Research Reports No.12 Abstract（2006）

12-01

Development of a new detergent based on French bean storage protein

Shigeru Utsumi
Graduate School of Agriculture, Kyoto University

Prior to this study, we had found that French bean 7S globulin exhibits excellent emulsifying ability and stability among various seed 7S and 11S globulins. Each constituent subunit of French bean 7S globulin has two N-linked glycans in analogy with soybean 7S globulin a and a' subunits. One of the two N-linked glycans is located in a homologous position to each other, but the other is in a completely different position, resulting in that the two N-linked glycans are adjacent to each other only in French bean 7S globulin subunit. In order to investigate whether this kind of characteristic of N-linked glycan contributes to the excellent emulsifying properties of French bean 7S globulin, we prepared recombinant French bean 7S globulin having no glycan and endoglycosidase-treated 7S globulin having one glycan per a subunit. Comparison of emulsifying properties of these modified versions with those of the native 7S globulin indicated that N-linked glycans are essential for the excellent properties, that the adjacency of two glycans is not important, and that structural features of protein moiety is also important. Development of a new detergent based on French bean 7S globulin requires no disturbance of these factors.

12-02

Effect of polysaccharides as food additives
on the mouse intestinal immunosystem

Toshihiko Toida and Shinobu Sakai
Graduate School of Pharmaceutical Sciences, Chiba University

Fucoidan, a complex sulfated polysaccharide purified from the brown seaweed, jelly coat of the sea urchin eggs and body wall of the sea cucumber, shows various biological activities and high affinity to the CD62L (L-selectin). We have already reported that sulfated polysaccharide such as chondroitin sulfate up-regulates the antigen-specific Th1 dominant immune response of murine splenocytes sensitized with ovalbumin in vitro [Sakai, S. et al., Immunol. Lett., 84, 211-216 (2002). Akiyama, H. et al., Biochem. J., 382, 269-278 (2004)]. In this study, the structural characterization using spectrochemical analyses have been carried out on a fucoidan sample isolated from the brown seaweed, and the effects of fucoidan on the immune response have also been investigated. Detailed structural analysis has been previously reported of different fucoidan samples showing various features of branched chain structures and sugar compositional persity. The fucoidan sample from the brown seaweed shows a typical 1D 1H NMR spectrum that is fucoidan; [-3Fuc-4(ｱOSO3-)a1-]n, and also suggests that the brown seaweed might slightly be containing other neutral saccharides (mostly Mannose, but also Glucose, Xylose, Rhamnose and Galactose) obtained by hydrolysis with trifluoroacetic acid. To examine the effects of fucoidan on the systemic and mucosal immune system, splenocytes and intraepithelial lymphocytes were obtained from antigen-sensitized mice, and were challenged with same antigen in the presence of fucoidan, and the cytokine levels in the medium of the cultured cells were measured. Fucoidan from the brown seaweed showed a significantly higher secretion of interferon gamma by antigen-sensitized splenocytes than that of a control group. Additionally, to evaluate the effect of fucoidan on the differentiation of lymphocytes, the cell surface markers on splenocytes and intraepithelial lymphocytes were analyzed using flow cytometry. This is the first demonstration that fucoidan structure impacts immunological activities on murine lymphocytes sensitized with antigen, and this finding may contribute a potential use of fucoidan in nutraceutical and/or supplements.

12-03

Analysis and functional estimation of processed metabolites
of plant polyphenols as food ingredients

Kanji Ishimaru, Sumei Huang
Faculty of Agriculture, Saga University

The leaves of two Euphorbiaceous plants (Sapium sebiferum and Phyllanthus urinaria), which contain a large amount of geraniin, were autoclaved. The concentrations of four polyphenol compounds (gallic acid, corilagin, ellagic acid and breviforin carboxylic acid) in the leaves remarkably increased, while that of geraniin decreased. These four polyphenols were presumed to be originated from the degradation of geraniin. DPPH radical scavenging activity of these four polyphenols seemed to be almost identical with that of geraniin. Taking into account the stability of these polyphenols in heating process, autoclaved leaves of these two plants seemed to be good materials for new food ingredients. Autoclaved leaves of S. sebiferum and P. urinaria were treated with four Bacillus species. In the process, the concentrations of four polyphenols were readily decreased and radical scavenging activities of the treated leaves also decreased. Autoclaved leaves of S. sebiferum were also treated with Penicillium sp., Fusallinia solani and Rosellina necafrix respectively. In the treatment, the concentrations of polyphenols in the leaves were gradually decreased, while their radical scavenging activities were not so much decreased. This result suggested the existence of some unknown metabolites having strong radical scavenging activity in the leaves.

