Enhancing Effect of a Roasting Treatment on the Radical Scavenging Activity of a Marine Brown Alga Polysaccharide (Alginic acid).

We found an effective roasting method that enhances the effects on various radical scavenging activities of polysaccharide (alginic acid) derived from the marine brown alga Lessonia trabeculata. These enhancing effects were observed by a roasting treatment under relatively high temperature conditions (160ºC and 180ºC), which were measured by (i) a stable radical compound diphenylpicrylhydrazyl (DPPH), (ii) a hydroperoxide generating system of linoleic acid autooxidation, and (iii) an opsonized zymosan (Opz)-induced oxygen radical generating system in human blood neutrophils. Although a significant enhancing effect of the roasting treatment on the radical scavenging activity of the alginic acid itself was not detected under relatively low temperature conditions (100ºC and 130ºC), the roasting treatment of a mixture of alginic acid and several specific amino acids caused considerable radical scavenging activities under the same roasting conditions. When alginic acid was roasted at relatively high temperatures (160ºC or 180ºC), the mixture of the alginic acid and specific amino acids exhibited much higher radical scavenging activities than did the alginic acid alone. The significance of this finding is discussed from the viewpoint of healthy food science.

Enhancing Effect on Radical Scavenging Activity of Edible Brown Alga, Laminaria japonica (Ma-konbu) by Roasting Treatment.

Various edible algae have been traditionally consumed as healthy food stuffs in Asian countries such as China, Korea and Japan, and roasting treatments have been carried out on some of these edible algae for the improvement of their taste and flavor. In the present paper, we analyzed the effect of roasting treatments on the radical scavenging activity of a typical Japanese edible brown alga, Laminaria japonica (L. japonica, Ma-konbu). The effect was estimated by a stable radical compound, diphenylpicrylhydrazyl (DPPH) and a chemiluminescence assay of superoxide anion generation using hypoxanthine (HPX) and xanthine oxidase (XOD) system. Weak but significant radical scavenging activities against the DPPH radical and superoxide anion were observed in the water extract of L. japonica when it was roasted at 130-150ºC. Very strong radical-scavenging activities were detected under much higher temperature conditions (180-200ºC). The enhancing effect of the roasting treatment on the radical scavenging activity was highly associated with the roasting-induced increase of polyphenol and tannin in the L. japonica extract. The sugar concentrations in the L. japonica extracts under different roasting conditions were significantly, but not strongly associated with their radical scavenging activities. The protein concentrations in the L. japonica extracts, however, were not associated with their radical scavenging activities under different roasting conditions. Furthermore, the extracts of L. japonica roasted under higher temperature conditions (180-200ºC) caused strong radical scavenging effects on the generation of opsonized zymosan (Opz)-induced reactive oxygen species (ROS) generation in human blood neutrophils, which was measured by chemiluminescence assay. These experimental results suggest that the roasting treatment of L. japonica causes an enhancing effect on the radical scavenging activity in the extract of this alga, and is associated with the increase in various radical scavenging substances in the extract. The significance of this finding is discussed from the viewpoint of healthy food science.

Potent immunomodulating effects of bran extracts of traditional Japanese millets on nitric oxide and cytokine production of macrophages (RAW264.7) induced by lipopolysaccharide.

To estimate the potent immunomodulating effects of different types of traditional Japanese millet, we analyzed the effect of bran extracts of foxtail millet (Awa in Japanese), barnyard millet (Hie) and proso millet (Mochi-kibi) on nitric oxide (NO) and inflammatory cytokine production induced by lipopolysaccharide (LPS) in a mouse macrophage cell line (RAW264.7 cells). All methanol extracts of these millet brans showed suppressive activities against the production of NO and inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 in LPS-stimulated macrophages, which were not responsible for their cytotoxic activities. These immunosuppressive activities were roughly proportional to the contents of the phenolic compounds in their extracts. Especially, the extract of proso millet exhibited the strongest immunosuppressing effect, which was associated with the highest content of phenolic compound. However, the extracts did not exhibit significant suppressive effects on the production of an anti-inflammatory cytokine, IL-10, in the same macrophage culture system. These results suggest that traditional Japanese millets have potent immunomodulating activities against the production of NO and cytokine production in activated macrophages.

Effects of an environmental endocrine disruptor, para-nonylphenol on the cell growth of Euglena gracilis: association with the cellular oxidative stress.

