Orally Disintegrating Films Containing Propolis: Properties and Release Profile

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Orally Disintegrating Films Containing Propolis: Properties and Release Profile JOSIANE GONC¸ALVES BORGES, ROSEMARY APARECIDA DE CARVALHO Department of Food Engineering, Faculty of Animal Science and Food Engineering, University of S˜ao Paulo, Pirassununga, S˜ao Paulo, Brazil Received 14 December 2013; revised 20 December 2014; accepted 22 December 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24355 ABSTRACT: The objective of this work was the production and characterization of orally disintegrating films of gelatin and hydrolyzed collagen containing the ethanol extract of propolis. The films were produced by casting with different concentrations of hydrolyzed collagen with and without the extract. The mechanical properties, mucoadhesive properties, swelling degree, in vitro release kinetics, stability of active compounds, Fourier transform infrared spectroscopy (FTIR), and antimicrobial activity of the films were evaluated. The films with the highest concentration of hydrolyzed collagen were less resistant and more elastic, and films containing the extract were more resistant than the control. In addition, the films with the extract showed higher mucoadhesion, which is important for ensuring the release of active compounds in the oral cavity. Generally, all formulations showed a high swelling capacity, which may have contributed to the quick release also demonstrated by the release kinetics model. Interactions between the extract compounds and the polymeric matrix were observed by FTIR spectroscopy, which may have contributed to an improvement in the mechanical properties. Films containing the extract had good stability and effective antimicrobial properties against Staphylococcus aureus, which shows that these films can potentially be used to C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci release active compounds in the oral mucosa.  Keywords: oral drug delivery; stability; polymeric drug delivery systems; mathematical model; kinetics

INTRODUCTION Currently, there is growing interest in developing delivery systems for active compounds in the oral cavity because this is an attractive region either for the local and systemic application.1 Thus, a growing number of studies on the development of new mechanisms for oral release have been performed, including tablets,2 gels,3 sprays,4 pastes,5 patches,6 wafers,7,8 and films.9,10 Orally disintegrating films have the advantage of ease of handling and transportation because they are thin and flexible,11 and they also have good acceptance by patients with difficulty in swallowing.12 These films are mainly composed of polymers, plasticizers, and the active compound of interest, and other ingredients may be incorporated such as flavorings and sweeteners to increase patient acceptance of the films.13 In the production of orally disintegrating films, gelatin has great potential because of its film-forming ability;11 furthermore, it is a natural polymer with good mucoadhesive properties.14 Several studies have used gelatin-based films incorporated with active compounds for different applications.15,16 Natural products have been used for many years in popular medicine as teas and infusions and have gained more prominence because of consumer desire to replace synthetic products with natural ones.17 Many plants, spices, and other natural sources are rich in active compounds such as phenolic compounds, which have interesting properties such as antimicrobial,18,19 antioxidant,20,21 anti-inflammatory,22 and anticarcinogenic activities.23 Correspondence to: Rosemary Aparecida De Carvalho (Telephone: +551935654355; Fax: +551935654284; E-mail: [email protected]) Journal of Pharmaceutical Sciences  C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association

Propolis is a natural substance composed mainly of organic acids, phenolic compounds, some enzymes, vitamins, and minerals.24 Studies show that propolis has great potential for use against pathogenic bacteria and fungi25–27 in addition to its anti-inflammatory properties,19 making its incorporation into orally disintegrating films an interesting proposal for a local application. The objective of this work was the production and characterization of orally disintegrating films from gelatin and hydrolyzed collagen containing the ethanol extract of propolis.

