Printing ethanol pomegranate extract in films by inkjet technology

Industrial Crops & Products 140 (2019) 111643

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Printing ethanol pomegranate extract in films by inkjet technology a

a

T

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Josiane Gonçalves Borges , Vitor Augusto dos Santos Garcia , Denise Osiro , ⁎ Rosemary Aparecida de Carvalhoa, a Department of Food Engineering, University of São Paulo, Faculty of Animal Science and Food Engineering, Av. Duque de Caxias Norte, 225, CEP 13635-900, Pirassununga, SP, Brazil b University Center of the Guaxupé Educational Foundation, Av. Dona Floriana, 463, CEP: 37800-000, Guaxupé, MG, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Punica granatum Carboximetilcelulose tape-casting Oral films

This work aimed to use a printing technique for the incorporation of ethanol mesocarp of pomegranate extract (EMPE) into carboxymethylcellulose films obtained by tape-casting to produce oral films with natural active compounds. The EMPE was evaluated in relation to the concentration of phenolic compounds and the potential use as an ink solution (surface tension and viscosity). The substrates were printed with 1–4 layers and the films were characterized in terms of color parameters, surface pH, disintegration time, atomic force microscopy, infrared spectroscopy, in vitro release and stability of the phenolic compounds for 196 days. The EMPE presented ideal characteristics in relation to surface tension and viscosity for use as a printing solution and concentration of phenolic compounds was 51.3 ± 1.2 mg galic acid/mL of extract. In addition, it showed great affinity with the films of carboxymethylcellulose. The films with 2, 3 and 4 printing layers showed more stability of the phenolic compounds and no changes were observed in the surface pH of the films. The disintegration time was < 30 s (1–4 layers), however an increase in the disintegration time was observed with the largest number of layers printed. In relation to the FTIR spectra, no band variations were observed with the increase in the number of printing layers. In conclusion, EMPE is a potential source of phenolic compounds and the printing in films based on carboxymethylcellulose is an innovative technique to convey these compounds of interest, mainly due to the high stability of the phenolic compounds.

1. Introduction

(Buanz et al., 2011). Different matrices may be used for printing the compounds of interest, such as hydroxypropylcellulose (Genina et al., 2013b), polyvinyl alcohol (Buanz et al., 2015), carboxymethylcellulose (Buanz et al., 2015). Among the polymers, carboxymethylcellulose is widely used in industrial food and pharmaceutical sectors (Leal et al., 2018), considered a natural, non-toxic and biodegradable polymer (Wang and Somasundaran, 2005). In literature, there are several reports of the use of the printing technique for the production of films incorporated with theophylline, acetaminophen and caffeine (Sandler et al., 2011), rasagiline mesylate (Genina et al., 2013b), rasagiline mesylate and tadalafil (Janßen et al., 2013), naproxen (Hsu et al., 2013), propranolol hydrochloride (Vakili et al., 2016), sodium picosulfate (Wimmer-Teubenbacher et al., 2018). However, there is a lack of studies regarding the incorporation of active compounds obtained from natural sources using the printing technique. Phenolic compounds are secondary metabolites found widely in plants (Lu et al., 2018), being commonly used due to their beneficial health effects, as well as other active components of natural products

Currently, casting is a common technique used in the production of oral disintegration films, mainly for commercialized products. However, this technique has some limitations, such as long drying time and difficulty in controlling the thickness due to different evaporation rates of the solvent in the films (Low et al., 2013). In addition, the active compounds must be incorporated in the middle of the film production process, which may lead to its degradation throughout the production process (Genina et al., 2013b). Furthermore, when the compounds are dispersed in the polymer matrix, they can cause changes in the mechanical properties of this material (Buanz et al., 2015). Alternatively, a printing technique can be used for the production of oral films. This method of printing consists of the deposition of the active compound on the polymer matrix with the use of a printer (Buanz et al., 2011). A great advantage of this technique is that the same matrix can receive multiple layers, so that the appropriate dosage of the active principle can be controlled according to the patient's needs

⁎ Corresponding author at: Department of Food Engineering, University of São Paulo, Faculty of Animal Science and Food Engineering, Av. Duque de Caxias Norte, 225, CEP 13635-900, Pirassununga, SP, Brazil. E-mail address: [email protected] (R.A.d. Carvalho).

https://doi.org/10.1016/j.indcrop.2019.111643 Received 4 January 2019; Received in revised form 31 July 2019; Accepted 1 August 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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phenolic compounds, viscosity, surface tension and contact angle between the extract and the substrate.