12-04

Studies for physiological functionality of anthocyanins
using DNA microarray analysis

Takanori Tsuda
Department of Food and Nutritional Sciences, Chubu University

Adipocyte dysfunction is strongly associated with the development of obesity and insulin resistance. It is accepted that the regulation of adipocytokine secretion or the adipocyte specific gene expression is one of the most important targets for the prevention of obesity and amelioration of insulin sensitivity. Recently, we demonstrated that anthocyanins, which are pigments widespread in the plant kingdom, have the potency of anti-obesity in mice and the enhancement adipocytokine secretion and adipocyte gene expression in adipocytes. In this study, we have shown for the gene expression profile in human adipocytes treated with anthocyanins (cyanidin 3-glucoside; C3G or cyanidin; Cy) and clarified anit-diabetic effect of anthocyanins using diabetic mice. The human adipocytes were treated with 100 mM C3G, Cy or vehicle for 24 h. The total RNA from the adipocytes was isolated and carried out GeneChip microarray analysis. Based on the gene expression profile, we demonstrated the up-regulation of adiponectin and down-regulated PAI-1 and IL-6 genes by the treatment of adipocytes with C3G or Cy. Dietary C3G significantly reduced serum glucose level using diabetic mice (KKAy mice). These data have provided an overview of the gene expression profiles in adipocytes treated with anthocyanins and demonstrated anthocyanins have potentially important functions in adipocytes related with obesity and diabetes that merit further investigation.

12-05

Characteristics for Change of Color of Microcapsules
Containing Dyes Sensitive to pH Change
Due to Rottenness and Deterioration of Food in Liquid

Masato Tanaka
Department of Chemical Engineering, Faculty of Engineering, Niigata University

It is tried to prepare microcapsules containing dyes sensitive to pH change in food due to rottenness and deterioration.
In the experiment, kinds of the shell material and the bridging agent were changed and it was investigated how these factors affected the characteristics of microcapsules.
Microcapsules prepared were found to change in color according to the pH change.

12-06

Content of sulfur dioxides in herbal materials
obtained from the Japanese market

Yukihiro Goda
pision of Pharmacognosy, Phytochemistry and Narcotics, National Institute of Health Sciences

Sulfur dioxides and sulfites are registered in "The Japan's Specifications and Standards for Food Additives" mainly used as bleach and anti-oxidants, and Food Sanitation Law prohibits the use to sesame, legumes and vegetables. In China, sulfur fumigation is performed for the purpose of bleaching, drying, insecticide and antibacterial to some crude drugs. Recently, it has been reported that large quantities of sulfur dioxides are detected from sulfur fumigated crude drugs. In the course of our study of the survey of impurity in herbal materials, we analyzed the content of sulfur dioxides for 24 kinds of crude drugs (5 companies, 118 herbal materials) purchased from the Japanese market. By using modified Rankine method, more than 1,000ppm of sulfur dioxides was detected from three crude drugs (Dioscorea Rhizome, Gastrodia Tuber, Fritillaria Bulb) and also more than 500 ppm of sulfur dioxides was detected from seven crude drugs (Puerariae Radix, Lilii Bulbus, Zingiberis Rhizoma, Asparagi Tuber, Platycodi Radix, Mori Cortex, Forsythiae Fructus). Since herbal materials such as Dioscorea Rhizome, Puerariae Radix, Lilii Bulbus, Zingiberis Rhizoma are used as food and food additives, sulfur dioxide content of these materials should be noted at the viewpoint of food safety.