MATERIALS AND METHODS Materials Porcine gelatin type A (260 Bloom/40 MESH/Lot LFP7466 P 11) and hydrolyzed collagen (B50) were purchased from GELITA ˜ Paulo, Brazil). The plasticizer used was sorbitol Brazil Ltd. (Sao ˜ Paulo, Brazil), and 12-type resin (Star Rigel Raf(Nuclear, Sao ˜ Paulo, Brazil) was used for production of the ethanolic fard, Sao extract of propolis. Production and Characterization of the Ethanol Extract of Propolis The ethanol extract of propolis was produced with a ratio of 30 g of resin per 100 mL of ethyl alcohol (80%) according to Nori et al.28 The extraction was performed under agitation (500 rpm) for 30 min at 50°C. After this period, the solution was refrigerated for 24 h, and then the supernatant was filtered to obtain the ethanol extract of propolis. The concentrations of phenolic compounds in the ethanol extract of propolis were characterized using the Folin–Ciocalteau method.29 The extract was dissolved in absolute ethanol (1:1000), and an aliquot of 0.5 mL of this solution was placed in a tube containing 2.5 mL of Folin–Ciocalteu reagent (1:10). Borges and De Carvalho, JOURNAL OF PHARMACEUTICAL SCIENCES

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After 5 min, 2 mL sodium carbonate solution (4%) was added. The solution was left to stand for 2 h, and the reading was performed at 740 nm using a spectrophotometer (SP-22 UV; Biospectro, Curitiba, Brazil) and expressed in milligram of gallic acid per gram of extract. Production of Orally Disintegrating Film The orally disintegrating films were produced using the casting technique as described by Borges et al.30 with a constant mass of gelatin and hydrolyzed collagen (mG + mHC = 2%, w/v) and plasticizer concentration (0.6%, w/v). The mass of hydrolyzed collagen was varied (0%, 10%, 20%, and 30%, w/w) in relation to mG + mHC . The previously hydrated (30 min, room temperature) gelatin and hydrolyzed collagen were solubilized at 50°C (10 min), and sorbitol (solubilized in water) was incorporated into the solution under magnetic stirring. The film-forming solution was kept at 50°C for 10 min. The extract (4%, w/v) was added to the filmogenic solution under stirring using an ultraturrax (IKA T-25) at 6000 rpm (1 min). The filmogenic solution was then poured in a plate and dried in forced-circulation oven (Marconi AM-035) at 30°C for 24 h. The thickness was kept constant by controlling the mass–area ratio. Before analyses, films were conditioned in desiccators containing a saturated salt solution of NaBr (RH = 58%, 25 ± 2°C) for 5 days. For Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy, films were conditioned in desiccators containing silica. Mechanical Properties The mechanical properties were determined using the tensile test according to American Society for Testing and Materials (ASTM) standards31 using the TA.XT Plus texture analyzer (Stable Microsystems SMD, England). Samples of film (100 × 25 mm2 ) were fixed in the probe at an initial distance of 100 mm. The test speed was set at 50 mm/min until breakage. The tensile strength at break (MPa), elongation (%),elastic modulus (MPa/%), and the area under curve (AUC) were determined by Exponent software. The energy at break (normalized to the film’s volume) was determined using the following equation: EB =

AUC V

as the mucoadhesive force. The analysis was performed for all formulation with 10 replicates each. Swelling Degree The swelling degree was determined gravimetrically using a phosphate buffer solution (pH 6.8) following the method described by Mohamed et al.34 Film samples (2 cm diameter) were immersed in 30 mL of phosphate-buffered saline at 37 ± 1°C. The samples were collected with the help of a net support at intervals of 30 s and weighed. The analysis was performed for all formulation. The swelling degree was determined using the following equation: SD =

Release In Vitro The in vitro release was measured according Perumal et al.35 for formulations with ethanol extract of propolis. Samples of the orally disintegrating films (22 × 35 mm2 ) were immersed in a phosphate buffer solution (pH 6.8) at 37 ± 1°C and stirred at 100 rpm. At different times (0, 2, 3, 4, 5, 10, and 15 min), aliquots (0.5 mL) of this solution were collected to determine the concentration of phenolic compounds by the Folin–Ciocalteu method,29 and it was replaced with 0.5 mL of fresh dissolution media after every aliquot. The analysis was performed in triplicate. Release Kinetics The release kinetics for the phenolic compounds were evaluated using the following mathematical models: zero order,36 Higuchi,37 Korsmeyer and Peppas,38 and Peppas and Sahlin,39 as shown in Table 1, using the computer program Statistica (version 11). Comparisons between the release profiles of the films were made by difference factor (ƒ1)40 and similarity factor (ƒ2)41 using the following equations: n 

