Ryusuke Tanaka

Inhibitory effects of brown algal phlorotannins on secretory phospholipase A2s, lipoxygenases and cyclooxygenases

The inhibitory effects of brown algal phlorotannins on secretory phospholipase A2s (sPLA2s), lipoxygenases (LOXs) and cyclooxygenases (COXs) were determined with an in vitro assay. Oligomers of phloroglucinol; eckol (a trimer), phlorofucofuroeckol A (a pentamer), dieckol (a hexamer) and 8,8′-bieckol (a hexamer) isolated from the brown alga Eisenia bicyclis had pronounced inhibitory effects on sPLA2 from porcine pancreas and bee venom (IC50 100–200 μM). The phlorotannins inhibited LOX activity more effectively than the well-known LOX inhibitors; resveratrol and epigallocatechin gallate. 8,8′-Bieckol, the strongest LOX inhibitor in this study, inhibited soybean LOX and 5-LOX with IC50 values of 38 and 24 μM, respectively. Negligible or very weak effects of the phlorotannins on COX-1 and COX-2 were found, except for an inhibitory effect of dieckol on COX-1 (74.7%) and of eckol on COX-2 (43.2%) at 100 μM.

Accumulation of hydroxy lipids in live fish infected with fish diseases

Abstract Lipid hydroperoxide (LPO) and hydroxy lipid levels of diseased fishes were investigated. Although significant differences in LPO levels were not always observed between normal and diseased fish, hydroxy lipid levels in muscle and liver were significantly higher in diseased fish than in uninfected normal fish cultured with the same feed in the same cage or in wild normal fish. Hydroxy triglyceride levels of diseased yellowtails with streptococcal infection were significantly elevated in ordinary and dark muscle, and especially in the liver; 1072±78 in diseased fish compared to 632±45 (nmol/g tissue) in normal fish cultured with the same feed. In this case, the polyunsaturated fatty acid contents of the diseased fish were decreased. This was notable, especially with respect to the decrease of docosahexaenoic acid. These trends, namely, increased accumulation of hydroxy lipids in the liver of diseased fish and decreased levels of polyunsaturated fatty acids, were observed in sweetfish with ulcer disease, globefish with Heterobothrium okamotoi infection and/or emaciation disease, red sea bream infected with edwardsiellosis and/or Vibrio anguillarum , yellowtail with jaundiced disease, and carp with cold water disease.

SIMULTANEOUS IDENTIFICATION OF 4-HYDROXY-2-HEXENAL AND 4-HYDROXY-2-NONENAL IN FOODS BY PRE-COLUMN FLUORIGENIC LABELING WITH 1,3-CYCLOHEXANEDIONE AND REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

In this study, we developed a reversed-phase high-performance liquid chromatographic method with fluorometric detection for the simultaneous quantitation of 4-hydroxy-2-hexenal (HHE) and 4-hydroxy-2-nonenal (HNE) in foods by using 1,3-cyclohexanedione reagent. This method provides good resolution and reproducibility. Chromatography is conducted with a gradient system using a mobile phase of methanol–water–tetrahydrofuran (75:25:1, v/v) (A) and acetonitrile (B). The gradient employed is 5% B to 25% B in 25 min; 25% B to 80% in 30 min; isocratic at 80% B in 40 min. The flow rate is 1.0 mL/min. Fluorescence detection is performed at an exciting wavelength of 385 nm and an emission wavelength of 450 nm. The calibration curves for HHE and HNE concentrations are linear from 0.08 to 14 and 0.08 to 5.2 µmol/mL, respectively. The limits of detection and quantitation for HHE are 27 and 160 nmol/mL and those for HNE are 30 and 149 nmol/mL, respectively. Validation data show that the assay is sensitive, specific, accurate, and reproducible for the simultaneous quantitation of HHE and HNE. This rapid method is, therefore, appropriate for quantitating HHE and HNE in fatty foods such as fish and animal meats.

