CENTRO DE CIÊNCIAS DA SAÚDE
CURSO DE GRADUAÇÃO EM FARMÁCIA
GABRIELA SUASSUNA BEZERRA
CHITOSAN-BASED COPAIBA OIL CARRIER POLYMERIC SYSTEMS: OBTAINING, CHARACTERIZATION AND ANTIFUNGAL ACTIVITY IN
VITRO
NATAL, RN
GABRIELA SUASSUNA BEZERRA
CHITOSAN-BASED COPAIBA OIL CARRIER POLYMERIC SYSTEMS: OBTAINING, CHARACTERIZATION AND ANTIFUNGAL ACTIVITY IN
VITRO
Trabalho de Conclusão de Curso
apresentado ao Curso de Graduação em Farmácia da Universidade Federal do Rio Grande do Norte, como requisito parcial para obtenção do título de Bacharel em Farmácia. Orientador: Prof. Dr. Ádley Antonini Neves de Lima
NATAL, RN 2020
CHITOSAN-BASED COPAIBA OIL CARRIER POLYMERIC SYSTEMS: OBTAINING, CHARACTERIZATION AND ANTIFUNGAL ACTIVITY IN
VITRO
Trabalho de Conclusão de Curso
apresentado ao Curso de Graduação em Farmácia da Universidade Federal do Rio Grande do Norte, como requisito parcial para obtenção do título de Bacharel em Farmácia.
Orientador: Prof. Dr. Ádley Antonini Neves de Lima
Presidente: Prof. Dr. Ádley Antonini Neves de Lima, Orientador, UFRN
Membro: Prof.ª Drª. Waldenice de Alencar Morais, UFRN
Membro: Prof. Dr. Fernando Henrique Andrade Nogueira, UFRN
Discente: Gabriela Suassuna Bezerra, Curso de Graduação em Farmácia, UFRN
RESUMO
Copaifera multijuga Hayne é uma espécie típica do Brasil conhecida por produzir o óleo-resina de copaíba e sua ampla utilização pela medicina popular devido suas diversas propriedades farmacológicas, no entanto, seu caráter lipofílico dificulta sua utilização em novos sistemas farmacêuticos. Quitosana é um polímero de grande interesse na área farmacêutica pela sua biocompatibilidade, propriedades filmogênicas e bioadesivas, o que permite seu uso pela via tópica. Para obter sistemas poliméricos de quitosana com óleo de copaíba, foram utilizados dois métodos de obtenção de filmes, a incorporação direta do óleo na matriz polimérica e um sistema carreador microemulsionado. Foi realizada a análise das interações entre o óleo e a rede polimérica por técnicas de espectroscopia de infravermelho, termogravimetria e difração de raios-X, bem como a caracterização do sistema microemulsionado utilizado. A microemulsão obtida foi eficaz na formação de um filme carreador do óleo de copaíba, provocando mudanças no padrão físico-químico que comprovam uma incorporação adequada e incremento na difusão do óleo no meio de cultura, podendo se tornar uma alternativa terapêutica para tratamentos de injúrias da pele, uma vez que foram observados halos de inibição de crescimento em cepas de Candida spp.
ABSTRACT
Copaifera multijuga Hayne is a typical Brazilian species known for producing copaíba oil-resin and its wide use by popular medicine due to its various pharmacological properties, however, its lipophilic character makes its use in new pharmaceutical systems difficult. Chitosan is a polymer of great interest in the pharmaceutical area due to its biocompatibility, filmogenic and bioadhesive properties, which allows its use by topic. To obtain chitosan polymeric systems with copaiba oil, two methods of obtaining films were used, the direct incorporation of the oil in the polymeric matrix and a microemulsioned carrier system. The interactions between the oil and the polymeric network were analyzed by infrared spectroscopy, thermogravimetry and X-ray diffraction techniques, as well as the characterization of the microemulsioned system used. The microemulsion obtained was effective in the formation of a copaiba oil carrier film, causing changes in the physical-chemical pattern that prove an adequate incorporation and increase in the diffusion of the oil in the culture medium, and may become a therapeutic alternative for skin injury treatments, since growth inhibition halos were observed in strains of Candida spp.
