Antimicrobial activity of fusidic acid inclusion complexes

Journal Pre-proof Antimicrobial Activity of Fusidic Acid Inclusion Complexes Eleonora Marian, Bogdan Tita, Narcis Duteanu, Laura Vicas, Stefania Ciocan, Tunde Jurca, Liana Antal, Otilia Tica, Mariana Mureşan, Annamaria Pallag, Otilia Micle PII: S1201-9712(20)32181-0 DOI: https://doi.org/10.1016/j.ijid.2020.09.1465 Reference: IJID 4699 To appear in: International Journal of Infectious Diseases Received Date: 13 August 2020 Revised Date: 24 September 2020 Accepted Date: 24 September 2020 Please cite this article as: { doi: https://doi.org/ This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier. Antimicrobial Activity of Fusidic Acid Inclusion Complexes Eleonora Marian1, Bogdan Tita2, Narcis Duteanu3,*, Laura Vicas1, Stefania Ciocan3, Tunde Jurca1, Liana Antal1,*, Otilia Tica1, Mariana Mureșan1, Annamaria Pallag 1, Otilia Micle1 1 University of Oradea, Medicine and Pharmacy Faculty, Piata 1 Decembrie, 410028, Oradea, Romania; 2” Vasile Goldis” Western University of Arad, Pharmacy Faculty, 86 Liviu Rebreanu Street, 310045, Arad, 3 of Romania; University Politehnica of Timisoara, Faculty of Industrial Chemistry and Environmental Engineering, 2 -p ro Victoria Square, 300006, Timisoara, Romania; * fusidic acid cyclodextrin compounds characterization antimicrobial activity Jo     ur na lP Highlights re Authors for correspondence: narcis.duteanu@upt.ro, lianaantal@gmail.com 1 Antimicrobial Activity of Fusidic Acid Inclusion Complexes Eleonora Marian1, Bogdan Tita2, Narcis Duteanu3,*, Laura Vicas1, Stefania Ciocan3, Tunde Jurca1, Liana Antal1,*, Otilia Tica1, Mariana Mureșan1, Annamaria Pallag 1, Otilia Micle1 1 University of Oradea, Medicine and Pharmacy Faculty, Piata 1 Decembrie, 410028, Oradea, Romania; 2” Vasile Goldis” Western University of Arad, Pharmacy Faculty, 86 Liviu Rebreanu Street, 310045, Arad, 3 of Romania; University Politehnica of Timisoara, Faculty of Industrial Chemistry and Environmental Engineering, 2 -p ro Victoria Square, 300006, Timisoara, Romania; * ur na lP ABSTRACT re Authors for correspondence: narcis.duteanu@upt.ro, lianaantal@gmail.com Objectives: To synthesize and characterize the inclusion complexes of fusidic acid with β – cyclodextrin, followed by the evaluation of their antimicrobial activity against pure stain (staphylococcus aureus ATCC 25923) and against isolated staphylococcus from clinical cases. Methods: The desired compounds were synthesized using a molar ratio of fusidic acid: βcyclodextrin of 1:1. Synthesized compounds were analyzed by Fourier transform Infrared Spectroscopy Jo (FTIR), XRay Diffraction (XRD), Scanning Electron Microscopy (SEM) and also Thermal Analysis and the results confirmed the formation of inclusion compounds by fusidic acid with β-cyclodextrin. Results: Physical – chemical characterization confirmed the preparation of desired inclusion compounds, and the antimicrobial test confirmed that all compounds obtained have antimicrobial activity. Antimicrobial activity of freeze drying complex against Staphilococcus aureus is similar with pure fusidic 2 acid activity, being better than cefoxitin one. Similar behavior being observed against methicillin resistant sthapylococcus aureus, and sthaphilococcus epidermidis. Conclusions: In present work three different inclusion complexes of fusidic acid were prepared by using three different preparation methods. All inclusion complexes obtained represented good antimicrobial activity against different staphylococcus aureus stains. Antimicrobial activity of these new prepared compounds was observed to be better than the one of cefoxitin. of Keywords: fusidic acid, cyclodextrin, compounds characterization, antimicrobial activity ro 1. INTRODUCTION During last decade most of the research was focused on the synthesis and analysis of the physico- -p chemical, therapeutic properties of new pharmaceutical substances [1-4] or mixtures of active principles re extracted from different plant products [3, 5-9]. In human pathology an important role is played by staphylococcal infections, especially by Staphylococcus aureus, with a relatively wide spread – ranging ur na lP from minor skin infections to life threatening sepsis. In this context, the increasing number of staphylococcal strains resistant at