The oral administration of thermophile-fermented compost extract and its influence on stillbirths and growth rate of pre-weaning piglets

Food produced via fermentation with mesophilic bacteria has been used to confer health benefits. In contrast, mammalian physiological responses to the intake of thermophile-fermented products have not been thoroughly investigated. We examined the effects of administering a compost extract consisting of fermented marine animals with thermophiles, including Bacillaceae, to pregnant sows and piglets. Retrospective studies were performed on two different swine farms (n=330-1050 sows). The rate of stillbirth was markedly lower in all parities of the compost extract-fed group compared to those of the control group (p≦0.001). Additionally, the birth to weaning period of newborns was significantly shorter (p<0.0001), while the ratio of weanlings per liveborn piglets was increased by more than 6.5% in the compost extract-fed group. Thus thermophiles and their products in the compost extract might promote growth and reduce stillbirths of piglets during the birth to weaning period.

Elevation of lipid peroxide level and production of hydroxy lipids in cultured Hepa-T1 cells by oxidative stressors

In order to investigate the relationships between levels of lipid peroxides (LPO), hydroxy lipids (L-OH) and of antioxidative enzymes in live fish, cultured cells from tilapia liver (Hepa-T1) were treated with oxidative stressors, 2,2′-azobios(2-amidino-propane) dihydrochloride (AAPH) and H2O2. By treating with 5 mM AAPH, LPO and L-OH of the cells significantly increased time-dependently. In particular, L-OH increased from 7.6±0.7 to 23.6±1.8 nmol/mg protein after 2 h of treatment, and synchronously the glutathione peroxidase (GPx) activity of the cells increased from 259±106 to 1970±135 mU/mg protein. In the case of H2O2, however, no elevation of L-OH was observed. In this case, catalase (CAT) and superoxide dismutase (SOD) activities increased time-dependently. Especially, after 2 h exposure, the CAT activity increased remarkably from 217±21 to 2510±120 mU/mg protein, though the GPx activity did not change significantly. These results suggest that the elevation of the L-OH level is closely associated with the elevation of GPx activity, but not with that of CAT or SOD. However, when vitamin E-enriched Hepa-T1 cells were treated with AAPH and H2O2, the production of LPO and L-OH was suppressed significantly. The findings suggest that vitamin E also plays an important role in the levels of the oxidation products in vivo.

The anti-allergic effect of the ethyl acetate fraction from an Ecklonia kurome extract

To determine the anti-allergic effects of Ecklonia kurome, the ethyl acetate fraction from an E. kurome (EEK) extract was compared to the ethyl acetate fraction from an Eisenia arborea (EEA) extract and epigallocatechin gallate (EGCG) for inhibitory effects on inflammation in experimental animals, degranulation in inflammatory lymphocytes and enzymatic activities involved in allergic reactions. EEK was found to inhibit mouse ear oedema by inflammatory inducers (arachidonic acid, 12-O-tetradecanoylphorbol-13-acetate and oxazolone) in a dose-dependent manner, through both topical and oral administrations. EEK also inhibited degranulation in rat basophilic leukaemia cells and enzymatic activities (cyclooxygenase-2, soybean lipoxygenase and phospholipase A2) in a dose-dependent manner. The effects of EEK were generally comparable of those of EEA and EGCG. Therefore, E. kurome and the components in EEK have the benefit of being used not only in common foods, but also more widely as a functional food resource in anti-allergic foods.

Thermophile-fermented compost as a possible scavenging feed additive to prevent peroxidation

The extract of compost from fermented marine animals and thermophiles, including Bacillaceae, confers health benefits as a feed additive for fish and pigs. However, little research has explored how such compost extracts affect the physiological functions of the animals. In this study, the physiological effects of oral administration of the compost extract on the liver and muscle of rats are evaluated. After long-term administration of the compost extract in rats fed with either a normal diet or a high-fat diet over 3 months, accumulation of lipid peroxide and malondialdehyde, a marker of peroxidation, in the livers was reduced. Under such conditions, the unsaturated fatty acid composition in the liver was not significantly different in the rats fed either with or without the compost extract. In contrast, analyzes of 2,2-diphenyl-1-picrylhydrazyl (DPPH) revealed that free-radical-scavenging activity was increased in the livers of rats fed with the compost extract, although the extract itself had little of this activity. Glutathione, an antioxidant, was slightly increased following compost exposure. In addition, the levels of glutamate and glutamine, sources of glutathione, were slightly raised. Such a tendency was also observed in the muscle. Thus, thermophile-fermented compost can be a fermented feed additive to prevent peroxidation in the liver and muscle, and the effects of this additive may, in part, be associated with the retention of antioxidants and free amino acids within the organs.

