Mechanisms of transformation of the antioxidant kaempferol into depsides. Gamma-radiolysis study in methanol and ethanol

In this study, we irradiated the antioxidant kaempferol in ethanol and methanol solutions with gamma rays at doses ranging from 0.2-20 kGy. NMR and ES-MS spectroscopy were used to identify radiolysis products. Two depsides, [2-[(4'-hydroxybenzoyl)oxy]-4,6-dihydroxyphenyl](oxo) methyl acetate and [2-[(4'-hydroxybenzoyl)oxy]-4,6-dihydroxyphenyl](oxo) ethyl acetate, were the major compounds of kaempferol degradation in methanol and in ethanol, respectively. Other products formed in low concentrations were identified as [4-hydroxyphenyl](oxo) methyl acetate, [4-hydroxyphenyl](oxo) ethyl acetate, and depside [2-[(4'-hydroxybenzoyl)oxy]-4,6-dihydroxyphenyl](oxo) acetic acid. The formation of the latter was observed in both solvents. We propose degradation mechanisms that suggest that (.)CH(2)OH and CH(3)(.)CHOH, produced by solvent radiolysis, react with the 3-OH kaempferol group because of its high H-donor capacity. pi-Electron delocalization in the flavonoxy formed after the first H-transfer leads to C-ring opening and consequently to the formation of depsides. G calculation of the degradation products and of (.)CH(2)OH and CH(3)(.)CHOH radicals confirmed the proposed mechanism of kaempferol radiolysis. The rate constants for the reaction between kaempferol and these free radicals were also calculated. Formation of depside has also been observed in many studies of the oxidation of flavonoids; those studying human metabolism have suggested similar redox transformation of flavonols. The antioxidant activities of radiolysis products were evaluated and compared to those of kaempferol.     

Oxidation of flavonoids by hypochlorous acid: reaction kinetics and antioxidant activity studies

Flavonoids, plant polyphenols, ubiquitous components of human diet, are excellent antioxidants. Hypochlorous acid (HOCl), produced by activated neutrophils, is highly reactive chlorinating and oxidizing species. It has been reported earlier that flavonoids are chlorinated by HOCl. Here we show that flavonoids from flavonol subclass are also oxidized by HOCl, but only if the latter is in a large molar excess (≥?10). The kinetics of this reaction was studied by stopped-flow spectrophotometry, at different pH. We found that flavonols were oxidized by HOCl with the rate constants of the order of 10⁴–10⁵ M^?1 s^?1 at pH 7.5. Antioxidant activity of HOCl-modified flavonoids was measured by 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) method. Slightly higher antioxidant activity, compared to parent compounds, was observed for flavonols after their reaction with equimolar or moderate excess of HOCl whereas flavonols treated with high molar excess of HOCl exhibited decrease in antioxidant activity. The mechanism of flavonoid reaction with HOCl at physiological pH is proposed, and biological consequences of this reaction are discussed.     

Theoretical investigation of the formation of a new series of antioxidant depsides from the radiolysis of flavonoid compounds

This paper deals with the formation of a series of antioxidant depsides obtained from flavonoid solutions irradiated with gamma rays. These reactions take place in radiolyzed alcohol solutions, a medium that is very rich in many different highly reactive species and that hosts specific reactions. We focus on the first step of those reactions, i.e., reactivity of the solute (flavonoid) with the alkoxy radicals CH(3)O(*) and CH(3)CH(2)O(*) formed in methanol and ethanol, respectively, and their carbon-centered isomers: the 1-hydroxy-methyl ((*)CH(2)OH) and the 1-hydroxy-ethyl (CH(3)(*)CHOH) radicals. Among the different flavonoid groups of molecules, only flavonols are transformed. To establish the structure-reactivity relationship that explains why the radiolytic transformation occurs only for those compounds, the process is rationalized theoretically, with Density Functional Theory calculations, taking into account the solvent effects by a Polarizable Continuum Model and a microhydrated environment (one or two water molecules surrounding the active center). The first redox reaction, occurring between the flavonol and the reactive species formed upon irradiation of the solvent, is studied in terms of (1) the O-H bond dissociation enthalpy of each OH group of the flavonoids and (2) electron abstraction from the molecule. We conclude that the reaction, initiated preferentially by the alkoxy radicals, first occurs at the 3-OH group of the flavonol. It is then followed by the formation of a peroxyl radical (after molecular oxygen or superoxide addition). The different cascades of reactions, which lead to the formation of depsides via C-ring opening, are discussed on the basis of the corresponding calculated energetic schemes.     

