## 3 Results and Discussions
### In Vitro Bioactive Antioxidant Assays  
The UE extract of both _Centaurea_ plant were selected for the in vitro cell viability test based on the preliminary study of the antioxidant ability. The cytotoxicity of these extracts against human skin fibroblast HS-68 to increasing concentrations has been determined by evaluating viability of tested cell lines after 24 h using MTT assay. The results were expressed in terms of % of vitality cells compared to the control (non-treated cells).  
Treatment with UE extracts, in concentrations between 0.02 and 0.32 ug GAE/mL, has not induce any significant decrease in cells viability compared to non-treated cells, as shown in Figure 4. Our results appear to be well supported by previous investigations that report a hardly any or no cytotoxicity and antiproliferative effect of _Centaurea_ extracts at low dose against human cell lines, e.g., NALM-6, REH, NB4, KMM-1 [42], HepG2 [43], Caco-2 [44], and A549 [19].  
Remarkably, an increase of cell vitality (11.6 \(\pm\) 1.15%) was observed for cells treated with 0.16 ug/mL of UE extract of _C. sicula_, which indicate a proliferative effect towards this skin fibroblast cell line. Otherwise, the effect of ethanol (EtOH) on HS-68 cells has been evaluated and no effect has been observed. The concentration 0.16 ug/mL have been selected for both _C. sicula_ and _C. kroumiensis_ in order to evaluate the antioxidant ability in cells. The protective effect test of the UE _Centaurea_ extracts after the induction of oxidative stress was carried out for 24 h. Hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) is one of the ROS that attack cellular components, leading to cell damage and limiting their viability [45].  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Centaurea Specie** & **Variables** & **TPC** & **DPPH (IC\({}_{50}\))** & **RP (EC\({}_{50}\))** \\ \hline C. kroumiensis (Coss.) & TPC & – & \(-\)0.758 * & \(-\)0.646 * \\ \cline{2-5}  & DPPH (IC\({}_{50}\)) & – & – & 0.676 * \\ \hline C. sicula L. subsp sicula & TPC & – & \(-\)0.959 * & \(-\)0.846 * \\ \cline{2-5}  & DPPH (IC\({}_{50}\)) & – & – & 0.802 * \\ \hline \hline \end{tabular}
* Correlation is significant at the level.  
\end{table}
Table 2: Correlation (Pearson) and relationships between antioxidant capacity and (TPC) in _Centaurea_ extracts. Values are with a significance (\(p\) < 0.05).  
A significant decrease (\(21.45\pm 5.82\%\)) in cell vitality, determined by MTT test (\(p<0.05\)), was noted following the exposure of fibroblast cells to a well-known concentration of H\({}_{2}\)O\({}_{2}\) (Control + HP), as shown in Figure 5. However, cells treated with UE of both _Centaurea_ extracts exhibited a strong protective effect comparable to that of the natural antioxidant gallic acid (GAE) and to the synthetic antioxidant N-acetilcysteine (NAC). These findings indicate that these extracts possess therapeutic effects, such as inhibition lipid oxidation and the prevention of free radicals' damage, and could be developed as versatile adjuvant in pharmaceutical, food, and cosmetics applications.  
Figure 4: Percentage of vitality of HS-68 fibroblast cells, obtained with MTT test (each data is presented as mean \(\pm\) SD, \(n=5\)). Cells treated with UE extracts of (_C. kroumierienis_ and _C. sicula_) for 24 h to concentrations 0.02, 0.08, 0.16 and 0.32 ug/mL (0.1% \(<\) ethanol). Control (cells without treatment) (Different letters indicate significant difference (\(p<0.05\))).  
Figure 5: Effect of UE _Centaurea_ extracts on HS-68 fibroblast cells exposed to oxidative stress, induced by H\({}_{2}\)O\({}_{2}\). HP = hydrogen peroxide, Control cells, NAC = cells pretreated with the synthetic antioxidant NAC, GAE = cells pretreated with gallic acid, cells pretreated with UE extract of _C. kroumierienis_, cells pretreated with UE extract of _C. sicula_. (Different letters indicate significant difference (\(p<0.05\))).