Stematically changing the parameters as outlined by the experiment strategy. The parameters have been determined following PX-478 Inhibitor preliminary literature research [482]. The temperature ranged from 40 to 80 C, the pressure ranged from 10 to 20 MPa, and also the cosolvent ratio ranged from 1 to 2 . The color with the SC-CO2 extracts was from pale yellow to dark brown. The ethanol-insoluble portions had been markedly separated within the extracts. Their scents were reminiscent on the beginning material. 4.3. Determination of Ethanol Content material of Samples with Gas Chromatography (GC-FID) The analyses were carried out with an Agilent 6890N GC-FID (Santa Clara, CA, USA) technique equipped with a TR-WAX (Thermo Fisher Scientific, Waltham, MA, USA) capillary column (30 m 250 1.0 ). The GC oven temperature was programmed to improve from 60 (five min isothermal) to 240 C at 30 C/min (five min isothermal). High-purity hydrogen (five.0) was used as a carrier gas at 2.9 mL/min (29 cm/s) in constant stress mode. Vials were crimped as a way to lessen the loss of volatile species. Absolute ethanol (a.r., Molar Chemicals Kft., Hal 3-Chloro-5-hydroxybenzoic acid Description ztelek, Hungary) was used as a typical to determine the ethanol peak depending on retention time. For FID quantification, external common approach was applied. Then, 100 mg absolute ethanol was diluted with dimethyl sulfoxide (a.r., Molar Chemical substances Kft., Hal ztelek, Hungary) to achieve a final concentration of 10 mg/mL as a stock answer. The calibration curve covered the range 0.five to two.0 mg/mL. four.four. Thin Layer Chromatography-Direct Bioautography (TLC-DB) 4.4.1. Direct Bioautography The antibacterial effect of your clary sage extracts on P. aeruginosa (ATCC 27853) and methicillin-resistant Staphylococcus aureus (MRSA 4262) was screened within the laboratory on the Division of Healthcare Microbiology and Immunology (Healthcare College, University of P s, Hungary). For bioautographic assay, bacteria were grown in one hundred mL Brain eart Infusion Broth (BHI) (Sigma Aldrich Ltd., Darmstadt, Germany) at 37 C within a shaker incubator at a speed of 60 rpm for 12 h [53]. The bacterial suspension was diluted with fresh nutrient BHI to an OD600 of 0.four, which corresponds to roughly four 107 colonyforming units (cfu)/ mL [16]. 4.4.two. Thin Layer Chromatography without having Separation Chromatography was performed on five 10 cm silica gel 60F254 aluminum sheet TLC plates (Merck, Darmstadt, Germany). Chloroform (a.r., Molar Chemical substances Kft., Hal ztelek, Hungary) was chosen because the solvent since it dissolved the extracts effectively and had a higher tension. As a consequence of distinct extraction parameters, every single sample had a various ethanol content (Table S1). During sample preparation, each sample was corrected for its ethanol content material. From final options (final solution’s clary sage extract concentration in every sample: 10 mg/mL), 3.0 had been applied towards the TLC plate with Finnpipette pipettes (Thermo Fisher Scientific, Darmstadt, Germany); solvent controls were chloroform and absolute ethanol (a.r., Molar Chemicals Kft., Hal ztelek, Hungary), when the positive controls were vancomycin (Vancocin, ANI Pharmaceuticals, Baudette, MN, USA) against MRSA (stock: 50 mg/mL; 0.6 applied to the TLC) and gentamicin (Sandoz, Holzkirchen, Germany) against P. aeruginosa (stock: 80 mg/2 mL; 0.75 applied for the TLC plate). TLC separation was not performed, because the goal of this experiment was to examine the antibacterial activity of your extracts (not separated compounds).Molecules 2021, 26,12 of4.4.three. Post-Chromatographic Detectio.