Perturbation of the lipid phase of a membrane is not involved in the modulation of MRP1 transport activity by flavonoids

The expression of transmembrane transporter multidrug resistance-associated protein 1 (MRP1) confers the multidrug-resistant phenotype (MDR) on cancer cells. Since the activity of the other MDR transporter, P-glycoprotein, is sensitive to membrane perturbation, we aimed to check whether the changes in lipid bilayer properties induced by flavones (apigenin, acacetin) and flavonols (morin, myricetin) were related to their MRP1 inhibitory activity. All the flavonoids inhibited the efflux of MRP1 fluorescent substrate from human erythrocytes and breast cancer cells. Morin was also found to stimulate the ATPase activity of erythrocyte ghosts. All flavonoids intercalated into phosphatidylcholine bilayers as judged by differential scanning calorimetry and fluorescence spectroscopy with the use of two carbocyanine dyes. The model of an intramembrane localization for flavones and flavonols was proposed. No clear relationship was found between the membrane-perturbing activity of flavonoids and their potency to inhibit MRP1. We concluded that mechanisms other than perturbation of the lipid phase of membranes were responsible for inhibition of MRP1 by the flavonoids.     

ESR spin trapping of hydroxyl radicals in aqueous solution irradiated with high-LET carbon-ion beams

The aim of this study was to quantify the hydroxyl radicals (*OH) produced when aqueous solutions are decomposed by high-linear energy transfer (LET) 290 MeV/nucleon carbon-ion beams using an electron spin resonance (ESR) spectrometer. Aerated cell culture medium containing 200 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was irradiated with doses of 0 to 20 Gy with an LET of 20 to 90 keV/ micro m. We were able to obtain ESR spectra 10 min after irradiation, and the formation of *OH and hydrogen atoms was confirmed by radiolysis of deuterium oxide and ethanol containing DMPO. Our results showed that the yield of *OH by carbon-ion radiolysis increased in proportion to the absorbed dose over the range of 0 to 20 Gy. Furthermore, we discovered that the yield of *OH decreased linearity as LET increased logarithmically from 20 to 90 keV/ micro m. The generation of *OH by carbon-ion radiolysis at LETs of 20, 40, 60, 80 and 90 keV/ micro m was 64, 58, 52, 49 and 50%, respectively, of that for low-LET X radiolysis. These unique findings provide a further understanding of the indirect effect of high-LET radiation.     

Chemical features of flavonols affecting their genotoxicity. Potential implications in their use as therapeutical agents

Flavonls are natural compounds present in edible plants and possess several biological activities that can be useful in drug design. Conversely some of these compounds have been shown to be genotoxic to prokaryotic and eukaryotic cells. In this study we tried to establish the chemical features responsible for the genotoxicity of flavonols and to study the conditions that can modulate their genotoxicity namely pH, the presence of antioxidants and metabolism. We assessed the induction of revertants in Salmonella typhimurium TA98 and the induction of Chromosomal aberrations in V79 cells by eight different flavonols and one catechin in the presence and in the absence of metabolizing systems. We have also studied the generation of hydroxyl radical by these flavonoids using the deoxyribose degradation assay. The results obtained in this study suggest that flavonols having a free hydroxyl group at position 3 of the C ring, a free hydroxyl group at position 7 of the A ring and a B ring with a catechol or pyrogallol structure, or a structure that after metabolic activation is transformed into a catechol or a pyrogallol, are flavonols whose genotoxicity in eukaryotic cells depends on their autooxidation. These flavonols can autooxidize when the pH value is slightly alkaline, such as in the intestine, and therefore can induce genotoxicity in humans. Given the above mentioned considerations it is necessary to clarify the mechanisms and the conditions that mediate the biological effects of flavonols before considering them as therapeutical agents.     

