In vitro permeation through porcine buccal mucosa of caffeic acid phenetyl ester (CAPE) from a topical mucoadhesive gel containing propolis

In vitro permeation through porcine buccal mucosa of caffeic acid phenetyl ester (CAPE) from a topical mucoadhesive gel containing propolis

Fitoterapia 73 Suppl. 1 (2002) S44–S52 In vitro permeation through porcine buccal mucosa of caffeic acid phenetyl ester (CAPE) from a topical mucoadh...

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Fitoterapia 73 Suppl. 1 (2002) S44–S52

In vitro permeation through porcine buccal mucosa of caffeic acid phenetyl ester (CAPE) from a topical mucoadhesive gel containing propolis G.C. Ceschela,*, P. Maffeia, A. Sforzinia, S. Lombardi Borgiaa, A. Yasina, C. Ronchib a

Dipartimento di Scienze Farmaceutiche, Universita` di Bologna, Via San Donato 19y2, Bologna 4100, Italy b MonteResearch, Via Pisacane, Pero, Milano, Italy

Abstract Recent studies have shown that propolis has on the oral cavity appreciable antibacterial, antifungal and antiviral actions, as well as anti-inflammatory, anaesthetic and cytostatic properties. In light of these studies, an assessment of the diffusion and permeation of caffeic acid phenetyl ester (CAPE) through porcine buccal mucosa was considered useful as a possible application in the stomatological field. To do so, a mucoadhesive topical gel was prepared to apply to the buccal mucosa. The gel was formulated in such a way as to improve the solubility of the propolis, conducting to an increase of the flux. The mucosal permeation of CAPE from the formulation was evaluated using Franz cells, with porcine buccal mucosa as septum between the formulation (donor compartment) and the receptor phase chamber. The diffusion through the membrane was determined by evaluating the amount of CAPE present in the receiving solution, the flux and the permeation coefficient (at the steady state) in the different formulations at set intervals. Qualitative and quantitative determinations were done by HPLC analysis. From the results, CAPE allowed a high permeability coefficient in comparison to the coefficient of other molecules, as expected from its physical–chemical structure. Moreover, the developed gel improved the CAPE flux approximately 35 times more with respect to an ethanol solution formulated at the same gel concentration. The developed gel was also tested in order to evaluate the mucoadhesive behaviour and comfort *Corresponding author. E-mail address: [email protected] (G.C. Ceschel). 0367-326X/02/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 6 7 - 3 2 6 X Ž 0 2 . 0 0 1 9 0 - 9

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in vivo on 10 volunteers in a period of 8 h. The in vivo evaluation of mucoadhesive gel revealed adequate comfort and non-irritancy during the period of study and it was well accepted by the volunteers. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Propolis; Caffeic acid phenetyl ester; In vitro permeation; Porcine buccal mucosa; Mucoadhesive gel

1. Introduction Propolis is a resinous or wax-like beehive product that has been used by man since ancient times for its pharmaceutical properties. It is still used in folk medicine w1x, as a constituent of bio-cosmetic, health foods and for numerous other purposes w2,3x. The antibacterial, antiviral and antifungal activities are the most popular among the most extensively investigated biological actions of propolis w4–6x. Moreover, it was found that the ethanolic extract of propolis has some pharmacological activities, such as anti-inflammatory, anaesthetic and cytostatic w1,7,8x. These activities suggested its possible use in the local treatment of inflammatory conditions, especially in the stomatological field. For topical oral administration, the conventional formulations like lozenges, troches, gels, oral rinses or mouthwashes would be the simplest dosage forms for delivery of actives components through the mucosa of the oral cavity. However, these conventional dosage forms have the disadvantage of initial burst of activity followed by a rapid decrease in concentration w9,10x. Successful topical treatment of oral diseases is difficult at best for reasons related to the constant flow of saliva and the mobility of the involved tissues. Buccal mucoadhesive formulations which control the drug release are expected to overcome these problems w11x. The aim of the present work is to develop a new topical mucoadhesive formulation containing ethanolic extract of propolis. In doing so, a gel formulation was developed. In order to overcome problems due to the use of ethanol, which has an irritating effect on mucosal tissue, we tried dissolving propolis in solvents other than ethanol. To assess the effectiveness of the developed formulation an in vitro permeation study on standard Franz diffusion cells was carried out. Among the numerous propolis components, we considered only the CAPE permeation flux. CAPE was chosen as model component for reasons related to its intrinsic activity w12–18x and to the relatively simple determination in HPLC devices. At the end, an in vivo test was also performed in 10 safe volunteers in order to evaluate the mucoadhesion behaviour and the comfort in vivo. 2. Materials and methods 2.1. Materials Propolis was supplied by Carlo Sessa S.p.a. (Milan, Italy), hydroxypropyl cellulose (methocel K4MEP) by Eingenmann and Veronelli S.p.a. (Milan, Italy),


