Consumption of fish balls "tsumire" may help prevent liver function deterioration in aged rats.

Background: Possible health benefits of fish balls tsumire consumption on human health have been deduced based on the results of our earlier study investigating tsumire consumption by young (6-weeks-old) Sprague-Dawley rats. Aim: The objectives of this study were to evaluate the effect of tsumire intake on aged rats by feeding them a diet containing 5% dried tsumire. Methods: Biomarker levels and organ weight of 80-weeks-old Sprague-Dawley rats that were fed with tsumire were examined for potential health benefits. Results: Following 84 days of administering tsumire-containing diet, we found a decrease in some liver function parameters, such as the levels of AST, ALT, ALP and LAP, in the tsumire-fed rats compared to control rats provided with normal diet. In particular, significantly reduced LDH levels were observed in the experimental group. Conclusions: The results can be extrapolated to possible beneficial effects of consumption of tsumire on human health.

Effect of fish paste products, fish balls 'tsumire', intake in Sprague-Dawley rats.

The fish paste product, fish balls 'tsumire', is a traditional type of Japanese food made from minced fish as well as imitation crab, kamaboko and hanpen. Although tsumire is known as a high-protein and low-fat food, there is a lack of scientific evidence on its health benefits. Hence, we aimed to investigate the effects of tsumire intake on organ weight and biomarker levels in Sprague-Dawley rats for 84 d as a preliminary study. Six-week-old male Sprague-Dawley rats were divided into two groups: group I, fed normal diets, and group II, fed normal diets with 5 % dried tsumire. Throughout the administration period, we monitored their body weight and food intake; at the end of this period, we measured their organ weight and analysed their blood biochemistry. No significant differences were observed with respect to body weight, food intake, organ weight and many biochemical parameters between the two groups. It was found that inorganic phosphorus and glucose levels were higher in group II rats than in group I rats. On the other hand, sodium, calcium, amylase and cholinesterase levels were significantly lower in group II than in group I. Interestingly, we found that the levels of aspartate aminotransferase, alanine transaminase, lactate dehydrogenase and leucine aminopeptidase in group II were significantly lower than in group I, and that other liver function parameters of group II tended to be lower than in group I. In conclusion, we consider that the Japanese traditional food, 'tsumire,' may be effective as a functional food for human health management worldwide.

Effect of fish paste products "Hanpen" intake in Sprague-Dawley rats.

Fish paste product, "Hanpen," is a traditional type of Japanese food made from minced fish as well as imitation crab and kamaboko, and a marshmallow-like soft texture is characteristic of hanpen. Hanpen is known as a high-protein and low-fat food. However, there is a lack of evidence on its health benefits. The aim of this study was to investigate the effects of hanpen intake on organ weight and biomarker levels in Sprague-Dawley rats with diets consisting of hanpen for 84 days as an initial study. Male, 6-week-old Sprague-Dawley rats were divided into two groups: group I, fed normal diets, and group II, fed normal diets with 5% dried hanpen. Throughout the 84-day treatment period, we checked body weight and food intake, and after 84 days, we performed organ weight and blood biochemical analyses. No significant differences were seen in body weight, food intake, organ weight, and most biochemical parameters between group I and group II. Interestingly, total cholesterol (T-CHO) and high-density lipoprotein cholesterol (HDL-C) levels of group II were significantly higher than those of group I after administration for 84 days. Moreover, lactate dehydrogenase (LDH) level of group II was marked lower than that of group I, and other liver function parameters of group II tended to be lower than those of group I. As conclusion, "Hanpen," a Japanese traditional food, could be effective as a functional food for human health management worldwide.

Effects of Supplementation with Anti-Inflammatory Compound Extracted from Herbs in Healthy and Obese Cats.

