Bożena Tyrakowska

The influence of pH on antioxidant properties and the mechanism of antioxidant action of hydroxyflavones.

The effect of the pH on antioxidant properties of a series of hydroxyflavones was investigated. The pKa of the individual hydroxyl moieties in the hydroxyflavones was compared to computer-calculated deprotonation energies. This resulted in a quantitative structure activity relationship (QSAR), which enables the estimation of pKa values of individual hydroxyl moieties, also in hydroxyflavones for which these pKa values are not available. Comparison of the pKa values to the pH-dependent antioxidant profiles, determined by the TEAC assay, reveals that for various hydroxyflavones the pH-dependent behavior is related to hydroxyl moiety deprotonation, resulting in an increase of the antioxidant potential upon formation of the deprotonated forms. Comparison of these experimental results to computer calculated O-H bond dissociation energies (BDE) and ionization potentials (IP) of the nondeprotonated and the deprotonated forms of the various hydroxyflavones indicates that especially the parameter reflecting the ease of electron donation, i.e., the IP, and not the BDE, is greatly influenced by the deprotonation. Based on these results it is concluded that upon deprotonation the TEAC value increases (radical scavenging capacity increases) because electron-, not H*-, donation becomes easier. Taking into account that the mechanism of radical scavenging antioxidant activity of the neutral form of the hydroxyflavones is generally considered to be hydrogen atom donation, this implies than not only the ease of radical scavenging, but also the mechanism of antioxidant action changes upon hydroxyflavone deprotonation.

Prooxidant toxicity of polyphenolic antioxidants to HL-60 cells: description of quantitative structure-activity relationships

Polyphenolic antioxidants exhibited a dose‐dependent toxicity against human promyelocytic leukemia cells (HL‐60). Their action was accompanied by malondialdehyde formation, and was partly prevented by desferrioxamine and the antioxidant N,N′‐diphenyl‐p‐phenylene diamine. This points to a prooxidant character of their cytotoxicity. A quantitative structure‐activity relationship (QSAR) has been obtained to describe the cytotoxicity of 13 polyphenolic antioxidants belonging to three different groups (flavonoids, derivatives of gallic and caffeic acid): log cL50 (μM)=(2.7829±0.2339)+(1.2734±0.4715) E p/2 (V)−(0.3438±0.0582) log P (r 2=0.8129), where cL50 represents the concentration for 50% cell survival, E p/2 represents the voltammetric midpoint potential, and P represents the octanol/water partition coefficient. Analogous QSARs were obtained using enthalpies of single‐electron oxidation of these compounds, obtained by quantum‐mechanical calculations. These findings clearly point to two important characteristics determining polyphenol cytotoxicity, namely their ease of oxidation and their lipophilicity.

Changes in the content of health-promoting compounds and antioxidant activity of broccoli after domestic processing.

The effect of water- and steam-cooking on the content of vitamin C, polyphenols, carotenoids, tocopherols and glucosinolates, as well as on the antioxidant activity of broccoli, are reported. Flavonoids, phenolic acids, vitamins C and E, β-carotene, lutein, and glucosinolates in domestically processed broccoli were quantified using high-performance liquid chromatography (HPLC) methods; total polyphenols were determined with Folin–Ciocalteu reagent. The antioxidant capacities of broccoli extracts were evaluated using the Trolox equivalent antioxidant capacity (TEAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. The results indicated that steam-cooking of broccoli results in an increase in polyphenols, as well as the main glucosinolates and their total content as compared with fresh broccoli, whereas cooking in water has the opposite effect. Steam-cooking of broccoli has no influence on vitamin C, whereas cooking in water significantly lowers its content. Both, water- and steam-cooking of broccoli results in an increase in β-carotene, lutein, and α- and γ-tocopherols as compared with fresh broccoli. Similar effects of steaming and water-cooking of broccoli on their antioxidant activity were observed.

PH-Dependent Radical Scavenging Capacity of Green Tea Catechins

The effect of pH on the radical scavenging capacity of green tea catechins was investigated using experimental as well as theoretical methods. It was shown that the radical scavenging capacity of the catechins, quantified by the TEAC value, increases with increasing pH of the medium. Comparison of the pKa values to theoretically calculated parameters for the neutral and deprotonated forms indicates that the pH-dependent increase in radical scavenging activity of the catechins is due to an increase of electron-donating ability upon deprotonation. The data also reveal that the radical scavenging activity of the catechins containing the pyrogallol (or catechol) and the galloyl moiety over the whole pH range is due to an additive effect of these two independent radical scavenging structural elements. Altogether, the results obtained provide better insight into the factors determining the radical scavenging activity of the catechins and reveal that the biological activity of green tea catechins will be influenced by the pH of the surrounding medium or tissues.

TEAC antioxidant activity of 4-hydroxybenzoates

The influence of pH, intrinsic electron donating capacity, and intrinsic hydrogen atom donating capacity on the antioxidant potential of series of hydroxy and fluorine substituted 4-hydroxybenzoates was investigated experimentally and also on the basis of computer calculations. The pH-dependent behavior of the compounds in the TEAC assay revealed different antioxidant behavior of the nondissociated monoanionic form and the deprotonated dianionic form of the 4-hydroxybenzoates. Upon deprotonation the radical scavenging ability of the 4-hydroxybenzoates increases significantly. For mechanistic comparison a series of fluorobenzoates was synthesized and included in the studies. The fluorine substituents were shown to affect the proton and electron donating abilities of 4-hydroxybenzoate in the same way as hydroxyl substituents. In contrast, the fluorine substituents influenced the TEAC value and the hydrogen atom donating capacity of 4-hydroxybenzoate in a way different from the hydroxyl moieties. Comparison of these experimental data to computer-calculated characteristics indicates that the antioxidant behavior of the monoanionic forms of the 4-hydroxybenzoates is not determined by the tendency of the molecule to donate an electron, but by its ability to donate a hydrogen atom. Altogether, the results explain qualitatively and quantitatively how the number and position of OH moieties affect the antioxidant behavior of 4-hydroxybenzoates.