methicillin represent a major challenge for all medical services [10-14]. Because of the resistance problems of some anti-staphylococcal antibiotic a tendency was observed to return at treatment schematics with other anti-staphylococcal agents, among them being fusidic acid [15]. Fusidic acid is an active compound derived from the mycelia of Fusidium coccineum and it has been used as drug in the treatment of staphylococcal infections in the early 1960’s [16]. Experimental data proved that Jo its narrow spectrum of action includes Gram positive cocci and bacilli, such as: Staphylococcus aureus, most coagulase-positive staphylococci, Beta-hemolytic streptococci, Corynebacterium spp, and most Clostridium spp. It has also demonstrated that fusidic acid has no susceptible action on Enterococci [15]. Almost all gram negative bacteria are not susceptible to fusidic acid, except Neisseria and Moraxella species, plus the strain of Bacteroides fragilis group [16]. Experimental data proved that one important clinical use of fusidic acid is represented by its effect against methicillin resistant Staphylococcus aureus. 3 Such drug can be administered orally, intravenously, or it can be applied topically. Topic application fusidic acid may be administered in a large variety of preparations like ointment, cream, lotion or gel. The chemical name for fusidic acid is (2Z)-2-[(3R,4S,5S,8S,9S,10S,11R,13R,14S,16S)-16acetyloxy-3,11-dihydroxy-4,8,10,14-tetramethyl-2,3,4,5,6,7,9,11,12,13,15,16-dodecahydro-1Hcyclopenta[a]phenanthren -17-ylidene]-6-methylhept-5-enoic acid. The chemical formula of fusidic acid is presented in Figure 1. of Figure 1. Chemical structure of fusidic acid ro Recently many researchers have analyzed the resistance of isolated staphylococcus from different -p parts of the world at fusidic acid [10-15], in vitro activity [16] etc. The experimental data obtained proved that the drug efficiency is influenced by its administration mode. In this context, the high effectiveness and re administration safety can be optimized by using different molecular structures as drug carriers. Cyclodextrins represent a family of cyclic oligosaccharides derived from starch, which are obtained by ur na lP enzymatic hydrolysis, and which are used to improve the quality, efficacy and safety of different active drugs. Pharmaceutical industry uses cyclodextrins for controlling solubility, optimizing bioavailability, stabilizing drug substances, reducing adverse drug effects, reducing volatility, converting liquid drugs to crystalline forms, conducting chemical reactions, controlling fluorescence, using as chiral selectors or as carrier molecules. The enhancement of solubility in aqueous media is concurrent with the reduction in Jo toxicity and it should be noted that they do not destroy microbial cells or enzymes. In recent years different compounds were analyzed for inclusion of pharmaceutical substances with cyclodextrins: repaglinide [17], sulphonamidic diuretics [18-22], pentacyclic triterpenes [23], fosinopryl natrium [24], erythromycin [25, 26], terpenes [27]. In present study three different inclusion complexes of fusidic acid with β-cyclodextrin have been synthesized by using three different preparation methods, with a ratio fusidic acid: β-cyclodextrin - 1:1. After preparation each compound has been characterized by physical-chemical methods viz., Fourier4 transform infrared spectroscopy (FT-IR), X-Ray Diffraction (XRD), and scanning electron microscopy (SEM). After physical-chemical characterization was tested the antimicrobial activity of produced inclusion complexes was analyzed and compared with the pure fusidic acid activity against Staphylococcus aureus ATCC 25923 reference strain and staphylococcal isolates from clinical cases. 