## 4 Discussion
### The Influence of Flower Extracts on Antioxidant Cellular Status--THP-1 Cell Line  
The antioxidant properties of extracts from both clover species were confirmed in our in vitro studies of the THP-1 cell line. Due to the fact that fluorine inhibits the activity of antioxidant enzymes and causes excessive production of oxygen radicals [4; 24; 25], it served as the initiator of the oxidation process. It can be concluded that in a relatively low concentration (10 uM) fluorine leads to changes in the activity of antioxidant enzymes, disrupts the effective functioning of the antioxidant apparatus, causes inflammation, apoptosis and the necrosis of cells, which is also confirmed by numerous scientific reports [1; 24; 38; 44; 45; 46; 47]. The cations of many elements (calcium, magnesium, manganese, aluminium) can form insoluble complexes with fluorine, which may contribute to the inhibition of the activity of enzymes that depend on those cations. Fluoride ions may also bind to functional groups of amino acids surrounding the active centre of enzymes, resulting in their inhibition [1; 46].  
The addition of flower extracts to the culture resulted in a change in the activity of SOD, CAT and the antioxidant potential of macrophages. On the basis of the acquired results, it can be observed that fluorine that is introduced into a culture, even with plant extracts, causes the inhibition of the studied antioxidant enzymes. We have demonstrated that the addition of a clover extract (red or white) caused a decrease in the excessive oxygen oxidation caused by fluorine [39; 40; 41]. Therefore, flower extracts protect against the toxic fluoride in macrophages of the THP1 line, demonstrated by the statistical analysis (\(p<0.05\)). Our study confirms the findings of other authors who revealed the antioxidant properties of the red clover. The majority of scientific reports concerning the biological activity of _Trifolium_ species contain results of in vitro and in vivo studies on _Trifolium_[6; 37]. [PERSON] et al. also showed that _Trifolium pratense_ showed recovery of liver antioxidant enzymes and lipid peroxidation [48]. In a study on rats that were supplied with bisphenol A (BPA) and a water-ethanol extract from the red clover, it was observed that the extract prevented the influence of BPA on the peroxidation of lipids and had a protective effect [49]. Water extracts of the white clover, rich in phenolic compounds, had an influence on the reduction of lipid peroxidation and the normalisation of the total reduce glutathione in the liver [33].  
The red clover and the white clover contain numerous elements, such as calcium, chromium, iron, magnesium, manganese, copper, selenium and zinc [50]. The antioxidant mechanism of the extract may be associated with the content of manganese, zinc and copper--the cofactors for superoxide dismutase, which unblocked antioxidant enzymes and increased their activity. Moreover, numerous polyphenol compounds present in the extracts might have caused an increase in the activity of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) and contribute to the increase in the concentration of low molecular weight antioxidants (ascorbic acid, a-tocopherol) [51; 52; 53].  
It is also worth highlighting that the antioxidant properties of extracts of the less-known, but readily available clover studied on cell lines show a similar or stronger positive effect on health in comparison to other, often more popular plants, e.g., _Camellia_ extract or _Castanea cerenata_[54; 55].