Solubility of thymol in supercritical carbon dioxide and its impregnation on cotton gauze

J. of Supercritical Fluids 84 (2013) 173–181

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Solubility of thymol in supercritical carbon dioxide and its impregnation on cotton gauze Stoja Milovanovic, Marko Stamenic, Darka Markovic, Maja Radetic, Irena Zizovic ∗ University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, 11120 Belgrade, Serbia

a r t i c l e

i n f o

Article history: Received 17 July 2013 Received in revised form 11 October 2013 Accepted 14 October 2013 Keywords: Solubility Impregnation Supercritical carbon dioxide Thymol Cotton gauze Antimicrobial activity

a b s t r a c t Through this study, an attempt has been made to evaluate the solubility of thymol in supercritical carbon dioxide as well to investigate a prospect of its impregnation on cotton gauze on laboratory scale. Solubility of thymol in supercritical carbon dioxide was determined at temperatures of 35 ◦ C, 40 ◦ C and 50 ◦ C, and pressures ranging from 7.8 to 25 MPa (CO2 density range 335.89–849.60 kg/m3 ) using a static method. The solubility data were correlated using semi-empirical equations introduced by Chrastil, Adachi and Lu and del Valle and Aguilera. Taking into account obtained results, temperature of 35 ◦ C and pressure of 15.5 MPa were selected as operating conditions for the impregnation process. Impregnation of cotton gauze with thymol was performed in a cell using carbon dioxide as a solvent. Kinetics of the process was determined and modeled. Masses of thymol on cotton gauzes after 2 h and 24 h of impregnation were 11% and 19.6%, respectively. FT-IR analysis confirmed the presence of thymol on the surface of the cotton fibers. The impregnated gauze provided strong antimicrobial activity against tested strains of Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis and Candida albicans. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Thymol (2-isopropyl-5-methylphenol) is a natural monoterpene phenol abundantly present in essential oils of thyme, oregano and winter savory [1]. It has been reported that among the identified natural antimicrobial agents, thymol exhibited significant antimicrobial activity [2]. Its antimicrobial power was proven against Gram positive bacteria (e.g. Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Listeria monocytogenes, Bacillus cereus), Gram negative bacteria (e.g. Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica) and yeasts (e.g. Candida albicans, Saccharomyces cerevisiae), which were the most sensitive [3]. Thymol was shown to be efficient even against methicillin-resistant staphylococci [4] with minimum inhibitory concentration values determined by agar dilution method in the range of 0.03–0.06% (v/v). Thymol also acts as a powerful scavenger of reactive oxygen species and therefore has strong antioxidant properties [5,6]. Recently, it was found that thymol significantly improved inflammatory responses and promoted wound healing by reducing the edema and diminishing the influx of leukocytes to the injured area [7]. Therefore, it was suggested that thymol could be utilized as a promising compound in the treatment of inflammatory processes and wound healing.

∗ Corresponding author. Tel.: +381 11 3303 795; fax: +381 11 3303 795. E-mail address: [email protected] (I. Zizovic). 0896-8446/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.supflu.2013.10.003

Transdermal systems capable to deliver a bioactive agent into skin cutaneous/subcutaneous levels are of great interest for therapeutic treatments. A wound dressing system should provide flexibility, controlled adherence to the surrounding tissue, gas permeability and durability/biodegradability. In addition, wound dressing systems should have the capacity to absorb fluids exuded from the wounded area and simultaneously to control water loss [8]. The choice of the dressing material is crucial since its interaction with the wound may significantly influence the healing process. Hence, natural-based biodegradable and biocompatible materials are gaining increasing attention [9]. Cotton fibers have been in use for over five thousand years. Cotton gauze is widely used for hygienic purposes because of its natural softness, high hygroscopicity and heat retaining properties [10]. Therefore, cotton gauze is a natural material, which meets all requirements for a good wound dressing. Since supercritical carbon dioxide (scCO2 ) is not only known as a solvent for valuable compounds but also for its high diffusion ability in organic matter, the latter property can be exploited for impregnation of solid matrices with natural antibacterial agents [11]. There are few studies in literature that combine these processes [8,11–13]. In order to perform an efficient impregnation, data on solubility of active substance in scCO2 are necessary. The methods for experimental determination of solubility in supercritical fluids can be classified into three categories: static, dynamic and chromatographic methods [14]. Direct experimental determination of the solubility data at high pressure is time-consuming and it requires costly equipment. Desirable

