Accepted Manuscript Preparation and characterization of microparticles of βcyclodextrin/glutathione and chitosan/glutathione obtained by spray-drying
Vanessa Webber, Daniel de Siqueira Ferreira, Pedro Luis Manique Barreto, Valeria Weiss Angeli, Regina Vanderlinde PII: DOI: Reference:
S0963-9969(17)30804-9 doi:10.1016/j.foodres.2017.11.035 FRIN 7157
To appear in:
Food Research International
Received date: Revised date: Accepted date:
10 July 2017 13 November 2017 19 November 2017
Please cite this article as: Vanessa Webber, Daniel de Siqueira Ferreira, Pedro Luis Manique Barreto, Valeria Weiss Angeli, Regina Vanderlinde , Preparation and characterization of microparticles of β-cyclodextrin/glutathione and chitosan/glutathione obtained by spray-drying. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Frin(2017), doi:10.1016/ j.foodres.2017.11.035
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ACCEPTED MANUSCRIPT Preparation and characterization of microparticles of β-cyclodextrin/glutathione and chitosan/glutathione obtained by spray-drying Vanessa Webbera*, Daniel de Siqueira Ferreiraa, Pedro Luis Manique Barretob, Valeria Weiss Angelia, Regina Vanderlindea, c a
Institute of Biotechnology, University of Caxias do Sul, Francisco Getúlio Vargas, 1130, 95070-
Department of Science and Technology of Foods, Federal University of Santa Catarina, Rod.
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b
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560 Caxias do Sul, RS, Brazil
Admar Gonzaga, 1346 Itacorubi, 88034-001 Florianópolis, SC, Brazil
Reference Laboratory of Enology, Brazilian Wine Institute – IBRAVIN, Avenida da Vindima,
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c
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1855, Exposição, 95054-470 Caxias do Sul, RS, Brazil
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e-mail:
[email protected]
Abstract
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Reduced glutathione (GSH) is an efficient antioxidant on limitation of browning, of the loss of
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aromas and off-flavor formation in white wines. The encapsulation of GSH in a polymer system to be added in white wines may prolong its antioxidant action. The aim of this work was to prepare
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and characterize spray-dried microparticles using β-cyclodextrin (β-CD) or chitosan as polymers for encapsulation of GSH for its addition to wine to prevent oxidation. The microparticles obtained
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after the drying process were characterized regarding morphology, chemical interacction betwen GSH and polymers, thermal stability, microstructure, encapsulation efficiency and in vitro GSH release. SEM showed spherical microparticles, with wrinkled surfaces for β-CD/GSH and smooth surfaces for chitosan/GSH. A wide distribution of particle size was observed. In general, β-CD/GSH showed an average diameter smaller than the chitosan/GSH microparticles. FT-IR showed a possible interaction between GSH and both polymers. DSC and DRX showed that encapsulation process produced a marked decrease in GSH crystallinity. The encapsulation efficiency was 25.0% 1
ACCEPTED MANUSCRIPT for chitosan/GSH and 62.4% for β-CD/GSH microparticles. The GSH release profiles from microparticles showed that β-CD can control the release behaviors of GSH better than chitosan in a model wine. Cumulative release data were fitted to an empirical equation to compute diffusional exponent (n), which indicates a trend the non-Fickian release of GSH.
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Keywords: microparticles, chitosan, cyclodextrin, glutathione, wine.
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1. Introduction
Glutathione (γ-glutamyl-glycine cysteine; GSH) is a thiol with low molecular weight
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synthesized from amino acids by the sequential action of γ-glutamylcysteine and glutathione sintetase. GSH is capable of reacting with a free radical to form glutathione disulfide (GSSG or
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oxidized glutathione) which is devoid of antioxidant activity. The GSSG can be reduced by glutathione reductase, which uses NADPH as a substrate regenerating the GSH (Kritzinger, Bauer,
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& du Toit, 2013). However, GSH can be lost irreversibly in highly susceptible environments to
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oxidation and, in the absence of the enzyme and/or substrate, GSH remains in the oxidized form and cannot be reduced over again (Uhlig, & Wenderl, 1992).
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GSH is a natural component of many fruits which have an important role in inhibiting the oxidation of fruit juices, wines and other foods. In white wines, GSH plays an important role in
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protecting against browning and lossing flavor due to oxidative process (Kritzinger et al., 2013). GSH inhibits formation of dark polymers by avoiding polymerization which occurs from o-quinone compounds which are present in the wine due to the oxidation of phenolic compounds. GSH can react with the quinones and form the Grape Reaction Product (GRP, ex: 2-S-glutathionyl caftaric acid), which is not dark and less susceptible to oxidation (Singleton; Zaya, Trousadale & Salgues, 1984; Comuzzo & Zironi, 2013). The addition of GSH to wine has been studied to limit the oxidation and to reduce the 2
ACCEPTED MANUSCRIPT addition of sulfites (Roussis & Sergianitis, 2008; Hosry, Auezova, Sakr, & Hajj-Moussa, 2009; Sonni, Clark, Prenzler, Riponi, & Scollary, 2011a; Webber, Dutra, Spinelli, Marcon, Carnieli & Vanderlinde, 2014), which are toxic and allergenic (Comuzzo et al., 2013). Moreover, the addition of up to 20 mg L-1 of GSH in must and wine was recently included among the oenological practices recommended by the Organization Internationale de la Vigne et du Vin (O.I.V.) (Resolutions OIV-
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OENO 445 and 446-2015) in order to limit the browning and the lost of varietal aromas.
