Research Reports No.14 Abstract（2008）

14-01

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

Kanji Ishimaru

Faculty of Agriculture, Saga University

Green tea (Camellia sinensis) aqueous extracts were autoclaved and then fermented with three strains of fungi (Penicillium sp., Fusarium solani or Rosellinia necafrix) respectively. Gallate type catechins (epigallocatechin gallate, gallocatechin gallate, epicatechin gallate, catechin gallate) rapidly decreased at the first 7days of the fermentation. Non-gallate type catechins (gallocatechin, epigallocatechin, epicatechin and catechin) slightly increased during the first 7 days of the period, but they also gradually decreased in all the fermented extracts. Esterase activities originated from these fungi seemed to be strong during fermentation and they caused the rapid degradation of ester linkage of the gallic acid moiety of gallate type catechins. During the fermentation period of 35 days, the concentration of caffeine stayed at a high level (0.55- 0.75 mg/ml) in all of the extracts. Only in the extract treated with F. solani, rutin concentration increased up to 0.32 mg / ml on the 28th day of the fermentation period. From the tea extract treated with F. solani, blumenol B which has not been detected in various tea products was also isolated together with rutin.

14-02

Effect of polysaccharides as food additives on the mouse intestinal immunosystem.

Toshihiko Toida

Graduate School of Pharmaceutical Sciences, Chiba University

Alginate, an algal polysaccharide, is widely used in the food industry as a stabilizer, or as a thickening or emulsifying agent. As an indigestible polysaccharide, alginate may also be viewed as a source of dietary fiber. Previous work has suggested that dietary fibers may protect against the onset and continuation of a number of cardiovascular and gastrointestinal diseases. Recently, there have been many reports which describe the effects of alginate on anti cholesterol and anti hyper tension. Furthermore, it has been expected the effect of alginate on intestinal immune system. These biological functions of alginate are strongly related to its molecular weight. However, there is few chemical/enzymatic method to obtain alginate oligomers. Based on this background, we have developed a new photolytic degradation method for alginate.

14-03

Effect of addition of plant protein with protease inhibitory activity
on quality of Kamaboko

Masayuki Taniguchi

Department of Materials Science and Technology,
Faculty of Engineering, Niigata University

In manufacture of Kamaboko, several endogenous proteases autohydrolyse partially proteins in surimi (fish meat paste); the proteolysis by endogenous proteases in surimi causes Modori (softening) and therefore lowering of quality of Kamaboko. In this study, we examined prevention of the softening of Kamaboko using rice protein (oryzacystatin) with protease inhibitory activity.
Our previous study showed that polished rice contains rice protein (oryzacystatin) with protease inhibitory activity. SDS-PAGE analysis showed that autolysis of surimi (pollack) was affected by temperature, pH and NaCl concentration. By using a synthetic fluorescent substrate of protease, we found that the rice protein contains inhibitors toward papain like protease and that the extracts of surimi (pollack) showed papain like protease. On the basis of these results, we prepared model Kamaboko using surimi (pollack) as a starting material. When the rice protein was added to surimi paste with 0-4% NaCl, the breaking force and deformation of Kamaboko obtained were higher than those without the rice protein. Moreover, the whiteness of Kamoboko was improved by adding rice protein. Therefore, we clarified that the addition of the rice protein to surimi (pollack) was effective to prevent Kamaboko from Modori (softening) in the presence of NaCl. Further study on clarification of relationship between degree of inhibition of protease activity in surimi and physical properties of Kamaboko is necessary.