and extracts obtained from these products (Lansky and Newman, 2007). According to Lee et al. (2010), the pomegranate has a high phenolic content fruit widely used as an antipyretic analgesic. Ferrazzano et al. (2017) evaluated the antimicrobial activity of a hydroalcoholic extract of pomegranate peel and juice against cariogenic bacterium and observed that pomegranate extracts were efficient against the main cariogenic bacteria involved in tooth decay. In addition to antimicrobial activity, some studies have reported the use of pomegranate extracts due to the antioxidant activity of the extracts obtained from the different parts of the plant (Bekir et al., 2013; Elfalleh et al., 2012; Višnjevec et al., 2017). Hmid et al. (2018) evaluated the antioxidant capacity of 18 pomegranate cultivars, reporting that the area under cultivation had a strong influence on the concentration of antioxidant compounds present. Due to its high anti-inflammatory activity, interest has grown in its nutraceutical and functional potential (Viladomiu et al., 2013), for exemple antitumor (Zahin et al., 2010), healing (Nayak et al., 2013), as well as helping with oral health (DiSilvestro et al., 2009). Due to its anti-inflammatory properties associated with the anti-proliferative and pro-apoptotic effects, the phenolic compounds are potential treatments for inflammatory painful diseases such as mucositis (Varoni et al., 2012). Thus, the development of carrier systems for these compounds can increase their application, with a variety of functions due to the different properties presented by the extracts obtained from the pomegranate. This study aimed to produce oral films based on carboxymethylcellulose by tape-casting, incorporating an extract of the mesocarp of pomegranate using a printing technique as a source of phenolic compounds. Additionally, the release kinetics and the stability of the compounds present in the films of oral disintegration were evaluated.

2.3. Characterization of the extract as an ink solution 2.3.1. Total phenolic content The total phenolic content of the extract was diluted in hydro alcoholic solution (50%). Aliquots of the diluted samples (0.5 mL) were added to a tube containing 2.5 mL of Folin-Ciocalteu (1:10; v/v). After 5 min, 2.0 mL of sodium carbonate solution (7.5%) were added and the solution was left to stand for 2 h in the absence of light (Singleton et al., 1999) before measurement in a spectrophotometer (Digimed, DMESPEC, São Paulo, Brazil) at 740 nm. The values were converted to mg of gallic acid/mL of extract. The standard curve of gallic acid was obtained using concentrations between 0.016 and 0.08 mg of gallic acid/ mL. 2.3.2. Viscosity The viscosity of the EMPE samples was determined using a Stabinger Viscometer SVM 3000 (Anton Paar) cylinder geometry viscometer. The analysis was performed at 25 °C in triplicate. For analysis, 2.5 mL of the sample was used and the data of dynamic viscosity and kinematic viscosity solution were obtained directly from the equipment. In addition, as the printing solution standard, the four colors of Epson Commercial inks (Black, Cyan, Magenta and Yellow) that were purchased together with the printer EcoTank L375 (Epson, São Paulo, Brazil). The viscosity of the commercial inks was determined using the viscometer (DV2T-LV, Brookfield, São Paulo, Brazil), with a SC4-18 spindle, speed between 20 and 200 rpm with a 20 rpm interval every 30 s (25.0 ± 1.0 °C). The mean viscosity was determined by the mean value of the points with torque greater than or equal to 10%, using the Rheocalc T1.1.13 equipment software.

2. Materials and methods 2.1. Materials

2.3.3. Surface tension The surface tension of extract and commercial inks (Epson, São Paulo, Brazil) were determined using a tensiometer (Attension Sigma Force, Sweden) with platinum ring probe (Du Noi ring). The surface tension (mean of 5 readings) was obtained by the mathematical correction of Hud-Mason, by the equipment, using 15 mL samples. The analysis was performed at 25 ± 0.5 °C in triplicate.

Pomegranate fruits were collected in the Pirassununga (São Paulo/ Brazil) region and extracts were produced with absolute ethyl alcohol (Synth). For the analysis of phenolic compounds, the reagents FolinCiocalteau (Sigma-Aldrich), anhydrous sodium carbonate (Synth) and gallic acid standard (Sigma-Aldrich) were used. Carboxymethylcellulose (Denver) and glycerol (Synth) were used for the production of drug free films. The phosphate buffer solution (pH 6.8) was prepared according to Föger, Kopf, Loretz, Albrecht, and Bernkop-Schnürch (2008) with 8 g of sodium chloride (NaCl, Synth, Brazil), 0.2 g of potassium chloride (KCl, Synth, Brazil), 0.2 g of potassium dihydrogen phosphate (KH2PO4, Vetec, Brazil) and 1.536 g of sodium hydrogen phosphate (Na2HPO4, Synth, Brazil) per 1 L of distilled water and the pH was adjusted with 0.1 M hydrochloric acid (Synth).

2.4. Production of film by tape-casting technique The films were produced by the tape-casting technique using a spreader ZAA 2300 (Zehntner, Switzerland) with the aid of an universal applicator (ZUA 220). For the production of the films, constant concentrations of carboxymethylcellulose (4 g/100 g of filmogenic solution) and glycerol (20 g/100 g of polymer) were used. The polymer was previously hydrated (60 min, room temperature) and then solubilized at 80 °C (thermostatic bath MA-420, Marconi) for 30 min. Then, the previously solubilized plasticizer was added and the solution was maintained at 80 °C for 10 min. The film-forming solution was kept under vacuum for 12 h to remove bubbles, then spread on glass plates using an universal applicator (ZUA 220), with a casting speed of 25 mm/s and wet coating thickness of 3000 μm. The films were dried in a forced circulation oven (MA-035, Marconi) at 40 °C for 12 h. The thickness of the films was determined using a Mitutoyo digital micrometer (IP e 65 model, Mitutoyo, Japan).