2. MATERIALS AND METHODS Materials used in the present investigation, such as fusidic acid (FA) and -cyclodextrin, were of of analytical purity and used directly without any further purification after procurement from Sigma Aldrich GmbH, Germany. FTIR spectra were recorded using a JASCO 6100 FTIR spectrometer by scanning the ro spectral domain between 4000 – 400 cm–1 and a resolution of 4 cm–1, using the classical technique of KBr pellets. X-ray diffraction patterns were obtained using a X’Pert PRO MPD Diffractometer equipped with -p Cu anode X Ray tube, PixCEL detector and a vertical theta – theta goniometer. All XRD spectra were re recorded at room temperature in 2θ rage of 0 to 80 degrees. Scanning electron microscopy data were recorded by using FEI Quanta FEG 250 scanning electron microscope, equipped with a high stability ur na lP Schottky field emission gun, and a specimen room with 379 x 280 mm door size. TG/DTG/DTA data were recorded on Netzsch–STA 449 TG/DTA instrument into the temperature range of 20 to 500°C using a platinum crucible containing approximately 5 mg sample. All tests were carried out under dynamic nitrogen atmosphere, 20 mL min–1 and heating rate of 10 K min–1. In vitro test was performed on staphylococcal strains using prepared fusidic acid compounds by diffusion method according to CLSI recommendations [28]. For test solutions were prepared in which the Jo concentration of fusidic acid compounds was 10 µg / 10µl. In all in vitro test the reference strain Staphylococcus aureus ATCC 25923 and isolates from human clinical cases: two strains of Methicillinsensitive Staphylococcus aureus (MSSA), a strain of Methicillin-resistant Staphylococcus aureus (MRSA) and a strain of Staphylococcus epidermidis were used. For each bacterial strain standardized inoculum (0.5 Mc Farland) was plated onto Mueller-Hinton Agar (Oxoid), followed by fusidic acid discs (10 μg, Oxoid) and 6 mm sterile filter paper (HiMedia 5 Laboratories) impregnated with 10 μg of the three cyclodextrin fusidic acid complexes F1, F2, F3 were placed onto inoculated culture media. Cefoxitin discs (30 μg Oxoid) and sterile filter paper disks impregnated with distilled water were used as positive and negative control respectively. After incubation at 37°C for 24 h, the antibacterial effect was assessed by measuring the diameters (in millimeters) of the inhibition zones of each compound. Each test was done in triplicate and mean values were selected [29]. Statistical analyses of All experimental data were analyzed using a statistical analysis software. The results obtained are ro being presented as mean ± standard deviation (SD) in case of numeric variables or as percentages. Continuous normal distribution variables were reported as mean ± SD. Further continuous variables were -p analyzed for normalization and compared using Student t test and they were expressed by nominal, standard re and / or median deviation. Correlation degrees (r) between parameters studied has been evaluated by determination of Pearson correlation coefficient. A limit of Pearson coefficient lower than 0.05 was ur na lP considered statistically significant. Further, intergroup comparisons were performed by using Chi-square test to evaluate to what extent the difference between the independent and dependent variables are statistically significant. Later, it has been tested for the assumption (by controlling the independent variables) using simple logistic regression method, in order to identify the direct effect and possible Jo potential mistaken effects. 3. RESULTS AND DISCUSSIONS 3.1. Synthesis of β – cyclodextrin inclusion compounds Inclusion complexes were prepared by using three different techniques: kneading, coprecipitation and freezedryer method. In all cases obtained complexes have a molar ratio of fusidic acid:β-cyclodextrin as 1:1. 6 a. Kneading method – was used for preparation of complex designated as F1. For preparation of F1 inclusion complex 0.5675 g of β – cyclodextrin was triturated together with 0.2583 g of fusidic acid in a mortar by adding several drops of ethanol (0.5 mL each). The paste obtained has been triturated till the evaporation of used solvent. Entire process has been repeated several times for a total time of 40 minutes. b. Coprecipitation method – was used for preparation of complex designed as F2. In order to prepare F2 inclusion complex 0.2583 g of fusidic acid (5 10-4 M) was dissolved in 5 mL of ethanol. Also, 0.5675 g of β – of cyclodextrin (5 10-4 M) was dissolved in 5 mL of water (this amount of fusidc acid and β-cyclodextrin were used in order to obtain a molar ratio equal to 1:1). Over β–cyclodextrin aqueous solution fusidic acid alcoholic solution were ro added drop by drop under continuous stirring, after that solvents were evaporated at 50°C in a drying stove to obtain -p the product in form of powder. c. Freeze – dryer method – was used for preparation of complex designed as F3. Equimolar amounts of re fusidic acid and β – cyclodextrin were dissolved in water and mixed for 2 h at 35°C, wrapped in aluminum foil to ur na lP protect obtained solution from the direct light. After the equilibration period of 24 h, the clear solution obtained has been frozen and subsequently lyophilized in a freeze – dryer. 