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The effects of lemon flavonoids, as crude flavonoids prepared from lemon juice, were investigated in diabetic rats. The oxidative stress of eriocitrin (eriodictyol 7-O-beta-rutinoside) and hesperidin (hesperetin 7-O-beta-rutinoside) on streptozotocin-induced diabetic rats was investigated. Diabetic rats were given a diet which contained 0.2% crude flavonoids, 0.2% eriocitrin, and 0.2% hesperidin. After the 28-d feeding period, the concentration of the thiobarbituric acid-reactive substance in the serum, liver, and kidney of diabetic rats administered crude flavonoids, eriocitrin, and hesperidin significantly decreased as compared with that of the diabetic group. The levels of 8-hydroxydeoxyguanosine, which is exchanged from deoxyguanosine owing to oxidative stress, in the urine of diabetic rats administered eriocitrin and hesperidin significantly decreased as compared with that of the diabetic rat group. Crude flavonoids, eriocitrin, and hesperidin suppressed the oxidative stress in the diabetic rats. These results demonstrated that dietary lemon flavonoids of eriocitrin and hesperidin play a role as antioxidant in vivo.

Lipoprotein peroxidation, especially the modification of apolipoprotein B-100, has been implicated to play an important role in the pathogenesis of atherosclerosis. However, there have been few detailed insights into the chemical mechanism of derivatization of apolipoproteins during oxidation. In the present study, we provide evidence that the formation of the toxic pollutant acrolein (CH2=CH–CHO) and its conjugate with lysine residues is involved in the oxidative modification of human low density lipoprotein (LDL). Upon incubation with LDL, acrolein preferentially reacted with lysine residues. To determine the structure of acrolein-lysine adduct in protein, the reaction of acrolein with a lysine derivative was carried out. EmployingNα-acetyllysine, we detected a single product, which was identified to be a novel acrolein-lysine adduct,Nα-acetyl-Ne-(3-formyl-3,4-dehydropiperidino)lysine. The acid hydrolysis of the adduct led to the derivative that was detectable with amino acid analysis. It was revealed that, uponin vitro incubation of LDL with acrolein, the lysine residues that had disappeared were partially recovered byNe-(3-formyl-3,4-dehydropiperidino)lysine. In addition, we found that the same derivative was detected in the oxidatively modified LDL with Cu2+ and that the adduct formation was correlated with LDL peroxidation assessed by the consumption of α-tocopherol and cholesteryl ester and the concomitant formation of cholesteryl ester hydroperoxide. Enzyme-linked immunosorbent assay that measures free acrolein revealed that a considerable amount of acrolein was released from the Cu2+-oxidized LDL. Furthermore, metal-catalyzed oxidation of arachidonate was associated with the formation of acrolein, indicating that polyunsaturated fatty acids including arachidonate represent potential sources of acrolein generated during the peroxidation of LDL. These results indicate that acrolein is not just a pollutant but also a lipid peroxidation product that could be ubiquitously generated in biological systems.

Acrolein (CH 2 =CH—CHO) is known as a ubiquitous pollutant in the environment. Here we show that this notorious aldehyde is not just a pollutant, but also a lipid peroxidation product that could be ubiquitously generated in biological systems. Upon incubation with BSA, acrolein was rapidly incorporated into the protein and generated the protein-linked carbonyl derivative, a putative marker of oxidatively modified proteins under oxidative stress. To verify the presence of protein-bound acrolein in vivo , the mAb (mAb5F6) against the acrolein-modified keyhole limpet hemocyanin was raised. It was found that the acrolein-lysine adduct, N ɛ -(3-formyl-3,4-dehydropiperidino)lysine, constitutes an epitope of the antibody. Immunohistochemical analysis of atherosclerotic lesions from a human aorta demonstrated that antigenic materials recognized by mAb5F6 indeed constituted the lesions, in which intense positivity was associated primarily with macrophage-derived foam cells and the thickening neointima of arterial walls. The observations that ( i ) oxidative modification of low-density lipoprotein with Cu 2+ generated the acrolein-low-density lipoprotein adducts and ( ii ) the iron-catalyzed oxidation of arachidonate in the presence of protein resulted in the formation of antigenic materials suggested that polyunsaturated fatty acids are sources of acrolein that cause the production of protein-bound acrolein. These data suggest that the protein-bound acrolein represents potential markers of oxidative stress and long-term damage to protein in aging, atherosclerosis, and diabetes.

The reaction of lipid hydroperoxide with protein was investigated using an antibody, which was prepared using 15-hydroperoxyeicosatetraenoic acid (15-HPETE)-modified keyhole limpet hemocyanin as an immunogen. The obtained antibody recognized not only 15-HPETE-modified bovine serum albumin (BSA) but also 13-hydroperoxyoctadecadienoic acid (13-HPODE)-modified BSA. Glutaroyl-BSA adduct, which was prepared by the reaction of glutaric anhydride with protein, was also recognized by the antibody. The results revealed that the carboxyl terminus of lipid moiety in adducts was required for an appearance of the antigenicity. The cross-reactivity of phosphatidylcholine hydroperoxide-modified BSA (PCAOOH-BSA) with the antibody was examined. The antibody could not recognize the intact PCAOOH-BSA, whereas alkaline-treated modified BSA revealed the antigenicity. Furthermore, stearic acid at the 1 position in the phospholipid was liberated from the PCAOOH-BSA following treatment with 0.25 N NaOH. The result showed that the phospholipid moiety could be covalently bound to the protein molecule. The formation of esterified fatty acid-protein adduct during oxidation was confirmed using low-density lipoprotein (LDL). During oxidation of LDL by copper ion or 2,2'-azo-bis(2-amidinopropane)dihydrochloride, the formation of antigenic materials was observed in a time- or dose-dependent fashion. The antigenicity was significantly enhanced by the alkaline treatment on the oxidized LDL, suggesting that considerable amounts of oxidized esterified fatty acids can covalently react with apoprotein B-100 in oxidatively modified LDL.