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minimization of the experimental efforts can be obtained by introduction of the methods of correlation and prediction. There are two approaches in the literature dealing with a problem of representation and estimation of high-pressure solubility data: (1) application of an expression directly relating the concentration of solute to the density of supercritical solvent, and (2) application of an equation of state [15–17]. The equation of state models require many physical properties (macroscopic critical properties and sublimation pressure are needed for cubic equations of state as well as molecular parameters for perturbed equations), which are estimated by group contribution methods leading to erroneous correlations. On the other hand, semi-empirical equations, like density based models, require only available independent variables like pressure, temperature and density of pure supercritical fluid instead of solid properties. They are based on simple error minimization [17]. To the best of our knowledge, there are no data available in the open literature on solubility of thymol in scCO2 at temperatures below 50 ◦ C. Therefore, in this study, solubility of thymol in scCO2 was determined at different conditions using a static method. On the basis of obtained solubility data, optimum operating conditions for thymol impregnation on cotton gauze using scCO2 were selected. Kinetics of the process, as well as antimicrobial properties of the impregnated cotton gauze, was investigated. Since semi-empirical density-based models which provide a relationship between the solubility of the solute and the pure density of the solvent represent an efficient tool for the data extrapolation to process conditions that have not been acquired by the measurements, the aim of this work was also to determine which semi-empirical equation provides the best fit to solubility data of thymol in scCO2 . Density-based models introduced by Chrastil [18], Adachi and Lu [15] and del Valle-Aguilera [19] were employed. Those models were selected as the first and the simplest density based model (Chrastil), improved model regarding density dependence of solubility (Adachi-Lu) and improved model regarding the enthalpy of vaporization dependence on temperature (del ValleAguilera). 2. Materials and methods 2.1. Materials Thymol (purity >99%) was supplied by Sigma–Aldrich Chemie GmbH, Germany. Commercial CO2 (purity 99%) was purchased from Messer–Tehnogas, Serbia. Sterile cotton gauze with weaving density of 17 threads/cm2 was produced by Niva, Serbia. 2.2. Solubility determination Solubility of thymol was determined using a static method in a high-pressure view chamber (Eurotechnica GmbH) presented in Fig. 1. The view chamber is set up for observation of multiphase and interfacial behavior at elevated pressures and temperatures. The chamber has cylindrical interior with internal volume of 25 mL. Electrical heating jacket around the cell allows quick and uniform heating. Changes inside the cell can be recorded by USBCCD monochrome camera. Solid sample of thymol (1.00 g) in a glass container was placed inside the chamber. The stainless steal filter was placed at the top of the container in order to avoid the thymol precipitation in the container during the decompression. After reaching the desired temperature, CO2 was pumped into the system. When the desired pressure was attained, the system has been kept at the constant temperature and pressure for a certain time intervals. At the end of process, the valve V2 was opened and the pressure was slowly lowered to the atmospheric pressure (decompression speed was 0.33 MPa/min in all experiments). The mass of

Fig. 1. Schematic presentation of the high-pressure view chamber.

remained thymol in the glass was determined gravimetrically using an analytical scale with accuracy ±0.0001 g. The mass of dissolved thymol was subsequently calculated. Solubility of thymol in scCO2 was experimentally determined at temperatures of 35 ◦ C, 40 ◦ C and 50 ◦ C, and pressures ranging from 7.8 to 25 MPa (scCO2 density range 335.89–849.60 kg/m3 [18]). The time needed for the system to reach equilibrium was evaluated as well. All the experiments were performed in triplicate. 2.3. Correlation of solubility data Chrastil [19] introduced semi-empirical equation for relating solubility of the solute and density of supercritical fluids:



c = e1 exp a1 +

a2 T



(1)

