Umeo Takahama

Protective effect of quercetin and rutin on photosensitized lysis of human erythrocytes in the presence of hematoporphyrin

Photosensitized hemolysis of human erythrocytes by hematoporphyrin was suppressed by flavonols such as quercetin and rutin at submillimolar concentrations. The suppression of photohemolysis was accompanied by inhibition of lipid peroxidation by the reagents. Quercetin and rutin were photooxidized in the presence of hematoporphyrin and the photooxidation was partially suppressed by 1 mM NaN3, a quencher of singlet molecular oxygen. Flavonols were also oxidized by radicals formed during degradation of lauroyl peroxide. These results indicate that flavonols can function as antioxidants in biological systems by terminating radical chain reactions and removing singlet molecular oxygen. A pharmacological function of flavonols, decrease of the increased permeability and fragility of capillary, was discussed in relation to their antioxidative functions.

Oxidation of vacuolar and apoplastic phenolic substrates by peroxidase: Physiological significance of the oxidation reactions

Phenolic components and peroxidases are localized in vacuoles. Vacuolar peroxidase can oxidize phenolics when H2O2 is formed in vacuoles or tonoplasts, or when H2O2 formed outside of vacuoles is diffused into the organelles. In a mixture of phenolics containing a good and a poor substrate for peroxidase, a radical transfer reaction is possible from the radicals of the good substrate to the poor substrate, resulting in the enhancement of oxidation of the poor substrate. Phenoxyl radicals formed by peroxidase-dependent reactions are reduced by ascorbate in vacuoles. So, as long as ascorbate is present in vacuoles, the accumulation of oxidation products of phenolics is not significant. This suggests that ascorbate/phenolics/peroxidase systems in the vacuoles can scavenge H2O2. During aging, some phenolics are accumulated in vacuoles and the apoplast, and the accumulated phenolics are oxidized to brown components by peroxidase-dependent reactions. The brown components can produced O2− and H2O2 by autooxidation. The significance and the mechanisms of browning are discussed in tobacco leaves and onion scales.

Flavonoids and Some Other Phenolics as Substrates of Peroxidase: Physiological Significance of the Redox Reactions

2O2 without accumulating oxidation products of phenolics. Scavenging of H2O2 by the systems can proceed in vacuoles and the apoplast, because phenolics, AA and POX are normal components of the compartments. AA seems to control lignification because it reduces radicals of lignin monomers which are formed by POX-dependent reactions. On lignification, oxidation of sinapyl alcohol is enhanced by radicals of coniferyl alcohol and hydroxycinnamic acid esters when apoplastic POX rapidly oxidizes coniferyl alcohol and the esters but slowly oxidizes sinapyl alcohol. POX seems to participate in the browning of tobacco leaves and onion scales on aging. H2O2, which is required for the POX-dependent reactions, can be formed by autooxidation of the phenolics that are transformed to brown components. It is discussed that browning involves the formation of antimicrobial substances.

Generation of hydroxyl radical in isolated pea root cell wall, and the role of cell wall-bound peroxidase, Mn-SOD and phenolics in their production.