Antioxidant capacity of flavanols and gallate esters: pulse radiolysis studies

Reactivities of several proanthocyanidins (monomers of condensed tannins) and gallate esters (representing hydrolyzable tannins) with hydroxyl radicals, azide radicals, and superoxide anions were investigated using pulse radiolysis combined with kinetic spectroscopy. We determined the scavenging rate constants and the decay kinetics of the aroxyl radicals both at the wavelength of the semiquinone absorption (275 nm) and the absorption band of the gallate ester ketyl radical (400-420 nm). For most compounds second-order decay kinetics were observed, which reflect disproportionation of the semiquinones. In the case of the oligomeric hydrolysable tannins, pentagalloyl glucose and tannic acid, the decay kinetics were more complex involving sequential first-order and second-order reactions, which could only be resolved by kinetic modeling. A correlation of the reaction rates with hydroxyl radicals (k*OH) with the number of adjacent aromatic hydroxyl groups (i.e., representing catechol and/or pyrogallol structures) was obtained for both condensed and hydrolyzable tannins. Similar correlation for the reactions with azide radicals and superoxide anions are less obvious, but exist as well. We consider proanthocyanidins superior radical scavenging agents as compared with the monomeric flavonols and flavones and propose that these substances rather than the flavonoids proper represent the antioxidative principle in red wine and green tea.     

Mitochondrial permeability transition induced by 1-hydroxyethyl radical

Impairment of mitochondrial functions has been found in ethanol-induced liver injury. Ethanol can be oxidized to the 1-hydroxyethyl radical (HER) by rat liver microsomal systems. Experiments were carried out to evaluate the ability of HER to cause mitochondrial swelling as an indicator of the mitochondrial permeability transition (MPT). Electron spin resonance (ESR) spectroscopy was used to detect HER and to study its interaction with mitochondria. The ESR signal intensity of the spin adduct formed from alpha-(4-pyridyl-1-oxide) N-tert-butylnitrone (POBN) and HER generated from either a thermic decomposition of 1,1'-dihydroxyazoethane (DHAE) or a Fenton reaction system containing ethanol was markedly diminished by the addition of mitochondria, indicating an interaction between HER and mitochondria. Exposure of rat liver mitochondria to HER generated from either system caused swelling, as reflected by a decrease in absorbance at 540 nm, in a HER concentration-dependent and a cyclosporin A-sensitive manner. Mitochondrial swelling was also induced in the Fenton reaction system without ethanol. The DHAE-dependent generation of HER in mitochondrial suspension resulted in a decrease of membrane protein thiols and collapse of the membrane potential (delta psi). The swelling induced by HER was prevented by glutathione and vitamin E, but not by superoxide dismutase. Catalase did not prevent the swelling caused by the acetaldehyde/hydroxylamine O-sulfonate (HOS) system, but was inhibitory in the Fenton reaction system with or without ethanol. These results indicate that HER, as well as hydroxyl radical, can induce the MPT, and suggest the possibility that the collapse of delta psi caused by HER may, at least in part, contribute to impairment of mitochondrial function caused by ethanol and in ethanol-induced liver injury.     