2.2. Production of ethanol mesocarp of pomegranate extract (EMPE) The mesocarp was obtained by manual separation of pomegranate fruits and dried in a forced circulation oven (MA 037, Marconi, Brazil) at 40 °C until the mass was constant. The dried samples were ground and the granulometry was standardized using a 40 Mesh sieve and frozen at -18 ± 1 °C until use. The extract was produced using a hydro alcoholic solution (70%) as solvent, with a constant ratio of mesocarp powder:solvent of 1:10 (w:v). The extraction was performed under mechanical stirring at 500 rpm (RW 20 Digital, IKA, USA) for 30 min at 70 °C using a thermostatic bath (MA-127, Marconi, Brazil). After this, the solution was kept refrigerated for 24 h before the supernatant was filtered. The filtered supernatant was named EMPE. The extract samples were concentrated at 40% in rotaevaporador (RV-05, IKA, USA) and evaluated in relation to the concentration of

2.5. Contact angle The contact angle between the extract (EMPE) and the substrate (carboxymethylcellulose-based films) was determined by the drop method using an optical tensiometer (Attension, Theta Lite). Carboxymethylcellulose-based films (2 cm x 2 cm) were attached to the 2

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base of the equipment and one drop of the extract (200 μL) was deposited on the surface of the substrate. The contact angle was determined at time 0, 1 and 10 s. The analysis was performed in triplicate of 10 samples.

(2.54 × 5.08 cm²) were solubilized in 20 mL of hydroalcoholic solution (50%) under constant stirring (100 rpm) and temperature (25 °C) for 12 h on a shaker table (Shaker MA-420). Then, the samples were diluted and aliquots of the diluted samples (0.5 mL) were used to evaluated total phenolic compounds were evaluated according to Singleton et al. (1999) as described in item 2.3.1. These results of total phenolic concentration were used as the maximum value in the release profile (100%) for each number layer. In vitro release was performed according to Perumal et al. (2008). Samples of films (2.5 cm x 2.5 cm) were immersed in phosphate buffered saline (pH 6.8) prepared according to Föger et al. (2008) at 37 °C and kept under stirring at 100 rpm on a shaker table (Shaker MA-420). Aliquots (0.1 mL) were removed at different times (0.5, 1, 2, 5, 10, 20, 40, 60, 80, 100, 120 and 140 min) and the volume kept constant by replacing with buffer solution (volume equal to that withdrawn) at the same time as the aliquots were withdrawn. The concentration of phenolic compounds was determined by the Folin-Ciocalteu method (Singleton et al., 1999). The analysis was performed in triplicate. The release kinetics were evaluated as a function of the concentration of phenolic compounds using the mathematical models presented in Table 1. The Statistica software (Version 11) was used to determine the constants of the models. In the evaluation of the release profile, the difference factor (f1) and similarity factor (f2) according to Eq. 1 and 2, respectively, as proposed by Food and Drug Administration (FDA, 1997).

2.6. Printing of ethanol mesocarp of pomegranate extract (EMPE) The EMPE was printed on the substrate (carboxymethylcellulosebased films) using a piezoelectric printer EcoTank L375 (Epson, São Paulo, Brazil), with an ink tank system filled with extract. The substrates received from 1 to 4 printing layers in consecutive impressions of the extracts, with an interval of 10 min (ambient temperature) between the impressions to guarantee a better absorption of the extract by the matrix. Printing was performed on the “Premium Photo Paper Glossy” with “best” print quality in the “Black/White” option and the templates to be printed were prepared in Word 2013 (Microsoft Inc.). 2.7. Characterization of printed disintegration films 2.7.1. Color parameters The determination of the color parameters (luminosity, chroma a* and chroma b*) was performed using a Miniscan XE (HunterLab) colorimeter with a CIE Lab system (Comisson Internationale de Eclairage) according to Gennadios et al. (1996). 2.7.2. Atomic force microscopy The surface of the oral disintegration films was evaluated using an atomic force microscope (Solver Next, NT-MDT). Samples of the films (1 cm2) were fixed with double-sided tape and were analyzed in a semicontact mode using the NSG01 tip with a force constant of 5 N/m, resonance frequency of 150 kHz and with scanning rate of 0.3 Hz. The images were analyzed using the software Image Analysis 3.1.0.0.

n

n

f 1 = {∑ |Rt − Tt|/ ∑ Rt } 100 t=1

(1)

t=1

n

f 2 = 50 log{[1 + 1/n∑ (Rt − Tt )²]−0,5 100}

(2)

t=1

Where: Rt = is the percentage dissolved of reference at time point; Tt = the percentage dissolved of test at time point (t); n = number of time points.

2.7.3. Surface pH The surface pH of the films was determined according to Manhar, and Suresh (2013). Samples of the films (2 cm x 1 cm) were immersed in 15 mL of phosphate buffer solution (pH 6.8) and the pH was determined using a WTW pH meter (WTW3210 model, electrode Sensoglass - SC 22, Germany) after 30 min.