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Polysorbates 20 (Tween 20), ethyl alchool and proylene glycol by Polichimica (Bologna, Italy). All materials were used as received. 2.2. Formulation In order to improve the solubility of propolis in the formulation, we first performed solubility tests in different water-soluble solvents: ethanol, glycerol, ethylene glycol, propylene glycol, PEG 400 and diethylene glycol monoethyl ether (Transcutol P) (data not shown). From these preliminary studies, it was found that propolis is soluble in hydroxylic solvents and above all else, in ethanol and in propylene glycol. Ethanol has a very low viscosity and rapidly solubilized propolis, but this solvent causes mucosal irritations and for this reason it was not used in the formulation. Propolis is quite soluble in propylene glycol but with a very low dissolution kinetic, perhaps due to the high viscosity of this solvent. From the solubility test it was also found that surfactants, like polysorbate, significantly improve the propolis solubility. For this reason, a polysorbate 20 solution at 0.374% of concentration in propylene glycol was chosen as a gel solvent. In order to obtain a mucoadhesive gel formulation, many different polymers were tested as gelling and mucoadhesive agents: starch, hydroxypropyl methylcellulose, methylcellulose, Sepigel䉸. Their viscosity action and their mucoadhesive behaviour were evaluated in an inclined plane test. Many polymers showed hard gelling problems due to incompatibility with propolis and due to the fact that we used a non-aqueous solvent. Among the tested polymers, hydroxypropyl cellulose was chosen because it showed the best gelling and mucoadhesive properties. To formulate the gel, first propolis was solubilized in ethanol in order to obtain a rapid solubilization. Then the solution was mixed with a solution of polysorbate 20 0.374% in propylene glycol. The hydroxypropyl cellulose was dispersed in the resulting mixture. Ethanol was evaporated in a hot plate (40 8C) under magnetic stirring. Ethanol evaporation was checked by measuring the loss in weight by mean of a laboratory scale balance. Finally, the gel was obtained dropping the minimum water to formulate a gel. Generally, hydroxypropyl cellulose gels are obtained by heating a polymer solution to 60 8C; in our case the polymer was suspended in an non-aqueous solvent at 40 8C and then the gel was obtained dropping water at room temperature. Gel composition is shown in Table 1 while the developed gel is shown in Fig. 1. 2.3. Tissue preparation The porcine buccal mucosa is largely used for in vitro experiments because the permeability of this membrane is very similar to the human buccal tissue w19,20x. Porcine buccal mucosa with a fair amount of underlying connective tissue was surgically removed from the oral cavity of a freshly killed male pig obtained, on each study day, from a local slaughter house (CLAI, Imola, Bologna). The buccal mucosa was placed in ice-cold phosphate buffer 1:15 M. The connective tissue of the mucosa was carefully removed using fine-point forceps and surgical scissors.

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Table 1 Composition in percentage of the developed gel formulation Component

Formulation before ethanol evaporation (wyw%)

Final gel (wyw%)

Propolis Hydroxypropyl metylcelullose Polysorbate 20 Ethanol Propylene glycol Purified water

0.98 0.75 0.14 60.72 37.41 –

2.13 1.62 0.30 0.00 81.03 14.91

The cleaned buccal mucosa membrane was then placed in ice-cold phosphate buffer 1:15 M until it was mounted in the diffusion cells. 2.4. In vitro diffusion study The in vitro diffusion studies were carried out in standard Franz diffusion cells having 0.64 cm2 diffusion area w21,22x. The receptor compartment had a volume of 4.8 ml and was maintained at 37 8C by means of a water bath, circulator and a

Fig. 1. Developed gel.


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jacket surrounding the cells. The cells were filled with ethanol. The solution in the receptor compartments was continuously stirred at 600 rev.ymin using a Tefloncoated magnetic stirrer. The porcine buccal mucosa, which was approximately 1-mm-thick, was clamped between the donor and receiving compartments. To compare the effectiveness of the developed gel in promoting the permeation of CAPE, we also dissolved propolis in ethanol at the same concentration of the gel. One millilitre of the developed formulation and of the propolis ethanol solution was placed in the donor compartment. The amount of CAPE diffused through porcine buccal mucosa was determined by removing aliquots of 2 ml from the receptor compartments using a syringe and immediately replacing the same volume of ethanol (kept at 37 8C). The samples were transferred to volumetric flasks, and stored in a refrigerator until they were analysed. Sampling schedule was 0.5, 1, 2, 4 and 8. All experiments were carried out in triplicate. 2.5. Analyses CAPE in samples was determined using an HPLC device (Dionex, model P580) equipped with a variable-wavelength UV detector (Dionex, model UVD170S). A Nova-Pak C18 (150=3.9 mm, 4 mm, Waters) column was used. Elution was carried out at room temperature with a mobile phase consisting of methanolyacetonitrile 50:50 vyv and a flow rate of 1 mlymin. The detection wavelength was 325 nm. The injecting volume was 20 ml. In these conditions the retention time of CAPE was 3.5 min. We first determined the CAPE concentration into the propolis ethanolic extract and then into the aliquots from the receptor compartment of the Franz diffusion cells. 2.6. Data analysis For CAPE, absorption is a passive diffusion process and can be described by Fick’s law equation: (1)

JssdQr yAdt 2

where Js is the steady-state buccal mucosa flux in mgycm per h, dQr is the change in quantity of material passing through the membrane into the receptor compartment expressed in mg, A is the active diffusion area in cm2 and dt is the change in time. The steady-state flux of CAPE through the porcine buccal mucosa was calculated from the slope of the linear portion of the cumulative amount permeated through the membrane per unit area vs. time plot. To determine the permeability coefficient, we used the equation: KpsJs yCd


where Kp is the permeability coefficient, Js is the flux calculated at the steady-time and Cd is the donor concentration w23x.

