Beáta Veliká

Effects of humic acids in vitro

Humic acids are known for their overall positive health and productivity effects in animal feeding trials and, controversially, as an aetiological factor of cancer. We tried to assess the in vitro effect of humic acids from a selected source in Slovakia when used at recommended prophylactic dosage. We investigated antioxidant properties, enzymatic and non-enzymatic antioxidant defence system in liver mitochondria and cultured cancer cell lines in vitro. We observed a significant decrease in superoxide dismutase activity after humic acids treatment irrespective of dissolving in dimethyl sulphoxide or direct addition to mitochondria suspension in a respiration medium. Activities of other antioxidant enzymes measured, such as glutathione peroxidase and glutathione reductase, showed no significant differences from the control as well as the reduced glutathione content. Percentage of inhibition by humic acids of superoxide radical indicated lower efficacy compared with that of hydroxyl radical. Survival of six different cancer cells lines indicated that only the acute T lymphoblastic leukaemia cell line was sensitive to the tested humic acids. Despite relatively low solubility in aqueous solutions, humic acids from the selected source participated in redox regulation. By recapturing the radicals, humic acids reloaded the antioxidant defensive mechanism. Results from in vitro study conducted with humic acids from the natural source showed potential of these substances as promising immunity enhancing agents.

(E)-2-Benzylidenebenzocyclanones, part VIII: spectrophotometric determination of pK a values of some natural and synthetic chalcones and their cyclic analogues

UV–Vis spectrophotometry was used to determine acid dissociation constant (pKa) values of the natural flavonoids phloretin, phlorizin, naringenin, and naringin, as well as 4′-hydroxychalcone, 4′-(dimethylamino)chalcone, and their cyclic analogues. Comparison of the results with those previously reported for the natural flavonoids showed the applied method is a relatively straightforward and easy-to-perform technique for the determination of pKa values of compounds with relatively low solubility. Comparative analysis of the pKa values of the synthetic chalcones showed a strong correlation between the degree of conjugation and the acid strength of the respective compounds with different geometry. Our results provide further evidence that modification of the three-dimensional structure of open-chain bioactive compounds is the method of choice to modify not only their stereochemistry but also their physicochemical properties.Graphical abstract

Determination of Excited Singlet-State Dipole Moments of Methoxy and Dimethylamino Substituted Benzylidenebenzosuberones Using Solvatochromic Method

The solvent effects on the electronic absorption and emission fluorescence spectra for a series of chalcone cyclic analogues were studied. The singlet-state excited dipole moments and the ground state dipole moments of the cyclic chalcone analogues E-2- benzylidene-1-benzosuberone E-2-(4′-methoxybenzylidene)-1-benzosuberone E-2-(4′-dimethylaminobenzylidene)-1-benzosuberone were calculated by using solvatochromic shift method by means of equations using the variations of Stokes’ shift with the solvents dielectric constant and refractive index values. It was found that the excited state dipole moments calculated by the solvatochromic shift method were greater than the ground state dipole moments indicating a substantial redistribution of the pi-electron densities in a more polar excited state for each derivative.

Hydroxybenzoic acids and their derivatives as peroxynitrite scavengers.

A social challenge of the 21(st) century is to reduce the incidence of chronic diseases. A balanced diet rich in polyphenols could contribute to reduce the risk and to the prevention of diabetes, coronary heart disease, cancer, Alzheimers diseases and cataract(1). Hydroxybenzoic acids (HBA) and their derivatives, which are one of the substances responsible for these beneficial properties, are known mainly due to their antioxidant properties(2). They are effective scavengers of free radicals and reactive nitrogen species, such as peroxynitrite. Peroxynitrite is resulting from the reaction of nitric oxide with superoxide, causes lipid peroxidation and subsequent cellular damage and is responsible for the inactivation of many enzymes, activation of stress signalling pathways, release of proapoptotic factors, as well as cardiovascular dysfunction in septic schock(3). In this study we have tested 2-HBA, 3-HBA, 4-HBA, acetylsalicylic acid, 4-HBA methyl and propyl esters, 2,3-dihydroxybenzoic acid (DHBA), 2,5-DHBA, 2,4-DHBA, 2,6-DHBA, 3,5-DHBA, 3,4-DHBA, gallic acid and caffeic acid, by UV/VIS spectroscopy. The best ability to scavenge peroxynitrite was observed for gallic acid, 2,4-DHBA, 3,5-DHBA and caffeic acid. Improved comprehension of the complex relationship between the antioxidant properties of substances and their structure is important to understand their proper use in the prevention and treatment of diseases and for the detection of pathological processes. Monitoring and improved understanding of the antioxidant properties of hydroxybenzoic acid derivatives are important due to their frequent use in modern medical nutrition therapies.