The Effect of Catechol O-methylation on Radical Scavenging Characteristics of Quercetin and Luteolin—A Mechanistic Insight

The biological effect of flavonoids can be modulated in  vivo due to metabolism. The O-methylation of the catechol group in the molecule by catechol O-methyl transferase is one of the important metabolic pathways of flavonoids. In the present study, the consequences of catechol O-methylation for the pH-dependent radical scavenging properties of quercetin and luteolin were characterized both experimentally and theoretically. Comparison of the pKa values to the pH-dependent TEAC profiles reveals that O-methylation not only affects the TEAC as such but also modulates the effect of changing pH on this radical scavenging activity due to an effect on the pKa for deprotonation. The pH-dependent TEAC curves and computer calculated electronic parameters: bond dissociation energy (BDE) and ionisation potential (IP) even indicate that O-methylation of the luteolin catechol group affects the radical scavenging potential only because it shifts the pKa for deprotonation. O-Methylation of the quercetin catechol moiety affects radical scavenging capacity by both an effect on the pKa, and also by an effect on the electron and hydrogen atom donating properties of the neutral (N) and the anionic (A) form of the molecule. Moreover, O-methylation of a catechol OH-group in quercetin and luteolin has a similar effect on their TEAC profiles and on calculated parameters as replacement of the OH-group by a hydrogen atom. Altogether, the results presented provide new mechanistic insight in the effect of catechol O-methylation on the radical scavenging characteristics of quercetin and luteolin.

Content of trichodiene and analysis of fungal volatiles (electronic nose) in wheat and triticale grain naturally infected and inoculated with Fusarium culmorum

Four groups of cereal kernels were analyzed in terms of their volatile metabolite contents using GC/MS and the electronic nose. Analyses were conducted on 36 triticale breeding lines and 22 wheat breeding lines. Grain came from field samples inoculated with Fusarium culmorum and simultaneous non-inoculated samples-controls. All sample groups contained significantly varied levels of trichodiene (TRICH), a precursor for the formation of fusarium metabolites, with approx. two times higher concentration recorded in triticale. In inoculated samples TRICH concentration for wheat was on average six times higher and for triticale eight times higher than in non-inoculated samples. In the course of analysis using the electronic nose in tested groups of grain differences were observed in the profiles of detected volatile compounds. This resulted in a statistically significant distribution of investigated samples into four objects.

The role of quinone reductase (NQO1) and quinone chemistry in quercetin cytotoxicity.

The effects of quercetin on viability and proliferation of Chinese Hamster Ovary (CHO) cells and CHO cells overexpressing human quinone reductase (CHO+NQO1) were studied to investigate the involvement of the pro-oxidant quinone chemistry of quercetin. The toxicity of menadione was significantly reduced in CHO+NQO1 cells compared to wild-type CHO cells, validating the NQO1-overexpression in the CHO+NQO1 transfectant. Quercetin inhibited the proliferation of wild-type CHO and CHO+NQO1 cells to a similar extent without affecting cell viability, indicating that NQO1 enrichment of CHO cells did not provide increased protection. On the other hand, inhibition of NQO1 in both types of cells by dicoumarol significantly potentiated the inhibitory effect of quercetin on cell proliferation, revealing the role of NQO1 in cellular protection against quercetin. Altogether, these results can be explained by the hypothesis that both wild-type CHO and CHO+NQO1 cells contain sufficient NQO1 activity for optimal protection against the pro-oxidant effect of quercetin on cell proliferation. The results also point at a cellular NQO1 threshold for optimal protection against quercetin. This NQO1 threshold seems to be in the range of NQO1 activities already present in various tissues.

The influence of stereochemistry on the antioxidant properties of catechin epimers

The influence of stereochemistry on the radical scavenging activity of catechins was investigated by studying the effect of pH on the antioxidant properties of catechin epimers. The difference in the pH-dependent Trolox equivalent antioxidant capacity (TEAC) profiles was observed only in case of gallocatechin gallate (GCG) and epigallocatechin gallate (EGCG), indicating the influence of steric structure on the TEAC antioxidant activity of these galloyl moiety-containing catechins. Based on comparison of the pH-dependent TEAC values to theoretically calculated parameters, including homolytic OH bond dissociation energy and ionization potential (IP) as well as theoretically predicted structures of the most stable monoanions of GCG and EGCG, it was concluded that due to steric hindrance in GCG molecule, the IP value of GCG monoanion increases reflecting lower radical scavenging capacity of GCG in comparison with EGCG. It results in the difference in the pH-dependent TEAC profiles of these two catechin epimers at pH above 3.5. This effect does not occur for other pairs of catechin epimers of this study.

Volatile metabolites in various cereal grains

To date, studies on volatile metabolites in cereal grain have focused mainly on a single species. In this paper, results are presented of the analysis of volatile compounds in five cereal grain species (spring wheat, durum wheat, triticale, rye, oats and barley) based on representative sampling of at least 15 cultivars of individual species. Profiles of volatile compounds were determined using solid phase microextraction (SPME) and GC–TOF (time of flight mass spectrometry). Many of the volatile compounds were only present in single samples; however, several dozen were found in over 50% of samples and 46 volatiles were found in all samples. Among them there were six alcohols, 10 aldehydes and ketones, six terpenes, seven hydrocarbons and 11 benzene derivatives. The highest concentrations of these compounds were found in durum wheat, while the lowest were observed in triticale and rye.