The difference in the distribution of sesamin, sesamolin, sesaminol glucosides and sesamolinol components in representative varieties of sesame were compared. Quantitation was achieved by HPLC determination using an external standard. The most abundant lignan glucosides in sesame seed were sesaminol triglucoside. The content of sesaminol triglucoside in 100 g seeds ranged from 14.1 to 91.3 mg with an average value of 68.4 mg; that of sesaminol diglucoside from 8.2 to 18.3 mg with an average value of 11.6 mg; and that of sesaminol monoglucoside from 5.4 to 19.5 mg with an average value of 8.3 mg. The total content of sesaminol glucoside was 88.3 mg in 100 g of sesame seeds. Also, the sesamolinol content in 100 g sesame seed ranged from 17.6 to 28.5 mg, the average being 20.5 mg. Within

We investigated the antioxidative flavonoid glycosides in the peel extract of lemon fruit (Citrus limon). Six flavanon glycosides: eriocitrin, neoeriocitrin, narirutin, naringin, hesperidin, and neohesperidin, and three flavone glycosides: diosmin, 6,8-di-C-β-glucosyldiosmin (DGD), and 6-C-β-glucosyldiosmin (GD) were identified by high-performance liquid chromatography (HPLC) analysis. Their antioxidative activity was examined using a linoleic acid autoxidation system. The antioxidative activity of eriocitrin, neoeriocitrin and DGD was stronger than that of the others. Flavonoid glycosides were present primarily in the peel of lemon fruit. There was only a small difference in the content of the flavonoid glycosides of the lemon fruit juice from various sources and varieties. Lemon fruit contained abundant amounts of eriocitrin and hesperidin and also contained narirutin, diosmin, and DGD, but GD, neoeriocitrin, naringin, and neohesperidin were present only in trace amounts. The content of DGD, GD, and eriocitrin was especially abundant in lemons and limes; however, they were scarcely found in other citrus fruits. The content of flavonoid compounds in lemon juice obtained by an in-line extractor at a juice factory was more abundant than that obtained by hand-squeezing. These compounds were found to be stable even under heat treatment conditions (121°C, 15 min) in acidic solution.

The effects of lemon juice and its crude flavonoids on blood pressure were examined using spontaneously hypertensive rats (SHR). The 5% diluted lemon juice was orally administered in the diet to SHR, and they tended to have a lower systolic blood pressure than the control rats after 90 days. The systolic blood pressure of SHR fed a diet containing crude flavonoids from the juice for 16 weeks was significantly lower than that of the control group (p

The antioxidative substances contained in cacao liquor, which is one of the major ingredients of chocolate, were separated by column chromatography and high-performance liquid chromatography. Three major compounds were purified and two of them were identified by 1H, 13C NMR and mass spectra as (-)-epicatechin (EC) and (+)-catechin (CA). Their antioxidative activity was measured by monitoring the peroxide value of linoleic acid and the thiobarbituric acid-reactive substance values of erythrocyte ghost membranes and microsomes. EC and CA had strong antioxidative effects in all three methods, but one unidentified peak was found to be less effective. Additionally, we ana-lyzed the polyphenol concentration of cacao liquor extractions produced in several countries. The total polyphenol concentration was 7.0 to 13.0%, catechin concentration was 0.31 to 0.49%, and epicatechin con-centration was 0.35 to 1.68% in the extractions. It is believed that cho-colate is stable against oxidative deterioration on account of the presence of these polyphenolic compounds, and it is also expected to have a pro-tective role against lipid peroxidation in living systems.

The inhibitory effects of curcumin and two tetrahydrocurcuminoids on tumor promoter-induced oxidative stress in vitro and in vivo were investigated. Curcumin, tetrahydrocurcumin (THC) and dihydroxytetrahydrocurcumin (DHTHC) exhibited significant inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced O2-generation in differentiated HL-60 cells. The inhibitory activity of THC was weaker than that of curcumin. This tendency was the inverse of the results of previous studies on in vitro antioxidative activity against lipid peroxidation. The curcuminoids inhibited TPA-induced intracellular peroxide formation in differentiated HL-60 cells. THC exhibited much weaker inhibition of intracellular peroxide formation than curcumin, suggesting that this inhibition might be attributable to the inhibition of O2-generation. The inhibitory effects of curcuminoids on TPA-induced H2O2 formation in female ICR mouse skin were further examined using the double-TPA-application model. Each TPA application induces two distinct biochemical events, 1) recruitment of inflammatory cells to the inflammatory regions and 2) activation of oxidant-producing cells. Double pretreatment of mice with curcuminoids before each TPA treatment significantly suppressed double TPA application-induced H2O2 formation in the mouse skin. Coadministrations of curcumin with either first or second TPA treatment significantly inhibited H2O2 formation. In addition, THC tends to show weaker inhibitory activities than curcumin in bioassays related to tumor promotion, i.e., inhibition of tumor promoter-induced inflammation in mouse skin and Epstein-Barr virus activation. These tendencies were parallel to those in the tumor-suppressive potential of curcumin and THC in mouse skin, as previously reported. Thus, we concluded that curcuminoids significantly suppress TPA-induced oxidative stress via both interference with infiltration of leukocytes into the inflammatory regions and inhibition of their activation.

