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RESEARCH ARTICLE
Bactericidal Activity of Usnic Acid-Loaded Electrospun Fibers Evando S. Araújo1, Eugênia C. Pereira2, Mateus M. da Costa3, Nicácio H. da Silva4 and Helinando P. de Oliveira1* 1
Instituto de Pesquisa em Ciência dos Materiais, Universidade Federal do Vale do São Francisco, 48920-310, Juazeiro, BA, Brazil; 2Departamento de Ciências Geográficas, Centro de Filosofia e Ciências Humanas, Universidade Federal de Pernambuco, 50.670-901, Recife, PE, Brazil; 3Universidade Federal do Vale do São Francisco, 56330-990 Petrolina– PE, Brasil; 4Centro de Ciências Biológicas, Universidade Federal de Pernambuco, 50.670-901, Recife, PE, Brazil.
ARTICLE HISTORY Received: December 30, 2015 Revised: May 09, 2016 Accepted: May 11, 2016 DOI: http://www.eurekaselect.com/142241 /article
Abstract: Background: Usnic acid has been progressively reported in the literature as one of the most important lichen metabolites characterized by a rich diversity of applications such as antifungal, antimicrobial, antiprotozoal and antiviral agent. Particularly, antimicrobial activity of usnic acid can be improved by encapsulation of active molecules in enteric electrospun fibers, allowing the controlled release of active molecule at specific pH. Methods: Bactericidal activity of usnic acid-loaded electrospun fibers of Eudragit L-100 and polyvinylpyrrolidone was examined against Staphylococcus aureus using inhibition hales methodology.
Helinando P. de Oliveira
Results: The controlled release of active material at high pH is established after 10 minutes of interaction with media and results in reasonable activity against S. aureus, as detected by inhibition hales. Conclusion: The strong biological activity of usnic acid-loaded electrospun fibers provides a promising application for corresponding material as a bactericidal agent for wound healing treatment.
Keywords: Antibacterial, controlled release, electrospinning, enteric polymers, fibers, usnic acid. 1. INTRODUCTION The symbiotic relationship established between fungus and alga results in the formation of lichens, which makes use of photosynthesis from algae in order to provide energy to fungi. Lichens grow on rocks, tree trunks and soil and present extraordinary capacity of water absorption. Their associated natural products are present in perfumery, decoration, food, pollution control and monitoring and natural remedies [1]. Species of Cladonia (Cladoniaceae), Usnea (Usneaceae), Lecanora (Lecanoraceae), Ramalina (Ramalinaceae) and Parmelia (Parmeliaceae) genera represent rich source of usnic acid – a lichen secondary metabolite – with a typical yield of usnic acid extraction in order of 6%. The isolation of usnic acid was firstly reported in 1844 by Knop [2]. Since its first isolation [2], the usnic acid [2,6-diacetyl7,9-dihydroxy-8,9b-dimethyldibenzofuran-1,3-dione] has been considered as the most extensively studied lichen metabolite associated with commercial applications such as creams, toothpaste, deodorant, sunscreen and so on. *Address correspondence to this author at the Institute of Materials Science, Federal University of Sao Francisco Valley, 48920-310, Juazeiro, BA, Brazil; Tel: 55(74)21027644; Fax: 55(74)21027645; E-mail:
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These products make use of important properties assigned to usnic acid viz. antiviral (Influenza A) [3, 4], antimicrobial, antifungi [5], anti-inflammatory [6, 7], antiproliferative [8, 9], analgesic, antiprotozoal and antipyretic [10], and in the treatment of pulmonary tuberculosis [11]. In addition, health food supplements for weight reduction incorporate usnic acid as additive. Based on this diversity of possibilities, increasing research about usnic acid-based new materials follows a general trend in the development of natural products for biologic applications (such as bactericidal activity). An exponential growth in the number of documents between 1940 and 2010 (around 8904 publications) has been observed, if considered natural product and bacterial as keywords. Particularly, in the interval from 2000 to 2014, the publication of documents (articles, conference papers, review articles, book chapters, conference reviews and letters) and patents considering the term “usnic acid” has been distributed according to Fig. (1). The most relevant area was Agricultural and Biology followed by Biochemistry, Genetics and Molecular Biology (shown in the Fig. 1). An important aspect involves the interest of medicine in usnic acid derivatives.
© 2016 Bentham Science Publishers
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Fig. (1). Number of publications (in log scale) by means of different applications (the most cited) in documents or patents in the interval of 2000-2014.