14-04

Structures and Safety of Metabolites of Anthraquinones and Their Related Natural Products

Hiroshi Morita

Faculty of Pharmaceutical Sciences, Hoshi University

Anthraquinones are organic compounds whose structures serve as a basic building block for a number of naturally occurring plant pigments. The compounds have been used in the production of dyes and as a catalyst for the production of wood pulp. Madder color is a coloring agent extracted from the roots of Rubia tinctorum, which has been allowed as one of the existing food additives in Japan until recently. However, many positive data have been reported in mutation tests for lucidin and its related anthraquinones. There are many positive data, but most of them are in vitro test system. Therefore, we are interested in the structures of their metabolites and their safety.
On the other hands, we have been isolated some peptides from Rubia species such as R. akane and R. cordifolia. Some of the compounds showed a potent cytotoxicity against various cell lines. Our interest has been focused on structures and safety of the methabolites of anthraquinones and naphthoquinones containing in Rubia tinctorum, and also concentrations of these peptides in various Rubia species. Many Anthraquinones such as lucidin and naphthoquinones such as mollugin have been isolated from the roots of Rubia tinctorum. On the other hand, distribution and quantitative variations of peptides in Rubia species such as R. akane, R. cordifolia, and R. tinctorum were investigated by means of HPLC with ODS as the stationary phase and MeCN-H2O as the mobile phase.
The structures and their metabolites, and also structure - mutagenicity relationships of their metabolites are under investigation.

14-05

Efficacy of enzymatically modified isoquercitrin on allergic diseases

Toshio Tanaka

Department of Respiratory Medicine, Allergy and Rheumatic Diseases,
Osaka University Graduate School of Medicine

We investigated the efficacy of enzymatically modified isoquercitrin (EMIQ), a flavonoid, to relieve symptoms of pollinosis. In a paralle1-group, double-blind placebo-controlled study design, 20 subjects with Japanese cedar pollinosis took two capsules daily of 100mg of EMIQ or placebo for 8 weeks during the pollen season. Subjective symptoms and ADL scores were recorded every day, and QOL score was obtained every 4 weeks. Blood sampling was performed before and after the study to measure serum cytokines, chemokines, IgE, quercetin and oxidized biomarkers. During the entire study period, total ocular score and ocular itching score for the EMIQ group was significantly lower (p=0.05) than for the placebo group. When limited to the period, total symptom score for the EMIQ group such as total nasal score (p=0.06, week 4-5), nasal obstruction score (p=0.08, week 4-5), 1acrimation score (p=0.06, week 5-6), ocular congestion score (p=0.08, week 4-7) and ADL score (p=0.08, week 4-7), all tended to be lower. The levels of serum cytokines such as interleukin (IL)-4, IL-5, IL-12, IL-13, interferon (IFN)-g, eotaxin and IgE were not significantly downregulated by the intake of EMIQ but the serum concentrations of oxidized LDL and TARC was reduced. Therefore, intake of the quercetin glycoside EMIQ proved to be safe and effective for the relief of ocular symptoms caused by Japanese cedar pollinosis.

14-06

Comparative Investigation on the Safety Assessment of Food Additives

International information concerned the safety assessment of food additives was continued to collect based on the findings of last year. The information was focused on the flavor substances among food additives since its chemical structures are identified, contained very low amount of ppm or ppb levels in general food, and unknown toxicity. In the U.S. and EU, the safety assessment of flavor substances has been performed by the different category from other food additives. Actually, the TTC concept for the safety assessment of flavor substances has been utilized not only in JECFA but also FEMA in the U.S. and EFSA in EU.
However, in Japan the safety assessment of flavor substance has been performed as the same category of other food additives, requiring the data of chemical structure, genotoxicity test, and repeated administration toxicity test in animals.
On the other hand, alternative methods for 2-year-bioasseay for carcinogenicity which were described in the last year report, the regulatory agencies do not accept as the alternatives but the results are evaluated as the identified the hazard. New proposals are raised that the identification of genotoxicity and the cell proliferation in target organs in 13-week animal experiment may contribute to the carcinogenicity of chemicals and the other one is that the existence of thresholds of genotoxic carcinogens. The existence of threshold has the potential to create the future problems for the safety assessment of chemicals. The NOAEL is important in drug development because it is used in the selection of the first close of a new medicine to be administered to either human volunteers or patients. However, the NOAEL is influenced by various factors and can be difficult to determine because it is based upon evaluation of data from the multiple parameters that make up a toxicology study protocol.
Nevertheless, the follow up studies on the progress of alternative methods and NOAEL are necessary and it is now the time to re-construct Japanese decision tree for flavor substances based on the different category from other food additives.