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Microencapsulation is a physical method which allows droplets or particles to be trapped in
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a film formed by the wall material. So it establishes a physical barrier between the active material and other components of the product (Gharsallaoui, Roudaut, Chambin, Voilley, & Saurel, 2007).
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Encapsulation systems have been used for protecting sensitive ingredients against the environmental conditions such as light, oxygen, water and temperature that food undergoes during
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its processing and storage (Dziezak, 1998). These systems allow that a polymeric barrier protects the bioactive substance against oxidation conditions and they act as a controlled-release mechanism,
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resulting in improving substance stability (Dziezak, 1998; Koo, Lee, Kim & Lee, 2011). In this
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sense, the microencapsulation of GSH in a polymeric system could be able to protect the peptide against chemical and/or enzymatic oxidation, and to improve substance stability. This seems to be a
Trapani et al., 2010).
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promising alternative (Kafedjiiski, Föger, Werle & Bernkop-Schnürch, 2005; Lopedota et al., 2009;
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Chitosan is a widely used polymer for encapsulation of many proteins and for active compounds because it is non-toxic, biodegradable, biocompatible and with good mechanical properties (Koo et al., 2011). Chitosan is obtained by the reaction of deacetylation of chitin in alkaline environment. It is also a natural polymer extracted from the exoskeleton of shellfish and insects, which comprises the monomer units β-(1→ 4)-2-amino-2-deoxy-D-glucose and β-(1→ 4) 2-acetamide-2-deoxy-D-glucose. This natural polymer has a highly organized crystalline structure, a low chemical reactivity and it is insoluble in aqueous environment and in most organic solvents. 3
ACCEPTED MANUSCRIPT However, chitosan can be readily dissolved in diluted solutions of weak acids, such as acetic acid (Laranjeira & Fávare, 2009). Chitosan microparticles may be easily obtained by ionic gelation between cationic groups of chitosan and multivalent anions (Koo et al., 2011). Kafedjiiski et al. (2005) synthesized and characterized chitosan-glutathione conjugate and concluded that it may be capable of carrying hydrophilic macromolecules and of improving mucoadhesive properties and
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permeability of drug in vitro.
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Previous studies reported the inclusion of cyclodextrins (CDs) in the nanoparticle systems of
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chitosan and GSH (Jingou, Shilei, Weiqi, Danjun, Tengfei & Yi, 2011; Trapani et al., 2010). Moreover, studies showed that CD microparticles may adduce hydrophilic molecules with high
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efficiency (Jingou et al., 2011; Lopedota et al., 2009). CDs are obtained from starch and they are formed by a variable number of glucose joined by α 1,4-linkages. The most common cyclodextrins
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are α-CD, β-CD and γ-CD, with 6, 7 and 8 glucose units, respectively. They have the shape of a truncated cone with a hydrophobic centered cavity and a hydrophilic outer layer, thus, they are
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soluble in water and they are suitable for microencapsulation by molecular inclusion (Del Valle,
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2004). CDs are able to interact with the hydrophobic side chains of the peptide compounds or the proteins, and thereby prevent its degradation until certain extent (Irie & Uekama,1999). Lopedota
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et al. (2009) suggest a weak interaction between GSH and CDs, and the backbone of the peptide may be partially included in the CD cavity, while carboxylic acid groups, polar amine and the thiol
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group remain outside the cavity. Even though it is a weak interaction, it is able to prevent the degradation of GSH by endopeptidases (Garcia-Fuentes, Trapani, & Alonso, 2006). Due to the high GSH reactivity and the diversity of compounds present in wine, such as phenolic and aromatic compounds, the concentration of GSH reduces quickly after being added wine (Lavigne, Pons, & Dubourdieu, 2007; Webber et al., 2014). Therefore, an interesting alternative for the addition of GSH to wine is its protection in microcapsules. GSH could be released in a controlled way and react continuously with the wine compounds for a longer time 4
ACCEPTED MANUSCRIPT during bottle storage. In addition, microencapsulation of GSH could prevent GSH reaction or it could rapidly bind with other compounds in wine. Moreover, if GSH is slowly released it does not favor reduced oxygen conditions in the wine, and it could also prevent a negative aspect of the use of GSH in wine, which is inducing the formation of H2S (off-flavor) occurring under reduced conditions of oxygen and/or with high concentrations of copper (II) during aging in bottles (Ugliano
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et al., 2011).