Effects of an environmental endocrine disruptor, para-nonylphenol (NP) on the cell growth of a photosynthetic eukaryotic microorganism, Euglena gracilis were analysed under different cell culture conditions. Although NP did not show significant inhibitory effects on the cell growth of E. gracilis (Z and SM strains) under light culture condition, NP exhibited significant suppressive effects under dark culture condition. Exogenous supplementation with lipophilic antioxidants (α-tocopherol, ß-carotene or 6-O-palmitoyl-ascorbic acid) to E. gracilis caused strong preventive effects against NP-induced cell growth inhibition under dark culture condition, but hydrophilic antioxidants [ascorbic acid, glutathione and epigallocatechin gallate (EGCG)] did not show significant preventive effects. NP caused significant generation of reactive oxygen species (ROS) in E. gracilis under dark culture condition, but E. gracilis under light culture condition did not show significant increase in ROS generation. Supplementation with lipophilic antioxidants to E. gracilis caused significant suppressive effects against NP-induced cellular ROS generation under dark culture condition, but hydrophilic antioxidants did not show significant suppressive effects. Furthermore, the productivities of typical cellular antioxidants (α-tocopherol, ß-carotene and ascorbic acid) in E. gracilis under light culture conditions were much higher than those under dark culture conditions.

Potent antioxidant and radical-scavenging activity of Chenpi--compensatory and cooperative actions of ascorbic acid and citric acid.

Dried peels of Satsuma mandarin (Citrus unshiu Marcov.) have been used as traditional Chinese and Japanese medicine, which are called 'Chenpi'. In our present study, cold and hot water extracts of Chenpi exhibited a strong inhibitory activity against linoleic acid peroxidation and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical-scavenging activity compared with o-methanol extract. When these extracts were separated into ethanol-soluble(ES) and ethanol-precipitable fractions, the dominant antioxidant and radical-scavenging activities were detected in the ES fractions, which suggests that these antioxidant and radical-scavenging activities are responsible for water-soluble and low-molecular-weight substances. As possible active principles for antioxidant activities in the water extracts, the contents of ascorbic acid and citric acid in these extracts were measured, and the antioxidant and radical-scavenging activities of these substances were assayed at various concentrations. The experimental results indicate that the antioxidant activity against lipid peroxidation in the water extracts is dominantly associated with citric acid, and the DPPH radical-scavenging activity of the water extracts is majorly responsible for ascorbic acid, suggesting a compensatory action of ascorbic acid and citric acid in expression of the antioxidant and radical-scavenging activities of Chenpi.

Immunomodulating activities in bran extracts of Japanese red, black and brown rices.

To determine the potent immunomodulating activities of different types of Japanese rice bran, we analyzed the effects of extracts of red, brown and black rice brans on the cell proliferation and cytokine production of mouse immunocompetent cells by cell culture experiments. Methanol extracts of these rice brans showed suppressive activities against the proliferative response (3H-thymidine incorporation) of mouse spleen cells induced by concanavalin A (Con A) or lipopolysaccharide (LPS) in the cell culture experiments. Although the black and brown rice bran extracts showed suppressive effects on the production of interferon gamma (IFN gamma) or interleukin 6 (IL-6) in mouse spleen cells induced by Con A or LPS, the red rice bran extract exhibited stimulatory effects on the same cytokine-producing systems. Furthermore, when the effects of these extracts on the production of macropahage-derived inflammatory cytokines such as interleukin-1alpha (IL-1alpha) and tumor necrosis factor alpha (TNF-alpha) were assayed, the red rice bran extract caused a stimulatory effect on the IL-1alpha production from mouse macrophages induced by LPS, but did not show a significant effect on TNF-alpha production. However, the brown and black rice bran extracts exhibited significant inhibitory effects on the production of IL-1alpha and TNF-alpha in the same macrophage culture experiment. A possible mechanism of the immunomodulating activities of the rice bran extracts and the immunopharmacological significance of these findings are discussed.

Potent antioxidant and radical-scavenging activities of traditional Japanese cereal grains.

To estimate the preventive potential of Japanese traditional cereals against oxygen radical-related chronic diseases such as cardiovascular diseases and diabetes, antioxidant and radical-scavenging activities in the extracts of five Japanese traditional cereal grains were analyzed by using an assay system of lipid peroxidation and a radical compound, 1,1-diphenyl-2-picrylhydrazyl (DPPH). DPPH radical-scavenging activities in the extracts of Japanese cereal grains were divided into two groups. One group including Japanese sorghum, black rice and red rice showed strong radical-scavenging activities, but the other group including Japanese barnyard millet and foxtail millet did not exhibit significant radical-scavenging activities. The DPPH radical-scavenging activities of these extracts were closely correlated to the contents of phenolic compound in the extracts, but not to the sugar or protein content in the extracts. In contrast, all the methanol and water extracts of the cereal grains caused significant antioxidant activities against hydroperoxide generation in the peroxidation of linoleic acid, in which the water extracts of these cereal grains caused much higher antioxidant activities than the methanol extracts of the same cereals. These results suggest that Japanese traditional cereals contain qualitatively different principles associated with antioxidant and radical-scavenging activities, and possible principles responsible for the antioxidant and radical-scavenging activities in the cereal grains are discussed.