1. INTRODUCTION
Copaifera multijuga Hayne is a species of the genus Copaifera L. typically found in Brazil, more specifically in the Amazon region and considered one of the main producers of copaiba oil-resin. Its use in popular medicine is widely spread since the Brazilian colonization because it has several therapeutic properties that were observed by the indigenous and scientists of the time 1,2. It is composed of a variety of sesquiterpenes and diterpenes, with β-caryophyllene (Figure 1) being the majority constituent and one of those responsible for its therapeutic characteristics, as well as other compounds that contribute to this 2-4. A variety of pharmacological properties have already been described for this oil-resin, for example, anti-inflammatory activities 1,3,5,6, healing 1,3, antinociceptive 6,7 and antimicrobial 4,8,9.
Figure 1. Chemical structure of β-caryophyllene
Fonte: PUBCHEM. β-caryophyllene (C12H24). Disponível em: <https://pubchem.ncbi.nlm.nih.gov/compound/beta-
Caryophyllene>. Acesso em: 14 jul. 2020.
Skin lesions appear when the integrity of the tissue is compromised by trauma or burns, in such a way that it impedes the normal performance of its protective barrier function, allowing the colonization of the region by opportunistic microorganisms, such as strains of Candida spp. When the infection is established, the healing process is delayed, which may generate complications to the patient, considering that currently resistance to traditional medications is frequently reported 10-12.
Chitosan is a natural cationic polymer of great interest in the pharmaceutical field due to its biodegradability and biocompatibility. It is obtained from the deacetylation of Chitin - a polymer easily obtained in nature, from the shell of crustaceans - and easily soluble in slightly acidic aqueous solutions, which when submitted to a simple evaporation of the solvent (technique "casting") can form films, coatings and semi-permeable membranes. Currently, it is already a molecule regulated as an excipient by USP and of possible use for topical administration of drugs 13-15. Due to its filmogenic and bioadhesive properties,
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it is considered an effective biopolymer in topical treatments for skin lesions and tissue regeneration 15.
To incorporate the oil-resin of copaiba in an aqueous polymeric matrix based on Chitosan it is necessary to adopt some strategy that allows its solubilization and to obtain a homogeneous mixture, therefore, using a microemulsioned system can be an adequate option. Since microemulsion is a thermodynamically stable, insoluble drug carrier system, of spontaneous formation and optically transparent to translucent, capable of increasing its solubility, dissolution and bioavailability, which can be used by several routes of administration, among them the topical one. It is a liquid system composed of oil and water, with interfacial tension stabilized by the action of surfactants 16,17.
In this context, the objective of this work was to develop chitosan polymeric films with copaiba oil by means of direct incorporation and microemulsion methods, to perform physical-chemical characterization and to evaluate its possible use as a therapeutic alternative for topical treatments of skin injuries, by evaluating the antifungal activity against strains of Candida spp.
2. EXPERIMENTAL SECTION 2.1 MATERIALS
The copaiba oil (CO) was obtained in partnership with UFAM (Federal University of Amazonas), which was collected in the Ducke Forest Reserve of the National Institute of Amazonian Research (INPA), from trees of the Copaifera Multijuga Hayne species, in the summer of 2006. The Chitosan polymer was acquired from Sigma-Aldrich® (95% deacetylation degree and average molecular weight). The surfactant PEG-8 capric/caprylic glyceride (Labrasol®) was acquired from Brasquim. All experiments were performed using distilled water and analytical grade reagents, in addition to automatic pipettors, analytical balance, plastic plates, glassware, standardized strains and other utensils.
2.2 METHODS
2.2.1 Obtaining the Ternary Phase Diagram (TPD)
In order to determine the microemulsion region, the ternary phase diagram was developed using the aqueous phase titration method followed by visual inspection of the component mixture. Copaiba oil was used as oily phase (OF), Labrasol® as surfactant (S) and 1% acetic acid solution as aqueous phase (AF). The surfactant was added to the oily phase in the proportions 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1 (p/p), respectively. The aqueous phase was titrated using an automatic pipettor and a Vortex-type tube agitator in each of the proportions obtained, followed by visual observation of the systems. They were then homogenized in an ultrasonic cell switch (VibracellTM 75041, 13mm, 750 W, 20 KHz probe, Bioblock Scientific, France) with 1 minute cycles at 40% amplitude, interspersed with ultrasound bath for 1 minute for the removal of possible bubbles and kept at rest for later observation of the final aspect of the system. The results obtained were plotted in an equilateral triangle and, from this, a proportion of the components in the region of formation of transparent or translucent systems was selected for use in obtaining the polymeric films.