Studies on γ-Radiolysis of Sulfur Containing Amino Acids

In connection with flavor deterioration accompanied by food irradiation, the effect of γ-irradiation on sulfoxide amino acids in air free aqueous system was investigated. The major radiolysis products from S-n-propyl-l-cysteine sulfoxide (PCSO) were alanine, cysteic acid, dipropyl disulfide, etc., and from S-allyl-l-cysteine sulfoxide (ACSO) were S-allyl-l-cysteine, cystine, cysteic acid, etc., which were isolated chromatographycally and identified by using IR and mass spectrometry. The sulfoxide in ACSO was more easily reduced to sulfide than that of in PCSO, and the bond of S-C (β-carbon in alanine moiety) in ACSO was difficult to cleave. These differences observed between PCSO and ACSO in the radiolysis products and their yields indicate that the radiolysis degradation is considerably influenced by the structure of alkyl group. From the experiments with N₂O or KBr addition during irradiation, principal roles of the active species in irradiated water in the degradation processes were partly elucidated.     

Effects of Hyperthermic Conditions on the Reactivity of Oxygen Radicals

Generation and reactivity of superoxide (0₂^?) and hydroxyl (OH') radicals in enzymatic and radiolytic systems were investigated over the temperature range from 20^o-50^oC. The generation rate and reaction kinetics of both enzymatically and radiolytically produced superoxide radicals were determined by a cytochrome c reduction assay. For OH' radical reaction studies the degradation of hyaluronic acid was assayed. An increase in temperature leads to a greater reactivity of both radicals, but in the case of an enzymatic source a disproportionate increase in the rate of generation is observed. In the pulse radiolysis system, the reactivity of superoxide radicals was found to be stimulated 15-fold over the temperature range from 20^oC to 60^oC, although the activity of superoxide dismutase was only minimally increased (about 1.6-fold). The results are discussed with respect to the possible importance of active oxygen species to the biological effects of hyperthermia.     

Antimicrobial and antioxidant properties of chitosan enzymatically functionalized with flavonoids

Chitosan fibres were grafted with flavonoids using tyrosinase to produce reactive o-quinones which subsequently react with primary amino groups of the chitosan. The reaction mechanism using chemically different flavonoids (flavanols, flavonols, flavone, flavanone, isoflavone) was followed by UV/vis spectroscopy and the successful grafting was demonstrated by ATR-IR spectroscopy, pH potentiometric titration and reflectance measurements. An increase of antioxidant activity of functionalized chitosan fibres using well established methods was found depending on the type of the flavonoid used. In addition, some flavonoids increased antimicrobial activity of chitosan against Bacillus subtillis and Pseudomonas aeruginosa.     

Degradation of HER2/neu by apigenin induces apoptosis through cytochrome c release and caspase-3 activation in HER2/neu-overexpressing breast cancer cells

We have shown that exposure of the HER2/neu-overexpressing breast cancer cells to apigenin resulted in induction of apoptosis by depleting HER2/neu protein and, in turn, suppressing the signaling of the HER2/HER3-PI3K/Akt pathway. Here, we examined whether inhibition of this pathway played a role in the anti-tumor effect. The results revealed that treatment with apigenin induced apoptosis through cytochrome c release and caused a rapid induction of caspase-3 activity and stimulated proteolytic cleavage of DFF-45. Furthermore, apigenin downregulated cyclin D1, D3 and Cdk4 and increased p27 protein levels. Colony formation in the soft agar assay, a hallmark of the transformation phenotype, was preferentially suppressed in HER2/neu-overexpressing breast cancer cells in the presence of apigenin. In addition, a structure-activity relationship study indicated that (1) the position of B ring; and (2) the existence of the 3', 4'-hydroxyl group on the 2-phenyl group were important for the depletion of HER2/neu protein by flavonoids. These results provided new insights into the structure-activity relationship of flavonoids.     

