; accepted 19 November 2013) A formula including green tea extract (GTE) was developed as an active food packaging material. This formula was moulded to obtain an independent component/device with antioxidant properties that could be easily coupled to industrial degassing valves for food packaging in special cases. GTE components (i.e., gallic acid, catechins and caffeine) were identified and quantified by HPLC-UV and UPLC-MS and migration/diffusion studies were carried out. Antioxidant properties of the formula alone and formula-valve were measured with static and dynamic methods. The results showed that the antioxidant capacity (scavenging of free radicals) of the new GTE formula was 40% higher than the non-active system (blank). This antioxidant activity increased in parallel with the GTE concentration. The functional properties of the industrial target valve (e.g., flexibility) were studied for different mixtures of GTE, and good results were found with 17% (w/w) of GTE. This new active formula can be an important addition for active packaging applications in the food packaging industry, with oxidative species-scavenging capacity, thus improving the safety and quality for the consumer and extending the shelf-life of the packaged food. Keywords: active food packaging green tea extract (GTE); antioxidant evaluation; radical scavenger; UPLC-MS; HPLC-fluorescence; valves
Introduction Food oxidation is considered a major cause of quality deterioration, affecting both the nutritional, sensory quality and safety of foods and has thus been a challenge for food preservation. Active compounds and ingredients can be incorporated into packaging materials to provide several functions that do not exist in conventional packaging systems. Active packaging may carry antioxidants, antimicrobial agents, flavouring substances and/or nutrients. Due to the health concerns of consumers and environmental problems, current research on this type of packaging has focused on the use of natural components and biodegradable packaging materials (Suppakul et al. 2003; Nerín et al. 2006; Yingyuad et al. 2006; López et al. 2007; Gutiérrez et al. 2009; Rodríguez et al. 2010). Substances with antioxidant potential are available from a variety of natural sources or as synthetic chemicals. Green tea extract (GTE) serves as a rich source of polyphenol antioxidants, particularly catechins and has the status of a food additive (Fernández et al. 2000; Roy et al. 2010). The functionality of catechins has attracted the attention of researchers and consumers, mainly because of their availability and relatively low cost. They have been reported to have antioxidant activities (Graham 1992; Pedrielli et al. 2001; Martin-Diana et al. 2008; Dicastillo et al. 2011). The main compounds responsible for this antioxidant activity *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
are gallic acid and eight major catechins: (+)-catechin (C), (–)-epicatechin (EC), (–)-catechin gallate (CG), (–)-epicatechin gallate (ECG), (–)-gallocatechin (GC), (–)-epigallocatechin (EGC), (–)-gallocatechin gallate (GCG), and (–)-epigallocatechin gallate (EGCG) (Poon 1998; Zeeb et al. 2000; Zuo et al. 2002; Saito et al. 2007; Lin et al. 2008; Colon & Nerin 2012). Antioxidant capacity measurements are based on the oxidation reactions that involve free radicals and can be static or dynamic. It is known that the oxidation process is a radical reaction initiated by the presence of OH and O free radicals (Colon & Nerin 2012). As these free radicals are the main oxidation agents responsible for most organic reactions (Tang et al. 2005), their direct determination is very interesting because it would enable one to measure the antioxidant properties of compounds by quantitative determination of radicals. Auto-oxidation is a slow, free radicals-mediated process that can take several days (Ingold 1961). It is a chain reaction including induction, propagation and termination. Different methods can be applied in static and dynamic modes to measure the antioxidant properties. Among the static experiments, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical assay is routinely practised for the assessment of free radical scavenging potential of an antioxidant molecule and it is considered one of the standard and easier colorimetric methods for the evaluation of
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Food Additives & Contaminants: Part A antioxidant properties of a variety of compounds (Okada & Okada 1998; Sánchez-Moreno 2002). However, the static methods can be time-consuming. Dynamic methods, such as that developed by Pezo et al. (2006), are performed, reducing the induction period of the chain reaction either by high temperature or by increasing the oxygen supply. One of these tests, developed by Pezo et al. (2006), involves the generation of an atmosphere enriched in free radicals, which in contact with the antioxidant formula can provide the radical scavenging properties of the formula (Pezo et al. 2006; Nerín 2011). DPPH and the method of Pezo et al. (2006) have been used to measure the antioxidant properties of the new active packaging here described. Both methods work directly with the packaging material as it is as neither dissolution nor extraction of the material that are required. Quantitative measurements can be obtained in both cases, so that the quantitative confirmation of the antioxidant properties can be achieved. The aim of this work was to develop an antioxidant formula (film) based on GTE to be incorporated as an independent component/device into industrial valves for food packaging. The industrial valve can thus act as a degassing device and as an oxidant-species scavenger, i.e., an active food packaging system. The formula and the demonstration of the antioxidant performance are described.
