## 2 Materials and Methods
### Anti-Ageing Activity of Achillea millefolium Extract  
Skin ageing processes are often associated with the degradation of collagen and elastin fibres present in the skin, which results in the loss of its elasticity and firmness. The ageing process is also accompanied by the development of various types of pigmentation disorders, which result in the appearance of mottled pigmentation (senile lentigo) and melasma on the skin surface [35]. Therefore, the next stage of the study was to assess the ability of the _A. millefolium_ extract to inhibit the activity of collagenase and elastase, whose enzymatic activity is closely linked to the degradation of extracellular matrix proteins such as collagen and elastin. The results of the analyses indicated that the extracts showed a concentration-dependent ability to inhibit the activity of both endopeptidases. At a concentration of 5.0%, the extract inhibited elastase activity by 19.2%, while inhibition of collagenase was stronger and reached 43.8%. In the evaluation of the potential ability of the extract to inhibit hyperpigmentation disorders, 15.5% and 23.4% inhibition of tyrosinase activity was observed for 1% and 5% concentrations, respectively (Figure 7). Reactive oxygen species are an important factor negatively affecting skin cell function. Free radical reactions lead to the disruption of defence and repair mechanisms in the skin. ROS is also produced by UV radiation on the skin, and signs of photo-ageing include a reduction in the number of skin fibroblasts, collagen breakdown, or elastic changes in the dermis [36]. In light of the above, the effect of yarrow extract in vitro on intracellular ROS levels in both UVB-irradiated and non-irradiated keratinocytes and fibroblasts was assessed. _A. millefolium_ extracts, in the concentration range 0.01-5.0%, were shown to reduce ROS levels in both UVB-exposed and non-exposed cells. This effect was observed on both keratinocytes and fibroblasts. In most cases (except UVB-irradiated HDF cells), ROS levels  
Figure 7: Evaluation of the ability of yarrow extract at concentrations of 1.0% and 5.0% to inhibit the activity of collagenase, elastase, and tyrosine. *** \(p<0.0001\). were higher in cells treated with 10.0% extract than in control cells (untreated with extracts) (Figures 8 and 9). The results indicate that the yarrow extract can reduce oxidative stress in skin cells exposed to UVB radiation, protecting them from the negative effects of excessive ROS production in these cells. A slight increase in the number of free radicals in cells after exposure to a 10% concentration of the tested extract may be the result of a large number of  
Figure 8: Effect of yarrow extract at concentrations of 0.01–10.0% on intracellular reactive oxygen species (ROS) levels in non-irradiated and UVB-irradiated HaCaT cells. The control was HaCaT cells not treated with the extract. Data are presented as the mean \(\pm\) SD of three independent experiments, in which each extract concentration was tested in triplicate. ****\(p<0.0001\), ****\(p=0.0019\) versus the control. Figure 9: Effect of yarrow extract at concentrations of 0.01–10.0% on intracellular reactive oxygen species (ROS) levels in non-irradiated and UVB-irradiated HDF cells. The control was HDF cells not treated with the extract. Data are presented as the mean \(\pm\) SD of three independent experiments, in which each extract concentration was tested in triplicate. ****\(p<0.0001\), ****\(p=0.0095\), *\(p=0.0302\) versus the control. -phenolic compounds, such as flavonoids or phenolic acids. The literature data indicate that these phytochemicals act as antioxidants at lower concentrations, neutralizing free radicals. However, at higher concentrations, they may exhibit prooxidant activity and intensify oxidative stress by activating the Fenton reaction, autoxidation of phenolic compounds, disruption of redox homeostasis, and inducing a cytotoxic effect [33; 34; 37; 38; 39; 40].