12-07

Main and/or side effect of food additive compounds
on circadian clock gene expression in mouse liver

Shigenobu Shibata
Department of Pharmacology, School of Science and Engineering, Waseda University

It is well known that clock genes such as Per1 and Per2 operate molecular clock works of circadian system in mammals. Clock gene expression is observed not only in the suprachiasmatic nucleus, a main oscillator､but peripheral organs like liver, heart, lung and so on. Thus, clock genes appeared in the peripheral organs must possess some role in peripheral organs, for example clock system in the liver may be critical role in clock function such as cholesterol synthesis and drug metabolism.
There are many kinds of food additive chemicals for the purpose of anti-aging, anti-oxidation, artificial sweet and so on. Some of these chemicals have been reported to cause a toxic effect on liver function. Therefore, in the term of circadian clock system, we examined whether food additive chemicals cause the impairment of clock gene expression in the liver. On the other hands, induction of Per1 and Per2 genes in the liver by these chemicals may he1p to adjust the phase of spontaneous circadian rhythm in the liver.
Orthophenylphenol (OPP) and thiabendazole (TBZ) at a high dose which might affect liver function strongly increased Per1 and Per2 gene expression in the liver. OPP and TBZ at a small dose which is suggested as minimum regulated dose of daily intake, decreased Per1 and Per2 gene expression. Butylated hydroxyanisole (BHA), butylated hydroxytluene (BHT) and potassium sorbate (SK) did not show any strong effect on Per1 and Per2 gene expression in the liver. Administration of sodium benzoate (SB) is tendency to cause the reduction of Per1 and Per2 gene expression, although efficacy was weak in comparison with OPP and TBZ.
In the next experiment we applied tissue culture of liver to understand whether in vitro application of glycyrrhizinic acid (GA) affects circadian rhythm of luciferase activity of Bmall-luciferase transgenic mice. GA augmented the amplitude of circadian rhythm and also normalized the Free-running periods of rhythm; GA-treated tissue shows 24hr, while control tissues do 21hr. The present results strongly suggest that GA may be a helpful compound for normalization of abnormal dock oscillation such as aging and stress condition, and this effect may be away from its artificial sweet.

12-08

Study on the relationship of DNA polymerase inhibitory activity, anti-cancer activity,
anti-inflammatory activity and anti-oxidant activity by curcumin

Yoshiyuki Mizushina
Laboratory of Food & Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin University

We previously reported that a phenolic compounds, petasiphenol and curcumin (diferuloylmethane), were a selective inhibitor of DNA polymerase l (pol l) in vitro. The purpose of this study was to investigate the molecular structure and bio-activity (i.e., pol inhibitory activity, anti-inflammatory activity and anti-oxidant activity) relationship of curcumin and 13 chemically synthesized derivatives of curcumin. The inhibitory effect on pol l (full-length, i.e. intact pol l including the BRCA1 C-terminus (BRCT) domain) by some derivatives was stronger than that by curcumin, and mono- acetylcurcumin (compound 13) was the strongest pol l inhibitor of all the compounds tested, achieving 50 % inhibition at a concentration of 3.9 mM. These curcumin derivatives had anti-inflammatory activity as same tendency as pol l inhibitory activity, but compound 13 had not anti-oxidant activity. The compound did not influence the activities of replicative pols such as a, d and e. It had no effect on pol b activity either, although the three-dimensional structure or pol b is thought to be highly similar to that of pol l. Compound 13 did not inhibit the activity of the C-terminal catalytic domain of pol l including the pol b-like core, in which the BRCT motif was detected from its N-terminal region. MALDI-TOF MS analysis demonstrated that compound 13 bound selectively to the N-terminal domain of pol l, but did not bind to the C-terminal region. Based on these results, the pol l-inhibitory mechanism of compound 13 is discussed.

12-09

Development of simple and rapid method
for the determination of harmful chemical material pollution
through food container packaging and analysis of food pollution.

Hiroyuki Kataoka
School of Pharmacy, Shujitsu University

In recent years, a cheap and disposable plastic or paper are taken advantage in container and packaging of various foods, with persification of the eating habits and increase of processed foods. However, chemical material pollution to food through these container packaging is highlighted, and it is an urgent problem that we get safety of food and to prevent health hazard beforehand. In this study, simple and rapid method for the determination of chemicals related to a food container and packaging were developed in order to make clear the elution of chemicals from those container and packaging, permeability of chemicals for container packaging, and food pollution as mediation with a plastic and paper. In-tube SPME/LC/MS and fiber SPME/GC/MS methods were practical and solvent-free, and the reality of food pollution in marketing foods was investigated using these methods.

