## 3 Materials and Methods
### Laboratory Analysis
#### 3.2.4 Anti-Biofilm Activity Determination  
Activity in Static Conditions  
To assess the anti-biofilm activity of all 10 tested compounds against _H. pylori_ 199, the crystal violet staining method was applied [96]. Bacteria in a final density of 10\({}^{7}\) CFU/mL were suspended in 1 mL of BHI broth with 5% fetal calf serum and a gradient concentration (2-256 \(\mathrm{\SIUnitSymbolMicro\text{$\mu$g}}\)/mL) of the tested compounds, and were cultured for 3 days under microaerophilic atmosphere, 37 \({}^{\circ}\)C, and 50 rpm shaking. Then, the entire bacterial suspension was removed from each well, and the plates were dried and stained with a 0.1% crystal violet solution (Sigma-Aldrich, St. Louis, MO, USA) for 15-30 min. The dye was removed, and the wells were then rinsed twice with a phosphate buffer solution (PBS; Sigma-Aldrich, St. Louis, MO, USA) and flushed with 1 mL of 96% ethanol (Stanlab, Lublin, Poland). Two hundred microliters of crystal violet-stained alcohol was withdrawn from each well, transferred to a 96-well microtiter plate (Bionovo, Legnica, Poland), and measured for absorbance at OD\({}_{590}\) with an Asys UVM 340 microplate reader (Biochrom Ltd., Cambridge, UK). The negative control consisted of wells without bacteria (the absorbance of these samples was used each time to subtract background from the tested samples).  
Confirmation of the anti-biofilm activity of dioscin against _H. pylori_ 199 was performed under flow conditions created by the Bioflux 1000 system (Fluxion, San Francisco, CA, USA) [57]. To generate these conditions, 48-well low-shear plates composed of inlet and outlet wells connected by a capillary (Fluxion, San Francisco, CA, USA) were used. In the first stage, inlet wells were filled with 900 \(\mathrm{\SIUnitSymbolMicro\text{$\mu$L}}\) of BHI broth with 5% fetal calf serum and the desired concentration of dioscin (16 \(\mathrm{\SIUnitSymbolMicro\text{$\mu$g}}\)/mL, 32 \(\mathrm{\SIUnitSymbolMicro\text{$\mu$g}}\)/mL, and 64 \(\mathrm{\SIUnitSymbolMicro\text{$\mu$g}}\)/mL, representing \(\frac{1}{4}\times\) MIC, \(\frac{1}{2}\times\) MIC, and MIC, respectively). Wells without dioscin served as a positive control. In the next stage, the above-mentioned medium was flowed through capillaries with a pressure of 10 \(\mathrm{\SIUnitSymbolMicro\text{$\text{$\text{$\text{$\text{$\text{$\text{$ \text{$\text{$\text{$\text{$\text{$\text{$\text{$\text{$}}}}{}{}{}{}{}{}{{}{{}}{{{}}{{{}}{{}{{}{}{{}{}{}{{}{}{}{{}{}{}{}{{}{}{}{}{}{{}{}{}{}{}{}{}{}{}{}{}{}{{}{}{}{}{}{}{}{}{{}{}{}{}{}{{}{}{}{}{}  
of 0.1 dyne/cm\({}^{2}\). Following this, the flow of medium was interrupted, and the remaining medium from inlet wells was taken. The emptied inlet wells were filled with 100 \(\mu\)L of dye solution made from a LIVE/DEAD kit (Thermo Fisher, Waltham, MA, USA). The flow of 0.1 dyne/cm\({}^{2}\) was turned on again to provide a 1-h staining of bacterial microcolonies and biofilms. After this, photos from capillaries were obtained with an inverted Axio Observer 7 microscope (GmbH, Carl Zeiss, Jena, Germany). Both the degree of capillary overgrowth and the ratio of green/red fluorescence (the ratio of live/dead bacteria) was determined using the Bioflux Montage software (Fluxion, San Francisco, CA, USA).