BACKGROUND: Obesity has become a serious public health problem all over the world, and prevalence of obesity has increased in cats. Obesity is characterized by continuous low-grade inflammation based on oxidative stress by excessively produced reactive oxygen species (ROS). Supplementation with anti-oxidant and anti-inflammatory compounds is very effective to relieve the obesity condition. A plant extract mixture containing Rhus verniciflua and some other herbs, Rv-PEM01-99, shows anti-oxidant and anti-inflammatory effects in animals. The aim of this study was to evaluate the effects of supplementation with Rv-PEM01-99 as an anti-inflammatory compound in healthy and obese cats. MATERIALS AND METHODS: Ten healthy mix breed cats and four obesity disease cats were used. The healthy cats were randomly divided into control and test groups. Anti-inflammatory compound, Rv-PEM01-99, in which quercetin derivative is the main component, was supplemented to the healthy test group and the obesity disease cats at the dose of 100-120 mg/kg/day (2.5-3.0 mg/kg/day as quercetin) for 4 weeks. Metabolites, hormones and enzymes were measured before and after the compound supplementation. RESULTS: The anti-inflammatory compound supplementation decreased serum amyloid A (SAA) concentrations as inflammatory markers in both healthy and obesity disease cats. In obesity disease cats, plasma total cholesterol concentrations and AST and ALT activities decreased significantly after the compound supplementation. CONCLUSION: Quercetin derivative seems to have strong anti-inflammatory activities. In the healthy cats, anti-inflammatory compound supplementation decreased plasma NEFA and SAA concentrations. In the obesity disease cats, the compound supplementation may have alleviated obesity disease by relieving inflammation and improvement of lipid metabolism in livers.

Rhus verniciflua leaf extract suppresses obesity in high-fat diet-induced obese mice.

BACKGROUND: Obesity is a serious health problem in the world. We thought that the activity and safety of natural plants and/or foods are very important in the management of therapy for obesity. Rhus verniciflua (R. verniciflua) is also known as lacquer tree in Japan and Korea, and it is consumed as food ingredients and/or traditional herbal medicine. We prepared an extract from R. verniciflua leaves (Rv-PEM01-99) to develop a new functional food material and/or nutritional supplements. OBJECTIVE: This study evaluated the anti-obesity effects of the Rv-PEM01-99 in high-fat diet (HFD)-induced obese mice. DESIGN: Six-week-old male C57BL/6J mice were divided into three groups: group I (HFD group), group II (HFD + 1% Rv-PEM01-99 group), and group III (HFD + 2% Rv-PEM01-99 group). Throughout the 56-day treatment period, body weights of these mice were checked twice a week. After 56 days, blood biochemical analyses were performed. RESULTS: In animal studies, no death or abnormalities in food consumption were observed between groups I, II, and III. Body weight gain in the groups administered Rv-PEM01-99 was less than that in group I. In particular, body weight gain in group III was significantly less than that in group I after 52 days of Rv-PEM01-99 administration. In addition, intra-abdominal fat and leptin levels in group III were significantly lower than those in group I. HPLC and LC/MS analysis showed a quercetin derivative as an active compound in Rv-PEM01-99. CONCLUSION: Rv-PEM01-99, containing a quercetin derivative, showed anti-obesity effect in HFD-fed mice. It could therefore be useful as food material and/or nutritional supplement for management of obesity.

Changes in Plasma Metabolites Concentrations in Obese Dogs Supplemented With Anti-oxidant Compound.

The aim of this study is to discuss the effect of anti-oxidant supplement (Rv-PEM01-99, Kibun Foods, Inc., Tokyo, Japan) on changes in energy metabolism in obese dogs. 200 mg/kg/day of Rv-PEM01-99 (equivalent to 5 mg kg/day of quercetin derivative) were applied for 6 weeks to the Beagle dogs fed high fat diet (HFD) or control diet (CD). In the present study, body weight (BW) decreasing effect of Rv-PEM 01-99 in obese dogs was not clear. However, plasma alkaline phosphatase (ALP) activities at the end of experiment were significantly decreased compared to those at the start of experiment in obese dogs supplemented with Rv-PEM 01-99 (paired-t test, p < 0.05). In control dogs supplemented with Rv-PEM 01-99, Plasma malondialdehyde (MDA), and triglycerides (TG) levels and lactate dehydrogenase (LDH) activities were significantly decreased compared to those at the start of experiment (paired-t test, p < 0.05). From these findings, Rv-PEM 01-99 seems to be not harmful for dogs. Anti-lipid peroxide effect and liver function improvement are expected in the dogs supplemented with Rv-PEM 01-99.