12-10

Comprehensive analysis of gene expression induced by food additives using DNA microarray

Yuzuru Otsuka
Ochanomizu University, Institute of environmental science for human life

To establish a new method that can evaluate food additives safety and food additives function, we conducted the comprehensive gene expression profile analysis using a DNA micro- array. Mother milk is used for safe food, and beta-Naphthoflavone is used as an one of dangerous chemicals. HepG2 cells were incubated with hydrogen peroxide and RNA was extracted form the cells. The expression profile of the genes was analyzed by an ABI DNA micro-array and compared with the results of the profile obtained by mother milk addition or beta-naphthoflavone addition. One thousand and three hundred seventy two genes were upregulated, 1819 genes were down regulated treated by mother milk. CYP1A1 and GSTs were upregulated by beta-naphthoflavone treatment. We concluded that the method described here is useful method to evaluate the safety and function of the food.

12-11

Studies for safety assessment of additives used to foods and pharmaceuticals

Kunikazu Teshima
School of Nursing and Rehabilitation Sciences, SHOWA University

In recent years, consumer's consciousness about the safety securement for the marketed products have been so　enhanced　that the indication of names of the ingredients in the product should be necessary to the manufacturers or dealers of the products.
Indication of food additive for food products is required by law and only the active ingredients for pharmaceutical products.
However, indication for pharmaceutical excipients started from 2004 as voluntarily.
Consumers could judge to use safely the products based on indication of the ingredients, and sometimes they should ask a scientific basis of safety of the ingredient.
Many of a scientific report for this kind are published as an inpidual research report and systematic review are less.
About 300 food additives have been used as well as pharmaceutical excipients.
Therefore we organized a team for safety assessment of those items under the cooperation of both scientists for food additives and pharmaceutical excipients.
We put priority to summarize and translate into Japanese on the monograph by JECFA (Joint FAO/WHO Expert Committee on Food Additives), and others were searched by internet data bases.
A retrieval method of safety information of previous year has　been used.
As a result, safety information of 38 ingredients and 5 groups of flavor and fragrance materials has been collected in 2005.

12-12

Establishment of qualification and quantification method
for transgenes introduced to genetically modified and processed foods

Yoshihiro Ozeki
Faculty of Technology, Tokyo University of Agriculture and Technology

Japanese official method for detection of genetically modified (GM) foods has been developed and established to qualify and quantify mainly the GM raw foods without any processing. Here, model corn grits in which contained GM corns of MON810 and GA21 were processed using an extruder at several heat temperature with high pressure. The genomic DNAs were prepared from processed model grits and copy numbers of transgenes and starch synthase IIb (SSIIb) gene were measured by TaqMan PCR. It was observed that different copy numbers for SSIIb gene in the processed grits were detected between MON810 and GA21, supposing that the degree of fragmentation of genomic DNAs processed with simultaneous heat and high pressure treatments was different from each other. In order to investigate the rate of the fragmentation of genomic DNA by simultaneous heat and high pressure processing, model soybean seeds contaminated with GM soybean were processed with autoclaving and sampled at different processing time, then genomic DNAs were prepared and copy numbers of Lectin gene, High-Mobility Group (HMG) gene and transgene were measured. It was revealed that the rate of decrease of the copy numbers was exponential related to processing time and the rate was different according to not only the length but also the nucleotide sequences of the target DNAs for TaqMan PCR. When the primer sets corresponding to HMG gene and transgene showing the same rate of the decreases were used to measure the percentage of the contamination of GM soybean, the calculated percentage was nearly close to the real one irrespective of the processing time.

12-13

Breeding study toward red radishes containing anthocyanin in high content

Koji Tsuji
Graduate School of Pharmaceutical Sciences, Osaka University

As a part of our program toward breeding for red radishes containing anthocyanin in high content, the second generations of red radish by way of selective crossing among the first generations were cultivated and their anthocyanin contents were quantitated by HPLC. Consequently, the second generation was shown to maintain character to produce anthocyanin in higher content. In addition, two populations with remarkably high anthocyanin content were disclosed among the second generations.
On the other hand, cultivation of red radish in spring was also examined. Red radishes cultivated in spring were exhibited to differ in anthocyanin composition from those cultivated in autumn. Furthermore, some inpiduals, containing anthocyanin in larger amount than red radishes cultivated in autumn, were observed among the population extending flower stalks significantly.