14-07

Improvement of constipation and fecal impaction for female students
by daily taking in the pickled vegetables fermented with Lactobacillus brevis
containing gamma-aminobutyric acid

Yukio Takii

Department of Food Science and Nutrition, School of Environmental Sciences, Mukogawa Women's University

Non-invasive treatments for constipation of healthy subjects (56 female university students: 18.3 average years of age) were conducted by daily taking in the pickled vegetables for 2 weeks. The subjects were pided equally in two groups for taking pickles containing gamma-aminobutyric acid after fermented with Lactobacillus brevis or non-fermented pickles as placebo. Not great differences in blood fatty acids were observed between two groups. However, the groups fed with fermented pickles had not atypical presentation of fecal impactions and frustrating fecal incontinence was attenuated.

14-08

Evaluation of safety and physiological functionality of natural dietary factors by analyzing their effects on immune-related functions of intestinal epithelial cells.

Mamoru Totsuka

Department of Applied Biological Chemistry, The University of Tokyo.

Intestinal epithelial cells (IEC) maintain an intestinal barrier function, and also play an important role in regulation of the intestinal immune system. In the present study, we aimed to evaluate the safety and physiological functionality of dietary factors by analyzing their effects on immune-related functions of intestinal epithelial cells. For this objective, we tried to establish a novel evaluation system using mouse small intestinal epithelial cell (sIEC) lines we have established recently. Moreover, we analyzed the effect of dietary factors on cytokine secretion from these sIEC lines and a human colon IEC line Caco-2.
In the normal condition, sIEC are known to express MHC class II molecules essential for antigen presentation to CD4+T cells, however, the characteristics of antigen presentation by sIEC are yet to be elucidated. When we stimulated CD4+ intestinal intraepithelial lymphocytes (CD4+IEL) derived from DO11.10 mice with a mouse sIEC line in the presence of the specific antigen, we found that CD4+IEL showed much higher interferon-g (IFN-g) secretion despite much lower proliferative response than when stimulated with antigen presenting cells prepared from spleen cells. This effect was not observed for CD4+T cells from other lymphoid tissues. Since IFN-g is important in the maintenance of intestinal homeostasis, this experimental system seems to be useful for evaluating the effect of dietary factors on intestinal immunity.
Through the screening of dietary factors for their ability to modulate cytokine secretion from IEC, we found that melatonin suppressed IL-6 secretion induced by the stimulation with bacterial components or proinflammatory cytokines. Melatonin down-regulated the IL-6 production at a mRNA level, but did not affect the secretion and mRNA expression of chemokines, such as IL-8 and MCP-1. These results demonstrated the novel function of melatonin in the intestine.

14-09

Studies on the gene screening for antioxidants: Interactions with drug metabolizing and antioxidant genes.

Tomoyuki Terada

Laboratory of Biochemistry, Faculty of Pharmacy, Osaka Ohtani University

Aldo-keto reductases (AKRs) are a large superfamily of proteins having an NADPH-dependent reductase activity toward various carbonyl compounds (aldehydes, ketones or quinones). AKRs have been identified from various sources including prokaryotes and eukaryotes. AKRs are classified into a number of class in the aspects of their structures and functions with systematic name, that is, AKR1A1 to 15A1. The antioxidative function of AKR through the reductions of carbonyl compounds which are typical prooxidants has been presented by many researchers. The regulations of these AKR gene expression have also been characterized focusing on a typical marker of the antioxidative response in the cells. In the present study, we have characterized the mechanism of the gene-regulation of AKR1B10, a typical AKR, in the non-small lung cancer cell lines (A549 or H23). The result of RT-PCR showed a significant expression in only A549 cell lines. The 5'-upstream of AKR1B10 gene has been isolated and identified the presence of polymorphism of CT-rich region. So, for the purpose to clarify this polymorphism on the regulation of AKR1B10 gene expression, 5'-upstream of AKR1B10 gene (about 3000 bp) were introduced into the upstream of luciferase gene to construct reporter-plasmid. Using this reporter-gene assay system, the effects of oxidant or antioxidant (H2O2 or ethoxyquin) on the regulation of AKR1B10 gene expression were investigated. The results of reporter-gene assay gave us the following two results. The first, the polymorphism of CT-ich region of AKR1B10 gene did not show significant effect on the response against oxidant or antioxidan. The second, Nrf2 was able to activate the expression of AKR1B10 gene. To evaluate the antioxidant-effectiveness of various compounds, both results of AKR1B10 and GSTP1 gene-regulation which was in the present and in the last year reports) showed that both genes can be a useful marker for antioxidant responses of the cells.