2.7.7. Stability: phenolic compounds Samples of EMPE-printed films (2.54 × 5.08 cm²) were stored in desiccators at room temperature (25 ± 5 °C) containing saturated sodium bromide solution (relative humidity = 58%) and the concentration of phenolic compounds in the printed matrix was evaluated over time for a period of 196 days. For quantification of the phenolic compounds, the film samples (2.54 × 5.08 cm²) were immersed in hydro alcoholic solution (20 mL of 50% ethyl alcohol) to extract these compounds. The solutions containing the films were stirred (100 rpm) in a constant temperature (25 °C) for 12 h on a shaker table (Shaker MA420). Then, the samples were diluted and the total phenolic compounds were evaluated according to Singleton et al. (1999).

2.7.4. Disintegration time The disintegration time was determined according to the methodology described by Garsuch and Breitkreutz (2010). Samples of films (2.0 cm x 3.0 cm) were fixed in a slide frame and an aliquot (200 μL) of distilled water was deposited on the surface of the film (Janßen et al., 2013). The time required to form a hole in the film surface was defined as the disintegration time (TD). 2.7.5. Infrared spectroscopy FTIR spectra of EMPE and films were obtained using a Perkin Elmer spectrometer (Spectrum One FT-IR) containing the UATR accessory. Prior to the analysis, the film samples were stored in desiccator with silica for 10 days. 16 scans per sample were performed in the spectral range from 4000 to 650 cm−1 with a resolution of 4 cm−1. FTIR absorbance spectra were mathematically treated, baselines were corrected, followed by normalization by equalizing peak intensity at 1023 cm−1 (CeOeC stretch vibration) of 1. For a quantitative analysis, the absolute area of the region between 1800 and 870 cm−1 was calculated from the absorption FTIR spectra of CMC films with and without EMPE layers (Baker et al., 2014; Stuart, 2012). All the mathematical procedures were performed in the Origin Pro 9.0 software obtained from OriginLab.

Table 1 Mathematical models used to evaluate the kinetic profile of the release of phenolic compounds in films of oral disintegration. Release Model

Equations

Reference

Zero order

Mt M∞ Mt M∞ Mt M∞ Mt M∞

= K 0 t+ b

(Varelas et al., 1995)

= KH t + b

(Higuchi, 1961)

= K1tm + K2t2m

(Peppas & Sahlin, 1989)

Higuchi Peppas and Sahlin Korsmeyer and Peppas

=

Ktn

+b

(Korsmeyer and Peppas, 1981)

Note: (Mt/M∞) = fraction of drug released over time (t); (b) = initial concentration of drug in solution; (K0, KH e K) = kinetics constants; (n) = exponent of drug release; (K1) = constant related to Fickian diffusion mechanism; (K2) = constant related to erosion/relaxation mechanism (Case II); (m) = Fickian diffusion exponent.

2.7.6. Release in vitro Initially, the concentration of total phenolic compounds for each printing layer was determined. Samples of EMPE-printed films 3

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to the values observed for commercial inks (Epson), showing that the extract can be used as a printing solution both in terms of viscosity and surface tension. Varan et al. (2017) evaluated the printing technique to obtain adhesive films for local treatment of cervical cancers as a result of HPV infection and observed for printing solution containing paclitaxel (anticancer) and cidofovir (antiviral), a surface tension of 43.25 and 41.40 mN/m, respectively. Viscosity and surface tension are important parameters for the printability, because too low viscosity may cause dripping of the ink or too high viscosity may clog the nozzle, and the surface tension must be high enough for the dripping of ink from nozzles and the formation of drops (Varan et al., 2017).

2.8. Statistical analysis The data were statistically evaluated using analysis of variance (ANOVA) and in the case of a significant difference, the mean test using Duncan's test (p < 0.05) was performed using the SAS computational program 9.2. 3. Results and discussion 3.1. Characterization of the extract for printing 3.1.1. Total phenolic content The concentration of phenolic compounds in the extract after filtration was 33.0 ± 0.5 mg of gallic acid/mL extract and after concentration (removal of 40% of the initial volume) was 51.3 ± 1.2 mg galic acid / mL of extract showing a 65% increase in the concentration of phenolic compounds. Višnjevec et al. (2017) evaluated an ethanol and water extract of pomegranate from different regions of Istria and observed that the total phenolic content in the pomegranate exocarp and mesocarp were, on average, significantly higher in the ethanol extracts (23 and 16 mg/g, respectively) than in the water extracts (7.9 and 6.7 mg/g, respectively). According to Višnjevec et al. (2017), the difference in total phenolic of different pomegranate samples might not only be due to the different genotype, but also to different geological factors and weather conditions.