Antitumour and Apoptotic Effects of a Plant Extract Mixture Containing Rhus verniciflua and Other Herbs in Human Leukaemia Cells.

The apoptotic effects of a novel antitumour agent (Rv-PEM01) prepared from 6 kinds of herbs, including Rhus verniciflua were investigated using flow cytometry and western blot analysis. Rv-PEM01 induced apoptosis but not necrosis in MOLT-3, KG-1, and K562 human leukaemia cell lines. Further, Rv-PEM01-treated cells showed significantly upregulated expression of caspase-3 and 9 and cleaved caspase-3 and 9 compared to the control cells. Taken together, the results suggest that Rv-PEM01 induced apoptosis via the mitochondrial-mediated pathway, and is a potential natural anticancer agent and/or a functional food material.

Effects of Comb Tooth Cap Medicinal Mushroom, Hericium ramosum (Higher Basidiomycetes) Mycelia on DPPH Radical Scavenging Activity and Nerve Growth Factor Synthesis.

The goal of this study was to evaluate the antioxidant effects of and nerve growth factor (NGF) synthesis caused by Hericium ramosum mycelia. Wild mushroom fruiting bodies were collected from nature to isolate their mycelia. Pieces of H. ramosum fruiting bodies were plated onto 90-mm Petri dishes with potato dextrose agar medium to isolate their mycelia. Antioxidant activity was measured using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging activity in vitro; the ethanol extract from H. ramosum mycelia (63.11 µmol Trolox/g) was more potent than that of other mushroom mycelia extracts. There was a proportional relationship (R2 = 0.7929) between DPPH radical scavenging activity and total phenolic content in extracts of different mushroom mycelia. We investigated the ability of H. ramosum mycelia to inducing NGF synthesis in vivo. Oral administration of H. ramosum mycelia significantly increased concentrations of NGF in the hippocampus of intact mice. These results are the first concerning antioxidant activity and NGF synthesis of H. ramosum mycelia. These mushroom mycelia could be useful as food and/or nutritional supplements because of certain biological functions.

Novel urushiols with human immunodeficiency virus type 1 reverse transcriptase inhibitory activity from the leaves of Rhus verniciflua.

Two novel urushiols, 1 and 2, and two known urushiols, 3 and 4, were isolated from the leaves of Rhus verniciflua and were examined for their human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitory activity. The novel urushiols were found to be 1,2-dihydroxyphenyl-3-[7'(E),9'(Z),11'(Z)-pentadecatrienyl]-14'-ol (1) and 1,2-dihydroxyphenyl-3-[8'(Z),10'(E),12'(E)-pentadecatrienyl]-14'-ol (2) by spectroscopic analyses. The absolute configuration at C-14' in 1 and 2 was determined to be a racemic mixture of (R) and (S) isomers by ozonolysis. Compound 2 (IC50: 12.6 µM) showed the highest HIV-1 RT inhibitory activity among the four urushiols, being 2.5-fold more potent than the positive control, adriamycin (IC50: 31.9 µM). Although the known urushiols were isolated from the sap and leaves of R. verniciflua, 1 was exclusively present in the leaves, and higher amounts of 2 were found in the leaves than in the sap. Present findings indicate that the leaves of R. verniciflua represent a new biological resource from which novel and known urushiols may be prepared, and the possible use of novel urushiols as bioactive products.

[Antitumor effects of a plant extract mixture].