where c (kg/m3 ) is the concentration of a solute in dense gas,  (kg/m3 ) is density of the gas, T (K) is temperature and e1 , a1 and a2 are solubility coefficients with following physical meanings: one molecule of solute is assumed to associate with e1 molecules of the gas; constant a1 depends on the molecular weights of solute and solvent and also is a function of e1 ; constant a2 is proportional to the heat of solvation and heat of vaporization of the solute. Adachi and Lu modified Chrastil’s equation to improve its capability of data representation [16]. A modification was made by considering the quantity e1 to be density dependent: 2



c = e1 +e2 +e3  exp a1 +

a2 T



(2)

Del Valle and Aguilera [20] improved Chrastil’s equation in a way demonstrated by Eq. (3). To recompense for the change in the entalpy of vaporization with temperature, they proposed the following modification:



c = e1 exp a1 +

a3 a2 + 2 T T



(3)

On the basis of the solubility (experimentally determined or correlated) mole fraction of thymol in scCO2 can be expressed as [21]: y=

c c + (M2 /M1 )

(4)

where M1 and M2 are the molar weights of scCO2 and thymol, respectively. The model parameters were calculated by minimizing AARD function (Eq. (5)) using excel solver tool:





1   yi,exp − yi,cal  AARD =  yi,exp  × 100% n n

i=1

(5)

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175

Fig. 2. Autoclave engineers screening system: B, CO2 bottle; C, cryostat; P, high pressure liquid pump; E, extractor vessel; S, separator vessel.

where n is the number of experimental data points, yi,exp is the experimental value of the mole fraction of thymol in scCO2 for experimental point i, and yi,cal is the calculated value of the mole fraction of thymol in scCO2 corresponding to point i. 2.4. Supercritical impregnation Impregnation of cotton gauze with thymol was performed in the apparatus presented in Fig. 2. Autoclave engineers screening system is designed for small batch research runs using CO2 as the supercritical medium. Volume of the extractor is 150 mL. Heaters are supplied on the extractor vessel for temperature elevation. Maximum allowable working pressure is 413 bar at 238 ◦ C. Thymol was placed at the bottom of the vessel and the cotton gauze fitted in stainless steel supports was placed above it. Gauze/thymol ratio was 0.65 ± 0.05 g/g. In order to prevent splashing of thymol on the surface of the gauze, porous barrier with pore diameter of 0.09 mm was placed between the gauze and thymol. Operating pressure and temperature were selected from the obtained solubility data (35 ◦ C and 15.5 MPa). After putting thymol and the gauze inside the vessel, the temperature was elevated and CO2 was pumped into the system until the required pressure was obtained. After reaching the desired conditions, valve V2 was closed and the pump was turned off. System was kept at a constant temperature and pressure for desired time intervals. A slow decompression (0.33 MPa/min) was applied at the end of the process. The quantity of thymol impregnated was determined gravimetrically by measuring the impregnated gauze on an analytical scale with accuracy ±0.0001 g. All experiments were performed in triplicate. Chairat et al. [22] and Gamal et al. [23] applied the pseudo first (Eq. (6)) and pseudo second order (Eq. (7)) kinetic models on modeling of dyes adsorption onto cotton fiber: dqt = k1 (qe − qt ) dt

(6)

dqt = k2 (qe − qt )2 dt

(7)

where qe is the mass of substrate adsorbed per mass of cotton in equilibrium, qt is the time dependant mass of substrate adsorbed per mass of cotton, t is the impregnation time, and k1 and k2 are

the rate constants for the pseudo first-order and pseudo secondorder kinetics, respectively. The same equations are applied in this study for modeling of the kinetics of thymol impregnation on cotton gauze. However, it should be noticed that according to the static method applied in this study, this process included solubilization of thymol in scCO2 as well. The best fit is obtained by minimizing the residual sum of square (RSS), which can be expressed as follows:

RSS =

n 

[Yi − Ycal ]2

(8)

i=1

where Yi and Ycal are the values of qt obtained experimentally and by the model, respectively. 2.5. Characterization of the cotton gauze The surface morphology of cotton gauze fibers was followed by field emission scanning electron microscopy (FESEM, Mira3 Tescan). The samples of cotton gauze were coated with a thin layer of Au/Pd (85/15) prior to analysis. Fourier transform infrared (FT-IR) spectra of the control cotton gauze and the cotton gauze impregnated with thymol were recorded in the ATR mode using a Nicolet 6700 FT-IR Spectrometer (Thermo Scientific) at 2 cm−1 resolution, in the wavenumber range 500–4000 cm−1 . 2.6. Antimicrobial activity of impregnated gauze Antimicrobial activity of the cotton gauze was evaluated against Gram-negative bacteria E. coli ATCC 25922, Gram-positive bacterial strains S. aureus ATCC 25923, B. subtilis ATCC 6633, E. faecalis ATCC 29812 and fungus C. albicans ATCC 24433 using the standard test method ASTM E 2149-01 [24]. Microbial inoculum was prepared in the tripton soy broth (Torlak, Serbia), which was used as the growth medium for microbes while the potassium hydrogen phosphate buffer solution (pH 7.2) was used as the testing medium. Microbes were cultivated in 3 mL of tripton soy broth at 37 ◦ C and left overnight (late exponential stage of growth). 50 mL of sterile potassium hydrogen phosphate buffer solution (pH 7.2) was inoculated with 0.5 mL of a microbial inoculum. One gram of sterile

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Table 1 Mole fractions of thymol in scCO2 at 35 ◦ C, 40 ◦ C and 50 ◦ C in scCO2 density range from 335.89 kg/m3 to 849.60 kg/m3 . y × 103

P (MPa)

 (kg/m3 )

P (MPa)

◦

 (kg/m3 )

P (MPa)

◦

35 C 7.8 8.0 8.2 8.7 12.5 15.5

y × 103

335.89 426.85 545.32 637.35 777.71 822.08

8.5 9.0 10.0 13.0 16.0 21.0

 (kg/m3 )

◦

40 C

0.900 2.37 4.64 6.12 9.27 11.0

y × 103 50 C

0.837 1.26 4.49 6.77 8.65 12.3

356.78 487.29 629.75 744.09 795.79 849.60

10.0 11.0 14.0 16.0 25.0

1.88 3.05 7.05 8.15 14.7

385.35 504.10 673.58 723.17 834.97

T (◦ C)

AARD (%)

AARD%(%)

35 40 50 35 40 50 35 40 50

10.99 33.64 8.85 16.84 27.34 5.17 12.16 31.30 5.35

Table 2 Parameters of semi-empirical equations applied. Eq.

e1

e2

e3

a1

(1)

3.7876

–

–

−19.9815

(2)

(3)

5.0732

3.7404

−1.15E − 03

–

7.63E − 07

–

a2 −621.7667

−23.0883

−1427.9593

−19.3191

−754.6777

gause cut into small pieces was put in the flask and shaked for 2 h. 1 mL aliquots from the flask were diluted with physiological saline solution and 0.1 mL of the solution was placed onto a tryptone soy agar (Torlak, Serbia). After 24 h of incubation at 37 ◦ C, the zero time and two hour counts of viable microbial were made. The percentage of microbial reduction (R, %) was calculated using the following equation: R=

C0 − C × 100 C0

a3

(9)

where C0 (CFU – colony forming units) is the number of microbial colonies on the control cotton gauze and C (CFU) is the number of microbial colonies on the cotton gauze impregnated with thymol.