It has been established that the covalent modification of proteins occurs in vivo as a consequence of reaction with reactive lipids such as 4-hydroxy-2-nonenal (HNE). The fact that HNE occurs in blood under physiological as well as pathophysiological conditions suggests that erythrocytes undergo modification by HNE. Here we describe the immunochemical characterization of HNE-treated erythrocytes by demonstrating the Michael-type HNE addition to both membrane and cytosolic proteins. Exposure of erythrocytes to HNE (0-0.5 mM) for 2 h resulted in HNE labeling of multiple membrane proteins. Pretreatment of erythrocytes with a sulfhydryl reagent, N-ethylmaleimide (NEM), resulted in a significant decrease of HNE attached to the proteins, suggesting that HNE primarily reacts with the sulfhydryl groups of erythrocyte membrane proteins, whereas enhanced HNE labeling of the membrane proteins was observed when the erythrocytes were pretreated with H2O2 (0.1-5 mM) for 15 min. On the other hand, highly selective modification of a 30 kDa protein was observed in the hemolysates of erythrocytes treated with HNE. The protein, which represents a major intracellular target of HNE in erythrocytes, was identified as carbonic anhydrase, based on the observations that (i) a reverse-phase HPLC analysis of the chloroform/ethanol extract of HNE-treated erythrocytes detected two major proteins, which cross-reacted with anti-carbonic anhydrase antibody as well as with the anti-HNE adducts antibody, (ii) the chloroform-ethanol extraction of authentic carbonic anhydrase gave a similar HPLC pattern, and (iii) the HNE treatment of erythrocytes resulted in the partial inhibition of the carbonic anhydrase activity.

Malondialdehyde (MDA), a naturally occurring dialdehyde produced in the membrane lipid peroxidation, is known to react with lysine residues of proteins, but the MDA-lysine adducts generated in the proteins have not been characterized adequately. In the present study, we provide evidence that the enaminal-type MDA-lysine adduct, N(epsilon)-(2-propenal)lysine, is formed in human low-density lipoprotein (LDL) upon reaction with MDA or Cu2+. We found that the incubation of N(alpha)-acetyllysine with MDA generated N(alpha)-acetyl-N(epsilon)-(2-propenal)lysine as the predominant product. In addition, a polyclonal antiserum raised against the MDA-modified protein was found to contain antibody populations that could be purified by affinity gel prepared by covalent attachment of N(alpha)-acetyl-N(epsilon)-(2-propenal)lysine. It was concluded that the affinity-purified anti-N(epsilon)-(2-propenal) lysine antibody was highly specific to the enaminal derivative of both lysine residues and phosphatidylethanolamine, based on the observations that (i) MDA was the only aldehyde which generated immunoreactive materials in proteins; (ii) among structurally defined MDA-lysine adducts tested, the antibody recognized the enaminal adduct only; and (iii) immunoreactivity to N-(2-propenal)serine was still significant but much weaker than its reactivity to N-(2-propenal)ethanolamine. Furthermore, analysis of antibody recognition sites with a variety of N-(2-propenal)alkylamines revealed that the mono-specific antibody recognized the N-2-propenal-N-ethyl moiety [-(CH2)2-NH-CH=CH-CHO] of enaminal adducts. Determination by a competitive enzyme-linked immunosorbent assay demonstrated that N(epsilon)-(2-propenal)lysine accounted for 33.7 and 3.1% of the lysine residues that disappeared during in vitro incubation of LDL with MDA and Cu2+, respectively. These results suggest that N(epsilon)-(2-propenal)lysine represents a major form of MDA covalently attached to proteins.

To investigate the prevention of protein modification by food components, effects of natural antioxidants on in vitro modification of collagen by peroxynitrite were examined using a polyclonal antibody specific to 3-nitrotyrosine. To perform the assay with many samples at a time, the inhibitory effects of antioxidants were evaluated using an enzyme-linked immunosorbent assay. Polyphenols such as caffeic acid, curcumin, and flavonoids showed strong inhibitory effects on the formation of 3-nitrotyrosine in peroxynitrite-modified collagen. The inhibitory effects of caffeic acid and its related compounds on nitration of Tyr were then further investigated. Both caffeic acid, ferulic acid, and p-coumaric acid inhibited the modification, whereas cinnamic acid did not. By the treatment of p-coumaric acid with peroxynitrite, nitrated p-coumaric acid was detected in the reaction mixture using a liquid chromatograph-mass spectrometer (LC-MS). These results suggest that some phenolic antioxidants may prevent tissue i...

Membrane lipid peroxidation is known to produce various aldehydic compounds which cause a wide range of biological effects including heart disease, aging, and cancer. To investigate the effect of lipid peroxidation products on the expression of glutathione S-transferases (GSTs), which catalyze the conjugation of reactive chemicals with glutathione and play an important role in protecting cells, normal rat liver epitherial cells (RL34) were exposed to a variety of aldehydic compounds. We found that the GST activity in RL34 cells was induced by α,β-unsaturated aldehydes, such as acrolein (1.3-fold), crotonaldehyde (1.3-fold), 4-hydroxy-2-hexenal (HHE) (1.4-fold), and 4-hydroxy-2-nonenal (HNE) (1.7-fold). The induction of GST activity by HNE was time-dependent, reaching a plateau after 16 h. The immunoblot analysis using the polyclonal antibodies against GST isozymes demonstrated that GST-P (π-class), a well-known tumor marker, was significantly induced 16 h after the HNE treatment. Also, immunostaining for the presence of GST-P confirmed the enhanced expression of GST-P in the cytoplasm of the cells. Northern blot analysis revealed that the HNE treatment of RL34 cells for 1 h enhanced the expression of GST-P mRNA, which returned to the control level after 16 h. These data suggest that the induction of GST-P by HNE may represent an important cellular defense mechanism against oxidative injury.

Steamed soybeans (SSB) were fermented with 30 kinds of Aspergillus strains which have been used in manufacturing Japanese fermented foods and beverages. Methanol extracts from the fermented soybean...