2.2.2 Development of Chitosan Films with Copaiba Oil
All the films, regardless of the form of incorporation of the copaiba oil used, were obtained through the "casting" technique, which consists of molding the polymeric matrix from the evaporation of the solvent. The chitosan blank film (BF) was also obtained only with the 1% chitosan solution, by the same method.
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2.2.2.1 Direct Incorporation film (DIF)
Initially, an aqueous solution of 1% chitosan in 1% acetic acid was prepared, which was kept in magnetic stirring for 24 hours and then vacuum filtered. From this solution, an aliquot, sufficient to produce films of 10 g, was placed in a polystyrene plate. The copaíba oil was incorporated into the polymeric matrix using a 50% alcoholic solution, an amount equivalent to 500 mg of oil. The system obtained was kept in a rotary incubator at 40°C for 24 hours for complete film formation.
2.2.2.2 Microemulsioned film (MEF)
The Chitosan polymer was weighed at 1% (w/v), dissolved in the chosen microemulsion system from the phase diagram and maintained in magnetic stirring for 24 hours. The resulting solution was vacuum filtered. A polystyrene plate was used as a mold. In each plate 10 g of the obtained system was added, which was kept in a rotary incubator at 40 °C for 24 hours for complete film formation.
2.2.2.3 Neutralization of films
Using a NaOH 1M solution, all the polymeric films obtained (BF, MEF and DIF) were neutralized to remove the remaining acid residues from the drying process and leave them at suitable pH for use in the skin. Sufficient amount of the solution was added to the mold plate to immerse the film, keeping it in contact with the solution for 30 minutes.
The films were then washed with distilled water to remove the excess alkaline solution until a pH near 7,0 was reached. The films were kept at room temperature until completely dried and then stored in a closed container.
2.2.3 Physico-chemical characterization of Microemulsion 2.2.3.1 pH determination
The pH of the formulation was measured using a digital pHmeter, with an electrode and temperature sensor previously calibrated with 4,0 and 7,0 buffer solution, at a temperature of 25±0,5°C. The electrode was introduced directly into the formulation. The analysis was performed in triplicate.
2.2.3.2 Electrical conductivity
The electrical conductivity of the formulation was evaluated by means of a digital conductivimeter, previously calibrated with a calibration solution presenting a conductivity of 146.9 µS cm-1 at a temperature of 25±0,5°C. The electrode was introduced directly into the formulation. The analysis was performed in triplicate.
2.2.3.3 Refraction index
The refractive index was measured using an Abbé refractometer (Quimis®), measured with distilled water (IR=1.333), at a temperature of 25±0.5°C. The analysis was performed in triplicate.
2.2.3.4 Droplet Size, PDI and Zeta Potential
The average droplet diameter, polydispersion index (PDI) and Zeta potential were obtained by dynamic light scattering (DLS) analysis using the Zetasizer Nano ZS90 equipment (Malvern Instruments Ltd, France). The analyses were performed at a constant temperature of 25°C, with an incidence angle of 90°, and a wavelength beam of 633 nm. The samples were diluted (1:1000) before analysis. The zeta potential of the microemulsion was also measured after dilution of the samples (1:1000) in NaCl solution (1mmol/L).
2.2.4 Physico-chemical characterization of polymeric films 2.2.4.1 Fourier Transform Infrared Spectroscopy (FTIR)
The Shimadzu® equipment (IRPrestige-21) was used. The films were placed on a total attenuated reflectance accessory (ATR) and the reading was done in the region of 700 to 4000 cm-1, with 16 scans and in the resolution of 4 cm-1.