3.2. Physical – chemical characterization of produced complexes 3.2.1. Fourier-transform infrared spectroscopy Recorded FT-IR spectra for pure fusidic acid, β – cyclodextrin, and for synthesized complexes are Jo presented in Figure 2. Analyzing data presented in recorded FT-IR spectra the presence of functional groups present in structure of synthesized inclusion complexes was identified. While analyzing the FT-IR spectra recorded for pure fusidic acid a vibration located at 607 cm-1 associated with plane stretching vibration of C=C bonds was observed. Vibration located at 746 cm-1 is associated with the plane vibration of C=C–C bonds. The vibrations associated with plane stretching of C-H bonds are located at 850 and 913 cm-1. Presence of vibration located at 970 cm-1 is associated with stretching of C=C-H bonds, whereas the vibration located at 781 cm-1 is located as the vibration associated with the vibrations out of plane of C-H 7 bonds. An intense vibration is observed at 3446 cm-1, which was associated with stretching of O-H bonds [30]. Figure 2. FTIR spectra of fusidic acid, β-cyclodextrin and the inclusion compound obtained by: a) kneading (F1); b) coprecipitation (F2); c) freeze-drying (F3) of Some intense vibrations are observed at 1686 and 1742 cm-1, which are being associated with ro symmetric and asymmetric stretching of carbonyl groups. Intense bands located at 2869 and 2953 cm-1 are associated with symmetric and asymmetric of methyl groups and vibrations located at 1380 and 2953 cm-1 -p are associated with symmetric and asymmetric deformations of methyl and methylene groups. Vibration re located at 1260 cm-1 is associated with stretching vibration of C-O groups and vibration located at 1027 cm-1 is associated with primary alcohols stretching. ur na lP From FT-IR spectra recorded for β – cyclodextrin the presence of an intense band located between 3500 and 3300 cm-1 was observed, which is associated with symmetric and asymmetric stretching vibrations of O-H bonds. Other intense band is located in region between 3000 and 2800 cm-1, being associated with the stretching vibrations of methylene groups [31]. Intense band located at 1158 cm-1 has been associated with stretching vibrations of C-O groups and the band located at 1029 cm-1 is associated with the bending vibrations of O-H groups [32]. Jo From the FT-IR spectra recorded for the prepared inclusion compounds it can observe that stretching vibration of carbonyl groups has been moved at lower wave numbers because of the interactions between host and guest molecules. Such interactions between these molecules are possible due to the formations of hydrogen bonds between hydroxyl groups from β – cyclodextrin (host molecule) cavity and carbonyl group from fusidic acid molecule (guest molecule). In the FT-IR spectra recorded for the synthesized inclusion compounds a sharp peak located at 3400 cm-1 is observed, which is peak associated with stretching of O-H 8 groups bonded through hydrogen bonds. Vibrations associated with the stretching of O-H groups from water are located at 1636 cm-1 (F1 spectra), 1626 cm-1 (F2) spectra and 1636 cm-1 (F3 spectra). Vibrations associated with the planar deformations of OH groups are located at 1372 cm-1 in spectra of F1 inclusion compound, at 1380 cm-1 in spectra recorded for F2 inclusion compound, and at 1370 cm-1 in spectra recorded for F3 inclusion compound. Vibrations associated with stretching of C-O-C bonds are located in spectra recorded for F1 inclusion compound at 1173 cm-1, for spectra recorded in case of F2 inclusion compound is located at 1153 cm-1, and for F3 compound the vibration is also located at 1153 cm-1. Also, the stretching vibration of OH groups from secondary alcohols, characteristic for β–cyclodextrin, are of located at 1029 cm-1 in spectra of F1 inclusion compound, 1025 cm-1 in spectra of F2 inclusion compound ro and