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.

Heterodisaccharide 4-O-(N-acetyl-beta-D-glucosaminyl)-D-glucosamine is a specific inducer of chitinolytic enzyme production in Vibrios harboring chitin oligosaccharide deacetylase genes.

Vibrio parahaemolyticus KN1699 produces 4-O-(N-acetyl-beta-d-glucosaminyl)-d-glucosamine (GlcNAc-GlcN) as a major end product from chitin using two extracellular hydrolases: glycoside hydrolase family 18 chitinase, which produces (GlcNAc)(2) from chitin, and carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), which hydrolyzes the N-acetyl group at the reducing-end GlcNAc residue of (GlcNAc)(2). In this study, we clarified that this heterodisaccharide functions as an inducer of the production of the two above-mentioned chitinolytic enzymes, particularly chitinase. Similar results for chitinase production were obtained with other chitin-decomposing Vibrio strains harboring the CE family 4 COD gene; however, such an increase in chitinase production was not observed in chitinolytic Vibrio strains that did not harbor the COD gene. These results suggest that GlcNAc-GlcN is a unique inducer of chitinase production in Vibrio bacteria that have the COD-producing ability and that the COD involved in the synthesis of this signal compound is one of the key enzymes in the chitin catabolic cascade of these bacteria.

Production and secretion of a recombinant Vibrio parahaemolyticus chitinase by Escherichia coli and its purification from the culture medium.

An open reading frame encoding the chitinase gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome. An expression plasmid containing the gene was introduced into Escherichia coli cells, and recombinant chitinase (Pa-rChi) was produced and secreted into the culture medium with the aid of the signal peptide. Pa-rChi was purified and its substrate specificity was determined.

Identification of a novel endochitinase from a marine bacterium Vibrio proteolyticus strain No. 442.

Chitin binding proteins prepared from Vibrio proteolyticus were purified and the N-terminal amino-acid sequence of a protein from a 110-kDa band on SDS-PAGE was found to be 85-90% identical to the 22nd-41st residues of the N-termini of chitinase A precursor proteins from other vibrios. We cloned the corresponding gene, which encodes a putative protein of 850 amino acids containing a 26-residue signal sequence. The chitinase precursor from V. proteolyticus was 78-80% identical to those from Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio carchariae. However, the proteolytic cleavage site for C-terminal processing between R597 and K598 in the chitinase precursor of other vibrios was not observed in the amino acid sequence of V. proteolyticus, which instead had the sequence R600 and A601. Subsequently, full-length and truncated chitinases were generated in Escherichia coli. The specific activity of full-length chitinase expressed in E. coli was 17- and 20-folds higher for colloidal and alpha-chitins (insoluble substrate), respectively, than that of the C-terminal truncated enzyme. However, both recombinants showed similar hydrolysis patterns of hexa-N-acetyl-chitohexaose (soluble substrate), producing di-N-acetyl-chitobiose as major product on TLC analysis. We showed that the C-terminus of the V. proteolyticus chitinase A was important for expression of high specific activity against insoluble chitins.

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.

Production of a recombinant chitin oligosaccharide deacetylase from Vibrio parahaemolyticus in the culture medium of Escherichia coli cells.

An open reading frame (ORF) encoding chitin oligosaccharide deacetylase (Pa-COD) gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome and its sequence was analyzed. The ORF encoded a 427 amino acid protein, including the 22 amino acid signal sequence. The deduced amino acid sequence was highly similar to several bacterial chitin oligosaccharide deacetylases in carbohydrate esterase family 4. An expression plasmid containing the gene was constructed and inserted into Escherichia coli cells and the recombinant enzyme was secreted into the culture medium with the aid of the signal peptide. The concentration of the recombinant enzyme in the E. coli culture medium was 150 times larger than that of wild-type enzyme produced in the culture medium by V. parahaemolyticus KN1699. The recombinant enzyme was purified to homogeneity from culture supernatant in an overall yield of 16%. Substrate specificities of the wild-type and the recombinant enzymes were comparable.

Aglycon specificity profiling of alpha-glucosidases using synthetic probes.

We designed and synthesized hydrogen bond based probes 1-8 with the exception of known glycosidase inhibition mechanisms, and aglycon specificity of 11 different sources of alpha-glucosidases were investigated using their probes. Probe 4 (2,6-anhydro-1-deoxy-1-[(1-oxopentyl-5-hydroxy)amino]-D-glycero-D-ido-heptitol) showed a potent inhibition of S. cerevisiae alpha-glucosidase among all alpha-glucosidases. Probe 4 was found to be a competitive inhibitor for S. cerevisiae alpha-glucosidase with Ki 0.13 mM.