12-14

Development of a metabolomic analysis with LC FT-ICR MS
for the safety assessment of food ingredient.

Shigeo Takenaka
Graduated School of Life and Environmental Sciences, Osaka Prefecture University

Metabolomics is an emerging description for the safety assessment of food ingredient. Here we report a method with LC FT-ICR MS using amiodarone treated rat as a model. There hundred mg/kg of oral administration of amiodarone caused phospholipidosis. The plasma lipid was extracted and was analyzed both LC MS and LC FT-ICR MS. The lipid profile of the treated rat was clearly pided on PCA scores plot with LC FT-ICR MS with a small amount of the sample compared to the ordinary LC MS analysis. The biomarker candidates were also suggested on PCA loading plot. The results indicate that metabolome analysis with LC FT-ICR MS is applicable to the safety assessment of food ingredient.

12-15

Effects of soy peptides supplementation after high intensity bicycle exercise
on muscle glycogen resynthesis and exercise performance

Toshihito Katsumura, Ryotaro Kime, Norio Murase, Takuya Osada,
Kiyoshi Shiroishi, Kousuke Shimomura, Mikiko Anjo, and Ayaka Sato
Department of Sports Medicine for Health Promotion, Tokyo Medical University

Introduction
Nutrient supplementation after exercise is an important factor in the recovery process. Amino acids plus carbohydrate (CHO) supplements have been found to enhance the rate of muscle glycogen recovery and improve subsequent exercise performance. As gut transit time of soy peptides (SP) is faster than amino acids, SP supplementation may be more effective for quick muscle glycogen resynthesis. In addition, almost all studies have administered a high concentration of amino acids. Hence, the purpose of this study was to compare the effects of low contents of SP in combination with CHO intake versus CHO alone after high intensity endurance exercise on glycogen resynthesis and exercise performance. Also, we investigated blood amino concentrations to determine the ratio of free tryptophan over branched-chain amino acids (Free Trp / BCAA) as an index of central fatigue.

Methods
Eleven well-trained athletes [ten males and one female, age = 21.4 ｱ 0.4 yrs (Mean ｱ SE), VO_2max = 57.4 ｱ 2.9 mL/kg/min, eight triathletes, two bicyclists] participated in this study. The subjects performed 2 h of intense bicycle exercise at 70-50% VO2max intensities. Each beverage [placebo (P) = 4g CHO, SP in combination with CHO (SP+CHO) = 4g CHO, 1g SP] was taken after the exhaustive exercise.
Blood samples and visual analogue scales (VAS) of central fatigue were obtained at rest and during the recovery period. Two hours after finishing the intake of each beverage, the subjects performed a 10 km bicycle time trial test. The subjects repeated the same protocol at least 1 week later with the other beverage. Drink treatments were administered in a double-blind fashion using a randomized cross-over design.

Results
The blood BCAA concentration was significantly higher in SP + CHO treatment than P treatment at 20 min after each beverage intake (P < 0.001). The Free Trp / BCAA ratio was significantly increased after the 2 h exercise and decreased during the recovery period in both treatments (SP + CHO; 0.0529 ｱ 0.001, P; 0.0520 ｱ 0.002). However, the Free Trp / BCAA ratio was greatly reduced in the SP + CHO (0.0423 ｱ 0.001) treatment compared with the P treatment (0.0489 ｱ 0.001) (p = 0.06) at 20min after beverage intake but not at 120min after intake. The VAS of central fatigue also recovered greatly using the SP + CHO (-26.1 ｱ 3.0%) treatment compared with the P treatment (-15.0 ｱ 2.8%) (p = 0.08) at 20min after beverage intake but not at 120min. There were no significant treatment effects in blood insulin, glucose, or free fatty acid concentrations. The 10 km bicycle exercise time trial test had no significantly differences between CHO + MP treatment (571.6 ｱ 11.0 sec) and P treatment (580.3 ｱ 11.7 sec).

Conclusion
The present data show that small amounts of soy peptides reduce exercise induced central fatigue quickly, but are not effective for muscle glycogen resynthesis.