3. Results and discussion 3.1. Experimental results and correlation of the solubility data Thymol was placed in the View Chamber at ambient conditions in the form of powder, but during the vessel pressurization with CO2 , its transition into liquid state occurred. Time needed for reaching the equilibrium solubility was investigated first. Dependence of thymol concentration in scCO2 inside the view chamber on time at 40 ◦ C and 10 MPa is presented in Fig. 3. As can be seen, the time needed for reaching the equilibrium was 24 h, and therefore all the solubility measurements were performed at this exposure time. Experimentally determined mole fractions of thymol in scCO2 at temperatures of 35 ◦ C, 40 ◦ C and 50 ◦ C, and pressures ranging from 7.8 to 25 MPa (scCO2 density range from 335.89 kg/m3 to 849.60 kg/m3 ) are presented in Table 1 and Figs. 4 and 5. Modeling results are presented in Figs. 4 and 5. Parameters of the applied correlations are given in Table 2. Generally, Fig. 4 reveals that all the equations tested can be used to correlate the solubility of thymol in scCO2 . According to the AARD% values (Table 2) the highest accuracy was obtained in the case of del Valle–Aguilera equation, followed by Adachi-Lu and Chrastil equations. Fig. 4 also indicates that solubilities obtained at 35 ◦ C and lower pressures were comparable to those obtained at 50 ◦ C and higher pressures. Thus, the

–

–

4079.9090

17.82

16.45

16.27

temperature of 35 ◦ C and pressure of 15.5 MPa were selected for the impregnation process. Mukhopadhyay and De [25] measured solubilities of thymol in scCO2 at 50 ◦ C and 70 ◦ C and pressures up to 14 MPa using a static method. The authors obtained results at 50 ◦ C similar to those achieved in the current study. They reported thymol mole fraction values at 50 ◦ C ranging from 0.00083 (at 7.8 MPa) to 0.00721 (at 12.7 MPa) while the values obtained in this study for the temperature of 50 ◦ C ranged from 0.00188 (at 10 MPa) to 0.00705 (at 14 MPa) and 0.0147 (at 25 MPa). Sovova and Jez [21] measured solubilities of menthol (similar to thymol fine chemical) in scCO2 at temperatures in the range from 35 ◦ C to 55 ◦ C and pressures up to 11.56 MPa using a dynamic method in a flow type apparatus. The authors reported menthol mole fraction values at 35 ◦ C ranging from 0.00096 (at 7.52 MPa) to 0.0126 (at 8.11 MPa), while the values obtained for thymol in this study were in the range from 0.0009 (at 7.8 MPa) to 0.00612 (at 8.7 MPa) and 0.011 (at 15.5 MPa).

Fig. 3. Thymol concentration in scCO2 in the view chamber vs. time at 40 ◦ C and 10 MPa.

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Fig. 4. Solubility of thymol in scCO2 versus pressure data correlated by: (a) Chrastil equation, (b) Adachi–Lu equation and (c) del Valle–Aguilera equation.

3.2. Supercritical carbon dioxide impregnation The rate of static impregnation process under selected conditions (35 ◦ C and 15.5 MPa) is presented in Table 3 and Fig. 6. It is noticeable that the adsorption was fast in the first two hours, but afterwards its rate decreased. The masses of impregnated thymol after 2 and 24 h of impregnation were 11% and 19.6%, respectively. Modeling results are depicted in Fig. 6, while the parameters of the models are presented in Table 4. It is evident, from Fig. 6 as well as from the values of RSS, that the pseudo-first order kinetics is more appropriate for mathematical description of the process. It should be stressed that selected technique of static impregnation also included solubilization process of thymol in scCO2 . Consequently,

Table 3 Rate of thymol impregnation at 35 ◦ C and 15.5 MPa. No.

Time (h)

Amount of impregnated thymol (%)

1 2 3 4 5 6

1 1.5 2 6 12 24

1.74 4.85 11.0 13.1 18.2 19.6

kinetics of the combined solubilization–impregnation process is obtained. Chairat et al. [22] and Gamal et al. [23] reported values of kinetic rate constant k1 in the range from 0.0195 min−1 to 0.0662 min−1 for conventional methods of dyes adsorption onto cotton fiber. Those values are one order of magnitude lower than the value obtained in this study (Table 4) for the thymol impregnation using scCO2 . This is reasonable result due to the higher diffusivity of scCO2 into fibers comparing to aqueous solutions. 3.3. Cotton gauze characterization The FESEM images of untreated and impregnated cotton gauzes are presented in Fig. 7. Characteristic morphology of cotton fiber can be observed in Fig. 7a. Although the thymol crystallites on the surface of cotton fibers after 2 and 24 h long impregnation cannot be observed, the change in fiber morphology is evident. Fine pleats parallel to the cotton fiber axis have been formed and

Table 4 Values of the model parameters.

