Kaempferol is a flavonoid that occurs in tea and in many vegetables and fruits, including broccoli, cabbage, beans, grapes, apples, and strawberries. The efficacy of Kaempferol has been demonstrated in the treatment of breast, esophageal, cervical, ovarian, and liver cancers and leukemia, which very likely arises from its prooxidant properties and the activation of pro-apoptotic pathways. Indeed, this matter has already been the focus of a number of published studies and reviews. The aim of the present study was to elucidate the antioxidant vs. prooxidant properties of flavonoids in the presence of the redox-active metal, copper (II) ion, by means of the Fenton reaction. The specific motivation of this work is that, since an increased level of Cu(II) ions is known to be associated with many disease states such as neurological conditions (Alzheimer’s disease) and cancer, any interaction between these ions and flavonoids might affect the outcome of therapeutic uses of the latter. The structure of the Cu-kaempferol complex in DMSO was investigated by means of low temperature EPR spectroscopy, which confirmed the existence of at least two distinct coordination environments around the copper (II) ion. UV vis-spectra of kaempferol and its Cu(II) complex in DMSO revealed an interaction between the 5-OH (A ring) group and the 4-CO (C ring) group of kaempferol with Cu(II) ions. An ABTS assay confirmed that kaempferol acted as an effective radical scavenger, and that this effect was further enhanced in the form of the Cu(II)-kaempferol complex. Quantitative EPR spin trapping experiments, using DMPO as the spin trap, confirmed suppression of the formation of a mixture of hydroxyl, superoxide, and methyl radicals, in a Fenton reaction system, upon coordination of kaempferol to the redox-active Cu(II) ions, by 80% with respect to the free Cu(II) ions. A viscometric study revealed a better DNA-intercalating ability of the Cu-kaempferol complex than for free kaempferol, essential for conferring anticancer activity of these substances. The results of the viscometric measurements were compared with those from a DNA damage study of Cu-kaempferol complexes in a Fenton reaction system, using gel electrophoresis. At low concentrations of kaempferol (Cu–kaempferol ratios of 1:1 and 1:2), a very weak protective effect on DNA was noted, whereas when kaempferol was present in excess, a significant DNA-protective effect was found. This can be explained if the weakly intercalated kaempferol molecules present at the surface of DNA provide protection against attack by ROS that originate from the Fenton reaction involving intercalated Cu(II)-kaempferol complexes. Following the application of ROS scavengers, L-histidine, DMSO, and SOD, gel electrophoresis confirmed the formation of singlet oxygen, hydroxyl radicals, and superoxide radical anions, respectively. We propose that the prooxidant properties of Cu-kaempferol complexes may provide anticancer activity of these substances. When present in excess, kaempferol displays antioxidant properties under Cu-Fenton conditions. This suggests that kaempferol might prove a suitable candidate for the prevention or treatment of oxidative stress related medical conditions that involve a disturbed metabolism of redox metals such as copper, for example, Menkes disease, and neurological disorders, including Alzheimer’s disease. For the potential use of kaempferol in clinical practice, it will be necessary to optimize the dose size and critical age of the patient so that this flavonoid may be beneficial as a preventive drug against cancer and neurological disorders.

The beneficial effects of polyphenols, predominantly in the context of oxidative stress-related diseases such as cancer, cardiovascular diseases and neurological conditions including Alzheimer’s and Parkinson’s diseases, have been documented by a number of papers and reviews. The antioxidant/prooxidant properties of phenolic compounds are related mainly to the number and positions of hydroxyl groups and to their redox metal (Cu, Fe) chelating capacity. In this work we studied structurally distinct phenolic molecules such as myricetin, morin, 3′,4′-dihydroxy-flavone, taxifolin and 4-hydroxycoumarin, either alone or as interacting with Cu²⁺ ions. EPR and UV-Vis spectroscopy confirmed that the effective binding of cupric ions to phenolic compounds requires the presence of the 3-OH and 4-CO groups on the flavonoid C ring and unsaturated C2-C3 bond of the C-ring, which permits through-conjugation with the B-ring. An ABTS assay revealed that radical scavenging activities of phenolic compounds are related to their number of hydroxyl groups, planarity of the molecular skeleton, extent of delocalization and they decrease in the order: myricetin > morin > 3′,4′-dihydroxyflavone ~ 4-hydroxy coumarin > taxifolin. Absorption titrations indicate that copper ions can modulate the DNA binding affinity of flavonoids via the formation of their Cu-chelates. Gel electrophoresis measurements indicated that the protective effect of the phenolic compounds decreases in the order: 3′,4′-dihydroxyflavone > 4-OH coumarin > morin > taxifolin ~ myricetin. This can be explained by the fact that myricetin, taxifolin and morin form stable Cu(II) complexes capable of causing DNA damage via interaction with DNA and ROS formation via the Fenton reaction. Application of ROS scavengers revealed the formation of singlet oxygen, superoxide and hydroxyl radicals and their concerted synergistic effect on the DNA. The overall results suggest that the most pronounced DNA damage has been observed for flavonoids containing higher number of hydroxyl groups (including 3-OH group of the C ring), such as myricetin (six hydroxyl groups), morin and taxifolin (five hydroxyl groups) in the presence of Cu(II) ions. The proposed mechanism of action by which Cu(II) complexes of myricetin, morin and taxifolin interact with DNA predispose these substances to act as potential anticancer agents. The anticancer activity of phenolic compounds can be explained by their moderate prooxidant properties, which can boost ROS formation and kill cancer cells. Alternatively, slight prooxidant properties may activate antioxidant systems, including antioxidant enzymes and low molecular antioxidants such as glutathione and thus act as preventive anticancer agents.

