## 3 Results and Discussion
### The Effect on VERO Cells  
Due to the possibility of the use in the preparation of food and beverages, we focused on evaluating the effect of prepared green walnut timetures and their combinations with different spices on normal monkey epithelial kidney cells (VERO) as the kidney is the target organ of the elimination of substances from the body. VERO cells are used in testing the toxicity of chemical compounds as well as, to assess the biological safety of new substances used in therapy and in the food industry [35; 36]. The effect of tinctures on VERO cells was evaluated in real-time using the xCELLigence system that allows for continuous monitoring of cell status during the entire time of cell exposure to tested substances. The specific RTCA patterns observed in VERO cells exposed to tested tinctures and tinctures with spice additives are presented in Figure 3.  
Figure 2: Example chromatograms of tinctures 1 and 4. Peak numbers correspond to the numbering in Table 4.  
The proliferation of cells treated with samples 3, 4 and 5 was significantly inhibited with a growing concentration of samples (\(p<0.05\)). The highest concentrations in almost all tested samples caused a decline in CI which indicates that no cells adhered to the well bottom (CI = 0) or that inhibition of cell proliferation occured (CI does not change in time); cells are still alive but do not multiply [37]. There were also cases where the cells treated with the samples showed a higher proliferative activity than the control cells: sample 3 at the lowest concentration, sample 5 at the concentration of 250 \(\upmu\)g mL\({}^{-1}\). For sample 4, a significant reduction in the proliferation rate was observed even at the lowest tested concentration. Phenolic compounds present in walnuts show the ability to decrease the incidence of tumours which has been reported by many authors [21; 22; 23; 38]. Interestingly, lower concentrations (125 \(\upmu\)g mL\({}^{-1}\)) present in samples 2, and 5 did not affect cell proliferation which may indicate no cytotoxic effect and the potential for safe use of tinctures in beverages. Using the RTCA, various plant extracts were tested for their effect on cell viability. Various relationships were observed, e.g., for giant goldenrod (_Solidago gigantea_) extracts,  
Figure 3: Real-time monitoring of cell response after exposure to tested substances (CI). Significant differences compared to control cells: *\(p<0.05\), **\(p<0.001\), ***\(p<0.0001\). lower concentrations increased the adherence of intestinal porcine epithelial cells IPEC-1, while higher concentrations acted negatively [30]. _Viscum album_ extracts were tested on various tumour cell lines using this method. It was shown that for some cells these extracts are highly cytotoxic while for others, are not [22]. It also happened that the given extract significantly supports cell adherence, as in the case of the acetone dandelion (_Taraxacum officinal_) flower extract effect applied to RK13 cells [29]. For the evaluation of changes in cell viability by the measurement of metabolic activity, the MTS test was employed. This end-point analysis was conducted after 48 h of cell treatment. Almost all tested samples cause a decrease in metabolic activity, except for sample 4 (125 and 250 \(\upmu\)g mL\({}^{-1}\)) in which the change was not statistically important (\(p>0.05\)). The decrease in metabolic activity exhibits a dose-dependent trend. Changes in metabolic and proliferative activity 48 h after cell treatment are summarized in Table 5.  
The effect of the addition of spices on the metabolic activity was diversified and a clear effect was observed in the case of cloves addition (sample 4). Such a spice reduced the cytotoxicity as compared to the control timetures (1 and 2) and at lower concentrations, metabolic activity of cells was not significantly different from the value of untreated cells. The weakened cytotoxicity was also observed in samples 5 and 6, however, the changes were less pronounced.