Polymeric film containing pomegranate peel extract as a promising tool for the treatment of candidiasis

Abstract Polymeric films containing pomegranate peel extract (PPE) can act as a drug-delivery platform for topical treatment of candidiasis. The composition, mechanical resistance, and in vitro antifungal activity of a polymeric film containing PPE at 1.25 mg.mL−1 were investigated. Films were prepared using a solvent casting technique. The incorporation of PPE in the polymeric matrix gave rise to homogeneous, smooth, shiny, and yellowish-brown films. FTIR spectra of the film containing PPE showed differences without compromising the stability of the extract and the matrix. SEM analysis showed the existence of interruptions in the continuity of the films with extract, which promoted a reduction in the mechanical parameters without significantly changing the tensile strength and elongation at break. Films showed adequate mechanical properties and antifungal activity against Candida albicans, C. glabrata, C. krusei and C. tropicalis. Graphical Abstract


Introduction
Candida infection is one of the most common fungal infections worldwide. Among the different species, Candida albicans is still the most often isolated pathogenic species, followed by C. glabrata, C. parapsilosis, C. krusei, C. tropicalis (Bohner et al. 2021). Currently, four major classes of antifungal synthetic and semisynthetic drugs with activity against Candida species are available (CDC 2021). The persistence of this infection is associated with the failure of antifungal therapy and antimicrobial resistance (Bhattacharya et al. 2020). Thus, effective drug delivery using alternative drugs for improved management of persistent fungal infections at wound sites is essential.
Punica granatum L., popularly known as pomegranate is an important source of bioactive compounds. Anibal et al. (2013) showed that ellagic acid, punicalagin and gallagyldilacton are the major compounds involved in antifungal activity. Anibal et al. (2013) and Kumar et al. (2020) identified that the pomegranate peel extract (PPE) can cause changes in the cell wall, in the cytoplasmic membrane, resulting in fungal growth inhibition.
Polymer-based films have been emerging as promising pharmaceutical dosage forms for use as drug delivery systems for topical, mucosal, vaginal and oral use, mainly. Polymeric films are innovative dosage forms that are well accepted by patients and have advantages over conventional dosage forms. They are easy to prepare; are flexible and conformable; are easy to apply and remove when necessary; and, depending on the type of polymer used in its preparation, they may have mucoadhesive capacity and can be bio reabsorbed, which avoids the need for removal. They can act as matrices for the sustained release of the active ingredients, including medicinal plants derivatives (Bassi and Kaur 2017;Osmałek et al. 2021; € Ozakar and € Ozakar 2021). In the case of skin injuries, polymer-based films can act as a local physical barrier against the entry of microorganisms, remove excess exudates and allow gas exchange with the environment (Lam et al. 2018;Umar et al. 2020). The feasibility of polymeric films containing PPE in the treatment of skin and mucosal candidiasis was therefore evaluated in this study. The physicochemical properties and in vitro antifungal activity of polymeric films prepared from a mixture of poly(vinyl alcohol) (PVA), corn starch (S) and poly(acrylic acid) (PAA) in presence of 1.25 mg.mL À1 of the dry crude and water soluble extract of pomegranate peel.

