## 2 Results and Discussion
### Anti-Biofilm Activity  
To obtain a more complete picture of the activity of the tested compounds against _H. pylori_, we extended our screening to include the determination of anti-biofilm activity and the effect on bacterial morphology. Both biofilm formation [13; 14; 15] and transformation into coccoid morphotype [16; 17; 18] are phenomena related to the tolerance of _H. pylori_ to antimicrobial substances. Therefore, we assumed that ideally the tested compounds should be able to reduce the amount of biofilm without or with minimal stimulation of the spiral-to-cocoid transition (cocoid forms arise often intensively during exposure to antibiotics or environmental conditions unfavorable to bacterial physiology [16; 17; 18]). We observed that dioscin was characterized by the most intense inhibitory activity against biofilm formation under static conditions, i.e., MICs reduced this process by about half (up to 50.2 \(\pm\) 10.6%) (Figure 3). Importantly, at these concentrations, we detected a low level of coccoid _H. pylori_ forms (approx. 20%) (Figure S1). Other compounds at MIC values for which we noticed an ability to decrease the amount of _H. pylori_ biofilm include aescin (decrease to 52.3 \(\pm\) 2.2%) or amentoflavone (decrease to 84.5 \(\pm\) 4.4%) (Figure 3); however, for both of them, the number of coccoid forms was unsatisfactorily high (40-60%) (Figure S1). Figure 3: Anti-biofilm activity of 10 selected compounds of natural origin against _H. pylori_ 99. The amount of biofilm under stationary conditions was determined using the crystal violet staining method. The study was performed with triplicate biological replications (_n_ = 3). The _p_-value represented by ns, *, **, and ***, is equal to \(>\) 0.05, \(<\) 0.05, \(<\) 0.01, \(<\) 0.001, and \(<\) 0.0001, respectively. In line with our earlier deduction, which indicated only a few publications describing the antimicrobial activity of dioscin, in this case also, it was difficult for us to refer to other data in the literature. Despite this, in the original article by [PERSON] et al. (2018) [PERSON] et al. (2018), the ability of dioscin to interfere with a number of virulence factors of fungi from the _Candida_ genus has been proved. This mechanism covered not only their biofilm formation, but also morphological transformation (transition to filamentous forms) and secretion of lytic exoenzymes. This seemed to confirm our primary assumptions about the possibility of using dioscin in anti-virulence therapy against _H. pylori_. Surprisingly, for some substances--hypericin, ginseng Rg3, procyanidin C1, and zeaxanthin--we noticed a stimulating effect on the amount of _H. pylori_ biofilm. For example, for hypericin at a concentration equal to 2 \(\times\) MIC (256 \(\mu\)g/mL) the amount of biofilm increased to 184.4 \(\pm\) 16% compared to the control (Figure 3). In our opinion, these observations carry great research and clinical implications. They indicate that the primary selection of antimicrobials based only on the MIC measurement may be insufficient or even confusing (hypericin, next to amentoflavone, as the second most active antimicrobial substance in this study) and emphasize the need to determine additional antimicrobial parameters [46]. Moreover, the above observations throw a shadow on the widespread hypothesis that microorganisms are unable to generate resistance or activate defense mechanisms against plant extracts or plant-derived compounds, in agreement with others [47; 48; 49]. On the other hand, it has been shown by several research groups that the activity of phytochemicals may be modulated by a concentration-dependent formation of dimers/trimers/polymers and aggregation-related changes in the antimicrobial action of these molecules [50; 51; 52].