at 1026 cm-1 in case of F3 inclusion compound. The stretching vibration of C-O bonds, having a medium intensity, appeared in FT-IR spectra of -p fusidic acid at 1260 cm-1, while in β – cyclodextrin spectra appeared at 1158 cm-1 and in spectra recorded re for inclusion complexes appeared at 1262 cm-1 (F1), 1242 cm-1 (F2) and 1262 cm-1 (F3). Peaks with higher intensity located at 1742 and 1686 cm-1 in spectra of fusidic acid, associated with symmetric and ur na lP asymmetric stretching vibrations of carbonyl groups, are present also in the spectra recorded for inclusion complexes at lower wave numbers (1726 and 1637 cm-1 – F1, 1735 and 1656 cm-1 – F2, and at 1715 and 1636 cm-1 – F3). Displacement of peaks correlated with peaks intensity diminution in the spectra recorded for the inclusion compounds confirmed that the desired inclusion compounds were obtained. Presence of some displacements can be explained taking into account the interactions between host and guest molecules, Jo which lead at total or partial blockage of the vibration characteristics for pure substances. 3.2.2. X-Ray diffraction Further, the prepared complexes were characterized by recoding the XRD spectra, spectra which are presented in Figure 3. In order to prove this, the preparation of desired compounds the XRD spectra for pure fusidic acid and pure β – cyclodextrin were recorded. From XRD spectra recorded for pure 9 fusidic acid and pure β – cyclodextrin it can observe that the main peaks are matching the standard data for pure compounds, meaning that the analyzed products are pure, representing a high crystallinity. In case of XRD spectra recorded for sample F1 (spectra presented in Figure 3.a) the presence of peaks corresponding to both pure compounds can be observed. Simultaneously the appearance of some peak modification, consisting in some peak shifting or some peak intensity modifications was also evident. Presence of some modifications has been associated with the presence of some physical interaction between fusidic acid and of β – cyclodextrin. Based on this observation it can be concluded that the desired complex has been obtained. ro Figure 3. X Ray-diffraction of fusidic acid, β-cyclodextrin and of the inclusion compound obtained by: (a) -p kneading procedure (F1); (b) coprecipitation (F2); (c) freeze-drying (F3) re In case of XRD spectra recorded for sample F2 (spectra presented in Figure 3.b) a similar behavior can be observed, means that also in sample F2 the presence of physical interaction between fusidic acid and ur na lP β – cyclodextrin can be observed. Also, by comparing the XRD spectra recorded for compounds F1 and F2 some differences between these can be observed, which can be explained by considering the different preparation method. In case of XRD spectra recorded for sample F3 (spectra presented in Figure 3.c) a similar behavior, meaning that also in this compound the presence of some physical interactions between fusidc acid and β – cyclodextrin can be observed. Differences noticed between XRD spectra recorded for these three samples Jo can be explained by considering the different preparation method. Observed changes into the recorded XRD spectra for F1, F2, and F3 samples represent the proof of complexation between the guest molecule (fusidic acid) and the host one (β–cyclodextrin). 3.2.3. Thermal analysis 10 Recorded TG curves are presented in Figure 4. Based on TG curve recorded for pure fusidc acid it can be observed that this compound presents a continuous small weight loss (1.38%) into the temperature interval of 25.0 and 155.3°C, presenting a peak at 84°C. This weight loss is associated with removal of water adsorbed during manipulation. Further, into the temperature interval of 155.3 to 223.5°C partial thermal decomposition of fusidic acid was observed to be taking place (a mass loss of 10.80%). This loss could be correlated with fusidic acid melting (taking place at 183.7°C). All these processes are accompanied by some endothermic effects. While continuing the heating, in the range of 223.5 and 470.6°C the decomposition of fusidic acid (peak associated with this process is located at 359.3°C) was taking place. of This is also an endothermic process, being accompanied by a mass loss of 86.01%. At the end of thermal -p ro cycle, the residual