Experimental and Computational Studies on the Interaction of DNA with Hesperetin Schiff Base CuII Complexes.

The interactions with calf thymus DNA (CT-DNA) of three Schiff bases formed by the condensation of hesperetin with benzohydrazide (HHSB or L1H3), isoniazid (HIN or L2H3), or thiosemicarbazide (HTSC or L3H3) and their CuII complexes (CuHHSB, CuHIN, and CuHTSC with the general formula [CuLnH2(AcO)]) were evaluated in aqueous solution both experimentally and theoretically. UV-Vis studies indicate that the ligands and complexes exhibit hypochromism, which suggests helical ordering in the DNA helix. The intrinsic binding constants (Kb) of the Cu compounds with CT-DNA, in the range (2.3-9.2) × 106, from CuHTSC to CuHHSB, were higher than other copper-based potential drugs, suggesting that π-π stacking interaction due to the presence of the aromatic rings favors the binding. Thiazole orange (TO) assays confirmed that ligands and Cu complexes displace TO from the DNA binding site, quenching the fluorescence emission. DFT calculations allow for an assessment of the equilibrium between [Cu(LnH2)(AcO)] and [Cu(LnH2)(H2O)]+, the tautomer that binds CuII, amido (am) and not imido (im), and the coordination mode of HTSC (O-, N, S), instead of (O-, N, NH2). The docking studies indicate that the intercalative is preferred over the minor groove binding to CT-DNA with the order [Cu(L1H2am)(AcO)] > [Cu(L2H2am)(AcO)] ≈ TO ≈ L1H3 > [Cu(L3H2am)(AcO)], in line with the experimental Kb constants, obtained from the UV-Vis spectroscopy. Moreover, dockings predict that the binding strength of [Cu(L1H2am)(AcO)] is larger than [Cu(L1H2am)(H2O)]+. Overall, the results suggest that when different enantiomers, tautomers, and donor sets are possible for a metal complex, a computational approach should be recommended to predict the type and strength of binding to DNA and, in general, to macromolecules.

Encapsulation and Biological Activity of Hesperetin Derivatives with HP-ß-CD.

The encapsulation of insoluble compounds can help improve their solubility and activity. The effects of cyclodextrin encapsulation on hesperetin's derivatives (HHSB, HIN, and HTSC) and the physicochemical properties of the formed complexes were determined using various analytical techniques. The antioxidant (DPPH•, ABTS•+ scavenging, and Fe2+-chelating ability), cytotoxic, and antibacterial activities were also investigated. The inclusion systems were prepared using mechanical and co-evaporation methods using a molar ratio compound: HP-ß-CD = 1:1. The identification of solid systems confirmed the formation of two inclusion complexes at hesperetin (CV) and HHSB (mech). The identification of systems of hesperetin and its derivatives with HP-ß-CD in solutions at pHs 3.6, 6.5, and 8.5 and at various temperatures (25, 37 and 60 °C) confirmed the effect of cyclodextrin on their solubility. In the DPPH• and ABTS•+ assay, pure compounds were characterized by higher antioxidant activity than the complexes. In the FRAP study, all hesperetin and HHSB complexes and HTSC-HP-ß-CD (mech) were characterized by higher values of antioxidant activity than pure compounds. The results obtained from cytotoxic activity tests show that for most of the systems tested, cytotoxicity increased with the concentration of the chemical, with the exception of HP-ß-CD. All systems inhibited Escherichia coli and Staphylococcus aureus.

Spectroscopic Characterization and Biological Activity of Hesperetin Schiff Bases and Their Cu(II) Complexes.

The three Schiff base ligands, derivatives of hesperetin, HHSB (N-[2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene]isonicotinohydrazide), HIN (N-[2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene]benzhydrazide) and HTSC (N-[2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene]thiosemicarbazide) and their copper complexes, CuHHSB, CuHIN, and CuHTSC were designed, synthesized and analyzed in terms of their spectral characterization and the genotoxic activity. Their structures were established using several methods: elemental analysis, FT-IR, UV-Vis, EPR, and ESI-MS. Spectral data showed that in the acetate complexes the tested Schiff bases act as neutral tridentate ligand coordinating to the copper ion through two oxygen (or oxygen and sulphur) donor atoms and a nitrogen donor atom. EPR measurements indicate that in solution the complexes keep their structures with the ligands remaining bound to copper(II) in a tridentate fashion with (O-, N, Oket) or (O-, N, S) donor set. The genotoxic activity of the compounds was tested against model tumour (HeLa and Caco-2) and normal (LLC-PK1) cell lines. In HeLa cells the genotoxicity for all tested compounds was noticed, for HHSB and CuHHSB was the highest, for HTSC and CuHTSC-the lowest. Generally, Cu complexes displayed lower genotoxicity to HeLa cells than ligands. In the case of Caco-2 cell line HHSB and HTSC induced the strongest breaks to DNA. On the other side, CuHHSB and CuHTSC induced the highest DNA damage against LLC-PK1.