Many antioxidants have been found in spices and herbs, and some of them are well known as strong scavengers of active oxygen radicals. We have isolated active products, which markedly inhibited the formation of malondialdehyde (MDA from 2-deoxyribose and the hydroxylation of benzoate with the hydroxyl radical, from methanol extracts of allspice and clove. Pimentol from allspice, and biflorin and its isomer, abbreviated as clove3, from clove were identified as the active principles. These revealed strong activity as hydroxyl radical scavengers at a concentration of 2.0 microM. The antioxidative activities in an in vitro model system involving the rabbit erythrocyte membrane ghost were as strong as those of alpha-tocopherol at 200 microM. Such advanced glycation end products (AGE) as pentosidine are biomarkers of diabetes mellitus, and active oxygens have been suggested to be involved in the formation of AGE. The above-mentioned free radical scavengers effectively inhibited the formation of pentosidine in a model system of N alpha-t-butoxycarbonyl-fructoselysine and N alpha-t-butoxycarbonyl-arginine.

The effects of methanol extracts of 51 spices on ·OH scavenging were studied in detail. 2-Deoxyribose oxidation and sodium benzoic acid hydroxylation methods were used for detecting the scavenging activity of ·OH. Mustard varieties, thyme, oregano, clove, and allspice all exhibited strong ·OH-scavenging activity. In particular, 3 varieties of mustard had above 90% ·OH-scavenging activity with a 1 μg/ml concentration of their extracts. The ·OH scavenger of Brassica nigra (brown mustard) was isolated and purified by XAD-2 column chromatography and preparative HPLC, and was identified as a 3,5-dimethoxy-4-hydroxycinnamic acid methyl ester by MS, ‘H-NMR, and 13C-NMR. The 3,5-dimethoxy-4-hydroxycinnamic acid methyl ester was prepared by methylating of sinapic acid with diazomethane.

An antioxidant was isolated from the peel and juice of lemon fruit (Citrus limon BURM. f.). It was identified as eriocitrin (eriodictyol 7-rutinoside) of the flavanone glycoside by HPLC, 1H-NMR and 13C-NMR analyses. The purified eriocitrin was readily soluble in water, methanol, and ethanol. A water solution of 0.05% eriocitrin was weakly acidic (pH 4.2). Eriocitrin was found to be stable even at high temperature (121°C, 15 min) in acidic solution (pH 3.5). The distribution of eriocitrin in citrus fruits was found to be especially abundant in lemons and limes, however, it was scarcely found in other citrus fruits. In the case of lemon fruit, eriocitrin was primarily distributed in the peel (about 1,500 ppm) composed of the albedo (mesocarp), flavedo (epicarp), and pulp vesicles. It was also significantly present in the juice (about 200 ppm) but was not detected in the seed. Two varieties of lemon fruits, eureka and lisbon, almost had the same eriocitrin content. The antioxidative activity of eriocitrin in the linoleic acid autoxidation system was equal to that of α-tocopherol, and it was enhanced when used together with citric acid. The eriocitrin had a synergistic effect on α-tocopherol.

We demonstrated that a dietary supplement, Antioxidant Biofactor (AOB®), a fermented grain food, inhibits phorbol ester (TPA)-induced inflammatory responses. Pretreatment of mouse skin with ethyl acetate extract of AOB (0.5mg) significantly attenuated the hydrogen peroxide level in mouse skin without inhibition of edema formation. Also, exposure of AOB extract to differentiated HL-60 cells resulted in dose-dependent inhibition of TPA-induced superoxide generation, which was stronger than those of the raw materials of AOB. Our results suggest that AOB is effective for prevention of skin oxidative stress.

Nervous system controls all the organs in the living like a symphony. In this chapter, the mechanism of neuronal death in aged is discussed in relation to oxidative stress. Polyunsaturated fatty acid (PUFA) is known to be rich in the membranous component of the neurons and plays an important role in maintaining the neuronal functions. Recent reports revealed that oxidation of omega-3 and omega-6 PUFAs, such as docosahexaenoic acid (DHA) and arachidonic acid (ARA), are potent antioxidant but simultaneously, their oxidation products are potentially toxic. In this chapter, the existence of early oxidation products of PUFA is examined in the samples from neurodegenerative disorders and the cellular model. Accumulation of proteins with abnormal conformation is suggested to induce neuronal death by disturbance of proteolysis and mitochondrial function. The role of lipid peroxide and lipid-derived aldehyde adduct proteins is discussed in relation to brain ageing and age-related neurodegeneration.

Dopamine is the endogenous neurotransmitter produced by nigral neurons. Dopamine loss can trigger not only prominent secondary morphological changes, but also changes in the density and sensitivity of dopamine receptors; therefore, it is a sign of PD development. The reasons for dopamine loss are attributed to dopamine's molecular instability due to it is a member of catecholamine family, whose catechol structure contributes to high oxidative stress through enzymatic and non-enzymatic oxidation. Oxidative stress in the brain easily leads to the lipid peroxidation reaction due to a high concentration of polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA, C22:6/ω-3) and arachidonic acid (AA, C18:4/ω-6). Recent studies have shown that lipid hydroperoxides, the primary peroxidative products, could non-specifically react with primary amino groups to form N-acyl-type (amide-linkage) adducts. Therefore, based on the NH2-teminals in dopamine's structure, the aims of this chapter are to describes the possibility that reactive LOOH species derived from DHA/AA lipid peroxidation may modify dopamine to form amide-linkage dopamine adducts, which might be related to etiology of Parkinson's diseases.