12-16

Effects of BEETRED on pancreatic b cells in ALS mice

So far we have showed that 36ppm BEETRED-intake inhibited ALS mice from ALDM. And this effect of inhibition against ALDM had a dependence on the concentration of ingested BEETRED solution. Then, to uncover the one of effects of this pigment, ALS mice treated with 36ppm BEETRED for 1week, 3weeks and 4weeks until 7 weeks of age were investigated by administrating alloxan, inducing pancreatic b -cells to damage specifically. After drinking BEETRED solution, ALS mice were treated with alloxan. It was indicated that the effect of inhibition against ALDM had a dependence on period of time during BEETRED solution intake. Furthermore, ED50 calculations were made by using Probit estimation. The ED50 value in female showed 19.5ｱ3.05ppm and in male showed 76.4ｱ18.1ppm.

12-17

Establishment of agricultural plants producing high bioactive compounds

Koichiro Shimomura, Kaori Touno
Faculty of Life Sciences, Toyo University

New varieties of blueberries (Vaccinium sp.) were produced in Agricultural Technology Center (Gunma). Antioxidant capacity, measured by superoxide anion-scavenging (SOS) activity, and total anthocyanin contents in fruits of new 2 clones of blueberries, 14 highbush blueberry cultivars, and 12 Vaccinium oldhamii plants were evaluated. There were differences on SOS activity and anthocyanin contents of blueberry fruits among cultivars. The blueberry fruits (five cultivar) harvested in the city of D showed high anthocyanin contents. The harvest time of blueberry fruits and a kind of rootstock using for cultivation affected on SOS activity and anthocyanin contents in blueberry fruits. The new variety of blueberry fruits (Earliblue) had higher antioxidative activity (SOS activity) than the parent strain. However, the SOS activity of Otsububoshi fruits, new variety established by tissue culture technique, was not stable, depending on the harvest years, which means that it has to be continued to analyze the SOS activity and anthocyanin contents. There were no significant correlation between SOS activity and anthocyanin content on fruits of 14 blueberry cultivars and 12 V. oldhamii plants. B 48 and B 50 had high SOS activity and anthocyanin contents in V. oldhamii plants. We therefore suggest that those varieties may be suitable for a crossbreeding to produce a new variety of blueberry hybrid.

12-18

Identification of Myricae Cortex and Plantaginis Herba by DNA profiling

Hajime Mizukami
Graduate School of Pharmaceutical Sciences, Nagoya City University

Fruits, leaves and bark of Myrica rubra (Myricaceae) are used to prepare "Myrica Extract", a natural food additive. The dried bark of M. rubra has also been used as a crude drug, together with barks of M. esculenta and M. adenophora. The DNA profiling protocol based on the chloroplast trnK gene sequence, which was established in a previous year, was successfully applied to the samples obtained in the market as well as the herbarium specimens. Furthermore, a protocol for molecular authentication of Plantago herb, which was used as ctude drugs and also as herbal supplements, based on nucleotide sequences of the ITS region of rDNA was established.

12-19

Prevention of Thermal Inactivation and Aggregation of Sweet Protein
Thaumatin by Phosvitin and its Possible Preventive Substances

Naotoshi Matsudomi
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University

The effects of phosvitin (PV) on thermal inactivation and aggregation of sweet protein thaumatin were examined by determining soluble protein after heating 80ｰC for 15 min in pH 7. Thaumatin became insoluble on heating above pH 7, and the sweetness disappeared. In the presence of PV, the thaumatin insolubilization was suppressed, the sweetness was retained by the addition of 0.01% PV to 0.1% thaumatin. This indicates that thaumatin may be more stable to heat in the presence of PV. However, the preventive ability of phosvitin against thaumatin insolubilization was reduced by adding NaCl to a PV-thaumatin mixture on heating. In addition, native PAGE and gel filtration analyses showed that PV prevented an insolubilization of thaumatin through ionic interactions. The CD and intrinsic tryptophan fluorescence spectra of a PV-thaumatin mixture hardly change before and after heating at 80℃ and pH 7.0. This result suggests that PV protects heat denaturation of thaumatin due to the action of phosphoserine residues in PV as a polyelectrolyte.