3.1.4. Contact angle It was verified the reduction in the contact angle as a function of time (Fig. 1), indicating that the extract had a great affinity for the substrate, which favors absorption by the substrate, enabling the printing of several layers. Genina et al. (2013b) reported a contact angle for a rasagiline solution on hydroxypropyl methylcellulose based film immediately after deposition of the ink on the substrate of 34.4 ± 2.0°, which reduced over time. Genina et al. (2013a) evaluated the contact angle between the ink solution with caffeine e loperamide and the substrate (icing sheet) and observed that the angle decrease drastically during the first second, de 73.0° to 17.1° and 58.9–16.4, respectively, and the drop disappeared completely after 180 s. The authors related this with the absorption of the printing solution by the substrate. Vuddanda et al. (2018) observed a contact angle between the substrate (hydroxypropyl methylcellulose based film) and warfarin printing solution of 38.18 ± 1° after immediate deposition on the substrate. The droplet rapidly penetrated the surface of the substrate and the authors suggested that the warfarin was absorbed into the substrate matrix.

3.1.2. Viscosity According to Sandler et al. (2011), the viscosity needs to be lower than 20 mPa.s to print in a controlled way. The viscosity of the EMPE was similar to Magenta ink (Epson) but was significantly different from the 3 other colors (Black, Cyan and Yellow) of commercial inks (Epson). However, it is important to note that all solutions (extract and commercial inks) presented the same order of magnitude. In this way, the extract can be used as the printing solution for Epson printer. The viscosity of the EMPE was similar to those reported in the literature as being appropriate for the use in a printing technique. Varan et al. (2017) evaluated polycaprolactone nanoparticles and anticancer cyclodextin inclusion complexes for a piezoelectric inkjet printer (PixDro LP 50) and observed better values of dynamic viscosity between 6.6 e 6.8 mPa.s and surface tension between 41.40 and 43.25 mN/m. Similar results were reported by Genina et al. (2013b) for printing solution containing rasagiline mesylate dynamics viscosity of the ink (≤ 5 mPa.s) using a printer D1660 (Hewlett – Packard Inc.). The research involving the incorporation of natural active principles using the technique of printing are incipient, in addition different types of printers are being used which makes comparisons difficult and the establishment of parameters in relation to the solution used for printing.

3.2. Characterization of printed films 3.2.1. Color parameters Table 3 shows the color parameters (L *, chroma a * and chroma b *) of the carboxymethylcellulose-based films with a different number of printing layers. The reduction of the a * values and increase of the b * values demonstrates intensification of the yellow coloration, due to the greater number of printing layers and consequently, higher concentration of EMPE. The results corroborate the color of the films observed visually (Fig. 2). 3.2.2. Atomic force microscopy The 2D and 3D images of the carboxymethylcellulose-based films with and without printing can be seen in Fig. 3. The control (unprinted) film was observed to have a surface with reduced roughness (Fig. 3). The first printing layer on the film caused an increase surface roughness, which may have occurred due to the swelling of the polymer matrix after absorption of the first layer of extract. Nonetheless, the increase in the number of printing layers caused a reduction in the roughness of the surfaces, and the film with 4 layers had a similar surface to the control film (Fig. 3). These results may be related to the absorption of the extract in the polymer matrix with the increase of the printing layers. Results of the contact angle (Fig. 1) indicated that the extract was rapidly absorbed by the CMC matrix, so the reduction of the roughness may be due to the absorption of the extract.

3.1.3. Surface tension The values observed for surface tension (Table 2) were similar to the recorded in the literature for synthetic active principles used as a printing solution. De acordo com Sandler et al. (2011) the surface tension needs to be between 20–45 mN.m−1 for use as a print solution. On the other hand, considering the commercial ink used in the printer, the surface tension the surface tension (Table 2) of the EMPE are similar Table 2 Dynamic viscosity and Surface tension for extract ethanol mesocarp of pomegranate extract (EMPE) and commercial inks (Epson). Sample EMPE Black® ink Cyan® ink Magenta® ink Yellow® ink

Dynamic viscosity (mPa.s)

Surface tension (mN. m

c

d

3.35 3.69 3.65 3.44 3.57

± ± ± ± ±

0.03 0.12a 0.30a 0.20bc 0.22ab

3.2.3. Surface pH The values observed for the surface pH (Table 3), regardless of the number of printing layers, were close to the values in the neutral conditions of the saliva (pH 5.8–7.1), and the increase in the number of printing layers, that is, the concentration of extract in the matrix did not significantly alter the surface pH. Dosage of the oral mucosa with acid or alkaline pH may cause irritation to the buccal mucosa (Bahri-Najafi et al., 2014; Kumria et al., 2016; Manhar and Suresh, 2013), but

−1

26.4 27.6 27.0 26.6 28.9

± ± ± ± ±

)

0.2 0.1b 0.1c 0.1d 0.1ª

4

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Fig. 1. Contact angle between the ethanol mesocarpo of pomegranate extract and the substrate (carboxymethylcellulose based films) as a function of time: (a) 0 s, (b) 1 s, (c) 10 s. Table 3 Surface pH, disintegration time (DT), color parameters and concentration of phenolic compounds (Cphenolic) of carboxymethylcellulose based films with different number of printed layer of ethanol mesocarp of pomegranate extract. Layers