Phospholipids such as phosphatidylethanolamine and phosphatidylcholine play crucial roles in the biological system to maintain the cellular environmental condition. Despite that, oxidative stress targets these phospholipids containing polyunsaturated fatty acids and accompanies the oxidized phospholipids. Recent studies have been suggested that oxidized phospholipids have the relationship with inflammation and might induce the atherosclerosis formation by uptake of oxidized LDL through scavenger receptor as ligands. Red blood cells, which have been studied the bilayer model, are also modified by oxidative stress because hemoglobin can mediate and produce the reactive oxygen species, which leads to lipid peroxidation of biomembrane. In these oxidation processes of biomolecules, hexanoylation against phosphatidylethanolamine and phosphatidylserine, which has the primary amine and is the target of this modification, generates the oxidized membrane such as erythrocyte ghosts. This unique structure of phosphatidylethanolamine and phosphatidylserine is possibly the useful biomarker to evaluate the oxidation of biomembrane in vivo using liquid chromatography tandem mass spectrometry and monoclonal antibody.

We have determined that a biological molecule can be physically immobilized on a polymer containing an azobenzene (azopolymer) using irradiating light. We immobilized antibodies and antigens on the surface of an azopolymer coated glass slide (antibody array) to establish "on-chip ELISAs". The assays used the flat-surface of a glass slide and could be applied to both sandwich and competitive ELISAs. The sensitivity and accuracy of the on-chip ELISA were similar to a conventional ELISA using a polystyrene plate. Using the assay system, we proved that representative oxidative-biomarkers could be simultaneously measured from uL of urine. That should realize low-cost study on animal or human, and accelerate development of health-promoting foods. So, this new concept antibody array has promising applications in proteomic studies, and could be used to examine biomarkers to investigate health-promoting food.

Nervous system controls all the organs in the living like a symphony. In this chapter, the mechanism of neuronal death in aged is discussed in relation to oxidative stress. Polyunsaturated fatty acid (PUFA) is known to be rich in the membranous component of the neurons and plays an important role in maintaining the neuronal functions. Recent reports revealed that oxidation of omega-3 and omega-6 PUFAs, such as docosahexaenoic acid (DHA) and arachidonic acid (ARA), are potent antioxidant but simultaneously, their oxidation products are potentially toxic. In this chapter, the existence of early oxidation products of PUFA is examined in the samples from neurodegenerative disorders and the cellular model. Accumulation of proteins with abnormal conformation is suggested to induce neuronal death by disturbance of proteolysis and mitochondrial function. The role of lipid peroxide and lipid-derived aldehyde adduct proteins is discussed in relation to brain ageing and age-related neurodegeneration.

We have determined that a biological molecule can be physically immobilized on a polymer containing an azobenzene (azopolymer) using irradiating light. We immobilized antibodies and antigens on the surface of an azopolymer coated glass slide (antibody array) to establish “on-chip ELISAs”. The assays used the flat-surface of a glass slide and could be applied to both sandwich and competitive ELISAs. The sensitivity and accuracy of the on-chip ELISA were similar to a conventional ELISA using a polystyrene plate. Using the assay system, we proved that representative oxidative-biomarkers could be simultaneously measured from uL of urine. That should realize low-cost study on animal or human, and accelerate development of health-promoting foods. So, this new concept antibody array has promising applications in proteomic studies, and could be used to examine biomarkers to investigate health-promoting food.

Dopamine is the endogenous neurotransmitter produced by nigral neurons. Dopamine loss can trigger not only prominent secondary morphological changes, but also changes in the density and sensitivity of dopamine receptors; therefore, it is a sign of PD development. The reasons for dopamine loss are attributed to dopamine’s molecular instability due to it is a member of catecholamine family, whose catechol structure contributes to high oxidative stress through enzymatic and non-enzymatic oxidation. Oxidative stress in the brain easily leads to the lipid peroxidation reaction due to a high concentration of polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA, C22:6/ω-3) and arachidonic acid (AA, C18:4/ω-6). Recent studies have shown that lipid hydroperoxides, the primary peroxidative products, could non-specifically react with primary amino groups to form N-acyl-type (amide-linkage) adducts. Therefore, based on the NH2-teminals in dopamine’s structure, the aims of this chapter are to describes the possibility that reactive LOOH species derived from DHA/AA lipid peroxidation may modify dopamine to form amide-linkage dopamine adducts, which might be related to etiology of Parkinson’s diseases.

Phospholipids such as phosphatidylethanolamine and phosphatidylcholine play crucial roles in the biological system to maintain the cellular environmental condition. Despite that, oxidative stress targets these phospholipids containing polyunsaturated fatty acids and accompanies the oxidized phospholipids. Recent studies have been suggested that oxidized phospholipids have the relationship with inflammation and might induce the atherosclerosis formation by uptake of oxidized LDL through scavenger receptor as ligands. Red blood cells, which have been studied the bilayer model, are also modified by oxidative stress because hemoglobin can mediate and produce the reactive oxygen species, which leads to lipid peroxidation of biomembrane. In these oxidation processes of biomolecules, hexanoylation against phosphatidylethanolamine and phosphatidylserine, which has the primary amine and is the target of this modification, generates the oxidized membrane such as erythrocyte ghosts. This unique structure of phosphatidylethanolamine and phosphatidylserine is possibly the useful biomarker to evaluate the oxidation of biomembrane in vivo using liquid chromatography tandem mass spectrometry and monoclonal antibody.

The amino-carbonyl reaction (The Maillard reaction) of bovine lens crystallin, serum albumin or skin collagen with glucose was investigated to find effective means to prevent the formation of Advanced Glycation End Products (AGE) and induce the reversible solubilization of polymerized glycated proteins. The organic germanium compounds (Ge-132, 373, 385), derivatives of amino acids containing germanium as the linker of framework, were combined by the box titration method to determine the dose that would be most effective, compared with Aminoguanidine-HCl (AMG),α-tocopherol (VE), and pirenoxine (Catalin-K, CK). Although AMG suppressed the formation of AGE, effective concentrations were higher than 20 mM. Ge-385, when administered by itself at a low dose, induced the reversible solubilization of AGE made from crystallin, and albumin. The addition of any two reagents such as AMG, VE, CK and Ge-132 or 385 together to proteins lessened the effective range, and the peaks of smaller molecules in the profiles of HPLC and PAGE were quite remarkable. Examination was made of the effects of Ge-132 on the eyes of SAM mice, which show senescence accelerated cataracts at a relatively young age. The prevention of cataract-genesis and induction of reversible transparency of turbid lenses became evident following the administration of Ge-132 to the eyes 4 times a day. The mode of action of organic germanium compounds was demonstrated quite capable of disconnecting the sugar-parts from AGE by decarbonylation, resulting in the formation of glucosone and amino residues, and further leading subsequently to fewer AGE.