f1=

3

Mucoadhesive Properties In Vitro The mucoadhesive properties were evaluated with a TA.XT Plus texture analyzer as described by Bruschi et al.32 Chicken pouch was used to simulate the oral mucosa.33 Samples of films were fixed on the equipment platform, and the chicken skin was placed in a cylindrical probe with a diameter of 20 mm. The sample was compressed by the probe covered with the skin at a constant force (0.1 N) for 30 s to ensure uniform contact between the skin and the film. The sample and the skin were completely separated at a constant speed (1 mm/s), and the maximum force required for this separation was determined Borges and De Carvalho, JOURNAL OF PHARMACEUTICAL SCIENCES

(2)

where SD is the swelling degree (%, w/w), mi is the initial mass of sample (g), and mf is the wet mass of the sample at different times (g).

(1)

where EB is the energy at break (MJ/m ), AUC is the area under the load versus displacement curve (MJ), and V is the volume of the film (m3 ). The analysis was performed for all formulation with 10 replicates each.

mi − mf 100 mi

t=1

|Rt − Tt| n 

100

(3)

Rt

t=1

Table 1. Release Models Used to Evaluate the Release Kinetics of Phenolic Compounds in Orally Disintegrating Films Release Model Zero order32 Higuchi33 Peppas and Sahlin35 Korsmeyer and Peppas34

Equations Mt M∞ = K 0 t + b √ Mt M∞ = K H t + b Mt m 2m M∞ = K 1 t + K 2 t Mt M∞

= Ktn + b

Mt /M , fraction of drug released over time (t); b, initial concentration of drug in the solution; K0 , KH and 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. DOI 10.1002/jps.24355

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

and ⎫ −0.5 n ⎬  2 1 + (1/n) 100 f 2 = 50 log (Rt − Tt) ⎭ ⎩ ⎧ ⎨

(4)

t=1

where Rt is the percentage dissolved of reference at time point (t), Tt is the percentage dissolved of test at time point (t) and n is the number of time points. Stability Samples of the orally disintegrating films with ethanol extract of propolis were stored at room temperature in desiccators containing a saturated solution of NaBr (RH = 58%) with a constant light intensity (150.0 ± 28.5 lux). The stability was evaluated by determining the concentration of phenolic compounds present in the films at each storage time point (0, 1, 2, 4, 5, 6, 8, 10, and 12 weeks). The concentration of these compounds was evaluated using the Folin–Ciocalteu method.29 Fourier Transform Infrared Spectroscopy Spectroscopic analysis was performed with a PerkinElmer spectrophotometer (Spectrum One FT-IR) according to the methodology described by Vicentini et al.42 Sixteen scans were performed (400–4000 cm−1 ) with a resolution of 2 cm−1 for all formulation. The files were converted into numerical files and analyzed using the computer program FTIR Spectrum Software. Antimicrobial Activity The films were evaluated for antimicrobial activity against Staphylococcus aureus using the disk diffusion technique.43 An aliquot of 0.1 mL of bacterial solution (10−7 cells·mL−1 ) was spread in a Petri dish containing nutrient agar. Samples (2 cm diameter) were placed in the center of inoculated plates and incubated for 24 h at 37 ± 1°C. After this period, the inhibition halo (cm), considering the diameter of the film, was determined. The analysis was performed for all formulation in triplicate.

RESULTS AND DISCUSSION Characterization of the Ethanol Extract of Propolis The ethanol extract of propolis showed a phenolic compound concentration of 55.5 ± 1.6 mg gallic acid per gram of extract. The concentration of these compounds can vary with different