Polyphenolic compounds such as flavonoids are closely linked with therapeutical approaches in oxidative stress related diseases mainly because of their antioxidant and metal binding properties. The formation of metal ion complexes can significantly improve the biological activity of the parent flavonoid e.g. during DNA-intercalation process with consequences on its therapeutical functions. The aim of this work is to describe the interaction between the studied set of structurally different flavonoids (kaempferol, luteolin, fisetin and apigenin) and Cu(II) ions. Formation of stable Cu(II) complexes of kaempferol, luteolin, fisetin and apigenin is confirmed by UV-Vis spectroscopy in methanolic solution. Changes in the Vis-region clearly document the complexation of Cu(II) ions, what is further confirmed by the calculated TD-DFT electronic transitions. According to the calculated DFT energies the Cu acceptor site preference decreases in the order Cu1 > Cu2 > Cu3. The molecular docking studies demonstrate a better intercalating ability towards the DNA of Cu(II)–flavonoid complexes when compared to free flavonoids. In particular, Cu1–apigenin and Cu1–kaempferol are identified as the most promising intercalating agents. The obtained results indicate the mechanism by which studied Cu(II)–flavonoid complexes are formed and may interact with DNA, what predispose such substances to act as potential therapeutics with DNA as a target molecule.

Cooper(ii) complexes represent a promising group of compounds with antimicrobial and antifungal properties. In the present work, a series of Cu(ii) complexes containing the non-steroidal anti-inflammatory drugs, tolfenamic acid, mefenamic acid and flufenamic acid as their redox-cycling functionalities, and 1,10-phenanthroline as an intercalating component, has been studied. The antibacterial activities of all three complexes, [Cu(tolf-O,O′)₂(phen)] (1), [Cu(mef-O,O′)₂(phen)] (2) and [Cu(fluf-O,O′)₂(phen)] (3), were tested against the prokaryotic model organisms Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) and their antifungal activities were evaluated towards the yeast, Saccharomyces cerevisiae (S. cerevisiae). The antibacterial activity of both strains has been compared with the antibiotic Neomycin. The calculated IC₅₀ values revealed slight differences in the antibacterial activities of the complexes in the order 1 ∼ 3 > 2. The most profound growth inhibition of E. coli was observed, at its highest concentration, for the complex 1, which contains chlorine atoms in the ligand environment. The trend obtained from IC₅₀ values is generally in agreement with the determined MIC values. Similarly, the complex 1 showed the greatest growth inhibition of the yeast S. cerevisiae and the overall antifungal activities of the Cu(ii) complexes were found to follow the order 1 > 3 ≫ 2. However, for complex 2, even at the highest concentration tested (150 μM), a 50% decrease in yeast growth was not achieved. It appears that the most potent antimicrobial and antifungal Cu(ii) complexes are those containing halogenated NSAIDs. The mechanisms by which Cu(ii) complexes cause antibacterial and antifungal activities can be understood on the basis of redox-cycling reactions between cupric and cuprous species which lead to the formation of free radicals. The higher efficacy of the Cu(ii) complexes against bacterial cells may be due to an absence of membrane-protected nuclear DNA, meaning that on entering a cell, they can interact directly with its DNA. Contrastingly, for the complexes to interact with the DNA in yeast cells, they must first penetrate through the nuclear membrane.