Pseudo first-order Pseudo second-order

qe

k1 (h−1 )

k2 (h−1 )

RSS

0.2444 0.2444

0.199 –

– 1.293

0.00294 0.00339

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Fig. 5. Solubility of thymol in scCO2 versus CO2 density. Data correlated by: (a) Chrastil equation, (b) Adachi–Lu equation and (c) del Valle–Aguilera equation.

specific surface area was enlarged. Katayama et al. [10] obtained similar results in the investigation on the influence of scCO2 and water on the cotton fiber surface morphology. The wrinkles formation was explained by the difference in degasification speed (after decompression) between the surface and interior of cotton fiber, which induced the difference in shrinkage between these two segments. Results of the FT-IR analysis of control and impregnated cotton gauze during 24 h are presented in Fig. 8. Evidently, a broad band in the region between 3500 and 3200 cm−1 is observed in the FT-IR spectrum of control cotton fabric, which is assigned to the stretching vibrations of cellulosic OH group [26–29]. In addition, a broad band with a peak centered at 2898 cm−1 corresponds to C H asymmetric stretching [26]. The bands at 1429, 1368, 1317 and 1281 cm−1 originate from C H in plane bending vibrations, C H bending (deformation stretch) vibrations, C H wagging vibrations and C H deformation stretch vibrations, respectively [26,27]. The bends at 1337, 1247 and 1203 cm−1 corresponds to OH inplane bending vibrations [26]. The bands at 1160 and 1108 cm−1 are assigned to asymmetric bridge C O C [26], whereas the peak at 1053 cm−1 is ascribed to asymmetric in plane ring stretching [26]. The peak at 1030 cm−1 is related to C O stretching [26]. Finally, the peak corresponding to asymmetric out-of-phase ring stretching at C1 O C4 ␤-glucosidic bond appears at 900 cm−1 [25–29]. Observed bands are characteristic for cotton and fit well to literature data. The presence of CO2 (doublet at 2362 and

2334 cm−1 ) and adsorbed water (at 1650 cm−1 ) was also detected [26–31]. The FT-IR analysis of cotton gauze impregnated with thymol (24 h long impregnation) confirmed the presence of thymol on

Fig. 6. Experimental and modeling results for pseudo first-order (a) and pseudo second-order (b) kinetics.

S. Milovanovic et al. / J. of Supercritical Fluids 84 (2013) 173–181

Fig. 7. FESEM images of: (a) untreated gauze, (b) 2 h impregnated gauze, (c) 24 h impregnated gauze.

the surface of cotton fibers. The peaks in the FT-IR spectrum at 2957 and 2866 cm−1 can be assigned to asymmetric C H stretching vibration and deformation overtones of CH3 group, respectively [30,32]. The peaks at 1623 and 1447 cm−1 correspond to aromatic ring C C stretching vibrations [29,32]. Bands detected at 1286 and 1259 cm−1 are assigned to combination of OH deformation vibrations and C O stretching vibrations [29]. The peak at 1360 cm−1 is assigned to OH bending of phenolic group [33]. Band at 804 cm−1 is attributed to out-of-plane aromatic C H wagging vibrations [34]. 3.4. Antimicrobial analysis The results of antimicrobial analysis are presented in Table 5. The samples which were impregnated for 2 and 24 h were evaluated. Both samples exhibited strong antimicrobial activity against tested strains of E. coli, S. aureus, B. subtilis, E. faecalis and C. albicans providing maximum microbial reduction (99.9%) for all tested microorganisms. The exact mechanism of antimicrobial action of thymol has not been established yet. Some studies indicated that thymol has an ability to disrupt lipid structure of the bacteria cell wall, further leading to a destruction of cell membrane, cytoplasmic leakage, cell lysis and cell death [35,36].

Fig. 8. FT-IR spectra of control and impregnated cotton gauze.