14-10

Stimulation of polyhalogenated aromatic hydrocarbons excretion induced by annato

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. Here, we examined a natural color additive, annatto, to enhance the excretion of storage PHAHs in male mice by using 3H-TCDD as a model PHAH.

14-11

Development of functional vegetables such as anti-oxidative vitamin high-containing green vegetables

Hirobumi Yamamoto, Koichiro Shimomura

Faculty of Life Sciences, Toyo University

For the supply of safe and tasty functional vegetables to consumers, we investigated relationships between culture conditions or preservations of green vegetables and their tastes as well as their functions. We found that "Komatsuna" (Brassica campestris L. var. Komatsuna Makino) grown with farmyard manure were more tasty and contained higher amount of maleic acid as the flavor substance than those grown with complex fertilizers. We also found that number of soil microbes in farmyard manure-using soils were larger than those in complex fertilizers-using ones, and the taste of Komatsuna grown with Pseudomonas fluorescens in soils containing complex fertilizers were improved, suggesting that the symbiosis of the plant with soil microbes is important for the good taste of the vegetables. We also demonstrated that the content of b-carotene in Komatsuna was increased by the physical stresses such as the osmotic pressure. In these conditions, not only the cell/tissue structures but also supply of glucose as the precursor were important for the stimulation of b-carotene production. Moreover, we showed that accumulation of oxalate in spinach (Spinacia oleracea L.) was positively correlated with the heredity, and breed varieties of spinach established in Japan accumulated lower amount of oxalate compared with those in Europe. On the other hand, nitrate anion accumulation in spinach was rather correlated with the environmental conditions such as the composition and the amount of fertilizers.

14-12

Studies on excretion of plant sterols through ABCG5 and ABCG8 in rats

Ikuo Ikeda and Masaki Kato

Garduate School of Agricultural Science, Tohoku University

We previously observed that stroke prone spontaneously hypertensive rats (SHRSP/Izm) and WKY rats (WKY/NCrlCrlj) deposited more plant sterols in the body than Wistar rats. We then showed that the former two strains had a same mutation on ATP binding cassette transporter G5 (ABCG5). It has been suggested that ABCG5 and ATP binding cassette transpoter G8 (ABCG8) form a heterodimer and excrete plant sterols and cholesterol from intestinal cells to the intestinal lumen and from the liver to the bile. It has been thought that mutations of ABCG5 or ABCG8 impair their function and induce deposition of plant sterols in the body. In order to reveal the function of ABCG5 and ABCG8, we compared intestinal absorption and biliary excretion of plant sterols in SHRSP/Izm with those in Wistar rats. Lymphatic recoveries of campesterol and sitosterol were almost the same between SHRSP and Wistar rats. Administration of a liver X receptor (LXR) agonist, in which expression of ABCG5/ABCG8 is accelerated, significantly reduced lymphatic absorption of these plant sterols at the same extent in SHRSP and Wistar rats. These observations suggest that a mutation of ABCG5 in SHRSP does not influence lymphatic transport of plant sterols. Biliary excretion of plant sterols in SHRSP was not lower than that in Wistar rats. Excretion efficiency, in which biliary excretion of plant sterols was pided by their hepatic deposition, was lower in SHRSP than in Wistar rats, when LXR agonist was given. The observation suggests that function of ABCG5/ABCG8 is impaired in SHRSP. In either case, we could not show causes of deposition of plant sterols in SHRSP. Biliary excretion and deposition of plant sterols in WKY/Izm rats was almost the same as that in Wistar rats, but not in SHRSP. We revealed that WKY/Izm, unlike WKY/NCrl/Crlj, has no mutation in ABCG5.