mass was 1.81%. re Figure 4 – Thermogravimteric curves recorded for: a) – fusidic acid, b) – β-cyclodextrin, c) – F1 inclusion ur na lP complex, d) – F2 inclusion complex, e) – F3 inclusion complex. By heating up the β – cyclodextrin apparition of an endothermic process can be observed, which is associated with crystallization water loss (mass loss – 13.59%) in the temperature range of 50.0 - 114.7°C. Further increase of temperature had no notable effect on β – cyclodextrin molecule until 306.5 °C, when the thermal decomposition is taking place. The β – cyclodextrin thermal decomposition is a single stage endothermic process, which is taking place in the range of 306.5 – 377.2°C. During this stage the total mass Jo loss is 70.26%. At end of the thermal cycle a residual mass of 7.14 % was obtained. Analyzing thermogravimetric curves of the three produced inclusion compounds a similar behavior for all inclusion complexes can be observed, because only the preparation method was different. Thus, all synthesized complexes present three different thermal processes on the TG curve. From the recorded themogravimetric curves the existence of some differences regarding the specific temperatures, mass loss and residual mass are evident. Characteristic parameters of thermal behavior of studied compounds are 11 presented in Table 1. Based on data presented in Table 1 it can be observed that the freeze-drying complex have a different behavior than other two prepared inclusion complexes. Table 1. Values of thermal parameters obtained for studied inclusion complexes of 3.2.4. Scanning electron microscopy ro In Figure 5 the SEM pictures recorded for pure fusidic acid and for synthesized inclusion complexes are presented. Analyzing the SEM pictures presented in Figure 5 some changes into the structure of the -p compounds, building a multilayered structure, with channels and cavities can be observed. These re modifications represent a clear proof of inclusion complexes formation. ur na lP Figure 5. SEM pictures recorded for: (a) fusidic acid, (b) kneading complex, (c) coprecipitation complex, (d) freeze-drying complex. 4. Antibacterial activity Further the antibacterial activity of fusidic acid and inclusion complexes has been tested and Jo compared with the antibacterial activity of cefoxitin. The results of antibacterial activity determination are shown in the Table 2. The data obtained were statistically analyzed, in order to understand the importance of synthesized compounds as antibacterial drugs. Data obtained after statistical analysis are presented in Tables 3 to 5. Table 2. Antibacterial activity of the fusidic acid complexed with cyclodextrins 12 Table 3: Antibacterial activity of the fusidic acid complexed with cyclodextrins values obtained throughout statistical analysis Table 4: Mean values ±SD of Table 5: Correlations obtained after statistical analysis ro Fusidic acid zone diameter breakpoints of ≥ 24 mm, and < 24 mm are EUCAST criteria for the susceptible, and resistant category, respectively [33]. From data obtained after statistical analysis it can be -p concluded that the fusidic acid and its inclusion complexes with β–cyclodextrin meet the EUCAST criteria both on used reference strain and on microorganism isolates from patients, thus, exhibiting good re antimicrobial activity. Analyzing the inhibition zone (image not included in present paper), it can be ur na lP observed that for the inclusion complexes the largest one has been found for the strain of Staphylococcus epidermidis, being equal with the inhibition zone obtained for standard disc, data being similar with those obtained by Jones et al [34]. Staphylococcus epidermis is part of the normal flora of the human organism, having the same habitat as Staphylococcus aureus and is usually found on skin and human membranes. Although not routinely pathogenic, it can cause severe infections in patients with orthopedic/breast implants, immunosuppressed Jo or with catheter, as well as the basis of nosocomial infections in oncology and neonatal departments [35]. Considering all this information it is therefore important to find a combination with good antimicrobial activity, leading at an increased effectiveness against Staphylococcus infection. In comparison, the antimicrobial efficiency of inclusion