Physicochemical characterisation and mechanical strength
The incorporation of PPE at 12.5 mg.mL À1 in 100 mL of polymeric solution resulted in ten films containing the PPE at 1.25 mg.mL À1 each (PVA:S:PAA:PPE), that represent a unit-single pharmaceutical dosage form. Visual analysis of the films without PPE (PVA:S:PAA) were smooth, shiny, bubble free, continuous, free of phase separation and apparent domain formation. PVA:S:PAA:PPE films present a yellow-brown color but, the other characteristics remained unchanged ( Figure S1). The infrared spectroscopy test (FTIR) provides essential information about the chemical composition, miscibility and, compatibility between the film components in polymeric matrix (Wang et al. 2010). The FTIR spectra of pure PPE presented characteristic bands (He et al. 2019). Changes in the FTIR spectra of PPE based films, indicated the occurrence of intermolecular chemical interactions between the extract components and the polymers ( Figure S2) comparable to the results reported by He et al. (2019). Analysis of mechanical properties of the films were determined to provide information on the brittleness, toughness, and adaptability to the films in the application area. The parameters tensile strength (TS), elastic modulus (E), and % elongation at breakpoint (%EB) were measured. Polymeric films for use as drug delivery should be soft and resistant with moderate TS values and high E values, as observed in the present work (Table S1). According to the statistical analysis, only the elastic modulus had a significant difference after the incorporation of the PPE in pure polymeric matrix. Although, the mechanical resistance of PPE-film was maintained. Amal et al. (2015) prepared polymeric films with an aqueous extract of pomegranate peel that showed good tensile strength (45.36 ± 0.64 Mpa), which is necessary for use as a wound dressing. Dutra et al. (2017), produced polymeric films containing papain that presented values of TS, E, and %EB also consistent with the data of the present work and the films were considered appropriate for the use as drug delivery. Good mechanical parameters may result from the existence of chemical interactions between bioactive compounds in PPE and polymers, as confirmed by FTIR and, also observed by Fleck et al. (2016) in polymeric PAAhydrogel containing ethanolic PPE. SEM photomicrographs ( Figure S3) showed a continuous, compact, uniform-looking matrix with small pores in the PVA:S:PAA film while in the microstructure of PVA:S:PAA:PPE, discontinuity in the matrix was also observed contributing to the small variations in the values of its mechanical parameters.

Determination of MIC and investigation of antifungal activity
PPE showed antifungal activity in plate assays, against C. albicans at concentrations of 5 mg.mL À1 (10.18 mm) and 10 mg.mL À1 (10.15 mm) with MIC and MFC of 5 and 10 mg.mL À1 , respectively (Table S2). These results corroborate other data in the literature. Anibal at al. (2013) showed the antifungal effect of pomegranate extract on strains of Candida spp. obtaining a MIC of 125 mg.mL À1 . Serqu en et al. (2020) determined the susceptibility of strains of C. albicans to different concentrations of an ethanolic PPE, and MIC values ranging between 1.56 mg.mL À1 and 6.25 mg.mL À1 . We emphasise that the difference between the studies may be related to the chemical composition of the extract, methodology, culture conditions, and the microorganism evaluated (Bona et al. 2014).
The antifungal activities of PVA:S:PAA:PPE film (1.25 mg.mL À1 ) showed antifungal activity against C. albicans (14.67 ± 0.53 mm), C. glabrata (15.50 ± 0.57 mm), C. tropicalis (12.50 ± 1.20 mm), and C. krusei (20.25 ± 1.70 mm). PVA:S:PAA film showed no antifungal activity against the tested fungi. Several Candida species are responsible for cutaneous candidiasis being a common disease that affects all ages. Local skin or mucosal conditions such as increased humidity, occlusion, broken skin barrier, and altered microbiota often favor cutaneous Candida infections. The development of a new dosage form for topical administration of antifungal agents has many advantages over traditional dosage forms (cream, ointment, paste, gel, solution or powder), such as longer contact time between the active ingredient and the application site; the difficulty of removing the product; the possibility of modulating the release for an extended period; the better adherence to treatment; and the possibility of administering higher doses of antifungal at the site of infection (Lam et al. 2018;Kaur and Kakkar 2010). Despite the numerous reports in the literature on the development of conventional pharmaceutical dosage forms containing PPE, few studies report the preparation and evaluation of polymeric films based-PPE. Batista et al. (2020) observed antifungal activity of films solutions containing the same extract at 1.25% w/w, prepared using the electrospinning method.

Conclusions
The incorporation of 12.5 mg.mL À1 of total dry pomegranate peel extract in the PVA:S:PAA blend resulted in polymeric films as unit-single dosage containing 1.25 mg.mL À1 of PPE each. Films were smooth, transparent, shiny, light yellow and resistant to handling. The existence of chemical interactions between the PPE components and the polymers was observed, contributing to the formation of intact, uniform films with mechanical properties considered appropriate for use as pharmaceutical dosage form. PVA:S:PAA:PEE showed antifungal activity on the studied Candida spp. such findings suggest that the films containing this extract are a promising pharmaceutical dosage form for the treatment of cutaneous and mucosal candidiasis.

Disclosure statement
No potential conflict of interest was reported by the authors.