From the Physicochemical Characteristic of Novel Hesperetin Hydrazone to Its In Vitro Antimicrobial Aspects.

Microorganisms are able to give rise to biofilm formation on food matrixes and along food industry infrastructures or medical equipment. This growth may be reduced by the application of molecules preventing bacterial adhesion on these surfaces. A new Schiff base ligand, derivative of hesperetin, HABH (2-amino-N'-(2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene)benzohydrazide), and its copper complex, CuHABH [CuLH2(OAc)], were designed, synthesized and analyzed in terms of their structure and physicochemical properties, and tested as antibacterial agents. Their structures both in a solid state and in solution were established using several methods: FT-IR, 1H NMR, 13C NMR, UV-Vis, FAB MS, EPR, ESI-MS and potentiometry. Coordination binding of the copper(II) complex dominating at the physiological pH region in the solution was found to be the same as that detected in the solid state. Furthermore, the interaction between the HABH and CuHABH with calf-thymus DNA (CT-DNA) were investigated. These interactions were tracked by UV-Vis, CD (circular dichroism) and spectrofluorimetry. The results indicate a stronger interaction of the CuHABH with the CT-DNA than the HABH. It can be assumed that the nature of the interactions is of the intercalating type, but in the high concentration range, the complex can bind to the DNA externally to phosphate residues or to a minor/major groove. The prepared compounds possess antibacterial and antibiofilm activities against Gram-positive and Gram-negative bacteria. Their antagonistic activity depends on the factor-strain test system. The glass was selected as a model surface for the experiments on antibiofilm activity. The adhesion of bacterial cells to the glass surface in the presence of the compounds was traced by luminometry and the best antiadhesive action against both bacterial strains was detected for the CuHABH complex. This molecule may play a crucial role in disrupting exopolymers (DNA/proteins) in biofilm formation and can be used to prevent bacterial adhesion especially on glass equipment.

Structural and Spectral Investigation of a Series of Flavanone Derivatives.

Four flavanone Schiff bases (E)-1-(2-phenylchroman-4-ylidene)thiosemicarbazide (FTSC) (1), N',2-bis((E)-2-phenylchroman-4-ylidene)hydrazine-1-carbothiohydrazide (FTCH) (2), (E)-N'-(2-phenylchroman-4-ylidene)benzohydrazide (FHSB) (3) and (E)-N'-(2-phenylchroman-4-ylidene)isonicotinohydrazide (FIN) (4) were synthesized and evaluated for their electronic and physicochemical properties using experimental and theoretical methods. One of them, (2), consists of two flavanone moieties and one substituent, the rest of the compounds (1, 3, 4) comprises of a flavanone-substituent system in relation to 1:1. To uncover the structural and electronic properties of flavanone Schiff bases, computational simulations and absorption spectroscopy were applied. Additionally, binding efficiencies of the studied compounds to serum albumins were evaluated using fluorescence spectroscopy. Spectral profiles of flavanone Schiff bases showed differences related to the presence of substituent groups in system B of the Schiff base molecules. Based on the theoretically predicted chemical descriptors, FTSC is the most chemically reactive among the studied compounds. Binding regions within human and bovine serum albumins of the ligands studied are in the vicinity of the Trp residue and a static mechanism dominates in fluorescence quenching.

Chelating ability and biological activity of hesperetin Schiff base.

Hydrazone hesperetin Schiff base (HHSB) - N-[(±)-[5,7-dihydroxy-2-(3-hydroxy-4-methoxy-phenyl)chroman-4-ylidene]amino]benzamide has been synthesized and its crystal structure was determined. This compound was used for the formation of Cu(II) complexes in solid state and in solution which were characterized using different spectroscopic methods. The analyses of potentiometric titration curves revealed that monomeric and dimeric complexes of Cu(II) are formed above pH7. The ESI-MS (electrospray ionization-mass spectrometry) spectra confirmed their formation. The EPR and UV-visible spectra evidenced the involvement of oxygen and nitrogen atoms in Cu(II) coordination. Hydrazone hesperetin Schiff base can show keto-enol tautomerism and coordinate Cu(II) in the keto (O(-), N, Oket) and in the enolate form (O(-), N, O(-)enol). The semi-empirical molecular orbital method PM6 and DFT (density functional theory) calculations have revealed that the more stable form of the dimeric complex is that one in which the ligand is present in the enol form. The CuHHSB complex has shown high efficiency in the cleavage of plasmid DNA in aqueous solution, indicating its potential as chemical nuclease. Studies on DNA interactions, antimicrobial and cytotoxic activities have been undertaken to gain more information on the biological significance of HHSB and copper(II)-HHSB chelate species.