14-13

Basic Study of Dissolution Behavior of Silicates in Liquid Food

Teruhisa Fujimaki

Chemistry pision, Kanagawa Prefectural Institute of Public Health

Ensuring food safety is an issue shared by all countries in the world. Particularly in Japan that has a low food self-sufficiency rate, there is a high level of interest in the safety of imported food. Many food additives are still not allowed to be used in Japan despite the fact that their safety is universally established and that they are in wide use worldwide. In Japan, reviews of the safety of undesignated additives are under way. One example of such additives is silicates. In order to conform to international standards, the Food Safety Commission is evaluating the health effects of silicates and is considering designating silicates as a safe additive. In its evaluation of silicates, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) has not specified any allowable daily intake (ADI) for silicates because they are insoluble in water.

In Europe and North America, silicates are mainly used as an anti-caking agent in table salt and substitute salts. For this reason, it can be assumed that salts and other substances using silicates as anti-caking agent are extensively utilized in cooking, together with various food ingredients, and that they are mixed into various liquid foods, such as table vinegar.

In this study, the author used food-simulating solvents and other agents to study the dissolution behavior of various elements contained in silicates (sodium aluminosilicate, calcium silicate, and magnesium silicate) and insoluble minerals (activated white clay, granite porphyry, and diatomite) that have already been permitted to be used as food additive. This study is different from the previous one in that some of the silicates used in this study meet the standards indicated in the fifth edition of the U.S. Food Chemicals Codex and that in addition to food-simulating solvents, artificial gastric juice was used as a dissolution solvent.

14-14

Chemical Analyses of the volatile components
from lavender (Lavandula angustifolia Mill) flowering tops

Five samples of lavender (Lavandula angustifolia Mill) including four different varieties were collected in Hokkaido Furano area at five different sites. The flowering tops of the samples were soaked into dichloromethane and the extracts were analyzed using GC/MS. Fifty four peaks were observed and 45 of them were identified or their reasonable structures were estimated. Most abundant components common to the all samples were linalyl acetate (1), linalool (2), lavandulyl acetate (3), coumarin (4), (E)-beta-farnesene (5), and 7-methyl coumarin (6). Statistical analyses (a clustering analysis, a principal component analysis) were performed on the abundance of the 54 components. The results were as follows: (Ⅰ) chemical profiles of the samples which belong to the same variety collected at the same site were essentially the same regardless their collecting time of the day, (Ⅱ) the different varieties of lavender showed different chemical profiles.

14-15

Inhibitory effect of natural food additives on cytochrome P-450 enzymes

Hideyuki Ito

pision of Pharmaceutical Sciences, Okayama University Graduate School of Medicines, Dentistry and Pharmaceutical Sciences

Interactions between food components and drugs have recently come into focus of interest. The interactions often occur as a result of inhibiting or inducing of the metabolic enzymes, cytochrome P450 (CYP). It is well known that concomitant intake with grapefruit juice increases the concentration of calcium antagonists in human by suppression of CYP3A4 activation. We investigated the inhibitory effects of 30 natural food additives on CYP3A4 or CYP2C9 activities. In vitro inhibitory effects on the metabolism mediated by CYP3A4 or CYP2C9 were determined on the basis of the hydroxylation of testosterone or diclofenac as substrates, respectively, using reversed-phase HPLC analysis. Among tested additives, Turmeric oleoresin and Kaoliang color exhibited potent inhibition on CYP3A4 and CYP2C9 activations. Tamarind and Monascus colors showed moderate inhibitory effects on CYP2C9. The inhibitory potencies of Palm oil and Marigold color on CYP3A4 were comparable to those of Turmeric oleoresin and Kaoliang color, respectively. These results suggested that constituents of a part of natural food additives may have the potential to inhibit the metabolism of drugs, so that further study should be investigated to clarify the clinical relevance.