2.2.4.2 Thermogravimetric Analysis (TG)
The analysis was performed in simultaneous thermogravimetric and calorimeter analyzer, model SDTQ600 (TA Instruments). The tests were performed using an alumina crucible with a dynamic nitrogen atmosphere (flow rate of 50 mL/min) at a heating rate of 10°C/min in the range of 25 - 600°C. About 3 mg were used for each sample.
2.2.4.3 X-Ray Diffraction (XRD)
XRD analysis was performed using a Bruker D2 Phaser (Massachusetts, USA) with radiation CuKα (λ = 1.54 Å) at a voltage of 30 kV and a current of 10 mA using a Lynxeye detector. The samples were scanned at room temperature for a period of 2 hours at a range of 5 to 45° to 0.05°/s.
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2.2.5 Antifungal activity evaluation
The antifungal activity for the films obtained was evaluated using the adapted Disco- diffusion method (Kirby-Bauer). Sabouraud agar were used as culture medium and 4 standards strains of Candida spp.
Four plates with inoculum equivalent to the 0,5 grade of the McFarland scale were prepared with the strains: Candida albicans ATCC 90028, Candida glabrata ATCC 2001, Candida tropicalis ATCC 13803 and Candida parapsilosis ATCC 22019.
The films were applied to the plates in a standard size (6 mm) after wetting in saline solution. The plates were incubated in an oven at 37 °C for 48 hours. Subsequently the formation of inhibition zones and their size were evaluated.
3. RESULTS AND DISCUSSIONS 3.1 Obtaining the ternary phase diagram
The TPD determines in which proportions of the components the microemulsion region can be reached. The choice of the appropriate surfactant, capable of reducing the interfacial tension between the oily and aqueous phases, is one of the main factors related to the stability of the system. Surfactant with an HLB (hydrophilic-lipophilic balance) range between 8-18 are used for the formation of oil/water (O/A) type microemulsions. Labrasol® (PEG-8 capric/caprylic glyceride) is a non-ionic surfactant characterized by advantages such as low toxicity and irritability, with HLB between 12-14, being therefore ideal for obtaining O/A microemulsions 16-19.
Figure 2 represents the TPD obtained from the visual observation of the behavior of the surfactant, oily phase and aqueous mixtures. The areas of microemulsion formation are highlighted in grey.
Figure 2. Ternary phase diagram. FA: aqueous phase, FO: oily phase and T:
surfactant.
Table 1 shows the proportions of the formulation components that were chosen (MEOC) from the diagram and that represents an O/A microemulsion, with a translucent and optically limpid aspect 16,17.
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Copaiba Oil 5%
Labrasol® 35%
Acetic Acid 1% 60%
Table 1. MEOC Composition.
3.2 Physico-chemical characterization of Microemulsion
3.2.1 pH determination
The pH of MEOC was evaluated in order to verify if the system would be able to solubilize the chitosan for further film formation. The value found was 3,65 ± 0,50, which represents a predominance of the slightly acid character of the aqueous phase, being then capable of solubilizing the polymer, giving the system the possibility of forming bioadhesive films after evaporation of the solvent 15.
3.2.2 Electrical conductivity
The electrical conductivity of a microemulsion helps in the identification of the type of system obtained, being the oil/water microemulsions characterized by their capacity to conduct electrical current, since their external phase is aqueous, a fact that is not observed in water/oil systems, since the oily external phase has insulating power. The value found was 116.05 ± 0.3 µS.cm-1, characterizing the MEOC as a conductive system, therefore, it is an oil/water microemulsion 17.
3.2.3 Refraction Index
From the reading in the Abbé Refractometer, the value of the refractive index obtained for the MEOC was 1,388, a value close to that specified for distilled water, which characterizes the system as optically clear and transparent to translucent 16,17.
3.2.4 Droplet Size, PDI and Potential Zeta
Table 2 presents the results obtained in the dynamic light scattering analysis (DLS).
Parameter Result
Droplet Size (nm) 225,3 ± 2,316
PDI 0,315 ± 0,034
Potential Zeta (mV) - 40
Table 2. Results of DLS analysis.