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Most of the previous studies about the preparation and characterization of nano and
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microparticles containing GSH are directed to the pharmaceutical field and previous studies do not discuss a formulation for GSH addition in wines (Kafedjiiski et al., 2005; Lopedota et al., 2009;
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Trapani et al., 2007; Trapani et al., 2010; Garcia-Fuentes et al., 2006; Jingou et al., 2011). Therefore, this work aims to prepare and characterize microparticles of chitosan and of β-CD
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containing GSH by spraying drying method for wine addition and evaluate the GSH release in a
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2.1. Material
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2. Material and methods
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model wine.
Reduced glutathione (≥ 98.0%, G4251), chitosan (low molecular weight, 75-85%
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deacetylated, viscosity: 20-300 cP, 1 wt. % in 1% acetic acid (25 °C, Brookfield)(lit.), soluble in dilute aqueous acid, supplier code: 448869 ALDRICH) and β-cyclodextrin (≥ 97.0%, optical activity: [α]20/D +162±3°, c = 1.5% in H2O, melting point: 290-300 °C (dec.)(lit.), solubility: 1 M NH4OH: soluble50 mg/mL, supplier code: C4767 SIGMA) were purchased from Sigma-Aldrich Brazil. 5,5-dithiobis (2-nitrobenzoic) acid used for quantification of GSH was also obtained from Sigma-Aldrich (≥98% BioReagent suitable for determining sulfifril groups D8130).
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ACCEPTED MANUSCRIPT 2.2. Preparation of β-cyclodextrin microparticles For the preparation of β-cyclodextrin microparticles, 4 g of GSH and 14 g of β-cyclodextrin were added in 500 mL of distilled water, resulting in an equimolar concentration of 13 mM of each component. After complete dissolution of the components under stirring, the mixture was dried in a spray-dryer (model B-290, Büchi) at inlet temperature of 200 °C (controlled), outlet temperature of
). The microparticles yield from the drying process was calculated by the following: yield =
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1
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72 °C, vacuum of 100% (35 m3 h-1 volume flow) and the flow pump (20% feed flow of 10 mL min-
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[(weight of the dried microparticles)/ (total weight of solids in solution before drying)] * 100.
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2.3. Preparation of chitosan microparticles
Chitosan microparticles were prepared according to the methodology of Koo et al. (2011) with
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some modifications. 5 g of chitosan were added to 250 mL of 2% acetic acid (pH = 4.5), and the solution was stirred for 12 hours until there was a complete dissolution of chitosan. This solution
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was diluted with 250 mL of distilled water. In 250 ml of this solution containing 2.5 g of chitosan it
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was added 200 ml of aqueous solution containing 12.5 g of GSH. The final mixture obtained was dried in a spray-dryer (B-290, Büchi) at inlet temperature of 130 °C (controlled), outlet temperature
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of 47 °C, vacuum of 100% (35 m3 h-1 volume flow) and pump flow 20% (feed flow of 10 mL min). The microparticles yield from the drying process was calculated by the following: yield =
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[(weight of the dried microparticles)/ (total weight of solids in solution before drying)] * 100.
2.4. Physicochemical characterization of microparticles
2.4.1. Morphological analysis Morphological evaluation of microparticles was performed by scanning electron microscopy. The microparticles were attached on an adhesive conductive carbon tape for 6
ACCEPTED MANUSCRIPT microscopy on a sample holder. The surface morphology of the particles was evaluated in a Shimadzu SSX-550 scanning electron microscope operating at 10 kV. All micrographs were obtained in the magnification of 1000 and 3000 times from the original.
2.4.2. Fourier Transform Infrared Spectroscopy – FTIR
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The spectra in the infrared region were obtained on a Nicolet model IS 10 Thermo Scientific
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spectrometer. This analysis was performed for the samples of GSH, β-CD, chitosan microparticles
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β-CD/GSH and chitosan/GSH, by mixing the components of the microparticles in the same proportions used in their preparation. For this analysis, samples were prepared in the form of tablets
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containing KBr (IR spectroscopic grade). The samples were scanned from 450 to 4000 cm-1 with a
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resolution of 1 cm-1.
2.4.3. Differential scanning calorimetry – DSC
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DSC curves were obtained with a DSC-60-Model DAC-60 Shimadzu. Aliquots of 5 mg of
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each sample were placed in aluminum pans. DSC measurements were performed by heating samples to 250 °C at a rate of 5 °C min-1. This analysis was performed for the samples of GSH, β-
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CD, chitosan microparticles β-CD/GSH and chitosan/GSH, by mixing the components of the
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microparticles in the same proportions used in their preparation.
2.4.4. Termogravimetric analysis – TGA The thermogravimetric curves of GSH, β-CD, chitosan, and these compounds dissolved and subjected to spray-dryer; and the microparticles (β-CD/GSH and chitosan/GSH) were obtained using a Shimadzu TGA-50 equipment, in temperature ranging from 23 °C to 800 °C, with a heating rate of 10 ° C min-1 under N2 with a flow of 50 mL min-1. The weight used was approximately 10 mg of the sample powder. 7
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2.4.5. X-ray diffractometry - XRD X-ray diffraction (XRD) analysis was performed with X-ray diffractometer Shimadzu XRD6000X. The samples were scanned using Cu Kα radiation at 2θ angle range from 5º to 40º, and
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with a scanning rate of 0.5º.