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-beta-D-glucoside). Cyanidin 3-O-beta-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-beta-D-glucosyl-3-oxo-2,3-dihydroben-zofuran+ ++ and protocatechuic acid. Based on reaction products, the antioxidative mechanism of cyanidin 3-O-beta-D-glucoside may be different from that of alpha-tocopherol; cyanidin 3-O-beta-D-glucoside would produce another radical scavenger, as it would break down the structure and scavenge the radicals.

8-Hydroxy-2'-deoxyguanosine (8-OHdG) is a mutation-prone (G:C to T:A transversion) DNA base-modified product generated by reactive oxygen species or photodynamic action. G:C to T:A transversions are observed in the p53 and ras genes of UVB-induced skin cancers of mice and in squamous and basal cell carcinomas of human skin exposed to sunlight. In the current study, 8-OHdG formation was evaluated in the epidermis of hairless mice after repeated exposure to UVB, and possible mechanisms involved were studied. Exposure of hairless mice to either 3.4 [2 minimal erythema dose (MED)] or 16.8 (10 MED) kJ/m2 of UVB three times a week for 2 wk induced a 2.5- or 6.1-fold increase, respectively, in the levels of 8-OHdG in DNA, compared to the unexposed controls. An immunohistochemical method using a monoclonal antibody specific for 8-OHdG showed stronger and more extensive staining in the nuclei of UV-irradiated epidermal cells than in those of nonirradiated cells. Western blots probed with antibodies against 4-hydroxy-2-nonenal-modified proteins confirmed the involvement of reactive oxygen species in the epidermal damage induced by chronic UVB exposure. 3-Nitro-L-tyrosine was detected in western blots in a concentration-dependent manner, suggesting that peroxynitrite derived from the reaction of nitric oxide and superoxide, both of which were probably released from inflammatory cells, was involved in modifying the DNA bases. Therefore, the formation of 8-OHdG after UVB exposure appears to be regulated by at least three pathways: photodynamic action, lipid peroxidation, and inflammation and may play a role in sunlight-induced skin carcinogenesis.

We report a novel type of fluorescent product derived from the reaction of the lysine residue with malondialdehyde (MDA). When the lysine-containing peptide ( N- acetyl-glycyl - l - lysine methyl ester ) was treated with MDA prepared by the acid hydrolysis of 1,1,3,3-tetramethoxypropane, the main fluorescent product, which corresponded neither to the 1-amino-3-iminopropene derivative (2) nor to the 4-methyl-1,4-dihydro-3,5-dicarbaldehyde derivative (3), was detected by reverse-phase HPLC. By analysis of its UV, NMR, and high-resolution FAB mass spectra, it was confirmed to be (1). This finding may provide a new clue to the formation mechanisms of fluorescent lipofuscin-like pigment.

4-Hydroxy-2-nonenal (HNE), one of the major products of membrane lipid peroxidation, has been shown recently to be present in a form covalently attached to proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate (Toyokuni, S., et al. (1994) Proc. Natl. Acad. Sci. USA 91, 2616–2620; Uchida, K., et al. (1995) Arch. Biochem. Biophys. 317, 405–411). In the present study, the mechanism of HNE cytotoxicity was studied using the renal tubular epithelial cells (LLC-PK 1 ), focusing on the protein modification and alteration of cellular redox status induced by HNE. Upon treatment with HNE for 2 h, the LLC-PK 1 cells were found to be resistant to the low concentration (10 μ M ) of HNE, while HNE at higher concentrations (≥50 μ M ) mediated cell death. The cytotoxicity of HNE appeared to be correlated with the HNE modification of cellular proteins. Among a number of proteins modified by HNE, a glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was detected as one of the major targets of HNE in the cells. On the other hand, exposure of LLC-PK 1 cells to HNE resulted in rapid reduction of cellular glutathione (GSH) levels, suggesting that HNE influenced primarily the redox status of the cells. Depletion of GSH with buthionine sulfoximine, a potent suppressor of GSH biosynthesis, before HNE treatment caused the cells to be sensitive to HNE cytotoxicity and to HNE modification of cellular proteins, whereas the increase in intracellular GSH levels by treatment with N -acetylcysteine before HNE treatment resulted in a dose-dependent inhibition of HNE-mediated protein modification. These results suggest that intracellular GSH is a determinant on cellular resistance against the HNE-mediated cytotoxicity.

The behavior of sulfites in beer during fermentation and storage after packaging was studied using a high-performance liquid chromatographic-fluorescence detection method that was developed for det...

An analytical method was based on the aldehyde-bisulfite (separated by HPLC) reaction with o-phthalaldehyde in the presence of ammonium acetate, producing a fluorescent compound. Detection limits of alkanal-bisulfite (from formaldehyde- to octylaldehyde-bisulfite) and 2-alkenal-bisulfite (from trans-2-hexenal- to trans-2-nonenal-bisulfite) ranged from 5 nM to 25 nM. The method was less sensitive for free sulfite (detection limit 100 μM) than for aldehyde-bisulfite. Increasing acetaldehyde-bisulfite with addition of 25 mM acetaldehyde to a beer sample showed free sulfite in the beer. Free sulfite and acetaldehyde-bisulfite were detected in commercial beers but other aldehyde-bisulfites were not.

