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The aim of this study was to develop and validate a sensitive RP-HPLC method for the determination of usnic acid, as a potential marker substance of the herbal product for oromucosal use being a complex mixture of plant extracts and an essential oil, including the extract of Usnea barbata. Analysis of usnic acid in the tested formulation was performed through an extraction with methanol, prior to injection onto the HPLC column (Zorbax Eclipse XDB-C18 600 Bar (4.6 mm×100 mm, 1.8 μm)). The employed gradient procedure of the solvents (phosphoric acid (pH 2.5) and acetonitrile), at flow rate of 0.1 mL min-1 allowed for the efficient and reproducible separation of usnic acid from the other compounds present in the investigated complex mixture. The established suitability, linearity, precision, accuracy and selectivity/specificity of this assay implied its applicability for the reliable examination in the stability study of the investigated multi-ingredient herbal preparation.

Supercritical CO2 extraction of Helichrysum italicum and impregnation of starch xerogels with the extract by using an integrated scCO2 extraction and impregnation process were performed at 350 bar and 40°C in order to produce biomaterials for possible oral intake of the extract. Xerogels produced by air-drying of acetogels and alcogels were used as carriers in the supercritical impregnation process. The effect of ethanol as a co-solvent, contact time, plant material/carrier mass ratio and xerogel preparation on the impregnation loading was studied. The highest impregnation loading (1.26±0.22%) was achieved after 5 h impregnation of the xerogel obtained from alcogel using pure scCO2 and plant material/ /carrier mass ratio of 10. Chemical analysis of the extracts showed that the addition of ethanol as co-solvent had a positive effect on scCO2 selectivity to terpene fraction and total flavonoids, while it lowered the total phenolic content. Despite the difference in chemical composition, both extracts expressed similar antioxidant activity according to the DPPH and FRAP methods. The integrated process was shown to be a feasible method for isolation and incorporation of bioactive components of H. italicum into starch xerogels.

Cellulose acetate films were impregnated with carvacrol using supercritical carbon dioxide. The supercritical impregnation process, conducted in a static regime at pressure of 21 MPa and temperature of 50°C, was optimized by variation in the processing time (30 and 120 min) and decompression rate (from 0.3 MPa/min to 36 MPa/min). Characterization of the obtained cellulose acetate films was performed by Atomic Force Microscopy and Differential Scanning Calorimetry. Effects of glycerol and carvacrol on the properties of the films were discussed. Release kinetics from the cellulose acetate film with 31.4% of carvacrol was investigated in a physiological saline solution. In addition, the Higuchi and Korsmeyer-Peppas release models fitted the carvacrol release curve well. Obtained cellulose acetate films impregnated with carvacrol can be of interest for the application in medicine as wound dressings considering their biocompatibility and biodegradability as well as their potential antimicrobial activity or in the food industry as an active food packaging.

This study discusses the possibility of immobilization of colloidal TiO2 nanoparticles (NPs) onto poly(ε-caprolactone) (PCL) beads and foams that could be utilized for the removal of textile dyes from water by photodegradation. PCL foams were fabricated by environmentally friendly treatment of PCL beads in supercritical carbon dioxide. PCL beads and foams loaded with colloidal TiO2 NPs were used as photocatalysts for the removal of the textile dyes C.I. Acid Orange 7 and C.I. Basic Yellow 28 from aqueous solutions (10 mg L-1) under illumination that simulated sunlight. Unlike the PCL beads, the PCL foams provided complete discoloration of the dye solution within 24 h of illumination. The PCL foams also exhibited excellent floatability that was maintained for more than four weeks. Additionally, their photocatalytic activity was preserved within three repeated photodegradation cycles, indicating that the floating photocatalyst provided superior photocatalytic activity compared to the non-floating PCL beads. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 172056 and Grant no. 45020]

The present work was aimed to study swelling kinetics of polylactic acid (PLA) and its impregnation with thymol in supercritical carbon dioxide (scCO2) medium. The influences of temperature and soaking time on the swelling kinetics and impregnation yield of PLA cylindrical disc and film were investigated. Swelling experiments were performed in a high pressure view cell at 10 MPa and temperatures of 40°C, 60°C and 75°C for 2 to 24 h. On the basis of swelling kinetics, pressure of 10 MPa and temperature of 40°C were chosen for supercritical solvent impregnation (SSI) of the PLA samples during 2 to24 h. The highest swelling extent was observed for the PLA monolith after 24 h treatment with pure scCO2 (7.5%) and scCO2 with thymol (118.3%). It was shown that sufficiently high amount of thymol can be loaded into both PLA monolith and film using SSI after only 2 h (10.0% and 6.6%, respectively). Monolith and film of PLA impregnated with thymol could be suitable for active food packaging and sterile medical disposables.