Cancer is the most common cause of death in Japan. Fundamental and clinical studies on cancer were conducted from the viewpoint of Western medicine so far. However, a sustained complete remission has not been achieved yet. In order to alleviate the side effects of anticancer drugs, some traditional herbal medicines (Kampo medicines) have been prescribed to cancer patients. We have been studying on antitumor substances in medicinal herbs and found an antitumor medicinal herb named Rhus verniciflua (lacquer, Urushi in Japanese). To investigate the antitumor effect in vitro, a plant extract mixture was prepared from six medicinal herbs containing lacquer. The plant extract mixture containing lacquer (Rv-PEM) inhibited the proliferation of several mouse and human tumor cell lines. Rv-PEM had more potent inhibitory effect on the proliferation of human leukemia cell lines (MOLT-3, KG-1) than on other tumor cell lines. The IC50 values of Rv-PEM on MOLT-3 and KG-1 cells were 0.208 and 0.293 mg/mL, respectively. After treating Rv-PEM to the tumor cells, DNA fragmentation and Caspase-3 and -9 activity increased in the treated cells. The mechanisms of the inhibitory proliferation activity of Rv-PEM would involve apoptosis of human leukemia cells (MOLT-3, KG-1, K-562) by the mitochondrial pathway.

Expression of the algal cytochrome c6 gene in Arabidopsis enhances photosynthesis and growth.

Photosynthetic plants convert light energy into ATP and NADPH in photosynthetic electron transfer and photophosphorylation, and synthesize mainly carbohydrates in the Calvin-Benson cycle. Here we report the enhancement of photosynthesis and growth of plants by introducing the gene of an algal cytochrome c6, which has been evolutionarily eliminated from higher plant chloroplasts, into the model plant Arabidopsis thaliana. At 60 d after planting, the plant height, leaf length and root length of the transformants were 1.3-, 1.1- and 1.3-fold those in the wild-type plants, respectively. At the same time, in the transgenic plants, the amounts of chlorophyll, protein, ATP, NADPH and starch were 1.2-, 1.1-, 1.9-, 1.4- and 1.2-fold those in the wild-type plants, respectively. The CO2 assimilation capacity of the transgenic plants was 1.3-fold that of the wild type. Moreover, in transgenic Arabidopsis expressing algal cytochrome c6, the 1-qP, which reflects the reduced state of the plastoquinone pool, is 30% decreased compared with the wild type. These results show that the electron transfer of photosynthesis of Arabidopsis would be accelerated by the expression of algal cytochrome c6. Our results demonstrate that the growth and photosynthesis of Arabidopsis plants could be enhanced by the expression of the algal cytochrome c6 gene.

Crystal structure of oxidized cytochrome c(6A) from Arabidopsis thaliana.

Compared with algal and cyanobacterial cytochrome c(6), cytochrome c(6A) from higher plants contains an additional loop of 12 amino acid residues. We have determined the first crystal structure of cytochrome c(6A) from Arabidopsis thaliana at 1.5 Angstrom resolution in order to help elucidate its function. The overall structure of cytochrome c(6A) follows the topology of class I c-type cytochromes in which the heme prosthetic group covalently binds to Cys16 and Cys19, and the iron has octahedral coordination with His20 and Met60 as the axial ligands. Two cysteine residues (Cys67 and Cys73) within the characteristic 12 amino acids loop form a disulfide bond, contributing to the structural stability of cytochrome c(6A). Our model provides a chemical basis for the known low redox potential of cytochrome c(6A) which makes it an unsuitable electron carrier between cytochrome b(6)f and PSI.

A novel microperoxidase activity: methyl viologen-linked nitrite reducing activity of microperoxidase.