14-16

Effect of guarana extract on endothelial cells and prostate cancer cell lines and on absorption kinetics of digestive nutrients

Shigeyuki Usui and Kazuyuki Hirano

Department of Clinical Pharmaceutics, Gifu Pharmaceutical University

We assessed effectiveness of guarana extract on human umbilical vein endothelial cells (HUVEC), human prostate cancer cell lines (LNCap, PC-3), and human colon cancer cell line (Caco-2). Cell proliferation of HUVEC, LNCaP, and PC-3 was suppressed depending on the concentration of guarana extract. LNCaP and PC-3 had a tendency to be more sensitive to the treatment with guarana extract on the proliferation than HUVEC. Results from the observations of nucleosomal DNA fragmentation and perinuclear condensation of chromatin in HUVEC suggest that guarana extract induce apoptosis. Caspase 3 was observed to activate in HUVEC during apoptosis induced by guarana extract. Garana extract was found to inhibit angiogenesis of HUVEC in vitro. The expression of aquaporin 3 (AQP3) was suppressed on the treatment with guarana extract. These observations suggest the possibility of new usage of guarana extract.

14-17

Biodynamics of Quercetin Glycosides and Their Participation in Oxidative Stress Control.

Junji Terao, Kaeko Murota and Yoshichika Kawai

Institute for Health Biosciences, the University of Tokushima Graduate School

Quercetin is a major flavonol-type flavonoid distributed in a wide variety of vegetables including onion. Numerous in vitro studies have revealed their perse biological effects, and antioxidant activity should, at least partly, be responsible for these effects. Qurcetin is commonly present as its glycoside form and converted into glucuronide/sulfate conjugates by phase-II enzymes during intestinal absorption. This is a detoxification process for xenobiotics to eliminate its toxicity. We found that these metabolites circulating in the human blood stream were mostly localized in plasma albumin fraction, but not LDL fraction. Onion consumption failed to enhance the antioxidant activity of albumin fraction against oxidized LDL formation, indicating that the level of quercetin metabolites bound to albumin is insufficient to exert their antioxidant effect in vivo. In contrast, we demonstrated that quercetin metabolites accumulated in human atherosclerotic lesions, but not in the normal aorta. An in vitro study suggests that quercetin metabolites are capable of suppressing foam cell accumulation by inhibiting the expression of scavenger receptors for oxidized LDL in macrophages. This inhibition may be involved in the mechansim for the prevention of cardiovascular diseases by the intake of quercetin-rich diet.

14-18

Effects of Some Polyphenols on the Formation of Free Radicals Formed in the Reaction Mixture of Rat Liver Microsomes/NADPH/ADP/Iron Ions

Hideo Iwahashi

Wakayama Medica1 University, School of Medicine

The reaction of rat liver microsomes with Fe3+, ADP and NADPH was examined using
EPR, HPLC/EPR and HPLC/EPR/MS combined use of spin trapping technique. A prominent EPR spectrum (aN=1.581mT and aHb=0.26mT) was observed in the complete reaction mixture. The EPR spectrum was hardly observed for the complete reaction mixture without rat liver microsomes. The radicals appear to be derived from microsomal components. The EPR spectrum was also hardly observed in the absence of Fe3+. Addition of some iron chelators such as EDTA, citrate and ADP resulted in the dramatic change in the EPR intensity. Iron ions seem to be essential for this reaction. For the complete reaction mixture with boiled microsomes, a weak EPR spectrum was observed, suggesting that enzymes participate in the reaction.
Five peaks were separated on the HPLC/EPR elution profile of the complete reaction mixture of rat liver microsomes with ADP, Fe3+ and NADPH. The retention times of the peaks 1 to 5 were 19.4, 22.5, 27.3, 29.8 and 31.4 min, respectively. To identify the radical adducts, HPLC/EPR/MS analyses were performed for the three prominent peaks. The HPLC/EPR/MS analyses showed that a new radical adduct, 4-POBN/hydroxypentyl radical, in addition to 4-POBN/ethyl radical adducts, forms in a reaction mixture of rat liver microsomes with ADP, Fe3+ and NADPH.
Addition of caffeic acid to the complete reaction mixture resulted in decrease of EPR signal intensity, suggesting that caffeic acid inhibits the formation of radical in the reaction of rat liver microsomes with Fe3+, ADP and NADPH.

