Stilbene compounds are specific inhibitors of the superoxide anion generation catalyzed by xanthine oxidase.

The inhibitory effect of xanthine oxidase (XO) reactions with stilbene compounds, 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging by stilbene compounds and superoxide anion (O2-) scavenging activity were examined. The inhibition of the O2- generation catalyzed by XO by stilbene compounds is stronger than the effect on uric acid formation. The suppression of the O2- generation with resveratrol was diminished by the addition of flavin adenine dinucleotide (FAD). The water-solubility and visible spectra (VIS) of the stilbene compounds in the presence of water-soluble flavin compounds indicated a π-π interaction between the stilbene compounds and the isoalloxazine in flavin compounds. These results indicate that stilbene compounds specifically bind the FAD site in XO so as to inhibit the O2- generation. In the case of piceatannol, it is deduced that the suppression of O2- generation is induced by this specific binding to the FAD site and the subsequent reduction of XO.

Effect of hydrogen peroxide on normal and acatalasemic mouse erythrocytes.

OBJECTIVES: Normal and acatalasemic mouse erythrocytes were used to clarify the relationship between oxidative damage in H2O2-treated erythrocytes and catalase activity. DESIGN & METHODS: Generation of hydrolysis-resistant erythrocytes and hemolysis were examined. The osmotic fragility test, the negative charges and the number of membrane-flickering erythrocytes among the H2O2-treated erythrocytes were investigated. RESULTS: Small amounts of hydrolysis-resistant mouse erythrocytes were generated by treatment with 0.1 mM H2O2, and the amount of acatalasemic erythrocytes was larger than untreated controls. Hemolysis in the acatalasemic erythrocytes was observed 30 min after the addition of the H2O2. A drastic increase in hydrolysis-resistant erythrocytes and a loss of membrane proteins in the acatalasemic erythrocytes were found as a result of the addition of 1 mM H2O2. Hemolysis in normal erythrocytes was observed at 3 mM H2O2. CONCLUSIONS: Catalase is a potent H2O2-scavenger even in acatalasemic mouse erythrocytes. It is concluded that the drastic increase of hydrolysis-resistant erythrocytes is induced by a loss of membrane function and is associated with the low catalase activity in these cells.

Characterization of the xanthine oxidase inhibitory activity of alk(en)yl phenols and related compounds.

The inhibitory activity of xanthine oxidase (XO) is a combination of uric acid formation inhibition and superoxide anion (O2-) generation suppression. The inhibition of uric acid formation by XO is useful for the screening of natural compounds that prevent gout, while the suppression of O2- generation is useful for treating oxidative stress. Many edible plants contain abundant phenolic compounds and alk(en)yl phenols, and some of these compounds display XO inhibitory activity. This review focuses on XO inhibitory activity since this activity is used to characterize natural products. Recently, it was demonstrated that the inhibitory activity could be characterized using assays for XO inhibition, the suppression of O2- generation, DPPH radical scavenging and O2- radical scavenging. The inhibitory activity was divided three reaction types. The first is XO inhibition, the second O2- generation suppression by modification of enzyme molecules and the third two forms of O2- scavenging. It was demonstrated that these three activities are related to both the hydroxy group arrangement in the phenol portion and the alk(en)yl chains. This characterization is useful for pursuing XO inhibitors and antioxidants in natural compounds.

Superoxide anion scavenging activity of alk(en)yl phenol compounds by using PMS-NADH system.

Anacardic acid C15:3 and cardol C15:3 sigmoidally suppressed superoxide anion (O2-) generation using xanthine oxidase. To study this suppression activity, anacardic acids, cardanols and cardols having different numbers of double bonds in alk(en)yl chains were prepared. The O2- scavenging activity and H2O2 formation from O2- using PMS-NADH were examined. Anacardic acids and cardols indicated sigmoidal O2- scavenging activity but cardanols did not. The O2- scavenging activity of anacardic acid C15:3 was weaker than the suppression activity using xanthine oxidase, but the scavenging activity of cardol C15:3 was quite similar to the suppression using xanthine oxidase. The H2O2 formation from O2- decreased by the addition of anacardic acids, cardanols and cardols but increased by the addition of gallic and caffeic acids. From these results, we deduced that the O2- suppression activity of xanthine oxidase reaction with cardols is the O2- scavenging activity and that anacardic acids and cardols are O2- scavengers having low prooxidant property.

Psychosocial Determinants of Fruit and Vegetable Consumption in a Japanese Population.

There is limited evidence in Japan regarding the psychosocial determinants of fruit/vegetable intake. We performed a cross-sectional study of people aged 18 years or older in four regions of Japan; 2308 (men: 1012, women: 1296) individuals who completed the questionnaires were included. We found that 24.8% of people were aware of the current recommendations for vegetables and 13.2% for fruit and that "ability to design meals" and "availability when eating outside of the home" were the most important factors related to self-efficacy and barriers to fruit and vegetable intake, respectively. People with high self-efficacy (OR: 3.16; 95% CI: 2.17, 4.60 for fruit; OR: 4.52; 95% CI: 3.08, 6.64 for vegetables) were more likely to consume more fruit and vegetables. People with high scores on attitude (OR: 1.54; 95% CI: 1.06, 2.24) and social support (OR: 1.59; 95% CI: 1.11, 2.27) were more likely to consume more fruit. People with high perceived barriers (OR: 0.69; 95% CI: 0.48, 0.98) were less likely to consume fruit. This study suggests a need to increase the general population's awareness of the fruit and vegetable intake recommendations; facilitating positive attitudes, self-efficacy, and social support for individuals and strengthening the ability of individuals to design meals with more vegetables and fruit might be useful intervention programs.

Suppression of superoxide anion generation catalyzed by xanthine oxidase with alkyl caffeates and the scavenging activity.

Alkyl caffeates are strong antioxidants and inhibitors of xanthine oxidase. However, it is unclear about the effect of caffeic acid and alkyl caffeates on superoxide anion (O2(-)) generation catalyzed by xanthine oxidase. Effects of caffeic acid and alkyl caffeates on the uric acid formation and O2(-) generation catalyzed by xanthine oxidase were analyzed. The scavenging activities of 1,1-diphenyl-2-picryhydrazyl (DPPH) radical and O2(-) generated with phenazine methosulfate (PMS) and NADH were examined. Caffeic acid derivatives equally suppressed O2(-) generation, and the suppression is stronger than inhibition of xanthine oxidase. Scavenging activity of O2(-) is low compared to the suppression of O2(-) generation. Suppression of O2(-) generation catalyzed by xanthine oxidase with caffeic acid derivatives was not due to enzyme inhibition or O2(-) scavenging but due to the reduction of xanthine oxidase molecules. Alkyl caffeates are effective inhibitors of uric acid and O2(-) catalyzed by xanthine oxidase as well as antioxidants for edible oil.

Protective effect of vitamin E against alloxan-induced mouse hyperglycemia.

BACKGROUND: Alloxan induces oxidative stress and hyperglycemia in animal models. Acatalasemic (catalase deficiency) mice are susceptible to alloxan-induced hyperglycemia. As the incidence of hyperglycemia induced by alloxan was reportedly improved when mice were fed a vitamin E supplemented diet, this protective effect was examined. METHODS: Acatalasemic and normal mice fed a vitamin E supplemented diet were treated with alloxan. The pancreas were examined with microscopy. We also isolated pancreatic islets of normal mice treated with alloxan. The glucose stimulated insulin secretion was examined. RESULTS: Vitamin E powerfully ameliorated the increase in apoptosis. Vitamin E increases insulin amounts secreted from pancreatic cells, but does not ameliorate the regulation of the glucose stimulated insulin secretion. CONCLUSIONS: It is suggested that the difference in the mice fed vitamin E supplemented diet is due to an increase of insulin secretion and that vitamin E supplementation may have a role in helping to slow the stages of diabetes mellitus.

Synthesis of Ester-linked Docetaxel-glycoside Conjugate and Its Application to Drug Delivery System using Immunoliposome Targeted with Trastuzumab.

Chemo-enzymatic synthesis of the ester-linked monosaccharide conjugate of docetaxel, 7-glycolyldocetaxel 2"-O-ß-D-galactopyranoside, was achieved by using lactase as a biocatalyst. The water-solubility and, EE and LE values for the liposome of 7-glycolyldocetaxel 2"-O-ß-D-galactopyranoside were much higher than those of docetaxel. The immunoliposome containing 7-glycolyldocetaxel 2"-O-ß-D-galactopyranoside showed effective suppression of tumor growth.

Inhibitory effects of cardols and related compounds on superoxide anion generation by xanthine oxidase.

5-Pentadecatrienylresorcinol, isolated from cashew nuts and commonly known as cardol (C15:3), prevented the generation of superoxide radicals catalysed by xanthine oxidase without the inhibition of uric acid formation. The inhibition kinetics did not follow the Michelis-Menten equation, but instead followed the Hill equation. Cardol (C10:0) also inhibited superoxide anion generation, but resorcinol and cardol (C5:0) did not inhibit superoxide anion generation. The related compounds 3,5-dihydroxyphenyl alkanoates and alkyl 2,4-dihydroxybenzoates, had more than a C9 chain, cooperatively inhibited but alkyl 3,5-dihydroxybenzoates, regardless of their alkyl chain length, did not inhibit the superoxide anion generation. These results suggested that specific inhibitors for superoxide anion generation catalysed by xanthine oxidase consisted of an electron-rich resorcinol group and an alkyl chain having longer than C9 chain.

Synthesis of glycosides of resveratrol, pterostilbene, and piceatannol, and their anti-oxidant, anti-allergic, and neuroprotective activities.

Resveratrol was glucosylated to its 3- and 4'-ß-glucosides by cultured cells of Phytolacca americana. On the other hand, cultured P. americana cells glucosylated pterostilbene to its 4'-ß-glucoside. P. americana cells converted piceatannol into its 4'-ß-glucoside. The 3- and 4'-ß-glucosides of resveratrol were further glucosylated to 3- and 4'-ß-maltosides of resveratrol, 4'-ß-maltoside of which is a new compound, by cyclodextrin glucanotransferase. Resveratrol 3-ß-glucoside and 3-ß-maltoside showed low 2,2-diphenyl-1-picrylhydrazyl free-radical-scavenging activity, whereas other glucosides had no radical-scavenging activity. Piceatannol 4'-ß-glucoside showed the strongest inhibitory activity among the stilbene glycosides towards histamine release from rat peritoneal mast cells. Pterostilbene 4'-ß-glucoside showed high phosphodiesterase inhibitory activity.

Chemical synthesis and tyrosinase inhibitory activity of rhododendrol glycosides.

The concise synthesis of rhododendrol glycosides 3-8, which are novel derivatives of (+)-epirhododendrin (1) and (-)-rhododendrin (2), has been achieved in six steps from benzaldehyde 9. The key reactions include aldol condensation and trichloroacetimidate glycosylation. From biological studies, it has been determined that synthetic derivatives of 1 and 2 possess potent tyrosinase inhibitory activity. Particularly, the inhibitory activity of cellobioside 8 (IC50=1.51µM) is six times higher than that of kojic acid. The R-epimers (4, 6, and 8) possessed more potent activity than the corresponding S-epimers (3, 5, and 7), indicating that tyrosinase inhibitory activity is significantly governed by stereochemistry of rhododendrol glycosides.

Effect of vitamin E on alloxan-induced mouse diabetes.

OBJECTIVES: Alloxan generates hydrogen peroxide in the body, and a small amount of alloxan administered to acatalasemic mice results in diabetes. D-α-Tocopherol (vitamin E) is an antioxidant which helps prevent excess oxidation in the body. In this study, we examined the effect of vitamin E on diabetes caused by alloxan administration in mice. METHODS: Mice were maintained on a vitamin E-deprived diet and supplemented diet, respectively, for 14 weeks. Alloxan was then intraperitoneally administered, and blood glucose, glucose tolerance and the insulin level in mouse blood were examined. RESULTS: Hyperglycemia was observed in the mice maintained on the vitamin E-deprived diet. The incidence of hyperglycemia in the mice maintained on the vitamin E-deprived diet was significantly higher than that in the mice maintained on the supplemented diet. The abnormal glucose metabolism caused by alloxan administration was ameliorated by the vitamin E-supplemented diet. CONCLUSIONS: It is deduced that vitamin E can prevent a decrease of insulin concentration in the blood in this mouse model.

Resveratrol inhibited hydroquinone-induced cytotoxicity in mouse primary hepatocytes.

Hydroquinone (1,4-benzenediol) has been widely used in clinical situations and the cosmetic industry because of its depigmenting effects. Most skin-lightening hydroquinone creams contain 4%-5% hydroquinone. We have investigated the role of resveratrol in prevention of hydroquinone induced cytotoxicity in mouse primary hepatocytes. We found that 400 µM hydroquinone exposure alone induced apoptosis of the cells and also resulted in a significant drop of cell viability compared with the control, and pretreatment of resveratrol to a final concentration of 0.5 mM 1 h before hydroquinone exposure did not show a significant improvement in the survival rate of the hepatocytes, however, relatively higher concentrations of resveratrol (≥1 mM) inhibited apoptosis of the mouse primary hepatocytes and increased cell viability in a dose-dependent manner, and in particular the survival rate of the hepatocytes was recovered from 28% to near 100% by 5 mM resveratrol. Interestingly, pretreatment with resveratrol for longer time (24 h), even in very low concentrations (50 µM, 100 µM), blocked the damage of hydroquinone to the cells. We also observed that resveratrol pretreatment suppressed hydroquinone-induced expression of cytochrome P450 2E1 mRNA dose-dependently. The present study suggests that resveratrol protected the cells against hydroquinone-induced toxicity through its antioxidant function and possibly suppressive effect on the expression of cytochrome P450 2E1.

Glycosylation of trans-resveratrol by plant-cultured cells.

Plant-cultured cells of Catharanthus roseus converted trans-resveratrol into its 3-O-ß-D-glucopyranoside, 4'-O-ß-D-glucopyranoside, 3-O-(6-O-ß-D-xylopyranosyl)-ß-D-glucopyranoside, and 3-O-(6-O-α-L-arabinopyranosyl)-ß-D-glucopyranoside. The 3-O-(6-O-ß-D-xylopyranosyl)-ß-D-glucopyranoside and 3-O-(6-O-α-L-arabinopyranosyl)-ß-D-glucopyranoside compounds of trans-resveratrol are both new. Incubation of plant-cultured cells of Ipomoea batatas and Strophanthus gratus with trans-resveratrol gave trans-resveratrol 3-O-ß-D-glucopyranoside and trans-resveratrol 4'-O-ß-D-glucopyranoside.

Acatalasemic mice are mildly susceptible to adriamycin nephropathy and exhibit increased albuminuria and glomerulosclerosis.

BACKGROUND: Catalase is an important antioxidant enzyme that regulates the level of intracellular hydrogen peroxide and hydroxyl radicals. The effects of catalase deficiency on albuminuria and progressive glomerulosclerosis have not yet been fully elucidated. The adriamycin (ADR) nephropathy model is considered to be an experimental model of focal segmental glomerulosclerosis. A functional catalase deficiency was hypothesized to exacerbate albuminuria and the progression of glomerulosclerosis in this model. METHODS: ADR was intravenously administered to both homozygous acatalasemic mutant mice (C3H/AnLCs(b)Cs(b)) and control wild-type mice (C3H/AnLCs(a)Cs(a)). The functional and morphological alterations of the kidneys, including albuminuria, renal function, podocytic, glomerular and tubulointerstitial injuries, and the activities of catalase were then compared between the two groups up to 8 weeks after disease induction. Moreover, the presence of a mutation of the toll-like receptor 4 (tlr4) gene, which was previously reported in the C3H/HeJ strain, was investigated in both groups. RESULTS: The ADR-treated mice developed significant albuminuria and glomerulosclerosis, and the degree of these conditions in the ADR-treated acatalasemic mice was higher than that in the wild-type mice. ADR induced progressive renal fibrosis, renal atrophy and lipid peroxide accumulation only in the acatalasemic mice. In addition, the level of catalase activity was significantly lower in the kidneys of the acatalasemic mice than in the wild-type mice during the experimental period. The catalase activity increased after ADR injection in wild-type mice, but the acatalasemic mice did not have the ability to increase their catalase activity under oxidative stress. The C3H/AnL strain was found to be negative for the tlr4 gene mutation. CONCLUSIONS: These data indicate that catalase deficiency plays an important role in the progression of renal injury in the ADR nephropathy model.

Synthesis and evaluation of bibenzyl glycosides as potent tyrosinase inhibitors.

Bibenzyl glycosides 1-6 were synthesized from 2,4-dihydoxybenzaldehyde and xylose, glucose, cellobiose or maltose. The key steps in the synthesis were the Wittig reaction and trichloroacetimidate glycosylation. Tests for tyrosinase inhibitory activity showed that all were significantly active, indicating that they are unique hydrophilic tyrosinase inhibitors. Bibenzyl xyloside 2 is a particularly potent inhibitor (IC(50) = 0.43 µM, 17 times higher than that of kojic acid). These results suggest that the hydrophilic cavity of tyrosinase might accommodate the bulky carbohydrate on the bibenzyl scaffold.

Enzymatic preparation of arbutin derivatives: lipase-catalyzed direct acylation without the need of vinyl ester as an acyl donor.

Direct and regioselective acylation of arbutin with aromatic or aliphatic acid using a lipase obtained from Candida antarctica in an organic solvent was investigated. We achieved the enzymatic synthesis of feruloyl arbutin and lipoyl arbutin without the need of vinyl ferulate and vinyl lipoate as acyl donors, respectively.

Evaluation of pyrogallol-induced cytotoxicity in catalase-mutant escherichia coli and mutagenicity in Salmonella typhimurium.

We evaluated pyrogallol cytotoxicity using Escherichia coli strains that express mammalian catalase gene derived from catalase mutant mice (Cs(b)) and wild-type (Cs(a)), and pyrogallol mutagenicity by Ames test. Pyrogallol was more toxic to Cs(b) rather than to Cs(a) (p < 0.05), while catalase, superoxide dismutase and ascorbic acid decrease the toxic effect. Pyrogallol also showed mutagenic effect (mutagenic index = 3.8 for 10 micromol pyrogallol/plate) while ascorbic acid (19.4% reduction, p < 0.001) and naringin (35.1% reduction, p < 0.001) played a protective role against it. Pyrogallol cytotoxicity and mutagenicity seem to be attributable, at least in part, to reactive oxygen species formation. This study also suggests that newly established catalase mutant E. coli is probably useful in hazard identification of oxidative chemicals.

Sensitization to alloxan-induced diabetes and pancreatic cell apoptosis in acatalasemic mice.

Human acatalasemia may be a risk factor for the development of diabetes mellitus. However, the mechanism by which diabetes is induced is still poorly understood. The impact of catalase deficiency on the onset of diabetes has been studied in homozygous acatalasemic mutant mice or control wild-type mice by intraperitoneal injection of diabetogenic alloxan. The incidence of diabetes was higher in acatalasemic mice treated with a high dose (180 mg/kg body weight) of alloxan. A higher dose of alloxan accelerated severe atrophy of pancreatic islets and induced pancreatic beta cell apoptosis in acatalasemic mice in comparison to wild-type mice. Catalase activity remained low in the acatalasemic pancreas without the significant compensatory up-regulation of glutathione peroxidase or superoxide dismutase. Furthermore, daily intraperitoneal injection of angiotensin II type 1 (AT1) receptor antagonist telmisartan (0.1 mg/kg body weight) prevented the development of alloxan-induced hyperglycemia in acatalasemic mice. This study suggests that catalase plays a crucial role in the defense against oxidative-stress-mediated pancreatic beta cell death in an alloxan-induced diabetes mouse model. Treatment with telmisartan may prevent the onset of alloxan-induced diabetes even under acatalasemic conditions.

One-procedure synthesis of capsiate from capsaicin by lipase-catalyzed dynamic transacylation.

Capsiate has a structure similar to capsaicin but no oral pungency. Furthermore, capsiate displayed antioxidant activity and inhibited angiogenesis and vascular permeability, and therefore, showed potential as a medicine and food supplement. Capsaicin is now commercially available, however capsiate is scarcely present in natural foods. We investigated the direct enzymatic conversion of a capsaicinoid to a capsinoid. It was observed that the rate of lipase-catalyzed acylation of vanillyl alcohol with nonanoic acid was faster than that of hydrolysis of N-vanillylnonanamide to vanillyl amide and nonanoic acid in n-hexane at 70°C. As a result, we performed a one-procedure synthesis of capsiate from capsaicin via lipase-catalyzed transacylation.