Dried tempeh is known to be significantly stable to lipid oxidation compared with unfermented soybeans (BS). A new antioxidant was isolated from the methanol extract of tempeh and identified as 3-hydroxyanthranilic acid (HAA) by UV, IR, EI-MS, and 1H-, 13C-, HMQC-, and HMBC-NMR techniques. HAA was effective in preventing autoxidation of soybean oil and soybean powder, while 6,7,4‘-trihydroxyisoflavone (a well-known antioxidant) was not. HAA also exhibited strong antioxidative activity in both water/ethanol and rabbit erythrocyte membrane ghost systems. HAA was not found in BS, but was produced during the incubations with Rhizopus oligosporus IFO 32002 and 32003. The amount of HAA reached a maximum at the stage of 2 days (acceptable), which had the strongest antioxidative activity. Keywords: Tempeh; fermented soybean product; antioxidant; 3-hydroxyanthranilic acid

Supercritical carbon dioxide (SC-CO 2 ) extraction was investigated for extraction of antioxidants from tamarind (Tamarindus indica L.) seed coat. Different combinations of pressure and temperature were used with and without ethanol as modifier. As temperature and pressure were increased, more antioxidants were extracted. Using ethanol as the modifier gave more extraction yield of antioxidant compounds. The SC-CO 2 extract with ethanol (30 MPa, 80 °C) was also a strong antioxidant for lard as well as linoleic acid autoxidation. These results suggest that the antioxidants included in tamarind seed coat can be extracted using SC-CO 2 and that they may be used for increasing the shelf life of foods by preventing lipid peroxidation.

Among the aldehydes that originate from the peroxidation of cellular membrane lipids, 4-hydroxy-2-nonenal (HNE) is thought to be largely responsible for cytopathological effects observed during oxidative stress. Taking advantage of the fact that HNE is very reactive with proteins and forms stable Michael addition-type adducts, a novel immunochemical procedure for quantifying "free" HNE has been developed. The method designated as "HNE-trapping ELISA" is based on the detection of HNE trapped by a protein that has been coated in the immunoplate. The HNE-derived epitopes generated in the coating protein are then detected by the ELISA using a monoclonal antibody (mAbHNEJ-2) specific to the haptenic groups of the HNE-protein conjugates. Using this method, we determined that a considerable amount of HNE was released from human plasma low density lipoproteins (LDL) treated with copper ions or endothelial cells.

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.

In order to develop a new type of antioxidative compound which has both the phenolic and beta-diketone moiety in the same molecule, we converted three known curcuminoids, curcumin (diferuloylmethane, U1), (4-hydroxy-3-methoxycinnamoyl)methane (U2), and bis-(4-hydroxycinnamoyl)methane (U3), which are the natural antioxidants of Curcuma longa L. (tumeric), to tetrahydrocurcuminoids (THU1, THU2, and THU3, respectively) by hydrogenation, and evaluated their antioxidative activity by using linoleic acid as the substrate in an ethanol/water system. Further, we used the rabbit erythrocyte membrane ghost and rat liver microsome as in vitro systems and determined the antioxidative activity of these curcuminoids. When we evaluated their antioxidative activity by these assays, it was found that THU1 had the strongest antioxidative activity among all curcuminoids in each assay system. THU1 has been reported to be one of the main metabolites of U1 in vivo [Holder et al., Xenobiotica, 8, 761-768 (1978)]. These results suggest that THU1 must play an important role in the antioxidative mechanism of U1 in vivo by converting U1 into THU1.

タマリンド種皮抽出物の抗酸化性を食品加工へ利用するための基礎について検討した.(1) タマリンド種皮抽出物は,リノール酸モデル系において強い抗酸化性を示し,クエン酸及びα-トコフェロールとの間に相乗効果を示した.(2) タマリンド種皮抽出物は,100℃,2時間の加熱に対して安定であり,pHの変化に対しては,pH 5.0で抗酸化性の低下が認められた.また塩化ナトリウム存在下では,10%溶液中でも抗酸化性は,70%以上の残存率を示した.(3) 実際の食用油脂としてラード及びコーンサラダ油にタマリンド種皮抽出物を添加したところ,いずれの油脂においても抗酸化性を示した.

Soy isoflavonoids have many useful properties. However, they are metabolized in vivo, including in humans. The effect of the metabolism of soy isoflavonoids on their properties is not fully understood. We have isolated the bacterial strain SY8519, which has been shown to metabolize daidzein to O-desmethylangolensin and to produce 2-(4-hydroxyphenyl)propionic acid from genistein. According to chiral HPLC analysis, the 2-(4-hydroxyphenyl)propionic acid obtained from the bacterium was optically active. To determine the absolute stereochemistry of the microbial product, we prepared (S)-2-(4-hydroxyphenyl)propionic acid from (S)-2-phenylpropionic and concluded that the microbial product had an R-configuration by chiral HPLC analysis. We also applied the metabolite to mouse adipocytes and found that 2-HPPA was less effective at reducing leptin secretion than the parent compound genistein. Our results suggested that 'O-desmethylangolensin-production' attenuates the effect of soy isoflavonoids by reducing not only the activity of daidzein but also that of genistein.