Potentiating effect of an endocrine disruptor, paranonylphenol, on the generation of reactive oxygen species (ROS) in human venous blood -- association with the activation of signal transduction pathway.

An endocrine disruptor, para-nonylphenol (NP), caused a dose-dependent stimulatory effect on the generation of reactive oxygen species (ROS) in human whole blood from 50 to 1000 microM, which was measured by chemiluminescence generation. ROS-scavenging enzymes such as catalase and superoxide dismutase, and the lipophilic antioxidative agents, alpha-tocopherol and beta-carotene, showed preventive effects on NP-induced ROS generation. To analyze the biochemical mechanism of NP-induced ROS generation in human blood, we investigated the effects of different types of metabolic inhibitors on the activation pathways of ROS generation. An NADPH-dependent oxidase inhibitor, diphenyl iodonium chloride (DPI), and a myeloperoxidase inhibitor, sodium azide (NaN3), showed remarkable inhibitory effects on ROS generation induced by NP, but an inhibitor against mitochondrial respiratory function, potassium cyanide (KCN), did not exhibit a significant effect. Furthermore, a phosphatidylinositol-3 (PI3) kinase inhibitor, wortmannin, and a tyrosine kinase inhibitor, protein phosphorylation inhibitor 1 (PP1), caused a strong suppression of NP-induced ROS generation. Selective protein kinase C inhibitor, Ro-32-0432, p38 MAP kinase inhibitor, SB-203580, and ERK MAP kinase inhibitor, PD 98059, showed significant suppressive effects on NP-induced ROS generation. In addition, when human blood was exposed to lower concentrations (5-50 microM) of NP, they did not cause the significant ROS generation by themselves, but the priming and synergistic effects of NP were detected by the addition of secondary stimulants, opsonized zymosan (OZ) or phorbol myristate acetate (PMA). The analysis of the priming and synergistic effects of NP on OZ- or PMA-dependent ROS generation by antioxidative substances and metabolic inhibitors showed similar results compared with those of human blood treated with NP alone. These results suggest that NP causes an enhancing effect by itself, or priming and synergistic effects on ROS generation in human blood with other inflammatory stimulants through the activation of signal transduction pathways such as protein kinase cascades.

Potent radical-scavenging activities of thiamin and thiamin diphosphate.

Various radical-scavenging activities of thiamin and thiamin diphosphate (TDP) were found in some in vitro experiments. Thiamin and TDP caused considerable suppressive effects on superoxide generation in hypoxanthine and xanthine oxidase system which was measured by a sensitive chemiluminescence method using 2-methyl-6-[p-methylphenyl]-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (MCLA), and their 50% inhibition (IC(50)) values were estimated to be 158 and 56 microM, respectively. They also showed the significant suppression against hydroperoxide generation derived from oxidized linoleic acid which was estimated by aluminum chloride method and their IC(50) values were calculated to be 260 and 46 microM. They further prevented the oxygen radical generation in opsonized zymosan-stimulated human blood neutrophils which was shown by chemiluminescence method using luminol, and their IC(50) values were calculated to be 169 and 38 microM. In contrast, they caused weak but significantly suppressive effects on the hydroxyl radical generation by Fenton reaction which was measured by electric spin resonance (ESR) method, their IC(50) values were calculated to be 8.45 and 1.46 mM respectively. These results strongly suggest a possibility that thiamin and TDP play as radical scavengers in cell-free and cellular systems.

Radical-scavenging activity of hot water extract of Japanese rice bran--association with phenolic acids.