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2.6. Encapsulation efficiency
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Encapsulation efficiency (EE%) was obtained from the subtraction of the concentration of GSH on the surface of the microparticles and the total GSH content in the particles after drying in a
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spray-dryer, divided by the theoretical GSH (GSH added in preparing microparticles) and it is expressed in percentage of encapsulated GSH, calculated using the equation EE = [(total GSH –
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superficial GSH)/(theoreticalGSH)]*100 (Koo et al., 2011; Quispe-Condori, Saldaña & Temelli, 2011).
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GSH concentration on the surface of the microparticles was performed in the adapted
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methodology by Quispe-Condori, Saldana and Temelli (2011), whereas GSH solubility in water is high and higher than the polymers. 4.662 mg of β-CD/GSH microparticles and 1.204 mg of the
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chitosan/GSH microparticles were weighed to result in a theoretical total concentration of 100 mg L-1 of GSH when dissolved in 10 mL. The microparticles were placed on a paper filter and
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percolated with 2 mL of distilled water to full flow to this beaker. The percolation procedure was performed five times. The quantification of the superficial GSH was performed by measuring GSH contained in distilled water which percolated the filter (n = 3) (accoding item 2.8).
2.7. GSH release study in vitro β-CD/GSH microparticles (46.62 mg) and chitosan/GSH microparticles (12.04 mg) were dispersed in 100 mL of model wine (100 mg L-1 of GSH) without stirring and the concentration of 8
ACCEPTED MANUSCRIPT GSH released was analyzed by spectrophotometry (accoding item 2.8) at 0.20; 0.50; 0.80; 1.00; 1.20; 2.80; 3.50; 4.00; 5.30; 6.00; 25.00 and 48.00 hours (n = 3). For the preparation of model wine, 5g L-1 of tartaric acid were added to a 12% hidroalcoholic solution The pH was adjusted at 3.5 with NaOH 1N. The concentration of tartaric acid was chosen to simulate the action of all the wine acids and to produce a ~ 4 g L-1 final acidity (Danilewicz, Seccombe, & Whelan, 2008; Papadopoulou, &
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Roussis, 2008).
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2.8. Analysis of GSH
GSH quantification was performed by spectrophotometry according to the adapted method
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by Rahman, Kode, & Biswas (2006). This method involves oxidation of GSH through diotio sulfhydryl reagent 5,5-bis (2-nitrobenzoic acid) (DNTB) to form the yellow 5-thio-2-nitrobenzoic
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acid (TNB), with the of absorbance at 412 nm. For this analysis, 0.4 mL of the sample, 1.6 mL of phosphate buffer solution (pH 7.5), 2.0 mL of DNTB solution were pipetted in quartz cuvettes of 10
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mm pathlength. After 1 min of reaction, the sample was analyzed at 412 nm in a Perkin-Elmer
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spectrophotometer of Lambda 40 Model. The solution DNTB was composed of 2 mg DNTB in 3 mL phosphate buffer. The phosphate buffer solution was prepared with Na2HPO4.12H2O (10 mM)
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in ultrapure water; which was adjusted to pH 7.5 by using orthophosphoric acid solution. The concentrations of GSH in the samples were calculated by using the analytical curve in wine model
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prepared at the same day that the samples were analyzed and in the following concentrations of GSH 10, 20, 30, 40, 50, 75, 100 mg L-1.
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Results and discussion
3.1. Physico-chemical characterization of microparticles
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ACCEPTED MANUSCRIPT 3.1.1. Morphological analysis Morphological analysis showed the presence of spherical particles with wide size distribution for both formulations (Figure 1). Comparing Figures 1a and 1c, it was observed that, in general, β-CD/GSH microparticles had a smaller average diameter than chitosan microparticles. SEM showed wrinkled surfaces for β-CD/GSH (Figure 1c) and smooth surfaces for chitosan/GSH
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(Figure 1a) microparticles. β-CD/GSH microparticles exhibited an external surface with no
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apparent cracks or porosity (Figure 1c), which can indicate a good core material protection. The
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morphologies presented in this study are in agreement with the ones found by Oliveira, Santana and Ré (2005) related to chitosan microspheres; and by Zhang, Chen, Li, He, Zhou and Cha (2008), and
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de Oliveira, Fatibello-Filho, Fernandes and Vieira (2009) related to chitosan and β-CD microparticles, both using spray-drying.
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Figure 1 (1b and 1d) shows the micrographs of wall materials (chitosan and β-CD, respectively) dried by spray-drying, without the addition of active compound. We have observed
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some differences by comparing the morphology of microparticles and polymers without active
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compound addition. The micrographs of chitosan/GSH microparticle showed a different morphology to chitosan polymer one, which showed continuous surface depressions. However, the
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morphology of chitosan/GSH microparticle was similar to the morphology of pure GSH, subjected to dilution in water and subsequent drying in a spray-dryer (Figure 1e), which may indicate a low
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interaction between chitosan and GSH. On the other hand, the morphology of β-CD/GSH microparticle was different from both β-CD, as GSH, subjected to the same process of microparticles drying. β-CD micrograph showed large cavities and a non-spherical shape, while GSH proved to be spherical and smooth, and the microparticles showed no regular spherical shape with some superficial depressions. These results demonstrate a possible interaction between polymer, β-CD, and the active compound, GSH.
