## 4 Discussion
coli_ were studied, the RG concentration of 25% resulted in slight bacterial stimulation, whereas RG 0.78% caused weakened lysis, that was also extended in time. These concentrations did not influence phage viability in the previous experiment, while they positively altered bacterial growth in the modified MIC test. Here, the results for RG 25% can be explained by the bacteria-stimulating effect of the extract and probable gain of phage-inhibitory activity in the mixing environment. Therefore, when the RG concentration was lower (0.78%), the extract phage-inhibitory influence was weaker, allowing for limited lysis, so the effect was also concentration-dependent. Unfortunately, there is limited knowledge on _Ruta gravelens_ activity on Gram-negative bacterial cells performed with modern experimental methods. Moreover, the tendency of differentiated plant activity on bacteria associated with the type of extraction and used plant parts is also observed. The study in which antibacterial activity of ethanol, methanol, chloroform and distilled water extracts of _R. gravelens_ leaves was tested by the disk diffusion methods, the methanol and chloroform extracts recorded better antibacterial activity than the ethanol extract, and the water extract did not exert any activity. Moreover, the authors also point out that the phytochemical analysis of different solvent extracts show considerable change in the nature of the chemicals [35]. Other authors demonstrate that aquatic, ethanolic and methanolic extracts likely of the whole _R. gravelens_ plant (not specified in the paper) have been almost equally effective against _E. coli_ (25 g/L MIC aquatic extract; 12.5 g/L MIC ethanolic extract; 25 g/L MIC methanolic extract) and _P. aeruginosa_ (25 g/L MIC aquatic extract; 25 g/L MIC ethanolic extract; 25 g/L MIC methanolic extract). The experiments were performed using disk diffusion and well diffusion methods [36]. Another study designed to screen various solvent extracts of _Ruta gravelens_ used leaves, stems and seeds of the plant, extracted by four different solvents, receiving ethanolic, methanolic, chloroform and aqueous extracts. In this work, the disk diffusion method showed that _B. subtilis_ demonstrated a high zone of inhibition (20 mm) at 200 mg/mL of methanolic extract, but _E. coli_ and _C. tropicalis_ did not show any zone of inhibition against any extract [37]. The lack of _R. gravelens_ plant extract activity against _E. coli_ was also found in the work of other authors, where methanol, petroleum ether, ethyl acetate and water-methanol extracts of the plant aerial parts were tested, also using the disk diffusion method [38]. This phenomenon was also proven in the works of other authors, in which hydro and hydroalcoholic extracts of _Ruta gravelens_ (tested by the disk diffusion method and serial microdilution method) did not show an inhibitory effect on the growth of studied bacteria (i.a. _E. faecalis_, _E. coli_, _K. pneumoniae_, _S. Typhi_) up to a concentration of 5 mg/mL [39]. Bearing in mind the reports showing the lack of antibacterial effect of the _R. gravelens_ extract concluded from outdated research methods, the stimulating effect of RG presented in our work is possible. Moreover, it has been reported that _R. gravelens_ compounds show stimulating activity on the growth of fungi in a concentration-dependent manner, when tested using a 96-well microtiter assay [40]. Even if there are no papers describing the influence of _R. gravelens_ on bacteriophages, the antiviral activity of the plant was confirmed on acyclovir-resistant HSV-1 [41], which can support our findings regarding the RG antiphage activity. Currently, the literature describes the simultaneous application of phages and plant extracts or the effects of extracts on phages at a limited degree, and even fewer works describe their interactions in the environment of bacterial hosts. Previous works have mainly focused on the plaque-forming ability of the phages after their contact with plant extracts [11; 42; 43; 44].