## 3 Materials and Methods
### Laboratory Analysis
#### 3.2.2 Antibacterial Activity Determination  
The designation of antibacterial activity of 10 selected, plant-originated compounds (dioscin, aescin, glycyrrhetinic acid, ginseng Rg3, procyanidin A2 and C1, hypericin, robinin, amentoflavone, and zeaxanthin; all from Sigma-Aldrich, St. Louis, MO, USA) against \(H\). _pylori_ J99 was made by checking their minimum inhibitory concentrations (MICs) [93; 94]. To obtain this, bacteria in a density of \(10^{7}\) CFU/mL were cultured in 12-well titration plates (Bionovo, Legnica, Poland) filled with 1 mL of Brain Heart Infusion broth (BHI; Oxoid, Dardilly, France) with 5% fetal calf serum (FCS; Gibco, Paisley, Scotland, UK) and a concentration gradient of the tested compounds (2-256 ug/mL). The titration plates were then followed for 3 days of microaerophilic incubation at 37 \({}^{\circ}\)C and 100 rpm shaking (MaxQ 6000, ThermoFisher, Waltham, MA, USA). Then, bacterial turbidity in each well of the plates was determined visually, and the lowest concentration with the lack of growth was considered as the MIC.  
3.2.3. Impact on the Morphology and/or Morphostructure  
The screening determination of the effect of high concentrations (64-256 \(\mathrm{\SIUnitSymbolMicro\text{$\mu$g}}\)/mL) of all 10 tested compounds on the _H. pylori_ morphology was performed using light microscopy combined with a Gram-staining [95]. For each sample, bacterial suspension in a volume of 50 \(\mathrm{\SIUnitSymbolMicro\text{$\mu$L}}\) was dropped onto a coverslip and stained by [PERSON]'s method. From each of the obtained preparations, 100 cells from three to five different view pools were counted and classified as spiral/rod-shaped or coccoid forms. Observations were made under the Olympus BX50 light microscope (Olympus Optical, Tokyo, Japan), using a 100\(\times\) oil immersion objective with numerical aperture of 1.3.  
In order to more accurately define the impact of dioscin on _H. pylori_ 199 cells, bacteria were exposed to different concentrations of the compound and their morphostructure was visualized by scanning electron microscopy [95]. For this purpose, each well of the 12-well titration plates was filled with 1 mL BHI broth with 5% fetal calf serum containing 16 \(\mathrm{\SIUnitSymbolMicro\text{$\mu$g}}\)/mL, 32 \(\mathrm{\SIUnitSymbolMicro\text{$\mu$g}}\)/mL, and 64 \(\mathrm{\SIUnitSymbolMicro\text{$\mu$g}}\)/mL of dioscin (representing \(\frac{1}{4}\times\) MIC, \(\frac{1}{2}\times\) MIC and MIC, respectively) and inoculated with _H. pylori_ (10\({}^{7}\) CFU/mL). Such plates were then followed for a 72-h incubation period at 37 \({}^{\circ}\)C under microaerophilic conditions. Untreated bacteria were a positive control. After incubation, each bacterial culture was centrifuged, and the pellets were fixed with 4% glutaraldehyde and post-fixed in 2% OsO4. Fixed samples were dehydrated in ethanol-acetone series and, air-dried, and a silver coating was sputtered on the bacteria surface, providing optimal conductivity. SEM microphotographs were obtained by a DualBeam SEM/Xe-PFIB FEI Helios G4 PFIB CXe microscope (Thermo Fisher Scientific, Eindhoven, Netherlands).