In this direction, Lauterwein et al. [12] reported the action of usnic acid against biofilm formation on polymer surface while other authors have demonstrated its action against Enterococcus faecalis [12, 13], Staphylococcus aureus [12, 14], Streptococcus mutans [14], Streptococcus pyogenes [15, 16] and against anaerobic bacteria [12, 17]. In order to improve the biological activity of material against bacteria, the incorporation of usnic acid in micro/nano fibers represents a new and promising strategy in order to circumvent aggregation of antibacterial agents and to improve biological action of natural product. The adequate combination of biological agent and polymer applied as a support for electrospun production of loaded fibers introduces a pivotal role in the optimization of performance of resulting encapsulated devices. Eudragit® L100 (carrier polymer) has been considered a potential candidate for application as a support polymer matrix applied in electrospinning. At alkaline pH, the repulsion established between negative charges of carboxylate groups affects the swelling ratio of resulting fiber. As a result, the diffusion of encapsulated species takes place at specific pH and active substance is released to the media [18, 19]. In a recent patent [20], our group reported the antibacterial activity of usnic acid encapsulated in fibers of enteric polymer. Effective antibacterial action was established against S. aureus (according results of Agar diffusion tests). In this work, we have studied the controlled release of usnic acid from electrospun fibers of enteric polymer (Eudragit® L100) and conventional polymer matrix (PVP), allowing the description of kinetics of controlled release of usnic acid in aqueous media and potential application as bactericidal agent. Results enable important application of usnic acid-loaded fibers as pH dependent release systems for controlled action of usnic acid.
2. MATERIALS AND METHODS Eudragit® L100 (Evonik, Germany), polyvinylpyrrolidone (Aldrich, USA), ethylic alcohol-99.5% (Vetec, Brazil), (+)- usnic acid (Sigma Aldrich) – for experiments of controlled release and usnic acid (extracted by Pereira´s group – UFPE) – for bactericidal activity analysis were used as received. 2.1. Samples Preparation Samples of type I – PVP electrospun fibers were prepared as follows: poly(vinyl pyrrolidone) is introduced in 10 mL of ethylic alcohol in a ratio of 2:3 in mass. Resulting solution was kept under intense stirring for 10 minutes until complete dispersion of polymer in alcoholic solution. After this step, 10 mg of usnic acid was dispersed in previous solution which was kept under intense stirring until homogeneous dispersion of components. Samples of type II – Eudragit® L100 fibers were prepared as follows: 1.3 g of Eudragit® L100 was introduced in 6 mL of ethylic alcohol solution and kept under stirring for 10 minutes. Usnic acid (10 mg) was introduced in previous solution and stirred until homogeneous dispersion of components. Corresponding concentrations of components for both solutions were defined according to adequate conditions for spinnability in experimental setup (viscosity of polymer support) and minimum bactericidal concentration (MBC) of pristine usnic acid. Standard experimental setup for fibers production is composed by tubular compartment of a syringe (6 mL) connected to a metallic needle maintained under fixed pressure. Flux of solution along dip of needle is fixed 166 µL/min. An external DC excitation of 15 kV is established between the needle and the grounded target, which are disposed at 10 cm of distance. Electrospinning of fibers takes place for five
Bactericidal Activity of Usnic Acid-Loaded Electrospun Fibers
minutes in which the ejection of solution is established in direction to target under described conditions. The measurement of controlled release of usnic acid is provided by dispersion of fibers (50 mg) into simulated GIT fluid (Intestinal Fluid, Simulated, TS, according procedure established at USP 36-Reagents) – 80 mL and maintained at continuous stirring (90 rpm). Aliquots of resulting solution were analyzed using UV-vis spectrophotometry at fixed interval of time to establish the kinetics of mechanisms. With this aim, Beer-Lambert law has been applied for identification of corresponding usnic acid concentration due to the characteristic absorption band centered at 287 nm. For agar diffusion test, bacterial cultures (stock solutions) were kept in nutrient agar at 4°C from which isolated colonies were dispersed in order to provide a turbidity of 0.5 in McFarland scale [21]. An aliquot of 100 µL of resulting solution was inoculated on Muller-Hinton agar plates. Electrospun fibers (viz. 50 mg of pristine electrospun Eudragit® L100 fibers, pristine electrospun PVP fibers, electrospun Eudragit® L100+usnic acid fibers and electrospun PVP+usnic acid fibers) were aseptically deposited on the Miller-Hinton agar medium and swabbed with corresponding bacteria (different S. Aureus ATCC strains) and incubated at 37°C for 24 h. SEM images were performed in a SEM Vega 3 Tescan with an accelerating voltage of 5 kV. The absorbance was measured in a Hach DR5000 spectrophotometer. 3. RESULTS AND DISCUSSION UV-vis spectrum of usnic acid is shown in the Fig. (2), in which it is possible to identify a characteristic absorption band centered at 287 nm, the typical signature of material.