Surface pH

DT (s)

Color parameters L*

0 1 2 3 4

6.8 6.8 6.8 6.8 6.8

20.2 25.2 25.9 27.6 30.3

± ± ± ± ±

1.2a 2.1b 1.7b 2.2b,c 2.3c

90.7 89.1 88.3 87.8 86.6

± ± ± ± ±

0.0d 0.3c 0.5bc 1.3b 0.2a

Cphenolic (mg/cm2) a*

b*

−0.9 ± 0.0a −8.9 ± 0.6b −11.7 ± 0.7c −13.0 ± 0.2d −13.5 ± 0.1d

2.4 ± 0.1e 25.2 ± 1.8d 37.7 ± 2.8c 46.9 ± 1.4b 53.9 ± 0.2ª

– 0.12 0.23 0.35 0.49

± ± ± ±

0.01a 0.01b 0.02c 0.03d

Different lowercase letters in the same column indicate significant difference (p < 0.05) between the means, by the Duncan test, using SAS 9.2 software.

Fig. 2. Carboxymethylcellulose films printed with ethanol extract of pomegranate mesocarp, being: (0) - control without printing; (1) - 1 printing layer; (2) - 2 layers of printing; (3) - 3 layers of printing; (4) - 4 layers of printing.

(HP Deskjet 460, Hewlett-Packard Inc.), with values of 23.3 ± 5.6 and 30.5 ± 4.6 s, respectively, for the two methods used. Using the casting technique, Sakuda et al. (2010) and Abdelbary et al. (2014) added with synthetic compounds, reported values of 38.5 and 50.3 s, respectively, for CMC-based films.

according to the determined surface pH values, regardless of the number of printing layers, the oral films will not cause changes to the oral mucosa. Daud et al. (2011)observed for disintegration films based on different macromolecules (Maltodextrin, pullulan, hydroxypropylmethylcellulose and polyvinyl alcohol) incorporating Zingiber offciale extract surface pH at 6.91 ± 0.85. Tedesco et al. (2017) for HPMC-based films incorporating peanut skin extract surface pH between 6.36 and 6.88. Abdelbary et al. (2014) produced CMC films incorporating the synthetic compound flupentixol dihydrochloride and reported pH between 6.0-6.8.

3.2.5. Fourier transform infrared (FTIR) The region between 3700 a 2500 cm−1 presents a broad band very influenced by the presence of humidity, which justifies the variation of its intensity. This intense band results mainly from the stretching vibrations (ν) OeH and NeH occurring between 3700 and 3000 cm−1. In this case, the presence of moisture contributes to the increase in the formation of hydrogen bonds with the OeH and NeH groups and therefore the widening of this band occurs. In addition, vibrations occur νC-H between 3000 and 2500 cm−1 (Movasaghi et al., 2007; TürkerKaya and Huck, 2017) (Figs. 4 and 5). On the other hand, the FTIR spectra of CMC films with and without EMPE layers, the absorbance in the region between 1800 and 900 cm−1 almost overlap (Fig. 6a), which proves the little influence due to the deposition of layers of EMPE. These spectra are quite similar to each other and show characteristic signals of pure EMPE. The second derivative spectra also show that the EMPE layers contribute almost nothing quantitatively and qualitatively. However, even if the contribution of the EMPE film is almost imperceptible to the final FTIR spectrum, one can determine this subtle

3.2.4. Disintegration time Table 3 shows the disintegration time of the carboxymethylcellulose films in relation to the number of printed layers, where a significant increase of the disintegration time was observed after the first printing layer. An increase in the disintegration time was observed with the largest number of printing layers, possibly due to the increase in extract concentration in the polymer matrix, possibly in the interior, as a function of the absorption of the extract. As noted in the AFM, the different printing layers resulted in changes in the surface of the films and these changes may have influenced the disintegration time. Similar values of the disintegration time were reported by Buanz et al. (2015) for oral-dispersible films based on polyvinyl alcohol and carboxymethylcellulose added with clonidine hydrochloride incorporated in the matrix (casting technique) or the printing technique 5

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Fig. 3. Atomic force microscopy images of the surface in 2 dimensions (2D) and 3 dimensions (3D) of the films based on carboxymethylcellulose printed with ethanol extract of the pomegranate mesocarp, with: (0) - control without printing, (1) - 1 printing layer; (2) - 2 layers of printing; (3) - 3 layers of printing; (4) - 4 layers of printing.