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ACCEPTED MANUSCRIPT 3.1.2. Infrared Fourier transform spectroscopy – FTIR Figure 2 shows FTIR spectra of pure GSH, chitosan, chitosan/GSH microparticle, and the components of the physical mixture of the chitosan/GSH microparticles. The obtained spectra for chitosan (Figure 2b), β-CD (Figure 3b) and glutathione (Figure 2a and 3a) are similar to the one obtained in previous studies (Jingou et al., 2011; Lopedota et al., 2009; Zhang et al., 2008). The
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spectrum of pure GSH (Figure 2a and 3a) showed strong absorption bands attributed to carbonyl
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and amide stretching (1713 cm-1 and 1600 cm-1, respectively) as well as the band related to the -SH group at 2525 cm-1 (Lopedota et al., 2009).
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The characteristic bands of chitosan are 3426 cm-1 (-NH2 and -OH stretching), 2922 cm-1
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and 2872 cm-1 (-CH stretching), 1601 cm-1 (-NH2 stretching), 1078 cm-1 (C-O-C stretching) and 601 cm-1 (stretching vibration of the pyranoside ring) (Figure 2b) (Lopedota et al., 2009). The chemical
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chitosan structure presents two polar groups which are able to form hydrogen bonds, the hydroxyl and the amino groups. In the spectrum of chitosan/GSH microparticle (Figure 2c), the band on the
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region of OH and NH stretching shifts to lower values (3300 cm-1), which suggests interactions via
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hydrogen bonds between the components in the microparticle. The spectrum of chitosan/GSH microparticles shows a superimposed band at 2525 cm-1, which refers to the -SH group of GSH.
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The spectrum obtained by the physical mixture of chitosan and GSH (Figure 2d) was similar to the spectrum of pure GSH and different from chitosan/GSH microparticle spectrum, suggesting a
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possible interaction between the polymer and GSH. FTIR spectra of pure GSH, β-CD, β-CD/GSH microparticle, and the components of the physical mixture of the β-CD/GSH microparticles were showen in Figure 3. Infrared spectrum of the β-CD/GSH microparticle was very similar to the pure polymer. The characteristic peaks of β-CD include 3400 cm-1 (-OH stretching), 2927 cm-1 (-CH stretching), 1640 cm-1 (H-O-H bending), 1400 cm-1 (-OH bending), 1156 cm-1 (-CO stretching), 1028 cm-1 (C-O-C stretch) and 950 cm-1 (skeletal vibration involving the α-1.4 connection) (Hu et al., 2012; Jingou et 11
ACCEPTED MANUSCRIPT al., 2011). The spectrum of β-CD/GSH microparticle shows a strong band at 1646 cm-1 attributed to H-O-H polymer bending, a weak band at 1713 cm-1 of carbonyl stretching of GSH and a weak band at 2525 cm-1 overlapping referring to the -SH group of GSH (Figure 3c). It suggests a possible interaction between polymer and GSH (Lopedota et al., 2009). The obtained spectrum from physical mixture of β-CD and GSH (Figure 3d) shows characteristic peaks of these two
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components, unlike the spectrum of β-CD/GSH microcapsule, which was more similar to pure β-
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CD spectrum, indicating the possibility of complexation between β-CD and GSH.
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3.1.3. Differential scanning calorimetry – DSC
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DSC profiles of chitosan, β-CD, GSH and of the two formulations of microparticles are shown in Figure 4. The profile of pure GSH exhibited a single endothermic peak at 196 °C,
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corresponding to the melting point of GSH. In the DSC profiles of microparticles containing GSH, the peak at 196 °C was not present, and the thermograms were similar to the polymers. These
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results, taken together, are in agreement with the results obtained by Lopedota et al. (2009), in
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which the encapsulation process also produced a pronounced decrease in GSH crystallinity and this provided a nearly amorphous state. This was probably caused by the intercalation of GSH chains
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and the polymer chains for the two systems, chitosan/GSH and β-CD/GSH. In the case of βCD/GSH system, another probable cause is the low concentration of GSH in the formulation of the
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microparticle when compared to the polymer concentration.
3.1.4. Thermogravimetric analysis – TGA Thermogravimetric analyzes were performed to evaluate the thermal stability of the microparticles as well as of its individual components, as shown in Figure 5. Standard GSH thermogravimetric curve showed no weight loss until 205 °C. Above 205 °C, it was observed a weight loss of 55% between 205 °C and 400 °C and, after 400 °C, a less 12
ACCEPTED MANUSCRIPT pronounced weight loss with indefinite end point, which was also observed by Baek, Choy and Choi (2012). In the first stage of chitosan decomposition there was an initial water reduction of 10% up to 100 °C. Between 100-280 °C, it showed a weight loss of 5%, and between 280-380 °C it showed a loss of 35%, corresponding to the compound deacetylation and depolymerization. Between 380-700
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°C, it showed a loss of 20%, and above 700 °C, the weight loss continued with an undefined level.