Antioxidative activity of tamarind (Tamarindus indica L.) seeds was investigated. An ethanol extract prepared from the seed coat exhibited antioxidative activity as measured by the thiocyanate and thiobarbituric acid (TBA) method, but there was no activity in the extract prepared from germ. The ethyl acetate extract prepared from seed coat had strong antioxidative activity. To determine antioxidative compounds in the seed coat chemically, preparative HPLC was carried out, then 2-hydroxy-3',4'-dihydroxyacetophenone (TAO), methyl 3,4-dihydroxybenzoate (TAI), 3,4-dihydroxyphenyl acetate (TA2), and (-)-epicatechin (TA3) were isolated from the ethyl acetate extract and identified

The antioxidative activity of the anthocyanin pigments cyanidin 3-O-β-D-glucoside (C3G) and cyanidin (Cy) was examined by using linoleic acid autoxidation, liposome, rabbit erythrocyte membrane, and rat liver microsomal systems. C3G and Cy had antioxidative activity in all systems. Cy had a stronger activity than C3G and the same activity as α-tocopherol (Toc) in the liposome and rabbit erythrocyte membrane systems. In the rat liver microsomal system, Cy and C3G exhibited stronger activity than Toc. These data suggest that the pigments may play an important role in the prevention of lipid peroxidation of cell membranes induced by active oxygen radicals in living systems as dietary antioxidants after ingestion

The mechanism of bisulfite adduct contribution to stability of beer quality was studied. Formaldehyde- and acetaldehyde-bisulffite adducts had only a poor oxidation potential around 0.93 V, which is the oxidation potential of free sulfite so that they lost their reducing activity for iodine. However, they inhibited the chemiluminescence production of beer and Cypridina lucifern analog dependent luminescence in beer, indicating that they inhibit the free radical reactions in beer. The acetaldehyde-bisulfite adduct inhibited haze formation and flavor staling of beer during storage. Therefore, it could be thought that aldehyde-bisulfite adducts have a radical scavenging activity and protect the free radical chain reactions during beer storage, leading to the stability of beer quality

Antioxidative activity of pea bean extract was examined. A TFA-ethanol extract prepared from red and black bean seed coat exhibited strong antioxidative activity. To determine the antioxidative role of the pigments in the seed coats, the pigments, RP0 (cyanidin 3-O-β-D-glucoside) and RP1 (pelargonidin 3-O-β-D-glucoside) from red bean and BP1 (delphinidin 3-O-β-D-glucoside) from black bean, were isolated. RPO showed strong antioxidative activity in the linoleic acid system at neutral condition (pH 7.0), while RP1 and BP1 exhibited no antioxidative activity at pH 7.0. In acidic conditions (pH 3.0 and 5.0), RP1 and BP1 showed strong antioxidative activity

有機ゲルマニウム化合物,とくにBis(2-carboxyethylgermanium)trioxide[(GeCH2CH2COOH)2O3,Ge-132],Bis(2-amino-2-carboxy-1-phenylethylgermanium)trioxide[GeCHPhCH(NH2)COOH2O3,Ge-373],Bis(2-amino-2-carboxyethylgermanium)trioxide[GeCH2CH(NH2)COOH]2O3,Ge385]の生理活性をinvitroモデル実験系および実験動物(糖尿病ラット)に投与して検討した.Ge-373は,DNAを断片化する作用を示した.この事実から,有機ゲルマニウム化合物が糖に対して特殊な作用を発揮するのではないかと考え,MaiUard反応(non-enzymaticamino-carbonylreaction)における効果の検討を行った.種々アミン酸とリボースを混合しAmadori転移およびAdvancedGlycationEndproducts(AGE)の生成を測定する実験系に,Ge-132またはGe-385を加え,AGE形成を蛍光分光光度計により測定した.有機ゲルマニウム化合物は,Amadori転移産物の形成は阻害しなかったが,AGEの形成を抑制した.とくにGe-385は,いったんできたAGEを可逆的に減少させる作用を発揮した.Streptozotocin(STZ)により誘発した糖尿病ラットにGe-132またはGe-385を100mg/kg/d経口投与し,糖尿病合併症に対する効果を観察した.Glycatedalbumin,fructosamineの低下,レンズ混濁の低下が観察された.糖と有機ゲルマニウム化合物,とくにGe-132との相互作用の様子をNuclearMagneticResonance(NMR)を用いて解析した.Ge-132は糖の1および2位のヒドロキシル基と反応し,糖一有機ゲルマニウム複合体を形成することが判明した.この反応がAmadori転移以降の反応を阻害し,AGEの可逆的可溶化を誘導する化学分子論的機序であろうと考えられる.

Methylglyoxal (MG), a precursor of advanced glycation end products, reacts rapidly with arginine residue in proteins. Three products, one fluorescent and two non-fluorescent adducts, have been so far identified. The fluorescent MG–arginine adduct, putatively identified as 5-methylimidazolone, has been shown to be identical to argpyrimidine. The non-fluorescent products were identified as the 5-hydro-5-methylimidazol-4-one and a novel MG adduct, which we named tetrahydropyrimidine (THP). THP was found to be formed from the reaction of guanidino group of arginine with two-MG molecules under physiological conditions. In the present study, to detect the THP adduct in vivo, we developed a monoclonal antibody (mAb5A3) directed to protein-bound THP. During preparation of the monoclonal antibody, hybridomas were selected by comparing the reactivities of the culture supernatant to THP using a competitive enzyme-linked immunosorbent assay. It was found that mAb5A3 specifically recognized THP as the dominant epitope. The presence of THP adduct was attested by immunohistochemical analysis of human atherosclerotic lesion, in which macrophage-derived foam cells were positively stained by the antibody. Our present results suggest that accumulation of THP occurs in vivo and may contribute to the progression of vascular diseases.

Dehydrosinapinic acid dilactone (1) was converted to thomasidioic acid (3) and (E,E)-2,3-bis(3,5-dimethoxy-4-hydroxybenzylidene)succinic acid (4) via an alpha,beta-unsaturated gamma-lactone type dimer (2) in phosphate buffer (pH 7.4). A study of the reaction mechanism was accomplished by observing the reaction of methyl ester of 2. The lignans (3, 4) were also prevented the peroxynitrite-mediated protein nitration.