Materials and methods Chemicals and reagents Green tea extract (GTE) was supplied by Taiyo Greenpower Co. Ltd (Jiangsu, China). Individual GTE components were also used as standards: gallic acid (CAS 14991-7); caffeine (58-08-2); (+)-catechin (>99.0% (HPLC), CAS 154-23-4) (C); (−)-epicatechin (>95.0% (HPLC), CAS 490-46-0) (EC); (−)-epicatechin gallate (>98% (HPLC), CAS 1257-08-5) (ECG); (−)-catechin gallate (>98% (HPLC), CAS 130405-40-2) (CG); (−)-epigallocatechin (>95.0% (HPLC), CAS 970-74-1) (EGC); (−)-gallocatechin (>98% (HPLC), CAS 3371-27-5) (GC); (−)-gallocatechin gallate (>98% (HPLC), CAS 4233-969) (GCG); and (−)-epigallocatechin gallate (>95.0% (HPLC), CAS 989-51-5) (EGCG). Agar (CAS 9002-180), β-cyclodextrin (CAS 7585-39-9), activated carbon (CAS 7440-44-0), methyl-cellulose (CAS 9004-67-5), sodium alginate (CAS 9005-38-3), and glycerol (CAS 56-81-5) were supplied by Sigma (Madrid, Spain); arabic gum (CAS 9000-01-5) was from Merck (Darmstadt, Germany). Reagent-grade methanol, formic acid (>98%) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma. Salicylic acid (>99%) and hydrogen peroxide (>34.5%) were from Scharlab (Barcelona, Spain). Polyethylene (PE) layers (35 µm thicknesses)
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were supplied from Goglio SpA (Daverio, Italy); silicone (food contact grade) was purchased from Sumbeart (Seville, Spain). Ultrapure water was obtained from a Milli-Q system (Millipore, Billerica, MA, USA). Apparatus The method developed by Pezo et al. (2006) was used to evaluate the radical scavenging properties of the different formula in dynamic mode. The method consists of generating a free radical atmosphere that passes through a tube where the antioxidant material is placed. The gas phase containing the free radicals is carried by dried air through the sample and then it bubbles into an impinger containing a salicylic acid solution (SA), which in the presence of free radicals is hydroxylated to give 2,5-dihydroxybenzoic acid (2,5-DHB) as a major compound and the 2,3-DHB isomer and cathecol as minor compounds. If free radicals are scavenged by the antioxidant sample, they do not reach the SA solution and then 2,5-DHB is not formed. Thus, the quantitative measurement of 2,5-DHB provides a quantitative measurement of the antioxidant capacity of the material under test. The values are usually compared with those found using blank samples and known antioxidant materials. The analysis of 2,5-DHB and the remaining salicylic acid, both with fluorescent properties, was performed in a Waters 2795 Series HPLC system (Milford, MA, USA) coupled to a Waters 474 fluorescence detector operating at the optimum excitation and emission wavelengths for both compounds (λex = 324 nm, λem = 448 nm). GTE components (catechins, caffeine and gallic acid) were analysed by a Waters 2795 Series HPLC system coupled to a Waters PDA 2996 photodiode-array detector. An Atlantis dC18 Waters reversed-phase column (100 mm long, 4.6 i. d., mm, 3 µm) was used. The mobile phases were water with 0.1% (v/v) of formic acid (eluent A) and methanol with 0.1% (v/v) of formic acid (eluent B). Flow rate was 0.5 ml min–1 and injection volume of 20 µl. The gradient system was 0–5 min, 10% B, 5–14 min, linear gradient from 10% to 20% B; 14–20 min, linear gradient 20% to 50% B; 20–22 min, linear gradient from 50% to 90% B, 22–26 min, 90% B; 26–30 min, linear gradient from 90% to 10% B. Post-run time was 10 min; column temperature was set at 25°C. The maximum absorbance of catechins, gallic acid and caffeine was detected at 275 nm for quantitative purposes with a data acquisition rate of 64 Hz. Gallic acid, caffeine, C, EC, CG, GC, ECG, EGC, GCG and EGCG were confirmed by comparing the retention time and spectral data with their pure standards. To evaluate the selectivity of this method, Empower chromatographic software was used for gathering data and processing the chromatograms. GTE components before and after the oxidation process were also analysed by UPLC-MS-TQ AQUITY (Waters). UPLC-MS conditions
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were as follows: an Acquity UPLC@ BEH C18 1.7 µm (2.1 × 100 mm) was used. Mobile phases were: eluent A (methanol with 0.1% formic acid) and eluent B (water with 0.1% formic acid). Flow rate was 0.3 ml min–1 and injection volume 10 µl. The gradient system was 0–6 min, 5% A–95% B, 6–8 min, 95% A–5% B, 8.10–10 min, 5% A–95% B. Run time was 10 min; sample temperature was set at 7°C and column temperature at 35°C. SIM mode was used for the mass spectra conditions. ESI in negative mode was used for all the catechins and gallic acid, while for caffeine ESI was used in positive mode. Selected m/z were 169.02 for gallic acid, 194.08 m/z for caffeine, 289.07 m/z for (+)-catechin and (−)-epicatechin, 305.07 m/z for (−)-gallocatechin and (−)-epigallocatechin, 441.08 m/z for (−)-epicatechin gallate and (−)-catechin gallate, and finally 457.08 m/z for (−)-epigallocatechin gallate and (−)-gallocatechin gallate. The linear dynamic ranges obtained were calculated with at least seven calibration points and the values are expressed as μg of standard mixture per g of methanolic solution. Linear range varied from 0.001 to 75.1 μg g−1 depending on the standards, so an LOQ of 0.001 μg g−1 (= 1 ppb) can be assumed. The analytical method performance was validated through the participation in an international inter-calibration exercise (Exercise I of the Dietary Supplements Laboratory Quality Assurance Program, NIST Interagency Report (IR7955) (see http:// nvlpubs.nist.gov/nistpubs/ir/2013/NIST.IR.7955.pdf). Formula development (first generation) Several types of “formulas” were prepared and tested prior developing the specific one. The term “formula” is used for the developed film, over which we obtained our “pellets” or “formula” pellet, which are used to assemble the formula“pellet”-valve industrial device. The formula consists of a mixture of GTE, an agglutinant and a plasticiser agent, mixed with ultrapure water. Different concentrations of GTE (50%, 70% and 80%, w/w), agglutinant agent (10% and 35%, w/w) and plasticiser agent (10%, 20% and 40%, w/w) were prepared and tested. The addition of agglutinant and plasticiser was necessary in order to provide some specific mechanical properties (e.g., elasticity) necessary for the further manipulation and storage of the formula. Many different agglutinant agents were used (agar, β-cyclodextrin, activated carbon, methyl-cellulose, arabic gum and sodium alginate), meanwhile only one plasticiser agent was used (glycerol). All agents used (additives) were listed as permitted substances for the production of plastic materials for food contact. Once the liquid viscous phase formula was ready it was poured into a mould (polytetrafluoroethylene (PTFE)) and left to cure for 24 h at RT (20–25ºC). The final film (1 mm thickness) produced by cast was cut in circles of 0.3 cm diameter in order to obtain the formula “pellet” as well as the modified industrial valve containing the “pellet”
(i.e., the “pellet-valve”) for the antioxidant activity experiments (static and dynamic). Formula development (second generation) A second-generation “formula” was produced with the aim of improving the elasticity and flexibility of the final product. In this case a silicone-base (food contact-grade) formula was used. Room temperature vulcanising (RTV) silicone rubber for food contact applications was selected for this purpose. It was a two-component system (silicone and a catalyst), colourless, low viscosity, medium hardness (39 shore A) and fast cured (24 h at 25°C). Different concentrations of GTE antioxidant (5%, 9%, 17%, 20%, 23%, 32%, 42% and 50%, w/w) were added into the silicone based “formula” in order to find the best equilibrium between mechanical (i.e., flexibility) and antioxidant properties of the final film. This silicone-based formula film is used as a new formula “pellet” for “silicone-based pellet-valve” experiments. The liquid viscous mixture was poured into a PTFE mould (18 × 10 cm), with 0.1 mm depth and left for 24 h at 30°C in an oven to be cured. After this the active film was detached from the plate and prepared for further use. From this film circles were cut to obtain the pellets to be inserted into the valve. Measurement of antioxidant properties Antioxidant activity of the different “formula” prototypes of first and second generations was explored in static and dynamic experiments, as below described. Static experiment For this kind of experiments the chosen method was the DPPH.(Blois 1958). This method is well established and widely used. It is based on the capacity of the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) to react (through the acceptance of an electron) and change to a stable molecule. In the radical form DPPH absorbs at 515 nm, but upon reduction by an antioxidant or a radical species, the absorption disappears. The test was run in two different ways: one visual and the other by quantitative measurement with the spectrophotometer. Figure 1 shows a detailed diagram of this experiment. The visual tests were carried out with the formula “pellet” alone and with the “pellet-valve”; meanwhile the spectrophotometric tests were performed only with the “pellet-valve” set-up. Samples (three and five replicates, respectively) were prepared using the industrial valve (i.e., a degassing one-way valve for roasted coffee packaging) with a formula “pellet” inside. Each valve was placed inside a 20 ml glass vial, without direct contact with a 5 ml solution of DPPH (50 mg kg−1) in methanol and sealed. Possible contact between the “formula” and the solution of DPPH has to
Food Additives & Contaminants: Part A Static experiment
Spectrophotometer
Visual pellet
pellet-valve
control
pellet-valve
92 h
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Figure 1.