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2.7. In vivo mucoadhesion test To determine the mucoadhesive potential of different polymers, several techniques were reported w24–26x, mostly involving the measurement of adhesive strength, used to classify polymers according to their adhesive properties. Bouckaert et al., w27x studied the correlation between the in vitro and in vivo mucoadhesive properties of buccal mucoadhesive Miconazole slow release tablets and they found that although a difference was observed in vitro between formulations, no difference was found in adhesion time or in biopharmaceutical parameters calculated in vivo, meaning that differences seen in vitro do not necessarily induce significant differences in vivo. On the basis of this finding, we performed the mucoadhesion test directly in vivo to verify the good behaviour of the developed gel. Ten healthy volunteers were instructed to finish breakfast (consisting of an Italian breakfast) no later than 09:00 a.m. Thirty minutes later, the mucoadhesive gel was administered. The gel was placed on the attached gingiva in the region of the right upper canine. A standard meal was given in the period of 240 until 270 min after administration of the tablet. During the experiments the volunteers were allowed to drink water ad libitum from 60 min after administration of the tablet. At 480 min after administration, the volunteers were asked to remove the residual gel. The volunteers were asked to record their remarks regarding their experience with the tablet, considering irritancy, taste, comfort, drymouth, salivation and heaviness. 3. Results and discussion 3.1. In vitro diffusion studies Permeation profiles of CAPE through porcine buccal mucosa from the ethanol solution and from the developed formulation are shown in Fig. 2. In Table 2 fluxes Js and permeability coefficients Kp of CAPE from the developed gel and from the ethanol solution are represented. In all the experiments a steady state flux was obtained, meaning that a balance in permeation was attained. The CAPE concentration in the ethanolic extract was found to be 10.44% pyp. The CAPE permeability coefficient across the porcine buccal mucosa from the ethanol solution can be considered as very good, as expected from its physical– chemical structure (Fig. 3). Otherwise there was a strong improvement in the CAPE permeability coefficient across the porcine buccal mucosa in the developed formulation in which CAPE permeation is approximately 36 times more than CAPE permeation from the ethanol solution. This result is quite interesting and the explanation of the enhancer effect of the gel can be found in a different partition of CAPE between the donor solution and the membrane. In fact, CAPE is held more in ethanol than in propylene glycol due to its better solubility in ethanol. That is why CAPE concentration into the membrane is much higher when CAPE is formulated in propylene glycol.


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Fig. 2. Permeation profiles of CAPE trough porcine buccal mucosa from ethanolic solution and from the gel. Table 2 Flux and Kp values of CAPE from the ethanol and from the gel formulation

Ethanolic solution of CAPE Gel

Flux of CAPE (Js) (mcg*cmy2*hy1)

Kp (cm2*hy1)

13.67 0.3748

0.00170 0.0621

Fig. 3. CAPE chemical structure.

Another explanation can be found as strictly an enhancer effect of polysorbate, which could fluidise the lipids or induce structural changes in proteins of the stratum corneum w28x. 3.2. In vivo mucoadhesion test In vivo evaluation of mucoadhesive gel revealed adequate comfort, and nonirritancy during the period of study. The mucoadhesive gel formulation was well accepted by the volunteers and no irritation was recorded.

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None of the volunteers reported dry mouth or severe salivation at the place of attachment. No side effects like heaviness were reported. The volunteers reported taste altering, but this phenomenon did not constitute a relevant problem. After application, the mucoadhesive gel forms a transparent film that moves into the oral cavity and lines all the oral mucosa. It was not necessary to remove the gel as it disappeared during the time of the in vivo study. 4. Conclusion CAPE and other propolis components are able to permeate the in vitro porcine buccal mucosa in Franz cells. That is why propolis may be used in the stomatological field for its antimicrobic and anti-inflammatory properties, as well as for its analgesic qualities. In particular, propylene glycol containing an amphoteric surfactant used to formulate the mucoadhesive gel could be useful both as a solvent and as an enhancer when used topically. The gel has good technological characteristics such as the high propolis solubility, the absence of ethanol that has irritating actions on the buccal mucosa and the capacity to improve the propolis flux across the mucosa. References w1 x w2 x w3 x w4 x w5 x w6 x w7 x w8 x w9 x w10x w11x w12x w13x w14x w15x w16x w17x w18x w19x w20x w21x w22x w23x

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