12-20

Stimulation of organochlorine chemicals excretion induced
by functional foods and food additives

Tsuyoshi Nakanishi
Department of Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University

Polyhalogenated aromatic hydrocarbons (PHAHs) which include polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) are environmental contaminants found in emissions resulting from combustion of fossil fuels; incineration of municipal, hazardous, and hospital wastes; and production of bleached paper. Human exposure to these compounds usually involves complex mixtures in the diet, particularly milk and other dairy products, fish and meat. These compounds elicit a variety of biological and toxic responses; induction of xenobiotics metabolizing enzymes, behavioral abnormalities, teratogenicity, immunotoxicity, thymus atrophy, reproductive disorders, epithelial disorders, wasting syndrome, hepato-toxicity and cancer. PHAHs tend to accumulate in liver and adipose tissue because of its high lipophilicity. In experimental animals, fecal excretion is the major route of excretion of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), which is the prototype and the most potent isomer of the PHAHs, following intratracheal instillation, oral gavage, intraperitoneal or intravenous administration. However, PHAHs are so hardly metabolized that tend to remain in the body for a long time. In human, TCDD has an estimated half life of approximately 7 years. Therefore, potential exposure of humans to PHAHs has aroused great concern about not only potential toxicity, but also how to eliminate accumulated-PHAHs from the body. However, there are currently few of clinical therapy substances for enhancing elimination of accumulated-PHAHs from the body. In our current study, we screened some potential ingredients of foods and food additives to enhance the excretion of storage PHAHs in male mice by using 3H-TCDD as a model PHAH.

12-21

Enzyme-modified isoquercitrin suppresses progression of
atherosclerosis and stabilizes the plaque: comparison with quercetin

Yoshiki Nishizawa
Department of Metabolism, Endocrinology and Molecular Medicine,
Osaka City University Graduate School of Medicine

Anti-atherogenic effect of enzyme-modified isoquercitrin (EMIQ), an antioxidant flavonoid, was compared with quercetin using apo E-deficient atherogenic mice. Male apo E -/- mice (7 week-old), were fed either with diet alone, quercetin (0.0115% in total diet: equivalent molar concentration as 0.026% EMIQ), or EMIQ (0.026% in total diet) for 14 weeks. Mice were fed with high fat diet throughout the experiment. At 20 week-old, mice were scarified and atherosclerotic legions in the aorta were measured by Oil-red O staining and morphometry. Atherosclerotic plaque lesion at aortic sinus was also determined by the histological analyses. Throughout the experiment, there were no significant differences in body weight, plasma total cholesterol, triglyceride and HDL cholesterol levels. In EMIQ-treated group, atherosclerotic legion area in whole aorta and plaque lesion area in aortic sinus were significantly less than those of control group. Histo-immunological analyses revealed that EMIQ inhibits accumulation of macrophage, increases collagen content and increase the numbers of differentiated smooth muscle cells, suggesting its potential roles in stabilization of the plaque. Quercetin tends to have similar anti-atherogenic effects as EMIQ, however, its effects did not reach statistical significance. Thus, EMIQ appears to have anti-atherogenic and plaque-stabilizing effects, with the effects stronger than quercetin.

12-22

Chemopreventive effect on prostate carcinogenesis by resveratrol
in transgenic rat for adenocarcinoma of prostate (TRAP) model.

Satoru Takahashi
Department of Experimental Pathology, Nagoya City University Graduate School of Medical Sciences

Resveratrol is a polyphenol found in some plants and various food products but its highest concentration is in red grape skin has been shown as new potential prostate cancer chemopreventive agents. To date, only prostate cancer cell lines data has shown that resveratrol blocked proliferation, induced apoptosis and also involved in various molecular pathways e.g. androgen pathway. Therefore, we performed an in vivo experiment to explore the effect of resveratrol in rat model and tried to elucidate its mechanisms for prostate cancer prevention. Three week-old TRAP rats were given resveratrol (50, 100 and 200 g/ml) in drinking water for 7 weeks. The percent areas of epithelium of each prostate lobe were quantitatively determined. There were no treatment-related changes with reference to body and prostate weights and water consumption. Resveratrol treatment significantly suppressed neoplastic lesion development about 14 % even at low dose (50 g/ml) in the ventral lobes and potential suppression effect in the lateral lobes but not in dorsal lobes. The number of apoptotic cells in the ventral prostate of resveratrol treated rats was significantly increased compared with those in the controls. We also found that resveratrol down-regulated the AR protein expression at posttranscriptional level. These results suggest that resveratrol could be developed as an chemopreventive agent for prostate cancer.

12-23

The behavior at the time of cooking processing
of the propylene glycol containing in noodles

Motohiro Nishijima
Jissen Women's University

It was examined how much propylene glycol (PG) used when creating Japanese noodles would remain in a product. PG was added and Japanese noodles were made so that it might become 2% at raw noodles. The rate of PG detected in each cooking process boiled, and was 6% in warm Japanese noodles 11% with noodles. Moreover, with cold Japanese noodles, it turned out that it is decreasing to 4%.

12-24

Fundamental studies for safety evaluation of insoluble minerals
that are existing food additives

Lava is cooled to become vitreous rock species, such as perlite, pitchstone, and obsidian. Additives prepared from these rocks as the basic material are grouped in the insoluble mineral substances within the list of the Existing food additives, and they are used as agents for food production (filter and precipitating agents). In particular, such substances are often used for the production of liquid food, such as beer, wine, and vinegar, and pharmaceutical compounds as a filter agent. As a basic study on the estimation for the safety of these insoluble mineral substances, we investigated the methods of the material test and elution test in 2003. Metal analyses by the elution tests demonstrated the elution of toxic arsenic and lead. In particular, a high level of arsenic was eluted from diatomite samples. In 2004, we performed material tests for 8 insoluble mineral substances, and 28 elements were possible to be measured from these substances. We also examined the contents of arsenic and other toxic metals in commercially available liquid food samples, for which diatomite and others are used as filter agents. Results revealed that the levels of all these toxic metals detected were very low and well below the problematic levels at the food-hygienical point of view. And, it was shown that the concentration of arsenic (V) increased after filtration in the measurement of the level of arsenic with different chemical forms in soft drinks before and after the application of filter agents. In 2005, we examined the standard method for activated earth, acid earth, and bentonite, for which standards would be established with revision for the eighth edition of the Japanese Standards of Food Additives. We also analyzed granite and green tuff by the same method. Furthermore, we performed the elution tests of 8 insoluble mineral substances by simultaneous analysis using ICP-MS (33 elements) according to the seventh edition of the Japanese Standards of Food Additives. As a result, although many elements were detected, the concentrations of As and Pb in all samples were below the talc standard levels. The concentrations of other elements eluted were also low, suggesting that the risks of these elements to human health are very low. Next we tried model experiments using beer samples, and found that no food-hygienically problematic metals were detected if the appropriate material, elemental composition, and elution type of insoluble mineral substances were used. It has been reported that the toxicity of arsenic compounds markedly vary with their chemical forms. Therefore, we developed a method for the analysis of organic arsenic compounds (monomethylarsonate, dimethylarsinic acid, trimethylarsinic oxide, and arsenobetaine) in wine and beer, for which diatomite containing a large amount of arsenic was used as a filter agent, and we analyzed the arsenic compounds with different chemical forms in wine and beer samples by this method. As a result, none of the 4 organic arsenic compounds were detected. Since the elution of arsenic from diatomite was suggested by the model experiments with winemaking processes scaled down from those in wineries, we evaluated the rinsing conditions of diatomite using organic acids, and found that the arsenic elution could be reduced using tartaric acid.

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Fundamental Safety Assessment of the Registered Existing Food Additives
(Natural Source Food Additives)

In 2005, the 450 items of existing food additives (natural source food additives) were registered by the Ministry of Health, Labor and Welfare. These additives were assessed the safety by existing research data and classified in following three categories.
1) The additives verified the safety by the several qualified scientific data: 247 items.
2) The additives recognized the verification would not be urgently executed from the nature of the source, process etc: 132 items.
3) The additives recognized the safety verification should be urgently executed, since the scientific results have not been sufficiently corrected: 71 items.
The fundamental safety assessment in this report was focused to the items classified to second category; the additives recognized the verification would not be urgently executed, since the original natural sources of the additives should be verified the safety properties as food materials and the components should not be contained any harmful　substances even these additives were classified as less necessity of the safety assessment.
The procedure of assessment was carried on the following properties of the additives.
1) The identification of the original natural sources （animal and plant) of the additives.
2) The survey of eating experience as food of the natural sources.
3) The investigation of the components included in the natural sources and the 　　　scientific safety data obtained from RTECS.
4) The survey on the commercial enzyme products.