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types of propolis, the region, and the environmental conditions in which the resin is collected,44 as well as the parameters of solvent used for the extraction.45 Ahn et al.46 observed values between 42.9 and 302.0 mg gallic acid per gram of extract for propolis from different regions of China. Potkonjak et al.47 observed values similar to those obtained in this study for an extract of propolis produced in Serbia. Borges et al.30 observed a lower concentration of phenolic compounds (35.18 mg/g of extract) for the ethanol extract of propolis using the same resin (Type 12) used in this study but with a different solvent (absolute ethanol –96%). Characterization of the Orally Disintegrating Films Mechanical Properties No significant difference (p > 0.05) between the thicknesses of the films was observed (Table 2), indicating that the thickness control was efficient. Generally, it was verified (Table 1) that increasing the hydrolyzed collagen concentration caused a significant reduction (p < 0.05) of the tensile strength, the energy at break, and the elastic modulus and increased elongation of the orally disintegrating films with and without the incorporation of extract. Thus, the increase in hydrolyzed collagen reduced the resistance of the films and the films turned more flexible. This effect may be due to the reduced cohesiveness of the polymer matrix caused by the low molecular weight of the hydrolyzed collagen. Similarly, Hoque et al.48 observed a reduction of tensile strength due to the reduced molecular weight of the gelatin for films produced with gelatin with different degrees of hydrolysis, which may have caused a reduction in the interaction between the gelatin chains. The incorporation of the ethanol extract of propolis (Table 2), on the other hand, increased the resistance of the films because the films containing the extract showed higher tensile strength, energy at break, and elastic modulus compared with the control film. We also observed an increase in the elongation of the films, but it was not significant (p > 0.05) for all concentrations of hydrolyzed collagen. Thus, the extract could confer functional properties to the films, and also increased the resistance of films (Table 2). According to Lam et al.49 films must have adequate flexibility, elasticity, good mucoadhesie properties, and resistance to break due to the stress of oral activity. In addition, greater mechanical resistance of films reflects their ability to withstand mechanical stress without damage during production, handling, and application.50

Table 2. Effect of Concentration of Hydrolyzed Collagen (CHC ) and Ethanol Extract of Propolis (CEEP ) on Thickness (TI), Tensile Strength (TS), Elongation (E), Energy at Break (EB), Elastic Modulus (ME), and Mucoadhesion (MA) in Orally Disintegrating Films CEEP (%) 0

4

CHC (%)

TI (mm)

TS (MPa)

E (%)

EB (J/m3 )

ME (MPa)

MA (N)

0 10 20 30 0 10 20 30

0.067 ± 0.005aA 0.068 ± 0.006aA 0.067 ± 0.006aA 0.067 ± 0.005aA 0.069 ± 0.004aA 0.070 ± 0.003aA 0.068 ± 0.006aA 0.070 ± 0.004aA

31.1 ± 1.9aB 28.6 ± 2.7bB 25.6 ± 1.9cB 18.5 ± 1.7dB 35.9 ± 2.5aA 32.6 ± 2.2bA 27.1 ± 1.9cA 25.9 ± 2.0cA

35.8 ± 2.8aB 39.6 ± 3.5bA 40.4 ± 3.5bA 41.1 ± 2.9bB 40.2 ± 3.4aA 41.4 ± 3.5aA 42.9 ± 3.9aA 45.9 ± 4.1bA

9.0 ± 1.8aA 7.6 ± 1.2bA 6.6 ± 1.2bA 4.8 ± 0.6cA 13.3 ± 1.7aB 10.3 ± 1.1bB 7.6 ± 0.6cB 6.3 ± 0.6cB

821.8 ± 95.3aA 746.7 ± 81.2bA 590.0 ± 59.9cA 491.5 ± 54.9dA 906.5 ± 86.3aB 847.8 ± 84.1aB 721.7 ± 84.6bB 595.3 ± 71.2cB

0.5 ± 0.1aA 0.6 ± 0.1aA 0.6 ± 0.1aA 0.6 ± 0.1aA 0.8 ± 0.1aB 0.8 ± 0.1aB 0.9 ± 0.1aB 0.9 ± 0.1aB

Different lowercase letters in the same column for each concentration of CEEP indicate a significant difference (p < 0.05) between different CHC , and different capital letters in the same column for each CHC indicate a significant difference (p < 0.05) between different CEEP by Duncan’s test using the SAS computer program. CHC , %, w/v in filmogenic solution; CEEP , %, w/w in mG + mHC . DOI 10.1002/jps.24355

