Supplementary material

Experimental

Aerial parts of Lycopus europaeus were collected from The Garden of Medical Plants, Faculty of Pharmacy in Bratislava. Harvested plant was 2 years old and was collected at the flowering time. The leaves were separated from the stems and flowers and dried at 32 – 35 °C. A voucher specimen (LE11ZLR) has been deposited at the Department of Pharmacognosy and Botany, Faculty of Pharmacy, Comenius University in Bratislava, Slovakia.

Water extract of dried leaves of L. europaeus was prepared according to Pharmacopoea Bohemoslovaca 4^th edition (1987). The infusion was lyophilised; the yield was 10.40 %.

Disk-diffusion method on Mueller-Hinton agar medium (OXOID, Great Britain) was used for detection of antimicrobial susceptibility of S. aureus strains to oxacillin, gentamicin, ciprofloxacin, vancomycin, cotrimoxazole, erythromycin and clindamycin, using commercial antibiotic disks (OXOID, Great Britain; CLSI 2009a). The susceptibility test results were interpreted according to the CLSI guidelines (CLSI 2010). Resistance to methicillin/oxacillin was confirmed by Penicillin Binding Protein 2a latex agglutination test (OXOID, Great Britain).

The minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) of LEL were tested by broth microdilution assay according to the CLSI (CLSI 2009b). LEL for antimicrobial activity testing was dissolved in sterile aqua pro injectione and filtration-sterilized. Serial geometric dilutions from 5000 to 156 µg.mL^-1 were prepared in the Mueller-Hinton Broth (OXOID, Great Britain). All tests were performed in three independent runs.

The LC-MS analyses were performed on an Agilent 1260 Infinity LC System (Agilent Technologies, Germany), equipped with a binary pump, an autosampler, a column thermostat, and a diode array detector (DAD), coupled to a quadrupole-time of flight (6520 Accurate-Mass QTOF) instrument equipped with an orthogonal ESI source (Agilent Technologies, Germany). HPLC separation of LEL was carried out on a Hypersil BDS-C₁₈ column (4.0×250mm, 5µm, Agilent Technologies, Germany) at 35°C and a flow rate of 0.4 mL/min. Water (pH 2.8 with HOAc) and MeCN were used as mobile phase A and B respectively. The following gradient program was used: 10% B (20 min), 20% B (25 min), 60% (50 min), 95% (62 min) and 10% (70 min). The ESI ion source parameters were as follows: capillary voltage: 3.5 kV, nebulizer: 40 psi (N₂), dry gas flow: 10 L/min (N₂), and dry temperature: 300 °C. The mass spectrometer was operated in an autoMS² mode where each negative ion MS scan (m/z 100-3000, average of 4 spectra) was followed by MS² scans (m/z 100-3000, average of 4 spectra, isolation window of 4 amu, collision energy 20 eV) of the two most intense precursor ions. Ions were excluded from analyses for 0.5 min after two MS² spectra had been acquired. Nitrogen was used as collision gas.

The UV wavelengths used for determining the phenolic compounds were 280 nm for flavonoids and 320 nm for phenolic acids. Phenolic compounds were identified by comparing their UV and mass spectra with literature and authentic standards when available and by measuring accurate m/z. The quantitative determination of LEL components was provided by the method of external standards. We used luteolin-7-O-glucoside for determination of flavonoids and rosmarinic acid for determination of phenolic acids. Chromatographic standards and standards of free radicals were purchased from Sigma-Aldrich, Germany.
Radical scavenging activities determined by decolorization of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) were measured spectrophotometrically (Nagy et al. 2006). The sample (200 μL) with 1.8 mL of DPPH solution (55 μmol) was incubated at room temperature for 30 min. The absorbance was read against a blank at 517 nm. The ABTS, 2,2 azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation decolorization assay was carried out, using an improved ABTS decolorization assay of Re et al. (1999). For the study the ABTS•+ solution was diluted with ethanol to an absorbance of 0.70 (± 0.02) at 734 nm and equilibrated at 30 °C. After addition of 2 mL of diluted ABTS solution to 20 μL of sample, the absorbance reading was taken exactly 6 min after initial mixing. Ferric-reducing antioxidant power assay (FRAP) was carried out according to the procedure of Benzie & Strain (1996). Briefly, the FRAP reagent was prepared from acetate buffer 300 mmol/L (pH 3.6), 10 mmol/L TPTZ solution in 40 mmol/L HCl and 20 mmol/L FeCl₃ × 6 H₂O. The FRAP reagent was prepared fresh daily. Fifty microliters of sample were added to 1.5 mL of the FRAP reagent. The absorbance of the reaction mixture was recorded at 593 nm after 5 min. The standard curve was linear between 100 and 1000 μmol ascorbic acid (AA). Results are expressed as ascorbic acid equivalents (AAE) in μmol AAE/g dry mass. All determinations were performed in triplicate.
Bacterial strains

Thirty-one clinical S. aureus strains and two cultures collection strains (ATCC 29213, and ATCC 43300) were included in the study. The clinical strains were isolated from infections associated with central venous, wound, and respiratory suction catheters, and from skin lesions of patients with atopic dermatitis and impetigo (Table S1). The strains were preserved in Brain-Heart Infusion with 20% glycerol at -20 °C and before the analysis were cultivated overnight at 37 °C on blood agar.

SC₅₀ - 50% scavenging concentration

References

Measure of ‘‘Antioxidant Power’’: The FRAP Assay. Analytical biochemistry, 239, 70-76.

Clinical and Laboratory Standards Institute. (2009a). Performance standards for antimicrobial disk susceptibility tests; Approved standard - Tenth edition. CLSI Document M2-A10.Wayne, Pennsylvania, USA.

Clinical and Laboratory Standards Institute. (2009b). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved standard – Eight edition. CLSI Document M07-A8. Wayne, Pennsylvania, USA.

Clinical and Laboratory Standards Institute. (2010). Performance standards for antimicrobial susceptibility testing: 20th informational supplement. CLSI Document M100-S20. Wayne, Pennsylvania, USA.

Nagy, M., Spilková, J., Vrchovská, V., Kontšeková, Z., Šeršeň, F., Mučaji, P. & Grančai, D. (2006). Free radical scavenging activity of different extracts and some constituents from the leaves of Ligustrum vulgare and L. delavayanum. Fitoterapia, 77, 395-397.

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. & Rice-Evans, C. (1999). Antioxidant activity applying an inproved ABTS radical cation decolorization assay. Free Radical Biology & Medicine, 26, 1231-1237.