The impregnation of differently prepared chitosan gels with thymol using supercritical carbon dioxide (scCO2) was studied in order to obtain new materials with added value without the use of organic solvents. The optimum conditions of supercritical impregnation (15 MPa, 35 °C and 24 h) were defined based on previously investigated thymol solubility in scCO2. Cross-linkers formaldehyde and glutardialdehyde were used to prepare chitosan hydrogels. The obtained hydrogels of chitosan were converted in acetogels and then dried with air or by using scCO2 in order to obtain xerogels and aerogels. The best results, in terms of morphology and yield of impregnation with thymol, have been achieved for the aerogel of chitosan (11.30%) which was obtained with cross-linker formaldehyde and dried with scCO2 at 15 MPa and 50 °C.

Antibacterial effects of plant extracts were examined using mixtures of extracts obtained using different technological processes: usnea extract was obtained using the process of supercritical extraction (NKE), angelica extract was obtained through supercritical extraction processes (NKE) and ultrasound extraction using ethanol (UZ), and thyme extract was obtained using the process of hydrodistillation (HD). Mixtures of the listed extracts were examined in various ratios: U (NKE) and T (HD) in a ratio of 1:1, U (NKE) and T (HD) in a ratio of 7:3, U (NKE), T (HD) and A (NKE) in a ratio of 2:2:1, and U (NKE), T (HD) and A (UZ) in a ratio of 2:2:1. The investigations covered 15 strains of bacteria of the genus Staphylococcus, Streptococcus and Enterococus, including the strains MRSA, VRE as well as reference strains of S. pyogenes ATCC 19615, S. agalactiae ATCC 27959 and S. aureus ATCC 11632. The antibacterial action of mixes of plant extracts was examined using the microdilution method in bouillon, and the examined mix concentrations were from 1.25 g/mL to 1280 g/mL. The strongest antibacterial effect was exhibited by mixes of usnea (NKE) and thyme (HD) in ratios of 1:1 and 7:3 with obtained MIC values from 5 g/mL to 160 g/mL, but the MIC value of the listed mixtures for the biggest number of strains amounted to 40 g/mL. A somewhat weaker effect was exhibited by the other examined extract mixtures with obtained MIC values of 10 g/mL to 320 g/mL. Based on the obtained MIC values and the results of previous investigations, it can be concluded that the examined mixtures of plant extracts exhibited a very strong antibacterial effect on the examined bacteria strains. .

The aim of the present study was to isolate and characterize antioxidant extracts obtained from dried leaves of rosemary (Rosmarinus officinalis L.) and sage (Salvia officinalis L.), originating from the southern Balkan Region. The antioxidant fraction was isolated from the plant material by supercritical carbon dioxide (SC-CO2) fractional extraction under a pressure of 30 MPa and at temperatures of 40 and 100°C. In the present study, kinetic data and yields of antioxidant extracts obtained from dried leaves of rosemary and sage under different conditions were determined. Electron spin resonance (ESR) spectroscopy assay on the ability of the extracts to scavenge stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and reactive hydroxyl radicals during the Fenton reaction trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) showed that the investigated extracts had antioxidant activity comparable to that of butylated hydroxyanisole (BHA) and commercial rosemary extract. The antioxidant fractions isolated at the higher temperature had higher antioxidant activities. A tentative analysis of the chemical composition of the antioxidant fractions obtained at the higher temperature was accomplished by LC-DAD and LC-MS analytical methods. Abietane-type diterpenoids, flavonoids and fatty acids were identified in the SC-CO2 extract of rosemary and sage.

Investigations of antibacterial effect of supercritical extraction of thyme (TNKE) and thyme extract obtained by hydrodistillation (THD) covered 18 bacteria strains from the genuses Staphylococcus, Streptococcus and Enterococcus, including the strains MRSA, the strains VRE, as well as the reference strains S.pyogenes ATCC 19615, S. agalactiae ATCC 27959, S. aureus ATCC 11632, and S.aureus ATCC 25923. The anibacterial effect of thyme extracts was examined using the microdillution method in bouillon, and the cytotoxic effect of thyme extract was examined on the VERO cell line. The obtained values for MIC THD extract ranged from 160μg/mL to 1280μg/mL for all bacteria strains covered by the investigations. The obtained values for MIC TNKE extract were 1280 μg/mL to 2560 μg/mL for all examined strains except for 1 strain of S. intermedius of 320 μg/mL. The examined extracts revealed a cytotoxic effect on the VERO cell line in concentrations higher than 40 μg/mL.

Supercritical fluid extraction is an extraction process realized with supercritical fluids, which are at a temperature and pressure above their critical temperature and critical pressure. This process has shown to be very efficient one for the isolation of different substances of medium molecular weights and molecules of relatively low polarity. The solubility of more polar substances in supercritical fluids can be improved by the addition of small amounts of other polar solvents (cosolvent) to the supercritical fluids, which is the main solvent in extraction process. The advantage of supercritical extraction compared to other extraction procedures (the application of classical organic solvents hydrodistillation, distillation with steam) is that SFE is usually performed at moderate temperature (e.g. with SF CO2 at 40-70°C) so it can be applied for the separation of different substances which are thermally unstable and have a larger vapour pressure. All of these facts indicate that SFE is of special interest for the food and pharmaceutical industry.