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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Similar results were observed by Borges et al.30 for films of gelatin and hydrolyzed collagen with 100 g ethanol extract of propolis/100 g protein mass; it was also noted that increasing the hydrolyzed collagen concentration caused a reduction of tensile strength and increased elongation. Hoque et al.48 evaluated the mechanical properties of films containing natural extracts (cinnamon, cloves, and star anise) in gelatin films and also observed increased resistance of films with increasing extract concentration, correlating this behavior to the interaction between the phenolic compounds present in the extracts with the gelatin matrix. Daud et al.51 evaluated the effect of different solvents and drying temperature on the mechanical properties of hydroxypropylmethylcellulose-based orally disintegrating films with Withania somnifera extract and observed lower resistance values for tensile strength than those observed for films with ethanol extract of propolis in this work. Mucoadhesive Properties The increase in the hydrolyzed collagen increased the mucoadhesiveness of the films (Table 2), but no significant difference (p > 0.05) was observed. However, the addition of ethanol extract of propolis caused a significant increase (p < 0.05) in mucoadhesiveness of orally disintegrating films related to control films (Table 2), independent of the concentration of hydrolyzed collagen. The process of mucoadhesion is the result of electrostatic and hydrophobic interactions and hydrogen bonding between the material and mucosa.52 Thus, because of its hydrophobic character, increasing the concentration of ethanol extract of propolis may have contributed to increased hydrophobic interactions between the orally disintegrating films and the mucosa. Different values for this parameter are reported in the literature because of the different macromolecules and additives used to form the matrix. Mohamed et al.34 found values between 0.080 ± 0.019 and 0.698 ± 0.064 N for mucoadhesive films of carboxymethylcellulose and hydroxypropyl cellulose, respectively, with diltiazem hydrochloride. Abruzzo et al.15 observed a value of 0.103 mN (10.3 dyne) for mucoadhesion for gelatin-based films with propranolol hydrochloride, which is much lower than the value found in this work. When films are developed to release active compounds into the oral cavity, it is necessary to have a high mucoadhesion capacity to ensure that the material stays in the mouth for enough time to provide a precisely measured drug dose to the application site.53 Swelling Degree The orally disintegrating films with and without the incorporation of ethanol extract of propolis showed high swelling capacity (Figs. 1a and 1b). It should be noted that for some samples, especially for the orally disintegrating films without incorporation of the extract, after 90 s, the matrix disintegrated (Fig. 1a), which precluded the determination of swelling capacity at later times. According to Gordon et al.,54 gelatin may have a high swelling degree in films because of their solubility and the polymeric structure formed. Abruzzo et al.15 correlated the high degree of swelling obtained for the gelatin films with the high number of amino acids in the ionized structure and, hence, the presence of free charges that favor the entry of water into the polymer matrix. Borges and De Carvalho, JOURNAL OF PHARMACEUTICAL SCIENCES

Figure 1. Effect of the concentration of the ethanol extract of propolis (CEEP ) on the swelling degree versus time for films with different concentrations of hydrolyzed collagen (CHC ), with (a) CEEP = 0% and (b) CEEP = 4%. Different letters on a vertical indicate a significant difference (p < 0.05) between the means for different concentrations of hydrolyzed collagen at the same time point. The difference between mean values was determined by the Duncan test using the SAS computer program. CHC , %, w/w in mG + mHC ; CEEP , %, w/v in filmogenic solution.

In relation to the increased concentration of hydrolyzed collagen, no change in the degree of swelling in the films without the ethanol extract of propolis was observed (Fig. 1a). The films with hydrolyzed collagen showed a higher swelling capacity in relation to the film without hydrolyzed collagen, possibly because of the low molecular weight of the collagen, which leads to lower cohesiveness of the matrix, favoring water diffusion in the matrix. However, increasing the concentration of hydrolyzed collagen in the matrix caused a reduction in the degree of swelling. These results are most likely related to the increased solubility of the hydrolyzed collagen and consequent erosion of the matrix. However, for films with ethanol extract of propolis (Fig. 1b), significant differences in swelling capacity were found as a DOI 10.1002/jps.24355

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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

tosan) showed higher swelling (352.7 ± 8.7%) compared with films based only on chitosan (190.0 ± 7.9%). Release In Vitro