The hydroxyl radical produced in the apoplast has been demonstrated to facilitate cell wall loosening during cell elongation. Cell wall-bound peroxidases (PODs) have been implicated in hydroxyl radical formation. For this mechanism, the apoplast or cell walls should contain the electron donors for (i) H(2)O(2) formation from dioxygen; and (ii) the POD-catalyzed reduction of H(2)O(2) to the hydroxyl radical. The aim of the work was to identify the electron donors in these reactions. In this report, hydroxyl radical (.OH) generation in the cell wall isolated from pea roots was detected in the absence of any exogenous reductants, suggesting that the plant cell wall possesses the capacity to generate .OH in situ. Distinct POD and Mn-superoxide dismutase (Mn-SOD) isoforms different from other cellular isoforms were shown by native gel electropho-resis to be preferably bound to the cell walls. Electron paramagnetic resonance (EPR) spectroscopy of cell wall isolates containing the spin-trapping reagent, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), was used for detection of and differentiation between .OH and the superoxide radical (O(2)(-).). The data obtained using POD inhibitors confirmed that tightly bound cell wall PODs are involved in DEPMPO/OH adduct formation. A decrease in DEPMPO/OH adduct formation in the presence of H(2)O(2) scavengers demonstrated that this hydroxyl radical was derived from H(2)O(2). During the generation of .OH, the concentration of quinhydrone structures (as detected by EPR spectroscopy) increased, suggesting that the H(2)O(2) required for the formation of .OH in isolated cell walls is produced during the reduction of O(2) by hydroxycinnamic acids. Cell wall isolates in which the proteins have been denaturated (including the endogenous POD and SOD) did not produce .OH. Addition of exogenous H(2)O(2) again induced the production of .OH, and these were shown to originate from the Fenton reaction with tightly bound metal ions. However, the appearance of the DEPMPO/OOH adduct could also be observed, due to the production of O(2)(-). when endogenous SOD has been inactivated. Also, O(2)(-). was converted to .OH in an in vitro horseradish peroxidase (HRP)/H(2)O(2) system to which exogenous SOD has been added. Taken together with the discovery of the cell wall-bound Mn-SOD isoform, these results support the role of such a cell wall-bound SOD in the formation of .OH jointly with the cell wall-bound POD. According to the above findings, it seems that the hydroxycinnamic acids from the cell wall, acting as reductants, contribute to the formation of H(2)O(2) in the presence of O(2) in an autocatalytic manner, and that POD and Mn-SOD coupled together generate .OH from such H(2)O(2).

Peroxidases in vacuoles of Vicia faba leaves

Abstract A basic peroxidase localized in vacuoles of mesophyll cells of Vicia faba was isolated by ammonium sulphate precipitation, cation exchange chromatography and gel filtration. The peroxidase had a pH optimum at 5 and absorption maxima at 404, 510 and 635 nm in the oxidized form. Its absorption maxima in the presence of 0.1 mM H 2 O 2 were 418, 523 and 558 nm. The M r of the enzyme was estimated to be 49 000 by sucrose density gradient centrifugation. The peroxidase oxidized flavonols and 3,4-dihydroxyphenylalanine (DOPA). The V max s were quercetin kaempferol > rutin (quercetin 3- O -rutinoside)>DOPA>robinin (kaempferol 3- O -rhamnogalactoside 7- O -rhamnoside). K m values were below 0.1 mM for flavonols and 6.7 mM for DOPA. The peroxidase could also oxidize flavonol glycosides which were contained in methanol extracts of the lower epidermis of Vicia faba leaves.

The presence of 4‐hydroxyphenylacetic acid in human saliva and the possibility of its nitration by salivary nitrite in the stomach

Human saliva contained 4‐hydroxyphenylacetic acid (HPA) (2–10 μM) and nitrite (60–300 μM). HPA was nitrated to 4‐hydroxy‐3‐nitrophenylacetic acid (NO2HPA) when HPA and sodium nitrite were mixed at pH 1.0. NO2HPA was also formed when saliva was incubated under acidic conditions. These results suggest that salivary HPA is nitrated to NO2HPA when saliva is swallowed into the stomach.

Hydrogen peroxide scavenging systems in vacuoles of mesophyll cells of Vicia faba

Abstract Vacuoles isolated from mesophyll protoplasts of Vicia faba contained ascorbate in addition to 3,4-dihydroxyphenylalanine (DOPA) and peroxidase. When a low concentration of H 2 O 2 (0.25 mM) was added to the vauole fractions, a slow decrease in the level of ascorbate was observed. When 2.5 mM H 2 O 2 was added to the vacuole fractions, a rapid decrease in the levels of ascorbate and formation of dopachrome were observed. The oxidation of ascorbate and the formation of dopachrome were inhibited by NaN 3 (10 mM), KCN (1 mM) and stimulated by tropolone (5 mM), suggesting the participation of peroxidase but not phenol oxidase in the reactions. Vacuolar peroxidase isolated from leaves of V. faba oxidized DOPA and ascorbate in the presence of H 2 O 2 . The oxidation of DOPA was nearly completely inhibited by ascorbate but the oxidation of ascorbate was stimulated by DOPA, suggesting the reduction of an oxidized intermediate of DOPA to DOPA by ascorbate. In fact, ascorbate reduced dopaquinone, a two electron oxidized DOPA.