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of stretching CeOeC and angular CeOeH of carbohydrates and in 880 cm−1 aromatic ring vibration (Abbas et al., 2017; Movasaghi et al., 2007; Türker-Kaya and Huck, 2017). 3.2.6. In vitro drug release The initial concentration of phenolic compounds increased with the number of printed layers (Table 3), which confirms the increase in the concentration of active compounds as a function of the increased number of impressions. Fig. 7 shows the release profiles of films printed with different printing layers of EMPE. As the compounds were deposited on the surface of the substrates, the analysis was performed on both sides of the substrates (unprinted and imprinted side). The release kinetics of phenolic compounds from the substrates (films based on carboxymethylcellulose) printed with different numbers of pomegranate mesocarp extract layers were evaluated by different mathematical models (Table 4). For all formulations the KorsmeyerPeppas, and Peppas and Sahlin models (Table 4) showed better fit considering phenolic compound release, with values of R² > 0.95. Considering the Korsmeyer-Peppas model, it was verified that the values for parameter k were close and values of n less than 0.45, except for the film with 1 printing layer and with the unprinted side in contact with the buffer solution (Table 4, n = 0.52). According Korsmeyer and Peppas (1981), values of n less than 0.45 indicate that the release of the active compound occurred by Fickian diffusion, while anomalous release is observed for values in the range 0.45 < n < 0.89, that is, the transport occurs either by swelling and by diffusion. Thus, in general, it can be concluded that the films presented a release profile predominantly of Fickian diffusion. This behavior may be related to the absorption of the extract in the polymer matrix after the printing process and the reduced thickness of the films, since the two sides presented a similar mechanism of release with the exception of the film with 1 printing layer. In the case of the film with only 1 printing layer the variation between the printed or unprinted side with the buffer solution may be related to the lower concentration of extract inside the polymer matrix, so that several processes simultaneously as erosion and swelling of the polymer matrix, in addition to diffusion, for the release of the active principle when the unprinted side was in contact with the butter solution. The same behavior was observed for the Peppas and Sahlin model, with a greater influence of the diffusion in the process of release of the active compounds, regardless of the number of layers or the side in contact with the buffer solution (Table 4). Buanz et al. (2015) evaluated the release kinetics for polyvinyl alcohol and carboxymethylcellulose films containing clonidine hydrochloride, and found by the HixsonCrowell model that the drug release occurred by erosion (both for the films produced by casting and those produced by the printing method), related to the presence of carboxy methylcellulose carboxy groups that increased the dissolution of the polymer, unlike that reported in this work, which showed Fickian diffusion. It was observed, in general, that, independently of the evaluated

Fig. 4. Infrared absorption spectra for carboxymethylcellulose based film printed with ethanol extract of pomegranate mesocarp, with: (0) - control without printing, (1) - 1 printing layer; (2) - 2 layers of printing; (3) - 3 layers of printing; (4) - 4 layers of printing. The spectra 0, 1, 2, 3 and 4 were normalized by matching the peak at 1023 cm−1 to 1.

Fig. 5. FTIR spectra of CMC films with 0, 1, 2, 3 and 4 EMPE printed layers.The spectra 0, 1, 2, 3 and 4 were normalized by matching the peak at 1023 cm−1 to 1.

influence on the intensity of the vibration bands by calculating the total area of absorption in the region between 1800 and 870 cm−1. This region shows intense vibration signals attributed to the chemical components present in the EMPE. It is observed in Fig. 6b that there was an variation in the value of the area of the absorption spectrum devido a presença do extrato on the CMC film. The FTIR spectrum of EMPE exhibits characteristic signals for its composition, as in ˜1736 cm−1 of stretching C]O phenolic compounds, in 1642 cm−1 of stretching C]O protein Amide I, in 1087 e 1038 cm−1

Fig. 6. FTIR spectra of CMC films with 0, 1, 2, 3 and 4 EMPE printed layers, being, a) Absorbance and second derivative FTIR spectra of the CMC films with 0, 1, 2, 3 and 4 EMPE printed layers of the region between 1800 and 870 cm−1. The spectra 0, 1, 2, 3 and 4 were normalized by matching the peak at 1023 cm−1 to 1. b) Absolute area over the spectra of films between the wavelengths 1800 and 750 cm−1.

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Fig. 7. Release of phenolic compounds from the carboxymethylcellulose based film imprinted with ethanol extract of the pomegranate mesocarp with different numbers of print layer (N), on both sides of the substrate, being: (a) with unprinted side in contact with the solution and (b) printing side in contact with the solution.

side, the films had a similar release profile (Table 5), despite the difference in the percentage of release of the phenolic compounds for the different printing layers (Fig. 7). According to the Food and Drug Administration (FDA, 1997), two dissolution curves are similar when they have f1 between 0 and 15 and f2 between 50 and 100. Considering the same number of printing layers it can be observed that there was a difference between the sides with and without printing in contact with the buffer solution only for ODFs with only 1 printing layer. For films with number of layers greater than 1, comparing the values of f1 and f2, similar release profiles were found. Possibly the results are related to the absorption of the matrix in the matrix the reduced thickness of the ODFs, and in the case of the ODFs with only one printing layer at the lower absorption in the extract in the polymer matrix (greater deposition in the surface), which corroborates with the greater roughness

observed (Fig. 3). These results corroborate with those observed in the kinetic models tested, since the film with 1 printing layer and the unprinted side in contact with the solution presented anomalous release while all other formulations presented Fickian diffusion. Buanz et al. (2015) compared polyvinyl alcohol and carboxymethylcellulose films with clonidine produced by casting and printing (1 layer) methods and observed for both methods the same release profile (f1 = 1.25 e f2 = 64.7). In other studies, Bonsu et al. (2016) for HPMC-based films produced by casting incorporated with diclofenac sodium reported that the films had Fickian drug diffusion by Higuchi kinetics model. Leal et al. (2018) evaluated CMC-based films (casting technique) incorporating with tannic acid and observed that the value of n was equal to 0.5 by the Korsmeyer-Peppas model (R² = 0.937), indicating that the main mechanism of release was Fickian diffusion, 8