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These results are similar to those obtained before (Nieto, Peniche-Cova & Padrón, 1991; Neto, Giacometti, Job, Ferreira, Fonseca & Pereira, 2005).
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β-CD thermogram showed three decomposition steps. The first step of 15% weight loss up
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to 120 °C related to water evaporation, which can also be observed in the DSC thermogram as the endothermic peak in the same temperature band (Figure 4c). The second thermal decomposition
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event (70%) started at 290 °C. This event was previously reported by other authors in the same temperature range (Barbosa et al., 2014). The mechanism involved in this transition phase is not
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clear yet, but it seems to be associated with the crystal hydration, which occurs with β-CD
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synthesis, and which may be present in different crystalline forms with a possible transition between them (Giordano, Novak & Moyano, 2001). The pseudomorphics prevalent forms for β-CD
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crystals are the decahydrate or dodecahydrate forms. The structural differences of these carbohydrates depend on the quantity of water molecules and on how they are disposed within
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cyclodextrin. This explains the melting and the decomposition of β-CD in temperatures above 300 ºC, and it is mainly related to the absence of a clear melting point peak before decomposing, (Barbosa et al., 2014) as it can be observed in the DSC curve (Figure 4c). Comparing the thermogravimetric curves of the microparticles components separately to the microparticles containing GSH, we can see that the chitosan/GSH microparticle thermogravimetric curve is initially similar to chitosan curve, having a water loss until the temperature of 100 °C. From this temperature on, chitosan/GSH microparticles thermogravimetric curve has a weight loss 13
ACCEPTED MANUSCRIPT with a similar profile to that of pure GSH dried in a spray-dryer. These results suggest that there was no protection against the degradation of GSH. However, a different behavior can be observed for the sample of β-CD/GSH microparticles. β-CD/GSH sample thermogravimetric curve shows that there is a loss of water until 100 °C, and from this temperature up to 290 °C, there is no weight loss as occurred for β-CD sample. Therefore, the GSH molecule was probably protected, preventing
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its degradation from 150 °C to 290 °C. Up to 320 °C, the degradation of β-CD/GSH microparticule
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was similar to the profile of pure β-CD. At 320 °C, a weight loss of 42% occurred in the microparticles, and thereafter weight loss remains less pronounced. β-CD/GSH microparticule
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thermogram was different from pure GSH and pure β-CD, which suggests an interaction occurred
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between the compounds, given a greater GSH thermal stability.
Comparing the two microparticle thermograms (Figure 5 c and d), we can observe that β-
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CD/GSH microparticle conferred greater stability with the increase of temperature than the
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3.1.5. X-ray diffractometry - XRD
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chitosan/GSH microparticle.
The diffractograms of chitosan, β-CD, GSH and of the chitosan/GSH and β-CD/GSH
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microparticles are shown in Figure 6. As reported in previous studies, chitosan showed an amorphous to partially crystalline state (Papadimitriou, Achilias & Bikiaris, 2012), presenting two
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characteristic peaks at 2θ, one at 11º, which corresponds to hydrate crystals and another at 20º, corresponding to anhydrous crystals (Figure 5 c) (Jingou et al., 2011; Papadimitriou et al., 2012). On the other hand, β-CD and GSH standards showed strong crystalline characteristics with several strong peaks in the diffractograms. The X-ray diffraction spectrum of the crystalline GSH was characterized by an intense peak at 2θ of 22.3º and two low intensity peaks at 2θ of 9º and 11º (Lopedota et al., 2009). In the X-ray spectra of the microparticles, these peaks are absent or greatly
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ACCEPTED MANUSCRIPT reduced, indicating that GSH can exist as a molecular dispersion in the polymeric microparticles (Zhang & Zhuo, 2005). In chitosan microparticles diffraction spectrum, the peak at 2θ of 20° of chitosan becomes even wider. It was already known that the width of the X-ray diffraction peak is related to the crystallite size, and an extended peak is generally a result of an imperfect crystal. Thus, the wide
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peak of microparticles may be a result of the crosslinking reaction between polymer and GSH,
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which can destroy the crystal structure of the compounds (Rokhade, Agnihotri, Patil, Mallikarjuna,
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Kulkarni, & Aminabhavi, 2006).
These results are in agreement with previous results obtained from the analysis of the same
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compounds (Jingou et al., 2011; Lopedota e al., 2009; Papadimitriou et al., 2012; Trapani et al., 2007), and it suggest that the microencapsulation process causes a reduction of GSH crystallinity
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and this process provides a quite amorphous state.
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3.2. Encapsulation efficiency
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The ability of chitosan and β-CD to retain GSH was determined by the encapsulation efficiency (Table 1). GSH showed lower affinity with chitosan than with β-CD, probably because of
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a low molar mass of peptide and the presence of only one negatively charged group on its structure (Trapani et al., 2010).