14-19

Status of cinnamic acid derivatives contained in foodstuff in vivo and its meaning

Kiharu Igarashi

Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University

Preventive effect of clovamide (caffeoyldopa) with acid-amide bonding (peptide bonding) and contained in cacao beans and coffee beans　or of its components, on carbon tetrachloride-induced liver injury in mice were determined. Increases in the serum ALT and AST activities by intraperitoneal injection of carbon tetrachloride were mitigated by the oral administration of clovamide and caffeic acid at 30 min before the injection of the carbon tetrachloride. Serum collected from mice administered clovamide and caffeic acid contained compounds showed similar UV spectra resemble with clovamide and caffeic acid as well as those compounds when analyzed HPLC with photodiode array detector, indicating that clovamide and caffeic acid absorbed from gastro intestinal tract and their metabolites acted to prevent liver injury induced by carbon tetrachloride. Increase in the activities of ALT and AST released from rat primary culture hepatocytes exposed to carbon tetrachloride, to the culture medium tended to be mitigated by pretreatment of the cells with clovamide and it's a component, caffeic acid, but not by DOPA, suggesting that caffeic acid moiety of clovamide is important to demonstrate of effect of clovamide.

14-20

Pomegranate fruit juice suppressed development of prostate cancers

Makoto Asamoto

Department of Experimental Pathology and Tumor Biology,
Nagoya City University Graduate School of Medical Sciences

Prostate cancer is the common malignancy in the world. However incidence of the disease is much difference between Western and Asia countries. It is well known fact that food habit effects the incidences of malignancy. To prevent the disease, the use of natural foods or agents present in diets is one of essential approaches. Recently, chemoprevention effects in several malignant diseases of pomegranate fruit juice (PFJ) was reported. The present study was undertaken to evaluate the effects of PJF on androgen dependent prostate carcinogenesis in the transgenic rats developing adenocarcinoma of the prostate (TRAP) model. We found that oral administration of PFJ in TRAP rats inhibited development of prostate cancers. To cast light on new mechanisms underlying the effects of PFJ on prostate carcinogenesis, we conducted an in vitro experiment. Treatment of LNCaP prostate cancer cells with 5% PFJ resulted in inhibition of viability, along with restoration of connexin 43 (Cx43) expression and cleaved caspase-3 induction. Furthermore, inhibition of Cx43 protein induction by PFJ with siRNA suppressed apoptosis identified with active caspase 3 induction. From these in vivo and in vitro results, PFJ may have chemopreventive activity in TRAP rats and LNCap cells, associated with induction of Cx43 expression and apoptosis.

14-21

Modulating effects of food lectins on the transport of natural food colors in human intestinal Caco-2 cell monolayers

Koji Muramoto

Department of Biomolecular Sciences, Graduate school of Life sciences,
Tohoku University

The effect of 16 lectins isolated from food stuffs on the transport pathways across human intestinal Caco-2 cell monolayers was investigated by using three model color compounds: lucifer yellow (LY) for the paracellular pathway, fluorescein (FL) for the monocarboxylic acid transporter-mediated pathway, and rhodamine 123 (RH) for the p-glycoprotein-mediated efflux pathway. We also examined the effect of lectins on the paracellular pathway by measuring the transepitherial electrical resistance (TER) value of Caco-2 cell monolayers. Lectins affected the transepithelial transport pathways in different manners, probably due to their specific binding affinity against Caco-2 cell monolayers. Proteomic analysis by two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry was conducted to explore a molecular basis for the effects of three lectins, SBA from soybean, WGA from wheat germ, and CGA from Japanese jack bean, on the transport pathways in Caco-2 cells. Nineteen protein spots were detected with differential expression by lectin treatments, and many of them were identified to be chaperon proteins or cytoskeleton related proteins.

14-22

Palliative effect of drinking on mental stress after solving numerical puzzle.