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Table 4 Mathematical models of kinetics of phenolic compounds release from carboxymethylcellulose films with different numbers of layers of the ethanol mesocarp of pomegranate extract. Mathematical Model Ordem Zero

Higuchi

Korsmeyer-Peppas

Peppas e Sahlin

Layers

Side

k

R²

kh

R²

kk

n

R²

k1

k2

R²

1

a b a b a b a b

0.011 0.014 0.015 0.020 0.014 0.015 0.013 0.014

0.878 0.4953 0.3198 0.4962 0.5447 0.5125 0.7475 0.5021

0.091 0.101 0.106 0.108 0.100 0.105 0.095 0.099

0.9784 0.9029 0.8288 0.8562 0.9187 0.8972 0.9738 0.9006

0.083 0.211 0.255 0.235 0.194 0.206 0.140 0.196

0.52 0.32 0.29 0.32 0.34 0.34 0.41 0.33

0.9791 0.9885 0.9731 0.9551 0.9822 0.9641 0.9894 0.9709

0.045 0.190 0.213 0.124 0.158 0.142 0.121 0.150

0.009 −0.014 −0.013 0.018 −0.006 0.003 −0.003 −0.002

0.9974 0.9939 0.9935 0.9963 0.9858 0.9778 0.9924 0.9773

2 3 4

Note: a = unprinted side in contact with the buffer solution; b = print side in contact with the buffer solution. Table 5 Difference factor (f1) and similarity factor (f2) for the release profile of phenolic compounds of carboxymethylcellulose films with different numbers of the ethanol mesocarp of pomegranate extract. Reference

f1 Test

Layers

1 2 3 4

1

2

3

4

Side

a

b

a

b

a

b

a

b

a b a b a b a b

0 17 22 22 16 19 10 15

20 0 7 9 3 8 11 7

29 8 0 5 9 6 17 10

29 10 5 0 9 5 17 10

19 3 8 9 0 7 8 4

24 9 5 4 7 0 13 8

11 10 14 14 7 12 0 8

18 6 9 9 4 7 9 0

a b a b a b a b

100 90 85 85 91 88 96 90

90 100 97 96 99 97 96 98

85 97 100 99 97 98 91 97

85 96 99 100 96 99 90 96

91 99 97 96 100 98 97 99

88 97 98 99 98 100 93 98

96 96 91 90 97 93 100 96

90 98 97 96 99 98 96 100

f2 1 2 3 4

Fig. 8. Stability of the phenolic compounds of the carboxymethylcellulose based film printed with ethanol mesocarp of pomegranate extract with different numbers of printing layer.

edible objects using a 3D printer, showing that the antioxidant capacity and total phenolic content reduced after 8 days of storage. In this way, it was observed that the printed films presented greater stability.

Note: a = unprinted side in contact with the buffer solution; b = print side in contact with the buffer solution.

4. Conclusion

similar to that overserved in this work.

The extract of the mesocarp of pomegranate can be used as a printing solution because it has adequate viscosity and surface tension, as well as affinity with the substrate developed based on carboxymethylcellulose. Additionally, the carboximelticellulose film was efficient for use as a printing support for incorporation of the extract. The films had a high concentration of phenolic compounds, reduced disintegration time (< 30 s) surface pH close to the mouth (6.8), as well as stability of the phenolic compounds for a period of 196 days. In this way, the printing technique can be considered as an innovative method for the incorporation of active principle from natural sources into polymer matrices once that was observed a good stability of the phenolic compounds during storage.

3.2.7. Stability: phenolic compounds It was observed that during the storage at room temperature (25 ± 5 °C) in the presence of light and controlled relative humidity (58%), the oral films of did not present great variation in relation to the concentration of phenolic compounds for a period of 196 days (Fig. 8), indicating that carboxymethylcellulose did not cause degradation of the phenolic compounds. In addition, there was a significant loss of these compounds in films with only one printing layer, with a reduction in the concentration of phenolic compounds of approximately 18.0%. The films with 2, 3 and 4 printing layers had a higher stability of the phenolic compounds, with a reduction of approximately 4.5%, 5.5% and 1.9% respectively, indicating that the more printing layers, the lower the loss of phenolic compounds under the conditions evaluated. Borges and De Carvalho (2015) observed for films based on gelatin and hydrolyzed collagen containing ethanolic propolis extract, that the concentration of phenolic compounds did not show variation after 12 weeks of storage. Severini et al. (2018) evaluated the stability of active compounds of the printed smoothie of selected fruit and vegetables as

Acknowledgment R.A. Carvalho thanks the National Council for Scientific and Technological Development (CNPq) for the productivity grant.

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