A strong radical-scavenging activity against a stable radical compound, 1,1-diphenyl-2-picrylhydrazyl (DPPH) was found in the hot water extract of Japanese rice bran. When the extract was treated with ethanol, a dominant radical-scavenging activity was observed in the ethanol-soluble (ES) fraction in a dose-dependent manner, but a weak radical-scavenging activity was detected in the ethanol-precipitable (EP) fraction. Their activities were proportional to the amounts of phenolic substances in each fraction. The phenolic substances in the ES fraction were efficiently separated by Amberlite XAD column chromatography and high performance liquid chromatography using an ODS column. The four major phenolic acids (ferulic, para-coumaric, para-hydroxybenzoic and vanillic acids) and four minor phenolic acids (caffeic, gentisic, protocatechuic and syringic acids) were detected in the HPLC system. Among these phenolic acids, protocatechuic, caffeic, ferulic and gentisic acids showed relatively strong radical scavenging activities (EC50: 8, 9, 29 and 75 microM, respectively) compared with the control antioxidants such as ascorbic acid and alpha-tocopherol (EC50: 93 and 134 microM). Para-coumaric, syringic and vanillic acids exhibited weak but significant radical-scavenging activities (EC50: 780, 2640 and 3250 microM). However, para-hydroxybenzoic acid did not show any significant effects even at 5 mM. Furthermore, a simulated mixture combined with these phenolic acids in comparable amounts in the ES fraction showed slightly weak radical-scavenging activity compared with that of rice bran extract. However, all the phenolic acids detected in the ES fraction did not show significant antioxidant activities against hydroperoxide generation in lipid peroxidation compared with that of a typical antioxidant such as ascorbic acid, which was estimated by the alminum chloride method. These results suggest that Japanese rice bran has a potent radical-scavenging activity against DPPH radical and this activity is associated with some phenolic acids in the ES fraction. The significance of this finding is discussed from the viewpoint of the protective role of rice bran against oxygen radical-induced chronic diseases.

Antioxidant and prooxidant activities of B group vitamins in lipid peroxidation.

The antioxidant and prooxidant activities of six B group vitamins against hydroperoxide generation in linoleic acid peroxidation were analyzed by the aluminum chrolide method. The B group vitamins were divided into three types by their antioxidant and prooxidant properties. The first type, including B1, B2 vitamin, nicotinic acid and folic acid, showed prooxidant activities in the early phase (1 week) of the linoleic acid peroxidation at concentrations of 2.5 microM-2.5 mM, but caused relatively strong antioxidant activities in the later phase reaction (3 weeks) at the same concentrations. The second type, such as B12 vitamin, did not cause significant effects in the early phases (1-2 weeks) of linoleic acid peroxidation, but exhibited significant antioxidant activities in the later phase reactions (3 weeks). The third type, such as B6 vitamin, exhibited significant antioxidant activity in the early phase reaction and caused strong antioxidant effects in the later phase reaction (3 weeks). Generally, the antioxidant activities of B group vitamins in the later phases of lipid peroxidation was much stronger than those in the earlier phases. These experimental results suggest a possibility that B group vitamins have both antioxidant and prooxidant effects on lipid peroxidation under different experimental conditions.

Effect of endocrine disruptor para-nonylphenol on the cell growth and oxygen radical generation in Escherichia coli mutant cells deficient in catalase and superoxide dismutase.

para-Nonylphenol (NP) had previously been found to have strong suppressive effects of growth of bacterial and yeast cells, and these effects were associated with NP-induced generation of radical oxygen species (ROS). In the present study, we determined that wild-type strains of Escherichia coli (CSH 7, SY-11, and IFO-3545) were resistant to NP compared with other sensitive microorganisms reported previously. To elucidate the relationship between NP-induced ROS generation and cell growth inhibition in more detail, we analyzed the effect of NP on cell growth and survival of wild-type and mutant E. coli strains deficient in ROS-scavenging enzymes such as catalase and superoxide dismutase (SOD). The SOD-deficient strain QC 774 (sod A- and sod B-) was much more sensitive to NP than wild-type (CSH 7) and catalase-deficient (UM 1 kat E- and kat G-) strains. As a comparative experiment, when hydrogen peroxide was applied to the same growth and survival assays, UM 1 cells were more sensitive to hydrogen peroxide than QC 774 and CSH 7. A chemiluminescence (CHL) experiment using MCLA (2-methyl-6-Lf-methylphenyl]-3,7-dihydroimidazc [1,2-alpha] pyrazin-3-one) reflecting predominantly superoxide generation showed that NP caused marked CHL generation in QC 774 cells, but not in CSH 7 and UM 1 cells. However, the CHL experiment using L-012 reflecting predominantly hydroxyl radical and hypochlorite did not exhibit significant CHL generation in QC 774 cells at the same concentrations of NP. Furthermore, supplementation with SOD prevented NP-induced ROS generation and cell survival inhibition of QC 774 cells, but the catalase and metal-chelating agent deferoxamine did not have significant effects. These results suggest that one of the primary actions of NP in cells is the generation of superoxide which may be responsible for NP-induced cell growth inhibition.

Enhancing effect of the endocrine disruptor para-nonylphenol on the generation of reactive oxygen species in human blood neutrophils.