Synergistic effects of flavonoids and ascorbate on enhancement in DNA degradation induced by a bleomycin-Fe complex

Flavonoids were examined for synergistic effects with ascorbate on enhancement of DNA degradation induced by a bleomycin(BLM)-Fe complex. The synergistic effects of flavonoids and ascorbate on DNA degradation induced by the BLM-Fe complex were observed to be greater with flavonoids such as isorhamnetin, kaempferol and morin, which accelerated oxidation more markedly in the presence, than in the absence of BLM. Conversely, myricetin and fisetin, which showed oxidation barely accelerated by the addition of BLM, inhibited DNA degradation promoted by ascorbate. Consequently, there was a good correlation between oxidation of flavonoids accelerated by BLM and the extent of DNA degradation promoted synergistically with ascorbate. Our previous studies indicated that oxidation of flavonoids accelerated by BLM and DNA degradation promoted by flavonoids were not correlated with Fe(III)-reducing activity of flavonoids. Those results suggest that Fe(III)-reducing activity of flavonoids is not the only factor determining DNA degradation-promoting activity induced by the BLM-Fe complex. On the other hand, in a Fenton reaction, degradation of 2-deoxy-d-ribose promoted by flavonoids was correlated to the Fe(III)-reducing activity of flavonoids. However, there was not a synergistic interaction between flavonoids and ascorbate in the degradation of 2-deoxy-d-ribose. Therefore, it is suggested that the synergistic DNA degradation caused by flavonoids and ascorbate in the BLM-Fe redox cycle arose from the difference in the reductive processes in which flavonoids and ascorbate mainly act.     

Effect of O-glycosilation on the antioxidant activity and free radical reactions of a plant flavonoid,chrysoeriol

Chrysoeriol and its glycoside (chrysoeriol-6-O-acetyl-4'-beta-D-glucoside) are two natural flavonoids extracted from the tropical plant Coronopus didymus. The aqueous solutions of both the flavonoids were tested for their ability to inhibit lipid peroxidation induced by gamma-radiation, Fe (III) and Fe (II). In all these assays chrysoeriol showed better protecting effect than the glycoside. The compounds were also found to inhibit enzymatically produced superoxide anion by xanthine/xanthine oxidase system; here the glycoside is more effective than the aglycone. The rate constants for the reaction of the compounds with superoxide anion determined by using stopped-flow spectrometer were found to be nearly same. Chrysoeriol glycoside reacts with DPPH radicals at millimolar concentration, but the aglycone showed no reaction. Using nanosecond pulse radiolysis technique, reactions of these compounds with hydroxyl, azide, haloperoxyl radicals and hydrated electron were studied. The bimolecular rate constants for these reactions and the transient spectra of the one-electron oxidized species indicated that the site of oxidation for the two compounds is different. Reaction of hydrated electron with the two compounds was carried out at pH 7, where similar reactivity was observed with both the compounds. Based on all these studies it is concluded that chrysoeriol exhibits potent antioxidant activity. O-glycosylation of chrysoeriol decreases its ability to inhibit lipid peroxidation and reaction with peroxyl radicals. However the glycoside is a more efficient scavenger of DPPH radicals and a better inhibitor of xanthine/xanthine oxidase than the aglycone.     

Pathway engineering for healthy phytochemicals leading to the production of novel flavonoids in tomato fruit

Flavonoids are a large family of plant polyphenolic secondary metabolites. Although they are widespread throughout the plant kingdom, some flavonoid classes are specific for only a few plant species. Due to their presumed health benefits there is growing interest in the development of food crops with tailor-made levels and composition of flavonoids, designed to exert an optimal biological effect. In order to explore the possibilities of flavonoid engineering in tomato fruits, we have targeted this pathway towards classes of potentially healthy flavonoids which are novel for tomato. Using structural flavonoid genes (encoding stilbene synthase, chalcone synthase, chalcone reductase, chalcone isomerase and flavone synthase) from different plant sources, we were able to produce transgenic tomatoes accumulating new phytochemicals. Biochemical analysis showed that the fruit peel contained high levels of stilbenes (resveratrol and piceid), deoxychalcones (butein and isoliquiritigenin), flavones (luteolin-7-glucoside and luteolin aglycon) and flavonols (quercetin glycosides and kaempferol glycosides). Using an online high-performance liquid chromatography (HPLC) antioxidant detection system, we demonstrated that, due to the presence of the novel flavonoids, the transgenic tomato fruits displayed altered antioxidant profiles. In addition, total antioxidant capacity of tomato fruit peel with high levels of flavones and flavonols increased more than threefold. These results on genetic engineering of flavonoids in tomato fruit demonstrate the possibilities to change the levels and composition of health-related polyphenols in a crop plant and provide more insight in the genetic and biochemical regulation of the flavonoid pathway within this worldwide important vegetable.     