complexes is better than the one obtained for products which use standard fusidic acid. 13 From all synthesized inclusion complexes with β - cyclodextrin, the one obtained by freeze-drying method (F3) present a good antibacterial activity against methicillin sensitive Staphylococcus aureus – 2 isolated from patients, having an inhibition diameter of 28.66 mm and relatively low antibacterial effect against methicillin resistant Staphylococcus aureus, having an inhibition diameter of 26.03 mm. Experimental data obtained were compared with the one obtained when cefoxidine has been used as antibacterial drug. In the case of synthesized inclusion complexes the inhibition diameter is near or higher than diameter obtained when 30 μg of cefoxidine has been used. Obtained results are encouraging and of should be extended to a larger number of bacterial strains. ro 5. CONCLUSIONS -p Three complexes were synthesized by the inclusion of fusidic acid with β-cyclodextrin through three re methods (kneading, coprecipitation, freeze-drying) using molar ratio 1:1. Further, the new synthesized inclusion complexes were characterized with FTIR spectrometry, X Ray-diffraction, thermal analysis and ur na lP scanning electron microscopy. Analyzing the FT-IR spectra was observed that the stretching vibration of carbonyl group was shifted at lower wave numbers, which is associated with the presence of interactions between host and guest molecules. In this case there is a possibility of apparition of some hydrogen bonds between hydroxyl groups from β – cyclodextrin cavity and carbonyl group belongings to fusidic acid molecule. Based on these observations it can be concluded that the desired inclusion complexes were synthesized. Jo Also, recorded XRD spectra confirmed that the desired compounds were produced. The thermal behavior of synthesized compounds represents a clear indication of the inclusion complexes formation. All prepared compounds present a similar behavior and a proof of inclusion complexes formation is represented by the displacement of decomposition temperatures at lower values. Scanning electron microscopy was used to confirm preparation of inclusion complexes of fusidic acid with β–cyclodextrin by using three different synthesis routes. 14 Microbiological analysis proved that all synthesized inclusion complexes present antimicrobial activities. From all this, the best antimicrobial activity was shown by the inclusion complex obtained by freeze-drying method. Present study confirms that synthesized inclusion complexes are suitable as antimicrobial drugs against Staphylococcus epidermidis. Funding – not required Competing interests – none Ethical approval – not required ro of Transparency declarations - None to declare -p Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that Jo ur na lP re could have appeared to influence the work reported in this paper. 15 REFERENCES Jo ur na lP re -p ro of 1. Mraz CE, Muresan M, Micle O, Vicas L, Pallag A, Coltau M, et al. Effect of vitamin d on carbonic anhydrase activity experimental reasearch in vitro and in vivo. Farmacia. 2012;60(2):264-71. 2. Marian E, Tita B, Vicas L, Fulias A, Jurca T, Tita D. Thermal Behaviour of Active Substance Versus Pharmaceutical Compounds from Erythromycin. Rev Chim. 2012;63(10):996-1000. 3. Benedec D, Hanganu D, Oniga I, Filip L, Bischin C, Silaghi-Dumitrescu R, et al. Achillea schurii Flowers: Chemical, Antioxidant, and Antimicrobial Investigations. Molecules. 2016;21(8):12. 4. 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In Vitro Antimicrobial Findings for Fusidic Acid Tested Against Contemporary (2008-2009) Gram-Positive Organisms Collected in the United States. Clinical Infectious Diseases. 2011;52:S477-S86. 35. Oliveira WF, Silva PMS, Silva RCS, Silva GMM, Machado G, Coelho L, et al. Staphylococcus aureus and Staphylococcus epidermidis infections on implants. Journal of Hospital Infection. 2018;98(2):111-7. 