Quercetin-Dependent Reduction of Salivary Nitrite to Nitric Oxide under Acidic Conditions and Interaction between Quercetin and Ascorbic Acid during the Reduction

A salivary component, nitrate, is reduced to nitrite in the oral cavity. Polyphenols in foods are mixed with nitrite in the saliva to be swallowed into the stomach. An objective of the present study is to elucidate reactions between a polyphenol quercetin and a nitrite under acidic conditions. Nitric oxide, which is formed by the reactions between nitrous acid and quercetin or ascorbic acid (AA), can be measured using an oxygen electrode in the saliva as well as a buffer solution. The initial oxidation of quercetin was inhibited by AA, and quercetin enhanced the oxidation of AA, suggesting AA-dependent reduction of quercetin radicals, which might be formed during the oxidation of quercetin by nitrous acid. On the basis of the above results, the usefulness of an oxygen electrode for the measurement of nitrite-dependent nitric oxide formation under acidic conditions is proposed and the possible mechanism of reduction of nitrous acid by quercetin and the interaction between quercetin and AA, which is a normal component in the gastric juice, for the reduction of nitrous acid is discussed.

Inhibition of photooxidation of α-tocopherol by quercetin in human blood cell membranes in the presence of hematoporphyrin as a photosensitizer

Photooxidation of alpha-tocopherol (alpha-TH) and photoperoxidation of lipids in blood cell membranes in the presence of hematoporphyrin (HP) as a photosensitizer were inhibited by quercetin. Half maximal inhibition for the photooxidation of alpha-TH was obtained at about 0.3 mM quercetin and that for the lipid photoperoxidation at about 1.5 microM quercetin. The difference of the half maximal inhibition may be due to the difference of mechanism of the inhibition between the two reactions. O2- and H2O2 hardly participated in the photooxidation of alpha-TH and 1O2 participated in the photooxidation only partially (about 5%). The electron transfer reaction from alpha-TH to excited HP was indicated by measuring ferricyanide photoreduction in the suspensions of alpha-TH in PBS solution in the presence of HP. The photooxidation of alpha-TH in PBS solution was inhibited by quercetin and vice versa. In the presence of linoleic acid in PBS solution, quercetin inhibited the photooxidation of alpha-TH and alpha-TH stimulated the photooxidation of quercetin. Based on the above data, as possible mechanisms of the inhibition of photooxidation of alpha-TH in blood cell membranes by quercetin, competition of quercetin with alpha-TH for excited HP and for radicals generated during lipid peroxidation and reduction of oxidized alpha-TH by quercetin are proposed. The antioxidative function of quercetin was enhanced by ascorbate even under conditions in which ascorbate functioned as a prooxidant when it was added alone. The enhancement is attributed to the functions of ascorbate to reduce the oxidized quercetin and of quercetin to inhibit ascorbate photooxidation.

COOPERATION OF QUERCETIN WITH ASCORBATE IN THE PROTECTION OF PHOTOSENSITIZED LYSIS OF HUMAN ERYTHROCYTES IN THE PRESENCE OF HEMATOPORPHYRIN

Abstract— Quercetin(20–100 μM) suppressed photohemolysis sensitized by hematoporphyrin, while ascorbate(10–100 μM) stimulated it. However, in the presence of 40 μM quercetin, ascorbate promoted the suppression. The suppression by quercetin was due to scavenging of both singlet oxygen generated by a photosensitized reaction and radicals generated by decomposition of lipid peroxides formed by a singlet oxygen‐dependent reaction. In scavenging, quercetin was oxidized and the oxidation was suppressed by ascorbate. Ascorbate was oxidized by illumination in the presence of quercetin. It is suggested that the cooperation of quercetin with ascorbate in photohemolysis is due to reduction of oxidized quercetin by ascorbate regenerating the flavonol.