With the obtained droplet size value of 225,3 ± 2,316 nm, the system can be classified as microemulsion, since these are presented with reduced values for this parameter, between 100 nm - 300 nm 17,19. The PDI takes into consideration the average droplet size,
the solvent refractive index, the measurement angle and the variation of the distribution. Low values of PDI represent a homogeneous distribution of the droplet size, as well as the result found for MEOC. The electrical potential around the droplet is called zeta potential, being determinant in the stability characterization of dispersed systems. Zeta potential equal to or greater than the modular value of 20 mV represents more stable systems due to the predominance of repulsion forces among the droplets, therefore, the MEOC, as it has a zeta potential equal to -40 mV, was presented as a stable system 16-19.
3.3 Physico-chemical characterization of polymeric films
3.2.1 Fourier Transform Infrared Spectroscopy (FTIR)
The infrared spectra for CO, BF, MEF and DIF are represented in Figure 3. The CO spectrum has absorption bands in 2950, 2925 and 2855 cm-1 - characteristic of stretch vibration C-H -, in 1643 cm-1 - characteristic of stretch vibration C=C of cycloalkene -, in 1447 cm-1 - characteristic of asymmetrical deformation C-H of methyl -, in 1367 cm-1 - characteristic of symmetrical deformation C-H of methyl - and in 886 cm-1 - characteristic of deformation outside the C-H plane of alkenes 20. Previous studies also showed similar wave number absorption bands for the CO, besides being characteristic bands of functional groups present in sesquiterpenes and diterpenes, such as β- caryophyllene, the majority compound of this oil-resin 2.
In the spectrum of BF there are absorption bands around 3300 cm-1 - concerning the overlapping of absorptions characteristic of OH and NH2 - in 2929 and 2867 cm-1 - characteristic of stretch vibration C-H - in 1645 cm-1 - characteristic of stretch vibration C=O of amide - in 1560 and 1545 cm-1 - characteristic of deformation NH2 -, in 1408 cm -1 - characteristic of symmetrical stretching of Carboxylic Acid -, in 1380 cm-1 - characteristic of symmetrical C-H deformation -, in 1151, 1065 and 1026 cm-1 - characteristic vibrations of C-O stretching of alcohol and phenol -, in 947 and 896 cm-1 - characteristic of C-H deformations, as was also observed in other studies with chitosan 15,20. In the spectra of MEF and DIF a predominance of the characteristic absorptions of BF and typical reduced CO absorptions is observed, indicating the incorporation of the oil in the polymeric matrix, an effect that was also observed in other studies with this polymer 13–15,21.
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Figure 3. Infrared Spectral: (A) copaiba oil, (B) BF, (C) MEF and (D) DIF.
COPAIBA OIL BLANK FILM
2950, 2925, 2855 cm-1 3300 cm-1 OH and NH2 Stretch vibration C-H 2929, 2867 cm-1 Stretch vibration C-H Deformation NH2 1643 cm-1 1408 cm-1
Stretch vibration C=C of Cycloalkene Symmetrical stretching of Carboxylic Acid
1447 cm-1 1380 cm-1
Asymmetrical deformation C-H of
Methyl Symmetrical deformation C-H
1367 cm-1 1151, 1065, 1026 cm-1
Symmetrical deformation C-H of Methyl Stretching vibrations C-O of Alcohol and Phenol
886 cm-1 947, 896 cm-1
Deformation outside the C-H plane of
Alkenes Deformations C-H
Table 3. Infrared absorption bands for copaiba oil and chitosan blank film.
3.2.2 Thermogravimetric Analysis (TG)
The Figure 4 presents the thermogravimetric curves for copaiba oil, blank film, MEF and DIF.
Figure 4. Thermogravimetric curves: (A) BF, (B) copaiba oil, (C) MEF and (D) DIF.
The Table 4 presents the temperature and loss of mass data of the analyzed samples. In the curve of the CO, there are 3 stages of thermal degradation, the first due to volatilization 2. In the curve of MEF, there are 3 stages, among which we can highlight the first that corresponds to the evaporation of water and acetic acid residues in the film, and the second that corresponds to dehydration, depolymerization and decomposition of the polymer 22. In the DIF curve, two stages of loss of mass were observed corresponding to the same events identified for MEF. In view of these results, a thermal protection of the polymeric films to the copaiba oil is observed, possibly due to the interaction between the chitosan polymeric units and the essential oil that delays the thermal degradation 22.
Sample Stage 1 Stage 2 Stage 3
CO Ti = 69 °C Tf = 200 °C Δm = 80% Ti = 215 °C Tf = 296 °C Δm = 12,15% Ti = 326 °C Tf = 441 °C Δm = 7,55% BF MEF DIF Ti = 37 °C Tf = 132 °C Δm = 19% Ti = 182 °C Tf = 375 °C Δm = 42% - - - Ti = 31 °C Tf = 146 °C Δm = 13% Ti = 244 °C Tf = 307 °C Δm = 24% Ti = 315 °C Tf = 413 °C Δm = 56,4% Ti = 40 °C Tf = 101 °C Δm = 12% Ti = 270 ºC Tf = 324 °C Δm = 28,4% - - -
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3.2.3 X-Ray Diffraction (XRD)
The diffractograms obtained for the polymeric films are represented in Figure 4. The XRD evaluates the crystallinity profile of the films obtained, thus, BF presented a low intensity crystalline reflection at 38°, which may be the result of residues of chitin, acetic acid and inorganic minerals in the polymeric matrix. While in MEF, the disappearance of this reflection and the presence of a diffraction halo at around 20° are observed, suggesting that interactions between the dispersed essential oil and the polymeric matrix occurred, reducing the packing of chitosan monomers. In the DIF, it was observed the maintenance of the pattern obtained for the BF, suggesting that the oil was not evenly dispersed in the matrix. Phenomena like this have already been observed in other studies with chitosan films 14,15,21,23.
Figure 4. Diffractograms of polymeric films: (A) BF, (B) DIF and (C) MEF.
3.2.4 Antifungal activity evaluation
The disc-diffusion test provides qualitative results. It is one of the simplest, most reliable and most used susceptibility methods by microbiology laboratories. Its basic principle is the diffusion of the compound on the surface of the agar 24, in this case, the diffusion took place from a standardized disc size of the polymeric films tested with the copaiba oil incorporated in its matrix.
Table 5 shows the results obtained after incubating the plates for 48 hours. It was observed that only MEF showed antifungal activity against the strains, with the result for the Candida albincans strain being the most significant, with a 12 mm halo of inhibition. This data is related not only to the sensitivity to copaiba oil, but also to the capacity of
diffusion in the culture medium, it was observed that the microemulsioned system provides a better diffusion in the medium, as it is a system that carries hydrophobic compounds 17,19. The antifungal activity observed is due to the presence of β-cariofilene as the majority constituent of copaiba oil, of which this pharmacological property has already been reported in respect to strains of Candida albicans, which is considered an opportunistic microorganism and responsible for several clinical complications in skin lesions 2,4,10–12. Candida albicans ATCC 90028 Candida glabrata ATCC 2001 Candida tropicalis ATCC 13803 Candida parapsilosis ATCC 22019
BF Resistant Resistant Resistant Resistant
DIF Resistant Resistant Resistant Resistant
MEF 12 mm 3 mm 2 mm 3 mm
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4. CONCLUSION
Using a microemulsion as a carrier system of liposoluble and slightly acidic aqueous compounds, it was possible to incorporate Copaiba essential oil into a polymeric matrix of Chitosan, obtaining polymeric films that can become a therapeutic alternative for topical treatment of skin injuries that become propitious to the development of Candida albicans. The DLS technique confirmed the obtaining of the microemulsioned system and the other techniques used in the characterization of MEF and DIF, demonstrated that the oil was adequately incorporated in the polymeric network through the two techniques, more specifically with the microemulsioned system, leading to changes in physical- chemical characteristics, such as prevalence of the absorption pattern in infrared, change in the crystalline/amorphic profile and in addition to improvement in thermal stability. However, the evaluation of the antifungal activity showed that the microemulsified carrier system increased the diffusion of copaiba oil in the culture medium and consequently produced a 12 mm halo of microbial growth inhibition, suggesting that the use of this system in the development of a topical use product will favor the desired efficacy and may become an alternative for the treatment of skin injuries.
Acknowledgements
The authors would like to acknowledge the support from the UFRN and CNPq.
Conflicts of Interest
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