Although para-nonylphenol (NP) is known as an endocrine disruptor, the immunologic effect of NP has been poorly analyzed. We found that NP from 5 to 50 microM caused a dose-dependent stimulatory effect on the generation of reactive oxygen species (ROS) in human blood neutrophils, which was measured by using a chemiluminescence reagent, luminol. Furthermore, ROS-scavenging enzymes such as catalase and superoxide dismutase and antioxidative agents alpha-tocopherol and beta-carotene showed strong preventive effects on NP-induced ROS generation. To analyze the biochemical mechanism of NP-induced ROS generation in human neutrophils, we investigated the effects of different types of metabolic inhibitor for the activation pathways of ROS generation in the cells. Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase inhibitor, diphenyl iodonium chloride and the myeloperoxidase inhibitor sodium azide (NaN3) showed remarkable inhibitory effects on ROS generation induced by NP, but an inhibitor against mitochondrial respiratory function, potassium cyanide (KCN), did not exhibit significant effect. Furthermore, the phosphatidylinositol-3 (PI3) kinase inhibitor wortmannin and the tyrosine kinase inhibitor protein phosphorylation inhibitor 1 (PP1) caused strong suppression against NP-induced ROS generation. The selective protein kinase C inhibitor Ro-32-0432, p38 MAP kinase inhibitor SB 203580, and ERK MAP kinase inhibitor PD 98059 also showed significant suppressive effects on NP-induced ROS generation. These results suggest that NP causes an enhancing effect on ROS generation in human blood neutrophils through the activation of signal transduction pathways associated with the respiratory burst function in these cells. Additionally, to examine in vivo effects of NP, we also analyzed the effects of NP itself and the synergistic effects of NP and a typical inflammatory agent, opsonized zymosan, on human whole blood including neutrophils.

Potent suppressive activity of fresh and dried peels from Satsuma mandarin Citrus unshiu (Marcorv.) on hydroperoxide generation from oxidized linoleic acid.

The effects of various extracts prepared from fresh and dried peels of Satsuma mandarin (Citrus unshiu Marcov.) on hydroperoxide generation from oxidized linoleic acid were compared under different extraction conditions. The cold-and hot-water extracts of fresh peels showed significant suppressive activity against hydroperoxide generation in a dose-dependent manner. However, the methanol or acetone extract of fresh peels did not exhibit significant suppressive effects. The commercially available ascorbic acids equivalent to their concentrations in the water extracts of fresh peels showed roughly equal antioxidative activities compared with those of the water extracts of fresh peels. Although the cold- and hot- water extracts of dried peels indicated a considerable reduction of ascorbic acid concentration, they exhibited much higher antioxidative activities than those of the fresh peels. The methanol extract of dried peels also showed significant antioxidative activities, but did not contain significant ascorbic acid. These results suggest that the fresh peels of Satsuma mandarin have potential antioxidant activities, and the drying treatment of fresh peels caused an enhancement of the antioxidant activity. The pharmacological significance of this finding is discussed.

Potent antioxidative and antigenotoxic activity in aqueous extract of Japanese rice bran--association with peroxidase activity.

To estimate the preventive potential of Japanese rice bran (Oryza sativa japonica) against the oxygen radical-related chronic diseases such as cardio-vascular diseases and cancer, antioxidative and antigenotoxic activities of the rice bran extracts were analyzed by using assay systems for lipid peroxidation and genotoxin-induced umu gene expression. When effects of the rice bran extracts under different extraction conditions on hydroperoxide generation from auto-oxidized linoleic acid were examined using aluminum chloride method, the water extract showed strong antioxidant activity, but the methanol and acetone extracts did not exhibit significant activity. The water extract of rice bran was divided into the ethanol-precipitable (EP) and supernatant fractions, and EP fraction showed the dominant antioxidant activity, but the supernatant fraction did not exhibit significant antioxidant activity. When the effect of EP fraction on umu C gene expression in SOS response associated with DNA damage in Salmonella typhimurium (TA 1535/pSK 1002) induced by 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was analyzed, it showed a dose-dependent suppressive activity against Trp-P-1-induced umu C gene expression. The bio-chemical analysis of EP fraction indicates that the major antioxidative and antigenotoxic activity of EP fraction is associated with a proteinous component with the molecular weight of more than 30 KDa. As a possible active principle for the antioxidative and antigenotoxic activity in EP fraction, the strong activity of an oxygen radical-scavenging enzyme, peroxidase was detected, and the purified horseradish peroxidase also caused the similar antioxidative and antigenotoxic activities. The significance of this finding is discussed from the viewpoint of the preventive role of rice bran against oxygen radical-related chronic diseases.