Figure 2. Release of phenolic compounds from orally disintegrating films as a function of time for different concentrations of hydrolyzed collagen (CHC ). CHC , %, w/w in mG + mHC .

function of time. The incorporation of ethanol extract of propolis, independent of the concentration of hydrolyzed collagen in the film, significantly reduced (p < 0.05) the swelling capacity of the films. This may be related to the interactions of phenolic compounds with the gelatin matrix, which hampered the entry of water into the matrix. Similar results were obtained by Mu et al.,55 who found values between 312.0% and 613.4% for the swelling of gelatin films plasticized with glycerol. For films containing different proportions of gelatin and chitosan, Abruzzo et al.15 found that films with higher concentrations of gelatin (80% gelatin and 20% chi-

Figure 2 shows that increasing the concentration of hydrolyzed collagen caused a faster release of phenolic compounds. This may be related to the low molecular weight of the hydrolyzed collagen, which makes the matrix more soluble, causing rapid disintegration and thus faster release of the active compounds present in the matrix. Figure 2 also shows that after 15 min, all the formulations showed a release of phenolic compounds greater than 80%. This time period was less than that observed by Juliano et al.,56 whose films based on alginate with the ethanol extract of propolis released 80% of the compounds only after 60 min. This result shows that the films based on gelatin and hydrolyzed collagen could be an alternative media for the rapid release of the active compounds of propolis. Release Kinetics The parameters for the mathematical models to study the release kinetics are shown in Table 3. For all formulations, the models that best represent the release kinetics of the phenolic compounds from the orally disintegrating films were the Korsmeyer and Peppas38 and Peppas and Sahlin39 models because high R2 values were observed independent of the concentration of hydrolyzed collagen. According to the model of Korsmeyer and Peppas,38 the k values increased with increasing concentrations of hydrolyzed collagen, indicating faster release of the active component. This may be related to the reduced molecular weight of hydrolyzed collagen, allowing the matrix to disintegrate faster and

Table 3. Effect of the Hydrolyzed Collagen Concentration (CHC ) on the Parameters of the Zero Order, Higuchi, Korsmeyer and Peppas, and Peppas and Sahlin release kinetics models and difference factor (ƒ1) and similarity factor (ƒ2) CHC (%) Release Model Zero order32

K0 R2 KH R2 K N R2 K1 K2 R2

Higuchi33 Korsmeyers and Peppas34

Peppas and Sahlin35

0

10

20

30

0.0600 0.9528 0.1215 0.7453 0.0356 1.1315 0.9938 –0.1492 0.1443 0.9819

0.0694 0.8448 0.1758 0.7260 0.0696 1.1074 0.9708 –0.2450 0.2239 0.9858

0.0701 0.8547 0.1810 0.7488 0.0777 1.0531 0.9721 –0.2368 0.2225 0.9986

0.0708 0.8132 0.1909 0.7121 0.0859 1.0140 0.9444 –0.2840 0.2524 0.9843

Test Formulation (CHC , % ) Release Factors

Reference Formulation (CHC , %)

ƒ1

0 10 20 30 0 10 20 30

ƒ2

0 – 19.20 20.42 22.07 – 30.90 29.23 27.10

10

20

30

23.76 – 4.14 5.40 30.90 – 65.51 57.45

25.66 4.20 – 4.89 29.23 63.51 – 59.57

28.31 5.60 4.99 – 27.10 57.45 59.57 –

CHC , %, w/w in mG + mHC ; –, Not evaluated. DOI 10.1002/jps.24355

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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