Explanation for the static experiment.
be avoided because otherwise the reaction occurs immediately, thus limiting the validation of the experiment. The absorbance (515 nm) was measured each hour with a spectrophotometer (Unicam Helios, Cambridge, UK), using quartz cuvettes 100-QS (1 × 1 × 4.5 cm) from Suprasil (Spain).
Dynamic experiment The dynamic experiment was carried out in accordance with the design developed by Pezo et al. (2006, 2008), although slightly modified. The modification was based on a change of the antioxidant film “container” adapted to the present conditions, as shown in Figure 2. In brief, this system consists of generating a known concentration of free radicals (from a nebulising hydrogen peroxide solution at a
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flow rate of 0.8 ml min−1) in the gas phase (total air flow = 470 ml min−1). The total flow passed through a quartz tube covered by ultraviolet (UV) lamps to supply the UV irradiation required for free radical formation and so produced a free-radical-enriched atmosphere that was carried through the valve with the formula-“pellet” (i.e., firstgeneration formula). Finally, the free radicals, which are not scavenged by the antioxidant material, are trapped in an aqueous solution of salicylic acid, which is converted into 2,5-dihydroxybenzoic acid (DHB). The final solution is measured by HPLC with highly sensitive fluorescence detection. Both types of formula (first and second generation) were measured under these dynamic conditions. In the first-generation formula only the three best mixtures (i.e., general appearance and mechanical performance of the film) were analysed; meanwhile with the silicone-based formula different GTE-antioxidant percentages were evaluated.
Release studies The diffusion experiments were carried out following the Moisan tests in a migration cell as suggested by Moisan (1980). The cell consists of two 10 × 10 cm aluminium plates of 1 cm thickness. These experiments were carried out using some selected first-generation formula films (e.g., methyl-cellulose and β-cyclodextrin), second-generation silicone-based formulas (10%, 20% and 30% w/w of GTE) and GTE film alone. The diffusion experiments were performed using a 10 × 10 cm sample for each of the formulas containing the GTE with 10 polyethylene (PE) layers (35 µm thickness each) stacked under pressure in a
Modified part
Air flow (470 mL min–1)
UV Lamps
Auxiliary air H2O2, flow 0.8 mL min–1)
Figure 2.
Nebuliser air
Modified apparatus developed for evaluating the radical scavenging properties of the different formula in dynamic mode.
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pile on the active formula with the following arrangement: Al plate/formula/PE-1/PE-2/ … /PE-10/Al plate). Each of these cells was closed using four screws and a dynamometric tool in order to apply a constant twisting force of 0.8 Nm (Canellas et al. 2010). The diffusion cells were kept at 30ºC for 1 month. At the end of the experiment each of the PE layers was put into a 15 ml vial and extracted with methanol for 15 min with ultrasonic assistance, according to the previous optimisation (Canellas et al. 2010); the methanolic extract was then transferred to a 2 ml vial and injected into the HPLC-fluorescence and UPLC-MS. Determination of the active compounds (gallic acid, caffeine and catechins) released from the new formula (second generation) was carried out by determining the specific migration into food simulants established by European Commission Regulation (EU) 10/2011. Specific migration is the amount of a specific component that migrates from the food contact material to the food in contact with it. Food contact materials must not transfer their components into the foods in unacceptable quantities (migration) (Dicastillo et al. 2011). Due to the likely future industrial applications (roasted coffee beans) two food simulants were used: 3% acetic acid (w/v) as an acidic food simulant and 20% of ethanol (v/v) as an alcoholic food simulant, both in distilled water. For this experiment three different concentrations of GTE were used (10%, 20% and 30% w/w) in the siliconebased films. Migration studies were conducted at 70ºC for 2 h, which simulate the hot filling conditions, as roasted coffee beans were the main target food (UNE-EN 2002, 2005). Double-sided, total immersion migration tests were performed as follows: 9 cm2 of each silicone-based formula sample and 15 ml of the simulant (area to volume ratio around 6 dm2 l–1) were placed in 20 ml glass vials. After the exposure, the various green tea components were analysed by UPLC-MS.
Sensory analysis Drinking water samples in contact with the active material for different exposure times (1, 2, 4, 6 and 10 days) at RT
were tested. A triangular test (UNE 1992) between different samples after exposure to the active material and with pure drinking water was carried out. The panellists had three bottles, two of them with drinking water and a third after exposure to the active material for different times. They had to determine the different one and to register the odour, taste and colour of the drinking water.
