# Co(II) Complex of Quercetin-Spectral, Anti-/Pro-Oxidant and Cytotoxic Activity in HaCaT Cell Lines
## 3 Results
### Antioxidant Activity  
The antioxidant activity of the Co(II) complex of quercetin was studied by means of DPPH and FRAP spectrophotometric assays. The first relied on the reduction of DPPH\({}^{\bullet}\) radicals by antioxidants. With the increase in the concentration of quercetin and the Co(II)-quercetin complex, the antiradical activity against DPPH\({}^{\bullet}\) increased too (Figure 6). The determined value of IC\({}_{50}\) for quercetin was 3.88 \(\pm\) 0.06 uM, whereas for the Co(II) complex it was 2.01 \(\pm\) 0.25 uM. This means that its complexation with Co(II) increased the antiradical properties of quercetin (measured in the DPPH assay). According to the data, both synthesized Cu(II) and Fe(II) complexes with quercetin showed stronger antioxidant activity compared to the flavonoid alone in the DPPH assay [16]. In another study, Co(II)-quercetin and Cd(II)-quercetin complexes showed a higher antioxidant activity measured in the DPPH assay than in the ligand, i.e., 74.20, 82.31, and 48.43%, respectively (after 30 min, C = 0.01 M) [17]. In the work conducted by [PERSON] et al., the DPPH\({}^{\bullet}\) radical scavenging activity of Fe(II)-quercetin after a 30 min reaction was approximately 97%, while for quercetin it was only about 85% (C = 20 uM) [15]. [PERSON] et al. reported a greater antioxidant activity of the Co(II)-quercetin complex compared to the flavonoid alone. For example, in the DPPH assay conducted by the researchers, the % of the DPPH\({}^{\bullet}\) remaining for quercetin was equal to around 80%, while for the complex it was around 50% (C = 4 uM) [20]. In their other work, Cu(II)-quercetin complex also showed higher antioxidant activity, measured in the DPPH assay, than the ligand, e.g., the percentage of DPPH\({}^{\bullet}\) remaining with the complex was around 50%, and for quercetin it was around 90% (C = 4 uM) [21]. However, there are also reports in the literature of reduced antioxidant activity of quercetin after complexation with metals. For example, in the work of [PERSON] et al., quercetin showed higher DPPH\({}^{\bullet}\) radical scavenging activity than the Pb(II)-quercetin complex. In their research, after 15 min of incubation, it was found that quercetin inhibits DPPH\({}^{\bullet}\) radicals by 4.08, 23.90, and 30.10%, while Pb(II)-quercetin only inhibits them by 1.2, 4.69, and 5.8%, at concentrations of 5, 10, and 15 uM, respectively [22]. In a study conducted by [PERSON] et al., a Sb(II)-quercetin complex was found to have lower antioxidant activity than the ligand alone. The IC\({}_{50}\) values obtained in the DPPH\({}^{\bullet}\) assay were equal to 13.46 mg/L and 3.78 mg/L, for the complex and the ligand, respectively [23]. Similar results as in the DPPH test were obtained in the ferric-reducing antioxidant assay FRAP (Figure 7), where the Fe(III)-TPTZ complex is reduced, using an antioxidant, to a Fe(II)-TPTZ complex. The reducing activity of studied compounds expressed in FRAP values were: 566.23 \(\pm\) 18.52 and 830.16 \(\pm\) 15.56 uM Fe\({}^{2+}\), respectively. Both selected tests differ in their mechanism of action. The FRAP assay is described as a totally SET (single electron transfer) reaction, where one electron is transferred from an antioxidant to reduce the number of metal ions or radicals [42], whereas the reaction of antioxidants with DPPH\({}^{\bullet}\) can be describe as having SET or hydrogen atom transfer (HAT) mechanisms (or the  
Figure 6: (a) The IC\({}_{50}\) values for quercetin (Q) and Co(II) complex of quercetin (Co-Q); (b) the scavenging of DPPH\({}^{\bullet}\) by Q and Co-Q. Mean values from three independent experiments \(\pm\) SD are shown. combination of them) [43; 44]. Moreover, the type of solvent and the pH of the environment do not affect HAT-type reactions, unlike in the SET reaction, which depends on the acidity of the environment [45]. The DPPH assay was conducted in a methanolic solution, whereas FRAP was more often in an aqueous environment, which may affect the rate of reaction and compound solubility.