consequently releasing the active compounds faster. Furthermore, it was observed (Table 3) that the films present values of n greater than 1 for the model of Korsmeyer and Peppas,38 regardless of the concentration of hydrolyzed collagen, which indicates that the release is controlled by Case II transport mechanisms, that is, the release of the active compound occurs mainly because of the relaxation process and matrix erosion. For the Peppas and Sahlin39 model (Table 3), the Fickian diffusion constant (K1 ) was negative, whereas the constant for the process of erosion/relaxation (K2 ) was positive, which shows a higher effect of the relaxation process on the release of the active compounds in relation to Fickian diffusion. Similar results were observed by Ghosal and Ray,57 who found negative and positive values for K1 and K2 , respectively, which may be related to the insignificant effect of Fickian diffusion on the release of the active compounds in relation to the effect of the relaxation process. In addition, it was observed (Table 3) for films with collagen that the difference factors (ƒ1) were lower than 15 and the similarity factors (ƒ2) were higher than 50. However, between films without and with hydrolyzed collagen the ƒ1 values were higher than 15 and the ƒ2 values were lower than 50. The dissolution profile was considering similar when ƒ1 is between 0 and 1540 and ƒ2 is higher than 50.41 These results indicated that the release profile of phenolic compounds was similar only between films with hydrolyzed collagen. So, the incorporation of hydrolyzed collagen changes the dissolution profile and can also, as was observed by the kinetics models, showed faster release of the phenolic compounds. These results agree with the swelling degree data because all formulations showed high swelling, which may contribute to the release of the active compound by relaxation. Furthermore, the higher the collagen concentration, the faster the disintegration of the films was, as was the swelling observed in the tests due to erosion, indicating that the two processes occur simultaneously and are more pronounced in films with higher hydrolyzed collagen concentrations. Stability The concentrations of phenolic compounds in the orally disintegrating films did not vary as a function of storage time (12 weeks), regardless of the formulation, indicating good stability of the active ingredient in the orally disintegrating films studied (Fig. 3). Daud et al.58 observed good stability of the active compounds even after 3 months of storage for orally disintegrating films based on hydroxypropylmethylcellulose with ginger extract. This shows that disintegrating films can be an alternative vehicle for active natural compounds. Fourier Transform Infrared Spectroscopy The spectra of the films exhibited the typical bands for gelatin films (Fig. 4), as previously reported in the literature.15,48 Typical absorption bands were observed at approximately 3280, 1630, 1539, and 1235 cm−1 , which correspond to amide A,59,60 amide I,60,61 amide II,61 and amide III,60 respectively. Displacement of the absorption bands of the spectra was not observed with increasing concentration of hydrolyzed collagen (Fig. 4). However, with the addition of the ethanolic extract of propolis, displacement of the bands near the amide A (3280– Borges and De Carvalho, JOURNAL OF PHARMACEUTICAL SCIENCES

Figure 3. Effect of storage time (weeks) on the concentrations of phenolic compounds in orally disintegrating gelatin films containing the ethanol extract of propolis (CEEP = 4%) and with different concentrations of hydrolyzed collagen (CHC ). Different letters indicate a significant difference (p < 0.05) between the mean concentration of phenolic compounds at different storage periods for the same formulation. The difference between mean values was determined by the Duncan test using the SAS computer program. CHC , %, w/w in mG + mHC ; CEEP , %, w/v in filmogenic solution.

3285 cm−1 ), amide I (1630–1633 cm−1 ), and amide III (1235– 1237 cm−1 ) was observed between the films with and without incorporation of the ethanol extract of propolis. This may indicate interaction between compounds in the ethanol extract of propolis and the matrix of the film. According to Ozdal et al.,62 phenolic compounds can interact with the polymer matrix proteins, suggesting that the shift in the absorption bands may be related to the interaction of these compounds with the gelatin matrix. Similar results were reported by Aewsiri et al.,63 who observed that the incorporation of oxidized phenolic compounds (tannins) in gelatin-based films caused displacement of the bands related to the amides I (1637–1635 cm−1 ), II (1541– 1535 cm−1 ), and III (to 1238–1236 cm−1 ) due to the reduction of molecular ordering. Yan et al.64 evaluated the addition of gallic acid and rutin to gelatin films and observed the displacement of amide III, 1243–1238 and 1239 cm−1 , because of the addition of gallic acid and rutin, respectively. The authors suggest that this shift was due to the interaction with the phenolic C–N–C group of the gelatin molecules. Hoque et al.48 observed displacement of amide A (3275–3277 cm−1 ) because of the incorporation of anise extract in gelatin-based films and correlated this displacement with the interaction of phenolic compounds present in the extract with the polymer matrix, specifically, the binding of phenolic compounds with the NH2 groups of the gelatin. Similarly, Wu et al.16 observed, the displacement of the amide A (from 3317 to 3302 cm−1 ) in gelatin films with green tea extract and suggested that the displacement was related to the hydrogen bonding of the OH group to the N–H groups of the gelatin. Zhang et al.65 evaluated chemical cross-linking gelatin with natural phenolic compounds by high-resolution NMR spectroscopy and the observed C–N bonds DOI 10.1002/jps.24355