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The resulting structure of neat electrospun fibers is shown in the Fig. (3). As we can see, regular structures (free of beads) are provided in association of long micrometric fiber structures (cylinders and stranded-like structures).
Fig. (3). SEM images of neat Eudragit® L100 electrospun fibers.
The morphology of electrospun fibers of usnic acid – loaded PVP is shown in the Fig. (4). As can be seen, negligible concentration of beads and regular cylindrical structures is observed along resulting electrospun sample, characterizing a regular and volumetric process of incorporation of additives during production of fibers. SEM images of electrospun fibers of usnic acid-loaded in Eudragit® L100 matrix reveals that stranded-like fibers dominate in the composition of resulting material, as shown in Fig. (5). Results of antibacterial activity of PVP-based and Eudragit® L100 fibers are summarized in the Fig. (6). Agar diffusion experiments indicates that pristine fibers of PVP and Eudragit® L100 have negligible action against growth of different ATCC strains of S. aureus while usnic acid-loaded fibers presented inhibition halos against bacteria, confirming the antibacterial activity of loaded material.
Fig. (2). Spectrum of absorbance of usnic acid in aqueous solution.
Corresponding variation in the intensity of absorbance absorption band centered at 287 nm versus concentration of commercial usnic acid presents a linear dependence which enables the use of Beer-Lambert law and direct identification of concentration from measurement of absorbance at 287 nm.
Based on these results, it is possible to establish that released usnic acid from fibers acts in inhibition of bacterial growth. In this direction, kinetics of release from fibers represents an important parameter relative to the effective action of encapsulated species. Typical diffusion of usnic acid along fiber walls tends to dictate the kinetics of overall release. The nature of process has been typically established by release curves, in which relative concentration of active material is plotted versus time. Results of controlled release in both fibers are shown in the Fig. (7). Fitting of experimental data reveals that typical Weibull function describes overall process for complete release of usnic acid from both electrospun fibers, which is completed established in a scale of time of 10 minutes. It is noteworthy that improved release of usnic acid is provided by Eudragit® L100 (corresponding concentration of 0.045 mg/mL in comparison with 0.040 mg/mL for usnic acid-loaded PVP matrix).
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Fig. (4). SEM images of usnic acid-loaded PVP electrospun fibers.
Fig. (5). SEM images of usnic acid-loaded Eudragit® L100 electrospun fibers.
Fig. (6). Inhibition hales of usnic acid–loaded fibers in comparison with pristine EDGT and PVP electrospun fibers.
It is noteworthy that released usnic acid concentration in this experiment is around the MBC of bacteria and the control in the kinetics (kill time of bacteria) can be controlled by two parameters, viz. relative concentration of enclosed usnic acid in fibers and pH of release media. An additional advantage of Eudragit® L100 matrix refers to the dependence with
pH. This behavior is established at pH 8.0. At acidic condition, the release of usnic acid to solution is negligible, which enables pH as an external control on release of active material, providing important application in medicine. It means that combined action of both parameters (usnic acid concentration and pH of release media) regulates the
Bactericidal Activity of Usnic Acid-Loaded Electrospun Fibers
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Fig. (7). Controlled release curves of usnic acid-loaded in PVP and EDGT L-100 fibers dispersed in aqueous solution (pH 8).
kinetics of bactericidal action since competition of parameters can be explored in order to define adequate activity at specific environment.
[4] [5]
4. CURRENT & FUTURE DEVELOPMENTS The encapsulation of usnic acid in enteric polymer by electrospinning technique provides a useful method for massive production of usnic acid – loaded fibers with improved antibacterial action. The process avoids aggregation of molecules and improves the available surface area for action of released molecules due to the advantages of 1-D nanostructures.
[6]
The preparation of mixed systems composed by usnic acid encapsulated in electrospun fibers of enteric polymers (EDGT) represents an important advance in direction to development of pH-controlled release antibacterial new materials. The incorporation of active material in nanostructure aggregates an important property, which can be explored as secondary therapy in the diabetics wound healing treatment.
[9]
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[12]
CONFLICT OF INTEREST The author(s) confirm that this article content has no conflict of interest.
[13]
ACKNOWLEDGEMENTS The authors wish to thank CNPq, FAPESB, FINEP, FACEPE and CAPES for financial support. REFERENCES [1] [2] [3]
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