## 5 Bioactivity of Secondary Metabolites in the _Leontopodium_ Taxa
### Antioxidant Activity  
The antioxidant activity can be described as the property of a given compound to inhibit or decrease the oxidation of its substrate in low concentrations [119]. An antioxidant \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Bioactivity** & \multicolumn{2}{c}{**Taxon/Extract/Bioactive Compound(s)**} & **Experimental Model** & **Effects/Applied Tests** & **Ref.** \\ \hline \multirow{4}{*}{Antifungal activity} & _L. alpium_ & _Candida glabrata_ & - & low inhibition on biofilm formation & [29] \\  & - & ethanolic extracts from different plant parts & _Candida glabrata_ & - & low inhibition on biofilm formation & [29] \\  & _L. leontopodioides_ & & & & \\  & - & crude extract from whole plant & _Trichophyta_ & - & low anti-dermatophyte activity & [116] \\ \hline \multirow{4}{*}{Antiparasitic activity} & _L. campestre_ & Human fibroblasts cells (HFF) & - & anti-Toxoplasma activity & \\  & - & root extracts-flavonoids, flavones and lignans & infected with tachyozoites from & - & anti-Toxoplasma activity & \\  & & _Toxoplasma gondii_ & - & (IC50 40.39 \(\mathrm{\SIUnitSymbolMicro mL}\)) & \\ \cline{2-6}  & & or & - & anti-Plasmodium activity & [32] \\ \cline{1-1}  & & synchronous cultures with & & & activity \\ \cline{1-1}  & & parasite _Plasmodium falciparum_ & - & (IC50 & \\ \cline{1-1}  & & 3D7 strain & & 19.705 \(\mathrm{\SIUnitSymbolMicro mL}\)) & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: _Cont._acts on free radicals that negatively affect biological systems by neutralizing them through electron donation or by breaking down processes [120]. In this respect, different ex vitro tests, such as the Briggs-Rauscher oscillating reaction or antioxidant capacity assay using different synthetical substrates, highlighted the antioxidant activity of _L. aluminum_ extracts or the isolated compounds. Among them, antioxidant activities were estimated using the Trolox equivalent antioxidant capacity (TEAC) method [5,7] or by using the DPPH synthetic radical [35,39]. Studies have also confirmed the antioxidant efficacy of leontopodic acid and chlorogenic acid, major compounds of _Leontopodium_ cell culture extracts. These compounds seemed to protect cellular DNA against oxidative damage induced in vivo, in a 3D assay, a method based on the measurement of the degree up to which a particular compound can protect DNA from free radicals [5,116]. In essential oils from the aerial part of two other species, _L. leontopodioides_ and _L. longifolium_, only weak DPPH scavenging activity was detected [34,37]. The ability of callus culture extracts and the main compounds, such as leontopodic and chlorogenic acids, to reduce the levels of reactive oxygen species (ROS) has also been demonstrated in in vitro model systems (Table 4), mostly represented by cultures of human fibroblasts and keratinocytes but also the other cell cultures as histiocytic lymphoma line (U937), pig kidney cells line (LLC-PK1) or human neuroblastoma cells line (SH-SYSY). The antioxidant activity of callus culture extracts was also demonstrated using the cytotoxicity test of hydrogen peroxide applied in the HaCaT line [34]. The viability of cells treated with 1% extract was comparable to that of N-acetyl cysteine used as a control antioxidant. The decrease in intracellular ROS was promoted, also, by extracellular vesicles derived from callus cultures treated with LED light, using the same cell line and UVB irradiation [18]. Regarding the antioxidant activity of leontopodic acid, two studies demonstrated its cytoprotective activity against mycotoxins, and only one highlighted its antioxidant activity. The first showed that leontopodic acid increased the activity of glutathione peroxidase in U937 cells treated with deoxynivalenol [6]. In the second study, leontopodic acid reduced ROS formation in LLC-PK1 cells exposed to ochratoxin A and in the SH-SYSY line exposed to amyloid \(\beta\) aggregates [121].