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A possible way to occur the conjugation of chitosan and GSH would be by the radical polymerization method, forming amide bond between carboxyl groups of GSH glycine and chitosan amine groups (Koo et al., 2011; Kafedjiiski et al., 2005). The encapsulation efficiency of chitosan/GSH microparticle was probably not greater because it was not added catalysts for the reaction, and the reaction time was shorter than in other studies (Koo et al., 2011; Kafedjiiski et al., 2005). In addition, the pH of the solution was not adjusted to values close to neutrality, which may have prevented the conjugation through radical polymerization. At pH 7.0, the glycine residue of 15
ACCEPTED MANUSCRIPT GSH is particularly mobile, while glutamine part of GSH is the most rigid (Krezel & Bal, 2003). Therefore, at pH 4.5 of the mixture of chitosan solution, with GSH solution in microparticles preparation, the mobility of the glycine should have been slightly reduced, hampering the connection between these two compounds. Koo et al. (2011) found encapsulation efficiencies between 20 and 65% in obtaining nanoparticles of chitosan/GSH prepared by ionic gelling method
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with sodium tripolyphosphate, with pH adjustment to 6.0 and several varying proportions of GSH
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and of tripolyphosphate sodium. Low encapsulation efficiency of chitosan/GSH microparticle
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compared with β-CD/GSH microparticle is also due to the fact that chitosan used in this study has a low molecular weight and, therefore, the GSH molecules are immobilized in closer positions, and
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thus GSH can self-oxidize through the formation of intramolecular disulfide bonds, presenting less free thiol groups (Atyabi, Moghaddam, Dinarvand, Zohuriaan-Mehr & Ponchel, 2008), considering
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that reducing agents were not added during microparticles preparation. Ikeda, Motoune, Matsuoka, Arima, Hirayama and Uekama (2002) used β-CD to form
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inclusion complexes with captopril, and the results showed total inclusion of this molecule in the
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cavity of β-CD with the carboxylic acid located within the cavity, and with the thiol terminal part out of the cavity. The inside part of β-CD cavity probably binds to a portion of the GSH L-
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glutamate side chain as it occurred with GSH and α-CD in a previous study by Garcia-Fuentes et al. (2006). Data obtained in this previous study using nuclear magnetic resonance (NMR) showed that
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the association between GSH and α-CD is in the range of 55-70 M-1 at 25 °C (Garcia-Fuentes et al., 2006), and despite some overlapping signals, it prevented an accurate measurement of the constant complexation between GSH and a chemically modified cyclodextrin (sulphobutyl ether-βcyclodextrin), Trapani et al. (2010) suggest a similar affinity. In a study by Cutrignelli, Lopedota, Denora, Laquintana, Tongiani, and Franco (2014), encapsulation efficiency of liposomes containing GSH or liposomes containing GSH/cyclodextrin complexes (β and chemically modified) was between 13.6 and 23.7%. 16
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3.3. Kinetics GSH release in model wine Generally, active compound release occurs due to polymeric network swelling, which happens when the aqueous environment enters network leading to its expansion. This polymer network becomes increasingly wider, which allows the release of the active compound into the
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external phase until it reaches the thermodynamic equilibrium. In this sense, GSH kinetic release
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depends on the following phenomena: diffusion of the polymer in aqueous environment,
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macromolecule relaxation kinetics, and the active compound diffusion through swollen polymeric network (Del Nobile, Conte, Incoronato, & Panza, 2008). The GSH release was tested for these
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samples: chitosan/GSH and β-CD/GSH. Figure 7 shows the GSH release at intervals ranging from 0.2 to 48 hours.
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In Figure 7 we can see that GSH release of chitosan/GSH is faster than β-CD/GSH microparticle. For chitosan/GSH microparticle, it occurs an initial phase of rapid release followed
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by a slower phase until it gets to an equilibrium. This initial of fast phase release can be attributed to
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GSH presence on the surface of the microparticle. This effect is called “burst effect”. The release of 90% of GSH from chitosan/GSH microparticles to the environment occurred on the first day. For β-
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CD/GSH microparticles, this release was slower, and up to the first hour, the concentration of GSH released to the environment was less than 10 mg L-1 (limit of quantification). Thus, β-CD/GSH
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microparticles had its burst effect lower than chitosan/GSH, indicating low GSH on the surface content in β-CD/GSH microparticles. Therefore, the release form occured by GSH diffusion from the matrix interior of β-CD via the swelling of the polymeric network. At 25.00 h release time the nearest released GSH concentrations were obtained by comparing the two formulations. Previous studies about GSH encapsulation into a polymeric chitosan, β-CD or chitosan/βCD systems aimed the pharmaceutical application of GSH and, therefore, evaluated its release in various synthetic environment, such as simulated gastric and intestinal environment (Lopedota et 17
ACCEPTED MANUSCRIPT al., 2009; Trapani et al., 2010; Jingou et al., 2011; Del Nobile et al., 2008). The quick-release profile observed for chitosan/GSH microparticles is assigned to the combined mechanisms of balanced complexation between chitosan and the active compound, and of the active compound diffusion through a chitosan thin layer (Trapani et al., 2010). Data from in vitro release of GSH in wine model of β-CD/GSH and chitosan/GSH
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microparticles were based appropriately in the Korsmeyer-Peppas model, which is used to
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determine the release mechanism of the initial portion of the active compound (Mt/M∞ ≤ 60%), by using the equation: Mt/M∞ = ktn. In this equation , “Mt/M∞” is the fraction of GSH released at a
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time “t”; “K” is the release rate constant, and “n” is the release exponent, an indicative of the drug
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release mechanism. The value of “n” is used to characterize the GSH release mechanism. The
X Time (h), which are shown in Table 2.
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values of “n”, “K”, and the coefficient of determination (R2), were obtained from the graphs Mt/M∞
For spherical particles, n = 0.43 corresponds to a Fickian release mechanism, indicating that
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the active compound is released by diffusion, and n = 0.85 corresponds to a Case II transport
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(relaxation), indicating the release by swelling. Moreover, “n” values between 0.43 and 0.85 correspond to a non-Fickian or anomalous transport, showing a superposition of two factors,
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diffusion and swelling (Ritger & Peppas, 1987; Siepmann & Peppas, 2001). The value of “n” for β-CD/GSH microparticle was greater than 0.45 and less than 0.85,
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indicating that GSH release followed a non-Fickian release. Therefore, beyond GSH release by diffusion, the expansion of the polymer chain occurred, leading to fluid penetration into the matrix (Jingou et al., 2011). β-CD/GSH microparticles have a slower release than chitosan/GSH microparticles, which is suggested by “K” values, once they indicate that GSH release rate was higher in chitosan/GSH microparticles. This is in agreement with the results of encapsulation efficiency (Table 1), which showed a lower interaction between GSH and chitosan than between GSH and β-CD. 18
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4.
Conclusions
It was possible to encapsulate GSH using both β-CD and chitosan. The methods used in this study are easy to prepare, does not involve the presence of surfactants and, in the case of β-
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CD/GSH microparticles, does not involve the presence of organic solvents. The characterization of
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microparticles proved an interaction between GSH and the polymers, and a microencapsulation of
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the active compound, as well as an improvement of the molecule thermal stability. β-CD was more efficient for encapsulating GSH, allowing the slow release of the molecule into wine model
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solution, and added protection of the molecule, giving better thermal stability for GSH. More research is necessary in order to improve the stability of GSH to release it more slowly in
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hydroalcoholic environment with low pH, such as wines and sparkling wines, as well as evaluate
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the feasibility of using GSH encapsulated in these products.
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Acknowledgment
To the Foundation for Research of Rio Grande do Sul State (FAPERGS), the National Council for
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Scientific and Technological Development (CNPq) for financial support.
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Table 1. Encapsulation efficiency (EE%) of microparticles formulations of chitosan/GSH and βCD/GSH Formulation
EE% (n=3) 25,0 ± 14,4
β-CD/GSH
62,4 ± 12,2
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Table 2. Parameters obtained for the equation Mt/M∞ = Ktn for the microparticles of β-CD and of chitosan, both containing GSH n
R2
β-CD/GSH
0,1826
0,64
0,89
Chitosan/GSH
0,5810
0,10
0,85
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Figure 2. Infrared spectra for (a) GSH (b) chitosan, (c) chitosan/GSH microparticles and (d)
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physical mixture chitosan and GSH
Figure 3. Infrared spectra for (a) GSH, (b) β-CD, (c) β-CD/GSH microparticles, (d) physical
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mixture of β-CD and GSH.
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Figure 4. DSC thermograms of (a) GSH (b) chitosan, (c) β-CD, (d) β-CD/GSH (e) chitosan/GSH (f) physical mixture of chitosan and GSH (g) physical mixture of β-CD and GSH
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Figure 5. Thermograms of samples (a) chitosan, (b) GSH (c) chitosan/GSH microparticles (d) βCD/GSH microparticles (e) β-CD
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(c), chitosan (d) and GSH (e)
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Figure 6. Diffractograms of chitosan/GSH microparticle (a), β-CD/GSH microparticle (b), β-CD
Figure 7. Release profile of GSH in model wine (12% alcohol solution, pH 3.5) at 25 °C (n = 3)
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Figure 8. GSH release versus the ratio Mt/M∞ as a function of time for β-CD/GSH and
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chitosan/GSH microparticle
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Graphical abstract
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ACCEPTED MANUSCRIPT Highlights Glutathione (GSH) was encapsulated using chitosan or β-cyclodextrin as polymers.
Microparticles characterization showed an interaction between GSH and polymers.
Microparticles formation produced a marked decrease in the GSH crystallinity.
β-cyclodextrin/GSH microparticles allowed the slow release of GSH into wine model.
The β-cyclodextrin was more efficient than chitosan to encapsulate GSH.
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