Takanori Tsuda

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.

Absorption and metabolism of cyanidin 3-O-β-D-glucoside in rats

We have clarified for the first time how cyanidin 3‐O‐β‐D‐glucoside (C3G), which is a potent antioxidant anthocyanin, is absorbed and metabolized in vivo. Rats were orally administered C3G (0.9 mmol/kg body weight), and C3G rapidly appeared in the plasma. However, the aglycon of C3G (cyanidin; Cy) was not detected, although it was present in the jejunum. Protocatechuic acid (PC), which may be produced by degradation of Cy, was present in the plasma and the concentration was 8‐fold higher than that of C3G. These results suggest that plasma PC and C3G may contribute to the antioxidant activity of the plasma. In the liver and kidney, C3G was metabolized to methylated C3G (methyl‐C3G), suggesting that C3G and/or methyl‐C3G act as antioxidants in the tissues.

Dietary cyanidin 3-O-β-d-glucoside increases ex vivo oxidation resistance of serum in rats

The effect of dietary cyanidin 3-O-β-d-glucoside (C3G), a typical anthocyanin pigment, on the generation of thiobarbituric acid reactive substances (TBARS) during serum formation ex vivo and susceptibility of serum to further lipid peroxidation was studied in rats. Rats were fed a diet containing C3G (2 g/kg) for 14 d. Feeding C3G resulted in a significant decrease in generation of TBARS during serum formation. The serum from the C3G-fed group showed a significantly lower susceptibility to further lipid peroxidation provoked by 2,2′-azobis (2-amidinopropane)hydrochloride or Cu2+ than that of the control group. No significant differences were observed in serum phospholipid, triglyceride, esterified cholesterol, and free fatty acid concentrations between the control and the C3G-fed groups. Concentrations of endogenous antioxidants remaining in the serum after blood coagulation were not affected by the C3G feeding. These results demonstrate that feeding C3G increases the ex vivo oxidation resistance of the serum without affecting serum endogeneous antioxidant levels, and reduces the TBARS generated during serum formation without changing the concentrations of serum lipids.

Mechanism for the peroxynitrite scavenging activity by anthocyanins

We show that anthocyanins can function as potent inhibitors of the formation of nitrated tyrosine in vitro, and clarified how pelargonidin (Pel), which has a mono‐hydroxyl group on the B‐ring, can scavenge peroxynitrite (ONOO−) by detection of the reaction products. Pel was reacted with ONOO−, then the reaction mixture was analyzed using high‐performance liquid chromatography (HPLC). The HPLC analyses showed two novel peaks assumed to be the reaction products. Based on the instrumental analyses, the reaction products were identified as p‐hydroxybenzoic acid and 4‐hydroxy‐3‐nitrobenzoic acid. Pel can protect tyrosine from undergoing nitration through the formation of p‐hydroxybenzoic acid and 4‐hydroxy‐3‐nitrobenzoic acid.

Increase in Oxidative Stress in Kidneys of Diabetic Akita Mice

New biomarkers for oxidative damage, were used to identify whether hyperglycemia caused oxidative stress in diabetic Akita mice. At 13 weeks of age, the tissues of these mice were obtained, and the levels of Nε-(hexanonyl)lysine (HEL) and dityrosine (DT) were measured, these being related to lipid peroxide-derived protein covalent modification and protein cross-linking. The levels of HEL and DT in the kidneys of Akita mice were significantly increased compared with the control mice without any accumulation of thiobarbituric acid reactive substances and 4-hydroxy-2-nonenal-modified protein. Immunopositive staining was clearly observed in the kidneys of the Akita mice when using the anti-HEL antibody or anti-DT antibody. These results suggest that hyperglycemia in Akita mice induced oxidative stress and increased these markers in the kidneys.

Oxidation products of cyanidin 3-O-β-d-glucoside with a free radical initiator

Recently, we have reported that anthocyanins show strong antioxidative activity, but no attention has been paid to anthocyanins from the viewpoint of the reaction mechanism of alkylperoxyl radicals; therefore, we investigated the reaction products of antioxidative anthocyanins (cyanidin 3-O-β-d-glucoside). Cyanidin 3-O-β-d-glucoside was reacted with 2,2′-azobis(2,4-dimet hylvaleronitrile) to generate the alkylperoxyl radicals, and the reaction products were isolated by high-performance liquid chromatography. The products were identified as 4,6-dihydroxy-2-O-β-d-glucosyl-3-oxo-2,3-dihydrobenzofuran and protocatechuic acid. Based on reaction products, the antioxidative mechanism of cyanidin 3-O-β-d-glucoside may be different from that of α-tocopherol; cyanidin 3-O-β-d-glucoside would produce another radical scavenger, as it would break down the structure and scavenge the radicals.

Inhibition of Lipid Peroxidation and Radical Scavenging Effect of Anthocyanin Pigments Isolated from the Seeds of Phaseolus vulgaris L.

Recently, there has been increasing interest in the protective biochemical function of natural antioxidants contained in dietary plants, which are candidates for the prevention of oxidative damage caused by oxygen-free radical species. However, little attention has been paid to the antioxidative activity of edible beans. This background prompted us to investigate the antioxidative activity of bean seeds chemically. Three anthocyanin pigments, cyanidin 3-O-β-D-glucoside (C3G), pelargonidin 3-O-β-D-glucoside, and delphinidin 3-O-β-D-glucoside were isolated and identified from the seed coat of Phaseolus vulgaris L. Among the isolated pigments tested, C3G had marked antioxidative activity in liposomal and rat liver microsomal systems, an inhibitory effect on malondialdehyde (MDA) levels by UV light irradiation, and radical scavenging effects against hydroxyl and superoxide anion radicals. On the basis of these results, C3G was reacted with a radical initiator in a model system and the antioxidative mechanism was discussed. Anthocyanin pigments are widely distributed in the human diet through cereal crops, beans, fruits, and vegetables, indicating that we ingest considerable amounts of anthocyanin pigments from plant-based daily foods, and these pigments may play an important role as dietary antioxidants for the prevention of oxidative damage in living systems.