Fluorescence Properties of Paracetamol During Interactions with Mitochondria

ABSTRACT Paracetamol interaction with rat liver mitochondria in respiration media in the presence of succinate was the focus of this experiment. Fluorescence of paracetamol in water was studied by three-dimensional synchronous fluorescence fingerprint (SFF) and by excitation emission matrix (EEM). The direct molecular interactions of paracetamol and mitochondria were studied by fluorescence polarization technique. The paracetamol fluorescence maximum of SFF was Δλ = 110/λex = 320 nm, Fmax = 508 nm, and EEM maximum was λex = (320 nm)/λem = 425 nm, Fmax = 508. The fluorescence polarization results showed nonsignificant elevation of fluorescence polarization after addition of paracetamol into mitochondria in comparison to the control mitochondria group without paracetamol at time point t = 0. Paracetamol probably covalently bound to the mitochondrial surface proteins at time point t = 0, but paracetamol also entered mitochondria, which was observed as nonsignificant decline of fluorescence polarization during 30 min in the paracetamol-treated group. The practical advantages of spectral techniques (EEM, SFF, fluorescence polarization) are high sensitivity, reproducibility, minimal quantity of material, and capability to measure the mitochondrial autofluorescence.

Prooxidative and fluorescence properties of paracetamol during interactions with mitochondria

This study was carried out to investigate isolated liver mitochondrial functions after paracetamol administration by monitoring of liver mitochondrial fluorescence properties as well as prooxidative properties of paracetamol. Paracetamol was administered to rat (in subtoxic 500 mg · kg −1 dose) in vivo. The effect of this dose was compared with the subtoxic and toxic dose of paracetamol added to mitochondria in vitro (1 and 1.5) mg paracetamol/mg mitochondrial protein. Subtoxic dose of paracetamol in vitro did not change mitochondrial fluorescence, but it significantly decreased mitochondrial fluorescence in vivo in comparison with control mitochondrial group. Toxic dose of paracetamol in vitro significantly decreased mitochondrial fluorescence. The enzymatic activity of superoxide dismutase (SOD) significantly decreased after paracetamol administration in vitro and in vivo. While both activities of glutathione peroxidase (GPx) and glutathione reductase (GR) significantly increased in dependence upon paracetamol doses. Our experiment showed, that paracetamol participates in formation of free radicals and confirms previous studies, in which paracetamol administration caused elevation of antioxidative enzymes activities in dependence on dose, which is considered therapeutically as subtoxic and toxic.

Kinetic measurements of peroxynitrite scavenging properties of hydroxybenzoates

Objective: Hydroxybenzoic acids and their derivatives belongs to phenols, which are characterized by salubrious properties, leading to a reduction of the incidence of chronic diseases. The most significant health related features of hydroxybenzoic acids and their derivatives are their antioxidant properties. Peroxynitrite is a reactive nitrogen compound which can cause lipid peroxidation with subsequent cellular damage. The aim of the present work was to determine the peroxynitrite scavenging property of different hydroxybenzoates, with regard to their chemical structure. Methods: In our study, we tested 14 hydroxybenzoic acids and a number of their derivatives in the reaction with peroxynitrite. The compounds were selected to have different number and different position of the hydroxyl group(s) attached to the benzene ring. In this way we ensured the study of different suitable model compounds by testing their antioxidant properties. The peroxynitrite scavenging properties of the model compounds were studied using kinetic spectrophotometric measurements. The reaction rate was calculated based on the decrease in peroxynitrite concentration. Results: The most significant peroxynitrite scavenging properties based on the reaction rate were detected in compounds with hydroxyl groups substituted in the positions meta and para: 3,4-dihydroxybenzoic acid, gallic acid and caffeic acid. Also, increased reaction rates were recorded in the reaction of 2,4-dihydroxybenzoic acid, acetylsalicylic acid and 2-hydroxybenzoic acid with peroxynitrite. Conclusion: Our results confirmed the thesis that the free radical scavenging properties are dependent on the position and the number of the hydroxyl group(s) in relation to the carboxylic functional group attached to the benzene ring. Better understanding of the relations between the structure and the action of these antioxidants may be helpful in their proper utilization during prevention and treatment of diseases.