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Figure 4. Infrared absorption spectra of orally disintegrating films with different concentrations of hydrolyzed collagen (CHC ) and different concentrations of the ethanol extract of propolis (CEEP ). (1) CHC = 0% and CEEP = 0%; (2) CHC = 0% and CEEP = 4%; (3) CHC = 10% and CEEP = 0%; (4) CHC = 10% and CEEP = 4%; (5) CHC = 20% and CEEP = 0%; (6) CHC = 20% and CEEP = 4%; (7) CHC = 30% and CEEP = 0%; (8) CHC = 30% and CEEP = 4%. CHC , %, w/w in mG + mHC ; CEEP , %, w/v in filmogenic solution.

Figure 5. Antimicrobial activity of orally disintegrating films with different concentrations of hydrolyzed collagen (CHC ) and different concentrations of the ethanol extract of propolis (CEEP ). (a) CHC = 0% and CEEP = 0%; (b) CHC = 10% and CEEP = 0%; (c) CHC = 20% and CEEP = 0%; (d) CHC = 30% and CEEP = 0%; (e) CHC = 0% and CEEP = 4%; (f) CHC = 10% and CEEP = 4%; (g) CHC = 20% and CEEP = 4%; (h) CHC = 30% and CEEP = 4%. CHC , %, w/w in mG + mHC ; CEEP , %, w/v in filmogenic solution.

between phenolic (caffeic acid) and gelatin in alkaline (pH 9.0) conditions. Antimicrobial Activity The films without the ethanol extract of propolis showed no antimicrobial activity (Fig. 5), and microbial growth could be observed even on the surface of the films. Films with the ethanol DOI 10.1002/jps.24355

extract of propolis, on the other hand, showed antimicrobial activity against S. aureus, with an average halo of 28.5 cm (Fig. 5). The antimicrobial activity presented by propolis is mainly because of the presence of phenolic compounds.66 Thus, these results indicate the stability of the active compounds of propolis after processing the films, confirming the results observed for the stability of the films. Borges and De Carvalho, JOURNAL OF PHARMACEUTICAL SCIENCES

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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Juliano et al.56 evaluated the incorporation of propolis in films based on alginate, agar, and alginate/chitosan and observed that the extract conferred antimicrobial activity against S. aureus, with an inhibition zone equal to 12.65 mm observed for film samples with an initial size of 6.5 mm. However, researchers found swelling of the films from 6.5 to 8.4 mm, which may have caused a larger inhibition zone for that film compared with the results of the present study. The S. aureus is a common human pathogen that can be associated with various diseases, for example, angular cheilitis,67 mucositis,68 and endodontic infections.69 So that, the use of propolis in film could be a good alternative to control this microorganism in oral cavity.

CONCLUSIONS It was observed that the ethanol extract of propolis produced the antimicrobial activity in the film as well as provided a better resistance matrix and increased mucoadhesiveness. Increasing the concentration of hydrolyzed collagen resulted in faster release of active compounds, demonstrating that adjusting the hydrolyzed collagen concentration can be used as an alternative to manipulating the release kinetics of the compounds. Phenolic compounds exhibited good stability in orally disintegrating films over 12 weeks. Thus, gelatin and hydrolyzed collagen-based orally disintegrating films show great potential as carriers of active, natural propolis compounds for release in the oral mucosa. And, due their antimicrobial activity, these films could be used to control oral infection.

ACKNOWLEDGMENT The authors gratefully acknowledge Capes for the research ˜ Paulo Research Founscholarship granted to J.G.B. and to Sao dation (FAPESP) for financial support.

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