To investigate the nitrite reducing activity of microperoxidases (mps) in the presence of methyl viologen and dithionite, the fragments C14-K22 (mp9), V11-L32 (mp22), and G1-M65 (mp65) containing heme were prepared by enzymatic hydrolysis of commercially equine heart cytochrome c (Cyt c), in which His is axially coordinated to heme iron, and acts as its fifth ligand. The nitrite reducing activity of mps was measured under anaerobic condition, and the nitrite reducing activity of mps increased with the cutting of the peptide chain. The activity of the shortest nonapeptide mp9 was approximately 120-fold that of Cyt c (104 amino acid residues) and 3.2-fold that of nitrite reductase (EC 1.7.7.1) from Escherichia coli. In the nitrite reduction by mp, nitrite was completely reduced to ammonia. We presumed that ferrous mps reduced NO2- to NO by donating one electron, the NO was completely reduced to NH4+ under anaerobic condition via ferrous-NO complexes as a reaction intermediate using visible spectra and ESR spectra, and this overall reaction was a 6-electron and 8-proton reduction. Sepharose-immobilized mp9 had a nitrite reducing activity similar to that of mp9 in solution, and the resin retained the activity after five uses and even 1-year storage. The mp will be able to use as a substitute for nitrite reductase.

Radiation-induced enhancement of nitrite reducing activity of cytochrome c.

Commercial cytochrome c (Cyt c) was irradiated with Co-60 gamma-rays in the dose range of up to 3.0 kGy to investigate the enhancement of the nitrite reducing activity of Cyt c. The optimum irradiation dose to induce nitrite reducing activity for 30 muM Cyt c solution was 1.0 kGy under an O(2) atmosphere. The nitrite reducing activity of Cyt c irradiated at this dose was approximately 45-fold that of unirradiated Cyt c and ca. 1.2-fold that of nitrite reductase. The irradiation treatment resulted in unfolding of the peptide chain, exposure of the heme group, oxidation of methionine to methionine sulfoxide, dissociation of the sixth ligand (Met), and occurrence of autoxidation in Cyt c. Sepharose-immobilized irradiated Cyt c had a similar activity to that in solution. The resin retained the activity after five uses even after 1 year of storage. The irradiated Cyt c will be able to be used as a substitute for nitrite reductase.

Appearance of nitrite reducing activity of cytochrome c upon heat denaturation.

The appearance of NO2- reducing activity of cytochrome c (Cyt c) upon heat denaturation was investigated with equine heart Cyt c. Denatured equine heart Cyt c (dCyt c), which was treated at 100 degrees C for 30 min, had NO2- reducing activity in the presence of dithionite and methylviologen in an aqueous solution under anaerobic conditions. In contrast, hemoglobin and myoglobin had no such activity under the same conditions. Using spectroscopic methods, we found that the appearance of this activity in the Cyt c was due to the following intramolecular changes: unfolding of the peptide chain, exposure of the heme, dissociation of the sixth ligand methionine sulfur, and appearance of autoxidizability. The dCyt c catalyzed NO2- reduction to NH4+ via ferrous-NO complexes, and this reaction was a 6-electron and 8-proton reduction. Sepharose-immobilized dCyt c had activity similar strength to that in solution. The resin retained the activity after five uses and even after storage for 1 year. On the basis of these results, we concluded that Cyt c acquired a new catalytic activity upon heat treatment, unlike to other familiar biological molecules.

Increasing the conformational stability by replacement of heme axial ligand in c-type cytochrome.

To investigate the role of the heme axial ligand in the conformational stability of c-type cytochrome, we constructed M58C and M58H mutants of the red alga Porphyra yezoensis cytochrome c(6) in which the sixth heme iron ligand (Met58) was replaced with Cys and His residues, respectively. The Gibbs free energy change for unfolding of the M58H mutant in water (DeltaG degrees (unf)=1.48 kcal/mol) was lower than that of the wild-type (2.43 kcal/mol), possibly due to the steric effects of the mutation on the apoprotein structure. On the other hand, the M58C mutant exhibited a DeltaG degrees (unf) of 5.45 kcal/mol, a significant increase by 3.02 kcal/mol compared with that of wild-type. This increase was possibly responsible for the sixth heme axial bond of M58C mutant being more stable than that of wild-type according to the heme-bound denaturation curve. Based on these observations, we propose that the sixth heme axial ligand is an important key to determine the conformational stability of c-type cytochromes, and the sixth Cys heme ligand will give stabilizing effects.