17 CH3 H3C COOH HO CH3 CH3 CH3 O C O CH3 HO CH3 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 Fusidic acid (b) -cyclodextrine coprecipitation -p Absorbance (u.a.) 4000 3500 3000 2500 2000 1500 1000 500 4000 -1 ur na lP Wavenumber [cm ] 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 (c) Fusidic acid  -cyclodextrine freeze-drying Jo Absorbance (u.a.) of (a) Fusidic acid -cyclodextrine kneading ro 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 re Absorbance (u.a.) Figure 1. Chemical structure of fusidic acid 4000 3500 3000 2500 2000 1500 1000 3500 3000 2500 2000 1500 1000 500 -1 Wavenumber [cm ] Figure 2. FTIR spectra of fusidic acid, βcyclodextrin and the inclusion compound obtained by: a) kneading (F1); b) coprecipitation (F2); c) freeze-drying (F3) 500 -1 Wavenumber [cm ] 18 Figure 3. X Ray-diffraction of fusidic acid, βcyclodextrin and of the inclusion compound obtained of by: a) kneading procedure (F1); b) coprecipitation ur na lP re -p ro (F2); c) freeze-drying (F3) a Jo b c d 19 Figure 4 – Thermogravimteric curves recorded for: a) – fusidic acid, b) – β-cyclodextrin, c) – F1 inclusion complex, d) – F2 inclusion complex, e) – F3 inclusion complex. b Jo a ur na lP re -p ro of e c d 20 Figure 5. SEM pictures recorded for: a) fusidic acid, b) kneading complex, c) coprecipitation complex, d) freeze-drying complex. Table 1. Values of thermal parameters obtained for studied inclusion complexes Thermal Temperature Complex Residual Tpic DTG/oC decomposition m% Process nature interval, oC mass % stages 25.0 – 97.6 64.3 7.94 of I Endothermic dehydration II Endothermic 97.6 – 233.4 182.2 ro Kneading 4.81 complex 6.01 decomposition 233.4 – 470.7 Endothermic -p III 317.9 81.24 25.0 – 111.4 70.5 ur na lP I re decomposition Coprecipitat II 111.4 – 223.4 182.6 I Jo Freezedrying II 223.4 – 479.6 25.0 – 125.6 125.6 – 222.8 319.6 80.0 182.1 dehydration Endothermic 4.91 ion complex III Endothermic 7.00 6.09 decomposition Endothermic 82.00 decomposition Endothermic 10.27 dehydration Endothermic 2.69 10.45 decomposition complex III 222.8 – 500.0 Endothermic 319.2 76.59 decomposition 21 Table 2. Antibacterial activity of the fusidic acid complexed with cyclodextrins Coprecipitation freeze-drying Fusidic acid Cefoxitin Complex (10 µg) Complex (10 µg) complex (10 µg) (10 µg) (30 µg) 26.00 28.00 29.00 30.50 26.50 30.00 30.16 26.00 27.00 MSSA (Methicillin-sensitive MRSA Methicillin-resistant 29.00 30.00 25.50 24.00 25.50 27.00 30.00 26.50 30.00 30.16 25.50 Staph. Epidermidis 23.33 25.50 25.33 27.50 27.16 23.00 25.00 27.00 30.50 26.50 26.50 27.50 28.00 31.00 27.50 30.50 31.00 27.00 31.50 27.00 27.00 28.50 28.00 28.50 27.50 28.00 28.50 24.50 27.50 26.00 25.00 Staphylococcus aureus 29.16 28.50 27.00 Staphylococcus aureus) 2 29.50 26.00 23.00 Staphylococcus aureus) 1 28.00 24.83 27.50 27.66 25.50 28.33 26.00 of (Methicillin-sensitive 27.50 30.00 20.50 30.00 30.16 20.00 ro MSSA 26.33 25.00 28.00 26.50 30.50 20.00 30.50 30.00 31.50 30.00 30.00 30.50 30.00 30.50 29.50 29.50 30.00 30.00 29.50 29.83 31.50 31.33 31.00 26.00 26.16 27.16 20.16 30.00 ur na lP re 29.50 30.00 -p Staph. aureus ATCC 25923 Kneading Table 3: Antibacterial activity of the fusidic acid complexed with cyclodextrins values obtained throughout statistical analysis Minimum Maximum Mean Std. Deviation Variance Statistic Statistic Statistic Statistic Std. Error Statistic Statistic Kneading 20 23.00 30.50 26.2995 0.52079 2.32902 5.424 Coprecipitation 20 25.00 30.00 27.7575 0.33275 1.48809 2.214 Freezing 20 25.50 31.50 28.3990 0.42010 1.87876 3.530 Fusidic acid 20 29.50 31.50 30.2990 0.10309 0.46105 0.213 Cefoxitin 20 20.00 30.50 25.8990 0.73899 3.30488 10.922 Valid N (listwise) 20 Jo N Table 4: Mean values ±SD 22 Mean Std. Deviation N kneading 26.2995 2.32902 20 coprecipitation 27.7575 1.48809 20 Freezing 28.3990 1.87876 20 Fusidic acid 30.2990 0.46105 20 Cefoxitin 25.8990 3.30488 20 cefoxitin 0.000 Sig. (2-tailed) N 20 20 Pearson Correlation 0.917** 1 Sig. (2-tailed) 0.000 N 20 20 cefoxitin 0.875** 0.007 0.649** 0.000 0.976 0.002 20 20 20 0.713** -0.041 0.361 0.000 0.864 0.118 20 20 20 1 -0.111 0.861** 0.642 0.000 Pearson Correlation 0.875** 0.713** Sig. (2-tailed) 0.000 0.000 N 20 20 20 20 20 Pearson Correlation 0.007 -0.041 -0.111 1 0.081 Sig. (2-tailed) 0.976 0.864 0.642 N 20 20 20 20 20 Pearson Correlation 0.649** 0.361 0.861** 0.081 1 Sig. (2-tailed) 0.002 0.118 0.000 0.733 N 20 20 20 20 Jo Fusidicacid 0.917** Fusidic acid ro freezing 1 re coprecipitating freezing ur na lP kneading coprecipitation -p Pearson Correlation kneading of Table 5: Correlations obtained after statistical analysis **. Correlation is significant at the 0.01 level (2-tailed). 23 0.733 20