Reactions of reducing radicals with ribonuclease

Radiation-induced reactions of hydrated electrons, formate- and ethanol radicals with ribonuclease were studied by pulse radiolysis and by electrophoresis. Initially formate radicals react rapidly and very specifically with the disulphide bonds of ribonuclease. This reaction leads to aggregation by intermolecular S-S-interchange, the process being more effective at pH 4, since formation and decay of S-S-.-radical anions increases with decreasing pH. With high doses additional unreducible aggregates are formed. Radical formation at the positively charged histidine residues seems to be involved. Hydrated electrons do not react as selectively as the formate radicals, but with several sites in native ribonuclease. Thus with low doses unreducible aggregates are formed. Electrophoresis shows that reaction of the electrons causes fragmentation of the peptide chain, when OH-radicals are scavenged. Very weak transient spectra and very little degradation result on reaction of ethanol radicals with ribonuclease.     

Scavenging effects of phenolic compounds on reactive oxygen species

Phenolic compounds are widely present in plants and they have received considerable attention due to their antioxidant property. In this article we report the results of a study of the reactivity of 10 selected phenolics (sesamol, three phenolic acids, three flavonols, one flavone, and two flavanones) with superoxide anion radical (O(2) (*)), hydroxyl radical (HO(*)) and singlet oxygen ((1)O(2)). The following generators of reactive oxygen species were used: 18-crown-6/KO(2)/dimethylsulfoxide (DMSO) or hypoxanthine/xanthine oxidase as sources of O(2) (*), the Fenton reaction carried out in a sodium trifluoroacetate (pH 6.15) for HO(*), and a mixture of alkaline aqueous H(2)O(2) and cobalt ions for (1)O(2). We have employed chemiluminescence, electron spin resonance spin trapping, and spectrophotometry techniques to examine an antioxidative property. All tested compounds acted as scavengers of various reactive oxygen species. The reactivity indexes (beta) for the reaction of the phenolic compounds with HO(*) were calculated.     

Reduction in free-radical-induced DNA strand breaks and base damage through fast chemical repair by flavonoids

This paper provides evidence that dietary flavonoids can repair a range of oxidative radical damages on DNA, and thus give protection against radical-induced strand breaks and base alterations. We have irradiated dilute aqueous solutions of plasmid DNA in the absence and presence of flavonoids (F) in a "constant *OH radical scavenging environment", k of 1.5 x 10(7) s(-1) by decreasing the concentration of TRIS buffer in relation to the concentration of added flavonoids. We have shown that the flavonoids can reduce the incidence of single-strand breaks in double-stranded DNA as well as residual base damage (assayed as additional single-strand breaks upon post-irradiation incubation with endonucleases) with dose modification factors of up to 2.0+/-0.2 at [F] < 100 microM by a mechanism other than through direct scavenging of *OH radicals. Pulse radiolysis measurements support the mechanism of electron transfer or H* atom transfer from the flavonoids to free radical sites on DNA which result in the fast chemical repair of some of the oxidative damage on DNA resulting from *OH radical attack. These in vitro assays point to a possible additional role for antioxidants in reducing DNA damage.     

Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin,proanthocyanidin, and flavonol levels during bilberry fruit development

The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented.