Shunro Kawakishi

Inhibition of lipid peroxidation and the active oxygen radical scavenging effect of anthocyanin pigments isolated from Phaseolus vulgaris L

No attention has been paid to anthocyanin pigments from the viewpoint of inhibitors of lipid peroxidation and scavengers of active oxygen radicals; therefore, we investigated the antioxidative, radical scavenging, and inhibitory effects on lipid peroxidation by UV light irradiation of three anthocyanin pigments, pelargonidin 3-O-beta-D-glucoside (P3G), cyanidin 3-O-beta-D-glucoside (C3G), and delphinidin 3-O-beta-D-glucoside (D3G), isolated from the Phaseolus vulgaris L. seed coat, and their aglycons, pelargonidin chloride (Pel), cyanidin chloride (Cy), and delphinidin chloride (Del). All pigments had strong antioxidative activity in a liposomal system and reduced the formation of malondialdehyde by UVB irradiation. On the other hand, the extent of antioxidative activity in a rat liver microsomal system and the scavenging effect of hydroxyl radicals (-OH) and superoxide anion radicals (O2-) were influenced by their own structures.

THE CONTRIBUTION OF PLANT FOOD ANTIOXIDANTS TO HUMAN HEALTH

Abstract We have been actively involved in the isolation and characterization of endogenous plant antioxidants that are believed to inhibit lipid peroxidation and offer protection against oxidative damage to membrane functions. Antioxidants have been isolated from conventional food sources, such as tea (green and black), sesame and wild rice, and also from other plant sources, such as rice hulls, and crude plant drugs. Data on new types of water-soluble and lipid-soluble plant antioxidants are provided, and the biological activity and functionality of these antioxidants are discussed.

The monoclonal antibody specific for the 4-hydroxy-2-nonenal histidine adduct

Monoclonal antibodies directed against proteins modified with the major membrane lipid peroxidation product, 4‐hydroxy‐2‐nonenal, have been established and characterized. The monoclonal antibodies specific for HNE‐modified proteins were raised by immunizing mice with a HNE‐keyhole limpet hemocyanin conjugate. The resulting five monoclonal antibodies (mAbs HNEJ‐1–5) recognized HNE‐modified bovine serum albumin (BSA), but not native BSA in Western blot studies. Of the five mAbs, HNEJ‐2 exhibited the highest affinity for HNE‐modified proteins and a much higher affinity for the HNE‐histidine adduct than the HNE‐lysine or HNE‐cysteine adducts. mAb HNEJ‐2 did not cross‐react with proteins that had been treated with other aldehydes, such as 1‐hexenal, 2‐hexenal, 4‐hydroxy‐2‐hexenal, 2‐nonenal, formaldehyde, or glutaraldehyde. These results suggest that the major epitope recognized by mAb HNEJ‐2 is the Michael addition‐type HNE‐histidine adduct.

Antioxidative and antimutagenic effects of theaflavins from black tea

Theaflavins, polyphenolic ingredients of black tea, were observed to inhibit in vitro lipid peroxidation in the erythrocyte membrane ghost and microsomal systems. Theaflavins also showed inhibition of DNA single-strand cleavage and mutagenicity, both induced by hydrogen peroxide. These results suggest that theaflavins scavenge radicals to produce antioxidative and antimutagenic effects. It was also found that the gallic acid moiety of theaflavins is essential for their potent antioxidative activities.

AGGREGATION OF COLLAGEN EXPOSED TO UVA IN THE PRESENCE OF RIBOFLAVIN: A PLAUSIBLE ROLE OF TYROSINE MODIFICATION

Riboflavin‐sensitized photodynamic modification of collagen led to significant formation of cross‐linked molecules. Sodium azide or l,4‐diazabicyclo(2,2,2)octane, which are known to be singlet oxygen quenchers, and catalase could not inhibit the modification. Surprisingly, the collagen modification was accelerated in the presence of superoxide dismutase. The aggregation was accompanied by the loss of tyrosine and histidine residues in the collagen. An inhibitory effect of dissolved oxygen on the modification of collagen was observed. Similarly, the loss of tyrosine residues in the irradiated collagen was inhibited in the presence of dissolved oxygen. Dityrosine formation was also observed with the loss of tyrosine. These results indicate that photodynamic modification of tyrosine probably contributes to the riboflavin‐sensitized cross‐linking of collagen through the formation of dityrosine.

2‐Oxo‐histidine as a novel biological marker for oxidatively modified proteins

We report a promising finding that oxidative modification of proteins by free radicals could be monitored by the formation of oxidized histidine that is detectable by reverse‐phase HPLC with electrochemical detection (HPLC‐ECD). When the N‐protected histidine derivative (N‐benzoyl‐histidine) was exposed to a free radical‐generating system (copper/ascorbate), a number of products were detected by HPLC‐ECD and the main product among them was found to be identical to N‐benzoyl‐2‐oxo‐histidine. The acid hydrolysis of N‐benzoyl‐2‐oxo‐histidine provided a single product (2‐oxo‐histidine) that was detected sensitively by HPLC‐ECD. Thus 2‐oxo‐histidine was indeed generated as the main product in the oxidatively modified proteins by free radicals. Taken together, 2‐oxo‐histidine may be a useful biological marker for assessing protein modifications under oxidative stress.

A novel mechanism for oxidative cleavage of prolyl peptides induced by the hydroxyl radical

Mechanism for the oxidative cleavage of proline-containing peptides induced by the hydroxyl radical (.OH) has been studied. Accompanying the oxidation of prolyl peptides, we discovered the formation of significant amounts of gamma-aminobutyric acid (GABA) in the acid hydrolysates of the oxidized peptides. GABA was assumed to be derived from the 2-pyrrolidone compound and, in addition, its generation led to the assumption that prolyl peptides were mainly cleaved at the proline residues by .OH, accompanied by the oxidative modification of proline by itself. Hence, in order to confirm this prediction, we undertook the reaction of proline with .OH using proline analogue (Z-proline) and isolated the 2-pyrrolidone compound as the major product. We proposed a novel mechanism for formation of the 2-pyrrolidone compound induced by .OH, which has been established for the first time in the oxidative cleavage of prolyl peptides.

Potent Antioxidative Isoflavones Isolated from Soybeans Fermented with Aspergillus saitoi

Two potent antioxidative isoflavones were isolated from soybeans fermented with Aspergillus saitoi by silica gel column chromatography and preparative HPLC, using ODS column, or in addition, Toyopearl HW-40 column chromatography. The purified AS-13 and AS-9B compounds were identified as 8-hydroxydaidzein (8-OHD) and 8-hydroxygenistein (8-OHG), respectively, by MS, and (1)H-NMR, (13)C-NMR and HMBC spectra. These isoflavones, which have an o-dihydroxy structure between the 7- and 8-position, each exhibited significantly stronger antioxidative activity than daidzein and genistein in both oil and lipid/aqueous systems. Furthermore, the antioxidative activity and the content of each isoflavone analog in soybeans with different fermentation periods were investigated. It is suggested from these results that AS-13 and AS-9B were produced from daidzein and genistein, respectively, by hydroxylation at the 8-position of each isoflavone structure. In addition, it is concluded that these isoflavones were also the principal antioxidants in potent antioxidative soybeans fermented with A. saitoi.

Role of Dietary Antioxidants in Protection against Oxidative Damage

Recently, much attention has been focused on studies which suggest the involvement of active oxygens and free radicals in a variety of pathological events, cancer, and even the aging process (21,30). Oxygen is indispensable for aerobic organisms including, of course, human beings; however, it is believed that oxygen also may be responsible for undesired phenomena (4). In particular, oxygen species such as hydrogen peroxide, superoxide anion radical and singlet oxygen, and other radicals, are proposed as agents attacking polyunsaturated fatty acid in cell membranes, giving rise to lipid peroxidation (3). Several reports have suggested that lipid peroxidation may result in destabilization and disintegration of cell membranes, leading to liver injury and other diseases, and finally, to aging and susceptibility to cancer. However, normal cell membranes do not undergo lipid peroxidation so severely in vivo because of the extremely efficient protective mechanisms against damage caused by active oxygens and free radicals. Such systems include enzymatic inactivation by, for example, superoxide dismutase, glutathione-peroxidase and catalase, as well as nonenzymatic protection of polyunsaturated fatty acid by physiological and biological antioxidants such as vitamin E, vitamin C, s-carotene, and uric acid (2). More recently, bilirubin (35) and carnocine (18) have been reported as being biologically significant antioxidants. In addition, several antioxidants have been reported to play an important role in the prevention of carcinogenesis related to active oxygen radicals, and in some cases, to extend the life span of animals (8).

Site-specific oxidation of angiotensin I by copper(II) and L-ascorbate: conversion of histidine residues to 2-imidazolones.

The reaction of a histidine-containing peptide (angiotensin I) with copper (II)/ascorbate under physiological conditions has been studied chemically. In the presence of a catalytic amount of copper(II) ion, ascorbate mediated the oxidative damage to the peptide via selective loss of the histidine residue. Furthermore, the reaction of copper(II)/ascorbate with the peptide gave two products (AGT-1 and AGT-2) selectively. From amino acid analysis of the modified peptides, it was found that either of the two histidine residues within the native peptide was modified. Amino-terminal sequence analysis indicated that AGT-1 and AGT-2 were modified at the His9 and the His6, respectively. In addition, the data of FAB-MS and 1H NMR suggested that the unknown residues (modified histidine) within AGT-1 and AGT-2 should have the 2-imidazolone structure. In order to confirm the 2-imidazolone residue in both modified peptides, they were hydrolyzed and analyzed by reverse-phase HPLC. The result demonstrated that the acid hydrolysis of modified peptides gave a product which was identical to authentic 2-imidazolone residue. Consequently, it was confirmed that the reaction of Cu(II)/ascorbate occurs specifically at the C-2 position of the imidazole ring of the histidine residue within a peptide.