Results and discussion Formula development The chemical composition of the first batch of the formula generation is shown in Table 1. The percentage of agglutinant agent added varies widely from 1.5% (w/w) in the agar mixtures up to 35% (w/w) in the activated carbon, βcyclodextrin, methyl-cellulose and arabic gum mixtures. The small amount of agglutinant and GTE in the agar mixtures is due to the need for higher amounts of water in the preparation. This first-generation formula film was too rigid and after some time of preparation (24 h) elasticity was completely lost. Only the methyl-cellulose formula film kept some kind of elasticity after this period. Moreover, during the development of the first-generation formula-“pellet” some mixtures were prepared with and without the plasticiser agent. Glycerol was used as a plasticiser agent in 10%, 20% and 40% (w/w), but only a little improvement was achieved in some of the mixtures containing arabic gum and methyl-cellulose. Samples with the highest amount of glycerol were discarded due to problems in the final curing step. As a consequence, further formula development had to be explored with the second-generation formula. The different chemical composition of the second-generation formula is shown in Table 2. In this new mixture no plasticiser was needed. This type of silicone is semi-liquid and needs the addition of a catalyst for curing. The ratio of the mixture had to be 10 to 1 (w/w), for a curing time of 24 h at 30°C. Different percentages of GTE were tested, as can be seen in Table 2, from 5%, 9%, 17%, 20%, 23%, 32%, 42% to 50% (w/w). Implementation of the silicone-based formula caused
Table 1. Chemical composition (%, w/w) and antioxidant activity (static method using DPPH) of the first-generation formula. Antioxidant activity (DPPH)a Agglutinate type
Agglutinate (%)
Green tea (%)
Plasticiser (%)
2h
24 h
48 h
96 h
120 h
Agar Activated carbon Activated carbon Sodium alginate β-cyclodextrin Β-cyclodextrin Methyl-cellulose Arabic gum
3 9 22 9 9 22 10 10
2.5 91 78 91 91 78 80 80
0 0 0 0 0 0 10 10
No No No No No No No No
No Yes No Yes No No No No
No Yes Yes Yes Yes Yes No No
No Yes Yes Yes Yes Yes Yes Yes
No Yes Yes Yes Yes Yes Yes Yes
Note: aColour change from violet to yellow, yes or no, at 2, 24, 48, 96 and 120 h.
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Table 2. Chemical composition (%, w/w) and antioxidant activity of the second-generation formula (silicone-based). Antioxidant activity (DPPH) Silicone/catalyst ratio (w/w) 10:1 10:1 10:1 10:1 10:1
Silicone plus catalyst (%) (w/w)
GTE (%) (w/w)
8h
18 h
36 h
38 h
64 h
96 h
95 91 80 68 100
5 9 20 32 Nob
No No No No No
No No No No No
No No Yesa Yesa No
No No Yesa Yesa No
No Yesa Yesa Yesa No
Yesa Yesa Yesa Yesa No
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Notes: aColour change in one of the three replicates. b No, silicone blank.
an enormous improvement in the final elasticity of the mixture, which remained stable for a long period of time (at least 2 months). According to the results obtained during the preparation of the first- and second-generation formulas, the silicone-based formula and thus the silicone-based “pellet” achieved the best results, good results were found with 17% (w/w) of GTE.
Antioxidant activity All the mixtures of first-generation formulas had excellent antioxidant activity in both types of studies (static and dynamic). Table 1 shows the antioxidant activity (static experiment-DPPH) of the most representative mixtures for the first-generation formula. Spectrophotometric measurements were done with the methyl-cellulose, β-cyclodextrin and arabic gum mixtures, and five replicates of each and a blank for each mixture were measured during 92 h. The results are shown in Figure 3. Table 2 shows the results of antioxidant activity (DPPH) for the silicone-based pellet. As can be seen, the antioxidant performance increases with the percentage of GTE. As DPPH is a supplier of free radicals, the exposure of a sample to DPPH means
that the sample can act as a scavenger of free radicals, which means antioxidant performance. The tests were carried out without direct contact between the siliconebased pellet and the DPPH solution. This reinforces the statement of radical scavenger activity of the siliconebased pellet. Figure 3 shows a comparison between the different formulas under test and it emphasises the similarities of performance within them. All behave as radical scavengers, although a slight increase of the methyl-cellulose formula antioxidant capacity is observed. Among the first-generation formula, only that of methyl-cellulose (formula-“pellet”) was selected for dynamic experiments. Antioxidant activity is reported as a percentage of hydroxylation of the salycilic acid (SA) to form DHB, where a lower percentage of hydroxylation of SA indicates higher antiradical scavenging efficiency. The percentage of hydroxylation is in fact an indirect measurement of the concentration of free radicals that cross the polymeric layer or the specimen exposed to the free radicals atmosphere generated in the device above described. Antioxidant activity was between 50% and 30%, with an average of 40%, using mixtures with 0.03 and 0.04 g of GTE, higher than the blank sample formula. This means
Figure 3. Antioxidant activity measured with DPPH (mean percentage of five replicates) (spectrophotometric measurements) of the selected first-generation formula. AG, arabic gum; MC, methyl-cellulose; and Cy, β-cyclodextrin.
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that the active sample exposed to the free radicals behaves as an antioxidant or free radical scavenger. The percentage of hydroxylation is always referred to as the amount of SA. Meanwhile, antioxidant activity for silicone-based formula was in the same range.
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Release and diffusion studies Diffusion Diffusion studies reveal a low or null mobility of catechins, lower than the LOQ (
Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Development of an active food packaging system with antioxidant properties based on green tea extract a
b
b
a
Daniel Carrizo , Giuseppe Gullo , Osvaldo Bosetti & Cristina Nerín a
Aragon Institute of Engineering Research (I3A), EINA, GUIA group, Department of Analytical Chemistry, University of Zaragoza, Zaragoza, Spain b
Goglio S.p.A., R&D Chemical Laboratory, Packaging Division, Daverio, Varese, Italy Accepted author version posted online: 25 Nov 2013.Published online: 05 Feb 2014.
To cite this article: Daniel Carrizo, Giuseppe Gullo, Osvaldo Bosetti & Cristina Nerín (2014) Development of an active food packaging system with antioxidant properties based on green tea extract, Food Additives & Contaminants: Part A, 31:3, 364-373, DOI: 10.1080/19440049.2013.869361 To link to this article: http://dx.doi.org/10.1080/19440049.2013.869361
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Food Additives & Contaminants: Part A, 2014 Vol. 31, No. 3, 364–373, http://dx.doi.org/10.1080/19440049.2013.869361
Development of an active food packaging system with antioxidant properties based on green tea extract Daniel Carrizoa, Giuseppe Gullob, Osvaldo Bosettib and Cristina Nerína* a
Aragon Institute of Engineering Research (I3A), EINA, GUIA group, Department of Analytical Chemistry, University of Zaragoza, Zaragoza, Spain; bGoglio S.p.A., R&D Chemical Laboratory, Packaging Division, Daverio, Varese, Italy
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(Received 20 December 2012; accepted 19 November 2013) A formula including green tea extract (GTE) was developed as an active food packaging material. This formula was moulded to obtain an independent component/device with antioxidant properties that could be easily coupled to industrial degassing valves for food packaging in special cases. GTE components (i.e., gallic acid, catechins and caffeine) were identified and quantified by HPLC-UV and UPLC-MS and migration/diffusion studies were carried out. Antioxidant properties of the formula alone and formula-valve were measured with static and dynamic methods. The results showed that the antioxidant capacity (scavenging of free radicals) of the new GTE formula was 40% higher than the non-active system (blank). This antioxidant activity increased in parallel with the GTE concentration. The functional properties of the industrial target valve (e.g., flexibility) were studied for different mixtures of GTE, and good results were found with 17% (w/w) of GTE. This new active formula can be an important addition for active packaging applications in the food packaging industry, with oxidative species-scavenging capacity, thus improving the safety and quality for the consumer and extending the shelf-life of the packaged food. Keywords: active food packaging green tea extract (GTE); antioxidant evaluation; radical scavenger; UPLC-MS; HPLC-fluorescence; valves
Introduction Food oxidation is considered a major cause of quality deterioration, affecting both the nutritional, sensory quality and safety of foods and has thus been a challenge for food preservation. Active compounds and ingredients can be incorporated into packaging materials to provide several functions that do not exist in conventional packaging systems. Active packaging may carry antioxidants, antimicrobial agents, flavouring substances and/or nutrients. Due to the health concerns of consumers and environmental problems, current research on this type of packaging has focused on the use of natural components and biodegradable packaging materials (Suppakul et al. 2003; Nerín et al. 2006; Yingyuad et al. 2006; López et al. 2007; Gutiérrez et al. 2009; Rodríguez et al. 2010). Substances with antioxidant potential are available from a variety of natural sources or as synthetic chemicals. Green tea extract (GTE) serves as a rich source of polyphenol antioxidants, particularly catechins and has the status of a food additive (Fernández et al. 2000; Roy et al. 2010). The functionality of catechins has attracted the attention of researchers and consumers, mainly because of their availability and relatively low cost. They have been reported to have antioxidant activities (Graham 1992; Pedrielli et al. 2001; Martin-Diana et al. 2008; Dicastillo et al. 2011). The main compounds responsible for this antioxidant activity *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
are gallic acid and eight major catechins: (+)-catechin (C), (–)-epicatechin (EC), (–)-catechin gallate (CG), (–)-epicatechin gallate (ECG), (–)-gallocatechin (GC), (–)-epigallocatechin (EGC), (–)-gallocatechin gallate (GCG), and (–)-epigallocatechin gallate (EGCG) (Poon 1998; Zeeb et al. 2000; Zuo et al. 2002; Saito et al. 2007; Lin et al. 2008; Colon & Nerin 2012). Antioxidant capacity measurements are based on the oxidation reactions that involve free radicals and can be static or dynamic. It is known that the oxidation process is a radical reaction initiated by the presence of OH and O free radicals (Colon & Nerin 2012). As these free radicals are the main oxidation agents responsible for most organic reactions (Tang et al. 2005), their direct determination is very interesting because it would enable one to measure the antioxidant properties of compounds by quantitative determination of radicals. Auto-oxidation is a slow, free radicals-mediated process that can take several days (Ingold 1961). It is a chain reaction including induction, propagation and termination. Different methods can be applied in static and dynamic modes to measure the antioxidant properties. Among the static experiments, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical assay is routinely practised for the assessment of free radical scavenging potential of an antioxidant molecule and it is considered one of the standard and easier colorimetric methods for the evaluation of
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Food Additives & Contaminants: Part A antioxidant properties of a variety of compounds (Okada & Okada 1998; Sánchez-Moreno 2002). However, the static methods can be time-consuming. Dynamic methods, such as that developed by Pezo et al. (2006), are performed, reducing the induction period of the chain reaction either by high temperature or by increasing the oxygen supply. One of these tests, developed by Pezo et al. (2006), involves the generation of an atmosphere enriched in free radicals, which in contact with the antioxidant formula can provide the radical scavenging properties of the formula (Pezo et al. 2006; Nerín 2011). DPPH and the method of Pezo et al. (2006) have been used to measure the antioxidant properties of the new active packaging here described. Both methods work directly with the packaging material as it is as neither dissolution nor extraction of the material that are required. Quantitative measurements can be obtained in both cases, so that the quantitative confirmation of the antioxidant properties can be achieved. The aim of this work was to develop an antioxidant formula (film) based on GTE to be incorporated as an independent component/device into industrial valves for food packaging. The industrial valve can thus act as a degassing device and as an oxidant-species scavenger, i.e., an active food packaging system. The formula and the demonstration of the antioxidant performance are described.
Materials and methods Chemicals and reagents Green tea extract (GTE) was supplied by Taiyo Greenpower Co. Ltd (Jiangsu, China). Individual GTE components were also used as standards: gallic acid (CAS 14991-7); caffeine (58-08-2); (+)-catechin (>99.0% (HPLC), CAS 154-23-4) (C); (−)-epicatechin (>95.0% (HPLC), CAS 490-46-0) (EC); (−)-epicatechin gallate (>98% (HPLC), CAS 1257-08-5) (ECG); (−)-catechin gallate (>98% (HPLC), CAS 130405-40-2) (CG); (−)-epigallocatechin (>95.0% (HPLC), CAS 970-74-1) (EGC); (−)-gallocatechin (>98% (HPLC), CAS 3371-27-5) (GC); (−)-gallocatechin gallate (>98% (HPLC), CAS 4233-969) (GCG); and (−)-epigallocatechin gallate (>95.0% (HPLC), CAS 989-51-5) (EGCG). Agar (CAS 9002-180), β-cyclodextrin (CAS 7585-39-9), activated carbon (CAS 7440-44-0), methyl-cellulose (CAS 9004-67-5), sodium alginate (CAS 9005-38-3), and glycerol (CAS 56-81-5) were supplied by Sigma (Madrid, Spain); arabic gum (CAS 9000-01-5) was from Merck (Darmstadt, Germany). Reagent-grade methanol, formic acid (>98%) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma. Salicylic acid (>99%) and hydrogen peroxide (>34.5%) were from Scharlab (Barcelona, Spain). Polyethylene (PE) layers (35 µm thicknesses)
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were supplied from Goglio SpA (Daverio, Italy); silicone (food contact grade) was purchased from Sumbeart (Seville, Spain). Ultrapure water was obtained from a Milli-Q system (Millipore, Billerica, MA, USA). Apparatus The method developed by Pezo et al. (2006) was used to evaluate the radical scavenging properties of the different formula in dynamic mode. The method consists of generating a free radical atmosphere that passes through a tube where the antioxidant material is placed. The gas phase containing the free radicals is carried by dried air through the sample and then it bubbles into an impinger containing a salicylic acid solution (SA), which in the presence of free radicals is hydroxylated to give 2,5-dihydroxybenzoic acid (2,5-DHB) as a major compound and the 2,3-DHB isomer and cathecol as minor compounds. If free radicals are scavenged by the antioxidant sample, they do not reach the SA solution and then 2,5-DHB is not formed. Thus, the quantitative measurement of 2,5-DHB provides a quantitative measurement of the antioxidant capacity of the material under test. The values are usually compared with those found using blank samples and known antioxidant materials. The analysis of 2,5-DHB and the remaining salicylic acid, both with fluorescent properties, was performed in a Waters 2795 Series HPLC system (Milford, MA, USA) coupled to a Waters 474 fluorescence detector operating at the optimum excitation and emission wavelengths for both compounds (λex = 324 nm, λem = 448 nm). GTE components (catechins, caffeine and gallic acid) were analysed by a Waters 2795 Series HPLC system coupled to a Waters PDA 2996 photodiode-array detector. An Atlantis dC18 Waters reversed-phase column (100 mm long, 4.6 i. d., mm, 3 µm) was used. The mobile phases were water with 0.1% (v/v) of formic acid (eluent A) and methanol with 0.1% (v/v) of formic acid (eluent B). Flow rate was 0.5 ml min–1 and injection volume of 20 µl. The gradient system was 0–5 min, 10% B, 5–14 min, linear gradient from 10% to 20% B; 14–20 min, linear gradient 20% to 50% B; 20–22 min, linear gradient from 50% to 90% B, 22–26 min, 90% B; 26–30 min, linear gradient from 90% to 10% B. Post-run time was 10 min; column temperature was set at 25°C. The maximum absorbance of catechins, gallic acid and caffeine was detected at 275 nm for quantitative purposes with a data acquisition rate of 64 Hz. Gallic acid, caffeine, C, EC, CG, GC, ECG, EGC, GCG and EGCG were confirmed by comparing the retention time and spectral data with their pure standards. To evaluate the selectivity of this method, Empower chromatographic software was used for gathering data and processing the chromatograms. GTE components before and after the oxidation process were also analysed by UPLC-MS-TQ AQUITY (Waters). UPLC-MS conditions
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were as follows: an Acquity UPLC@ BEH C18 1.7 µm (2.1 × 100 mm) was used. Mobile phases were: eluent A (methanol with 0.1% formic acid) and eluent B (water with 0.1% formic acid). Flow rate was 0.3 ml min–1 and injection volume 10 µl. The gradient system was 0–6 min, 5% A–95% B, 6–8 min, 95% A–5% B, 8.10–10 min, 5% A–95% B. Run time was 10 min; sample temperature was set at 7°C and column temperature at 35°C. SIM mode was used for the mass spectra conditions. ESI in negative mode was used for all the catechins and gallic acid, while for caffeine ESI was used in positive mode. Selected m/z were 169.02 for gallic acid, 194.08 m/z for caffeine, 289.07 m/z for (+)-catechin and (−)-epicatechin, 305.07 m/z for (−)-gallocatechin and (−)-epigallocatechin, 441.08 m/z for (−)-epicatechin gallate and (−)-catechin gallate, and finally 457.08 m/z for (−)-epigallocatechin gallate and (−)-gallocatechin gallate. The linear dynamic ranges obtained were calculated with at least seven calibration points and the values are expressed as μg of standard mixture per g of methanolic solution. Linear range varied from 0.001 to 75.1 μg g−1 depending on the standards, so an LOQ of 0.001 μg g−1 (= 1 ppb) can be assumed. The analytical method performance was validated through the participation in an international inter-calibration exercise (Exercise I of the Dietary Supplements Laboratory Quality Assurance Program, NIST Interagency Report (IR7955) (see http:// nvlpubs.nist.gov/nistpubs/ir/2013/NIST.IR.7955.pdf). Formula development (first generation) Several types of “formulas” were prepared and tested prior developing the specific one. The term “formula” is used for the developed film, over which we obtained our “pellets” or “formula” pellet, which are used to assemble the formula“pellet”-valve industrial device. The formula consists of a mixture of GTE, an agglutinant and a plasticiser agent, mixed with ultrapure water. Different concentrations of GTE (50%, 70% and 80%, w/w), agglutinant agent (10% and 35%, w/w) and plasticiser agent (10%, 20% and 40%, w/w) were prepared and tested. The addition of agglutinant and plasticiser was necessary in order to provide some specific mechanical properties (e.g., elasticity) necessary for the further manipulation and storage of the formula. Many different agglutinant agents were used (agar, β-cyclodextrin, activated carbon, methyl-cellulose, arabic gum and sodium alginate), meanwhile only one plasticiser agent was used (glycerol). All agents used (additives) were listed as permitted substances for the production of plastic materials for food contact. Once the liquid viscous phase formula was ready it was poured into a mould (polytetrafluoroethylene (PTFE)) and left to cure for 24 h at RT (20–25ºC). The final film (1 mm thickness) produced by cast was cut in circles of 0.3 cm diameter in order to obtain the formula “pellet” as well as the modified industrial valve containing the “pellet”
(i.e., the “pellet-valve”) for the antioxidant activity experiments (static and dynamic). Formula development (second generation) A second-generation “formula” was produced with the aim of improving the elasticity and flexibility of the final product. In this case a silicone-base (food contact-grade) formula was used. Room temperature vulcanising (RTV) silicone rubber for food contact applications was selected for this purpose. It was a two-component system (silicone and a catalyst), colourless, low viscosity, medium hardness (39 shore A) and fast cured (24 h at 25°C). Different concentrations of GTE antioxidant (5%, 9%, 17%, 20%, 23%, 32%, 42% and 50%, w/w) were added into the silicone based “formula” in order to find the best equilibrium between mechanical (i.e., flexibility) and antioxidant properties of the final film. This silicone-based formula film is used as a new formula “pellet” for “silicone-based pellet-valve” experiments. The liquid viscous mixture was poured into a PTFE mould (18 × 10 cm), with 0.1 mm depth and left for 24 h at 30°C in an oven to be cured. After this the active film was detached from the plate and prepared for further use. From this film circles were cut to obtain the pellets to be inserted into the valve. Measurement of antioxidant properties Antioxidant activity of the different “formula” prototypes of first and second generations was explored in static and dynamic experiments, as below described. Static experiment For this kind of experiments the chosen method was the DPPH.(Blois 1958). This method is well established and widely used. It is based on the capacity of the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) to react (through the acceptance of an electron) and change to a stable molecule. In the radical form DPPH absorbs at 515 nm, but upon reduction by an antioxidant or a radical species, the absorption disappears. The test was run in two different ways: one visual and the other by quantitative measurement with the spectrophotometer. Figure 1 shows a detailed diagram of this experiment. The visual tests were carried out with the formula “pellet” alone and with the “pellet-valve”; meanwhile the spectrophotometric tests were performed only with the “pellet-valve” set-up. Samples (three and five replicates, respectively) were prepared using the industrial valve (i.e., a degassing one-way valve for roasted coffee packaging) with a formula “pellet” inside. Each valve was placed inside a 20 ml glass vial, without direct contact with a 5 ml solution of DPPH (50 mg kg−1) in methanol and sealed. Possible contact between the “formula” and the solution of DPPH has to
Food Additives & Contaminants: Part A Static experiment
Spectrophotometer
Visual pellet
pellet-valve
control
pellet-valve
92 h
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Figure 1.
Explanation for the static experiment.
be avoided because otherwise the reaction occurs immediately, thus limiting the validation of the experiment. The absorbance (515 nm) was measured each hour with a spectrophotometer (Unicam Helios, Cambridge, UK), using quartz cuvettes 100-QS (1 × 1 × 4.5 cm) from Suprasil (Spain).
Dynamic experiment The dynamic experiment was carried out in accordance with the design developed by Pezo et al. (2006, 2008), although slightly modified. The modification was based on a change of the antioxidant film “container” adapted to the present conditions, as shown in Figure 2. In brief, this system consists of generating a known concentration of free radicals (from a nebulising hydrogen peroxide solution at a
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flow rate of 0.8 ml min−1) in the gas phase (total air flow = 470 ml min−1). The total flow passed through a quartz tube covered by ultraviolet (UV) lamps to supply the UV irradiation required for free radical formation and so produced a free-radical-enriched atmosphere that was carried through the valve with the formula-“pellet” (i.e., firstgeneration formula). Finally, the free radicals, which are not scavenged by the antioxidant material, are trapped in an aqueous solution of salicylic acid, which is converted into 2,5-dihydroxybenzoic acid (DHB). The final solution is measured by HPLC with highly sensitive fluorescence detection. Both types of formula (first and second generation) were measured under these dynamic conditions. In the first-generation formula only the three best mixtures (i.e., general appearance and mechanical performance of the film) were analysed; meanwhile with the silicone-based formula different GTE-antioxidant percentages were evaluated.
Release studies The diffusion experiments were carried out following the Moisan tests in a migration cell as suggested by Moisan (1980). The cell consists of two 10 × 10 cm aluminium plates of 1 cm thickness. These experiments were carried out using some selected first-generation formula films (e.g., methyl-cellulose and β-cyclodextrin), second-generation silicone-based formulas (10%, 20% and 30% w/w of GTE) and GTE film alone. The diffusion experiments were performed using a 10 × 10 cm sample for each of the formulas containing the GTE with 10 polyethylene (PE) layers (35 µm thickness each) stacked under pressure in a
Modified part
Air flow (470 mL min–1)
UV Lamps
Auxiliary air H2O2, flow 0.8 mL min–1)
Figure 2.
Nebuliser air
Modified apparatus developed for evaluating the radical scavenging properties of the different formula in dynamic mode.
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pile on the active formula with the following arrangement: Al plate/formula/PE-1/PE-2/ … /PE-10/Al plate). Each of these cells was closed using four screws and a dynamometric tool in order to apply a constant twisting force of 0.8 Nm (Canellas et al. 2010). The diffusion cells were kept at 30ºC for 1 month. At the end of the experiment each of the PE layers was put into a 15 ml vial and extracted with methanol for 15 min with ultrasonic assistance, according to the previous optimisation (Canellas et al. 2010); the methanolic extract was then transferred to a 2 ml vial and injected into the HPLC-fluorescence and UPLC-MS. Determination of the active compounds (gallic acid, caffeine and catechins) released from the new formula (second generation) was carried out by determining the specific migration into food simulants established by European Commission Regulation (EU) 10/2011. Specific migration is the amount of a specific component that migrates from the food contact material to the food in contact with it. Food contact materials must not transfer their components into the foods in unacceptable quantities (migration) (Dicastillo et al. 2011). Due to the likely future industrial applications (roasted coffee beans) two food simulants were used: 3% acetic acid (w/v) as an acidic food simulant and 20% of ethanol (v/v) as an alcoholic food simulant, both in distilled water. For this experiment three different concentrations of GTE were used (10%, 20% and 30% w/w) in the siliconebased films. Migration studies were conducted at 70ºC for 2 h, which simulate the hot filling conditions, as roasted coffee beans were the main target food (UNE-EN 2002, 2005). Double-sided, total immersion migration tests were performed as follows: 9 cm2 of each silicone-based formula sample and 15 ml of the simulant (area to volume ratio around 6 dm2 l–1) were placed in 20 ml glass vials. After the exposure, the various green tea components were analysed by UPLC-MS.
Sensory analysis Drinking water samples in contact with the active material for different exposure times (1, 2, 4, 6 and 10 days) at RT
were tested. A triangular test (UNE 1992) between different samples after exposure to the active material and with pure drinking water was carried out. The panellists had three bottles, two of them with drinking water and a third after exposure to the active material for different times. They had to determine the different one and to register the odour, taste and colour of the drinking water.
Results and discussion Formula development The chemical composition of the first batch of the formula generation is shown in Table 1. The percentage of agglutinant agent added varies widely from 1.5% (w/w) in the agar mixtures up to 35% (w/w) in the activated carbon, βcyclodextrin, methyl-cellulose and arabic gum mixtures. The small amount of agglutinant and GTE in the agar mixtures is due to the need for higher amounts of water in the preparation. This first-generation formula film was too rigid and after some time of preparation (24 h) elasticity was completely lost. Only the methyl-cellulose formula film kept some kind of elasticity after this period. Moreover, during the development of the first-generation formula-“pellet” some mixtures were prepared with and without the plasticiser agent. Glycerol was used as a plasticiser agent in 10%, 20% and 40% (w/w), but only a little improvement was achieved in some of the mixtures containing arabic gum and methyl-cellulose. Samples with the highest amount of glycerol were discarded due to problems in the final curing step. As a consequence, further formula development had to be explored with the second-generation formula. The different chemical composition of the second-generation formula is shown in Table 2. In this new mixture no plasticiser was needed. This type of silicone is semi-liquid and needs the addition of a catalyst for curing. The ratio of the mixture had to be 10 to 1 (w/w), for a curing time of 24 h at 30°C. Different percentages of GTE were tested, as can be seen in Table 2, from 5%, 9%, 17%, 20%, 23%, 32%, 42% to 50% (w/w). Implementation of the silicone-based formula caused
Table 1. Chemical composition (%, w/w) and antioxidant activity (static method using DPPH) of the first-generation formula. Antioxidant activity (DPPH)a Agglutinate type
Agglutinate (%)
Green tea (%)
Plasticiser (%)
2h
24 h
48 h
96 h
120 h
Agar Activated carbon Activated carbon Sodium alginate β-cyclodextrin Β-cyclodextrin Methyl-cellulose Arabic gum
3 9 22 9 9 22 10 10
2.5 91 78 91 91 78 80 80
0 0 0 0 0 0 10 10
No No No No No No No No
No Yes No Yes No No No No
No Yes Yes Yes Yes Yes No No
No Yes Yes Yes Yes Yes Yes Yes
No Yes Yes Yes Yes Yes Yes Yes
Note: aColour change from violet to yellow, yes or no, at 2, 24, 48, 96 and 120 h.
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Table 2. Chemical composition (%, w/w) and antioxidant activity of the second-generation formula (silicone-based). Antioxidant activity (DPPH) Silicone/catalyst ratio (w/w) 10:1 10:1 10:1 10:1 10:1
Silicone plus catalyst (%) (w/w)
GTE (%) (w/w)
8h
18 h
36 h
38 h
64 h
96 h
95 91 80 68 100
5 9 20 32 Nob
No No No No No
No No No No No
No No Yesa Yesa No
No No Yesa Yesa No
No Yesa Yesa Yesa No
Yesa Yesa Yesa Yesa No
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Notes: aColour change in one of the three replicates. b No, silicone blank.
an enormous improvement in the final elasticity of the mixture, which remained stable for a long period of time (at least 2 months). According to the results obtained during the preparation of the first- and second-generation formulas, the silicone-based formula and thus the silicone-based “pellet” achieved the best results, good results were found with 17% (w/w) of GTE.
Antioxidant activity All the mixtures of first-generation formulas had excellent antioxidant activity in both types of studies (static and dynamic). Table 1 shows the antioxidant activity (static experiment-DPPH) of the most representative mixtures for the first-generation formula. Spectrophotometric measurements were done with the methyl-cellulose, β-cyclodextrin and arabic gum mixtures, and five replicates of each and a blank for each mixture were measured during 92 h. The results are shown in Figure 3. Table 2 shows the results of antioxidant activity (DPPH) for the silicone-based pellet. As can be seen, the antioxidant performance increases with the percentage of GTE. As DPPH is a supplier of free radicals, the exposure of a sample to DPPH means
that the sample can act as a scavenger of free radicals, which means antioxidant performance. The tests were carried out without direct contact between the siliconebased pellet and the DPPH solution. This reinforces the statement of radical scavenger activity of the siliconebased pellet. Figure 3 shows a comparison between the different formulas under test and it emphasises the similarities of performance within them. All behave as radical scavengers, although a slight increase of the methyl-cellulose formula antioxidant capacity is observed. Among the first-generation formula, only that of methyl-cellulose (formula-“pellet”) was selected for dynamic experiments. Antioxidant activity is reported as a percentage of hydroxylation of the salycilic acid (SA) to form DHB, where a lower percentage of hydroxylation of SA indicates higher antiradical scavenging efficiency. The percentage of hydroxylation is in fact an indirect measurement of the concentration of free radicals that cross the polymeric layer or the specimen exposed to the free radicals atmosphere generated in the device above described. Antioxidant activity was between 50% and 30%, with an average of 40%, using mixtures with 0.03 and 0.04 g of GTE, higher than the blank sample formula. This means
Figure 3. Antioxidant activity measured with DPPH (mean percentage of five replicates) (spectrophotometric measurements) of the selected first-generation formula. AG, arabic gum; MC, methyl-cellulose; and Cy, β-cyclodextrin.
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that the active sample exposed to the free radicals behaves as an antioxidant or free radical scavenger. The percentage of hydroxylation is always referred to as the amount of SA. Meanwhile, antioxidant activity for silicone-based formula was in the same range.
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Release and diffusion studies Diffusion Diffusion studies reveal a low or null mobility of catechins, lower than the LOQ (