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Table 5 Antimicrobial activity of impregnated gauze. Sample

Microorganism

2 h impregnation

24 h impregnation

Number of bacterial colonies (CFU) Control gauze Gauze + thymol Control gauze Gauze + thymol Control gauze Gauze + thymol Control gauze Gauze + thymol Control gauze Gauze + thymol

E. coli S. aureus B. subtilis E. faecalis C. albicans

2.2 × 104 <10 4.4 × 104 <10 1.5 × 105 <10 5.4 × 105 <10 7.4 × 104 <10

4. Conclusions Solubility of thymol in supercritical carbon dioxide was measured at temperatures of 35 ◦ C, 40 ◦ C and 50 ◦ C, and pressures ranging from 7.8 to 25 MPa (scCO2 density range 335.89–849.60 kg/m3 ) using a static method. Obtained data were correlated by the semi-empirical equations introduced by Chrastil, Adachi and Lu and del Valle and Aguilera with the acceptable accuracy. On the basis of the solubility data, the process of supercritical impregnation of cotton gauze with thymol was proposed. Kinetics of the process was determined and modeled using pseudo first order and pseudo second order kinetic models at selected process conditions of 35 ◦ C and 15. 5 MPa. After 2 h of impregnation, mass of impregnated thymol was 11%, while its value after 24 h of impregnation reached 19.6%. Both samples exhibited strong antimicrobial activity against tested strains of E. coli, S. aureus, B. subtilis, E. faecalis and C. albicans providing maximum microbial reduction (99.9%) for all tested microorganisms. Results of the FT-IR analysis confirmed the presence of thymol on the surface of cotton fibers. According to the results of this study, supercritical impregnation with carbon dioxide was indicated as a feasible technique for impregnation of cotton gauze with thymol in order to obtain an efficient wound dressing with antibacterial properties. Further investigations on cytotoxic effects of the impregnated gauze in vitro on cell lines are needed. Acknowledgments Financial support of the Serbian Ministry of Education, Science and Technology Development (Projects III45017 and 172056) is gratefully acknowledged. We gratefully acknowledge Dr Bojan Jokic´ (University of Belgrade, Serbia) for providing FESEM measurements. References [1] M. Sokovic, L.J.L.D. van Griensven, Antimicrobial activity of essential oils and their components against the three major pathogens of the cultivated button mushroom, Agaricus bisporus, European Journal of Plant Pathology 116 (2006) 211–224. [2] A. Wattanasatcha, S. Rengpipat, S. Wanichwecharungruang, Thymol nanospheres as an effective anti-bacterial agent, International Journal of Pharmaceutics 434 (2012) 360–365. [3] S. Cosentino, C.I. Tuberoso, B. Pisano, M. Satta, V. Mascia, E. Arzedi, F. Palmas, In vitro antimicrobial activity and chemical composition of Sardinian thymus essential oils, Letters in Applied Microbiology 29 (1999) 130–135. [4] A. Nostro, A.R.M. Blanco, A. Cannatelli, V. Enea, G. Flamini, I. Morelli, A.S. Roccaro, V. Alonzo, Susceptibility of methicillin-resistant staphylococci to oregano essential oil, carvacrol and thymol, FEMS Microbiology Letters 230 (2004) 191–195. [5] R. Aeschbach, J. Loliger, B.C. Scott, A. Murcia, J. Butler, B. Halliwell, O.I. Aruoma, Antioxidant actions of thymol, carvacrol, 6-gingerol, zingerone and hydroxytyrosol, Food and Chemical Toxicology 32 (1994) 31–36. [6] I. Kruk, T. Michalska, K. Lichszteld, A. Kladna, H.Y. Aboul-Enein, The effect of thymol and its derivates on reactions generating reactive oxygen species, Chemosphere 41 (2000) 1059–1064.

R (%) 99.9 99.9 99.9 99.9 99.9

Number of bacterial colonies (CFU) 7.0 × 103 <10 5.0 × 103 <10 6.0 × 103 20 4.7 × 104 <10 6.0 × 104 <10

R (%) 99.9 99.9 99.9 99.9 99.9

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