Thermophile-fermented compost as a fish feed additive modulates lipid peroxidation and free amino acid contents in the muscle of the carp, Cyprinus carpio.

Recently, a compost fermented with marine animals with thermophilic Bacillaceae in a clean and exclusive process at high temperature was reported as a possible feed additive to improve the healthy balance in sea fish and mammals (i.e., pigs and rodents). Here, the effects of the oral administration of the compost on the muscle and internal organs of carp (Cyprinus carpio) as a freshwater fish model were investigated. The fatty acid composition was different in the muscle of the carp fed with or without the compost extract, but there was little difference in the hepatopancreas. The accumulation of triacylglycerols, cholesterol, lipid peroxide and hydroxyl lipids decreased in the muscle after the oral administration of the compost extract in the carps over 12 weeks, but the accumulation did not always decrease in the hepatopancreas. In contrast, free-radical-scavenging activities and the concentrations of free amino acids in the muscle did not always increase and was dependent on the dose of the compost at 12 weeks. The scavenging activities and part of free amino acid levels in the muscle of the carp were improved at 24 weeks after a high dose of compost exposure, and then the survival rates of the carp were maintained. Thus, the oral administration of thermophile-fermented compost can prevent peroxidation and increase the content of free amino acids in the muscle of the freshwater fish, depending on the dose and term of the administration, and may be associated with the viability of the fish.

Effects of Chilled Storage, Freezing Rates, and Frozen Storage Temperature on Lipid Oxidation in Meat Blocks from Cultured Bluefin Tuna Thunnus thynnus

ABSTRACT The effects of a short chilled storage period before freezing, frozen storage temperature, and freezing rate on lipid oxidation of bluefin tuna (Thunnus thynnus) meat during frozen storage were investigated. After 12-months storage, all samples had increased in peroxide value though they were less at the lower temperatures (−45 and −60°C). Peroxide values in all samples stored at −20°C increased after 3 months storage, particularly those processed and stored 51 h after harvest. The lowest increase in peroxide value occurred in the samples frozen rapidly 3 h after harvest. Vitamin E levels decreased faster during frozen storage at −20°C. There were no apparent differences in levels of triacylglycerides nor in n-3 fatty acid levels between treatments, storage periods, and storage temperatures. After 12-months storage, headspace oxidative volatiles were highest in samples stored at −20°C and lowest in those stored at −60°C. Lipid oxidation in tuna meat stored at −45°C is similar to that at −60°C, and rapid freezing rather than slow freezing should be used.

Parthenosporophytes of the brown alga Ectocarpus siliculosus exhibit sex-dependent differences in thermotolerance as well as fatty acid and sterol composition

In the filamentous brown alga Ectocarpus siliculosus, male and female sex is expressed during the haploid parthenosporophyte phase of the life cycle. Here, we found that male parthenosporophytes displayed thermotolerance whereas female specimens displayed severely reduced viability at 25 °C and 28 °C. Profiling of polyunsaturated fatty acids showed that n-3 and n-6 were the predominant species in male and female parthenosporophytes, respectively, and that the n-3/n-6 fatty acid ratio was not affected by a temperature change. Both male and female parthenosporophytes contained the sterols fucosterol, cholesterol, and ergosterol, but these were present at higher levels at 10-25 °C in female specimens than in males. Thus, these fatty acids and sterols would be expected to make the membranes more rigid in the female compared to the male, which is opposite to the paradigm that increased rigidity confers thermotolerance. Our results suggest that the sex-dependent thermotolerance in E. siliculosus parthenosporophytes is not explained by the relationship between membrane fluidity and differences in fatty acids and sterol compositions.