Nobuyuki Sakai

Department of Human Sciences, Kobe Shoin Women's University

Preceding studies adopted Uchida-Kraepelin Test or some other vision-guiding computer task for studying mental stress. However, these tasks require not only the cognitive (mental) task but also the physical movements and the participants' vigor, thus participants reported exhaustion and depression after finishing these tasks. In this study, a new delightful and convenient mental-stress-task was explored. After confirming the task's effect on mental stress, the methods that ease mental stress were explored. The author found that the SUDOKU puzzle is easy and participants-friendly mental-stress-task, that the sympathetic nervous system was activated and the negative emotions were expressed after solving three SUDOKU puzzles in 30 minutes, and that the stress caused by the task was weakened by drinking a glass of water or coffee.

14-23

Improvement usability and storage stability of oily functional food and supplement by powderization and tabletting

Yoshiaki Kawashima, Hiromitsu Yamamoto, Kohei Tahara

School of Pharmacy, Aichi Gakuin University

Supplemental foods have been paid to attention for preventing the disease and for keeping health resulting in reducing the medical expenses. Among such supplemental foods, there are some oily materials, like Vitamin E, DHA and so on. For such materials, it is very difficult neither to be formulated into dosage form such as tablet nor to be dispensed. We have developed the preparation method to powderize oily materials for tabletting and to improve the stability during storage.
Florite is a useful adsorbent for Vitamin E and other oily materials to be powdered form due to their pores structure on the surface. It was found that during tabletting resultant powdered oily materials with adsorbent, capping and lamination occurred. Whereas, Vitamin E tablet of granules prepared by dry granulation with a roller compactor was found to be sufficiently strong mechanically.
Furthermore, the stability of vitamin E in the tablet improved compared to vitamin E powder. The powder coated with the polymer on the surface can reduce degradation of Vitamin E significantly by reducing the diffusion of oxygen into particle.
It was found the present method was successfully enable to prepare the tablet with oily materials improved uniformity and stability.

14-24

Fundamental Study for Safety Evaluation of Food Additives with Optical Activities

Food additives include optically active compounds, such as amino acids, sugars, and organic acids. Some optical isomers of the optically active compounds exert clearly different effects on the body. However, the present component standards of food additives do not include tests for the accurate determination of the ratios of optical isomers (optical purity). Hence, an attempt was made to develop test methods for the accurate determination of optical purity.
1. Optical isomer analysis for amino acids
A sensitive and selective method for the determination of amino acids in foods such as the fermentation foods and soft drink has been developed by liquid chromatography-mass spectrometry (LC/MS). The LC separation was performed on a Chirobiotic TAG column (2.1 mm x 25 cm) with 0.05% acetic acid-ethanol (60:40) as the mobile phase. This analytical method was applied to fermentation foods for 20 kinds of amino acids measurement. This method is useful as an optical isomer analysis for amino acids in food.
2. An analysis of racemic amino acid food additives utilizing high performance liquid chromatography with ultraviolet detection
Amino acids are authorized food additives commonly used as seasonings, dietary supplements and quality-retaining agents. We have developed a chromatographic method of analyzing racemic acids using HPLC with UV detection. The HPLC conditions were as follows: column 1, SUMICHIRAL OA-5000 (particle type, 4.6 mm i.d.×150 mm) : column 2, OA-5000 (monolithic type, 4.6 mm i.d.×100 mm) : mobile phase A, 0.5 mM～2 mM CuSO4 solution : mobile phase B, 2 mM CuSO4 in water and isopropyl alcohol solution : flow rate, 0.8 mL/min or 1.0 mL/min : detection, UV 254 nm.
We separated each racemate of Asn, Cystine and Ser by using the combination of particle type column and monolithic type column. The other racemates were separated by using particle type column alone. The limits of detection were under 1 ■ochg/mL.
3. Determination of the Amount of Racemic 疝och-Amino Acids in Beverages by Chiral Ligand Exchange Chromatography
A simple and efficient method has been developed for determining of the amount of racemic 疝och-amino acids in beverages by chiral ligand exchange chromatography with ultraviolet detection. The chromatographic resolution of racemic 疝och-amino acids was performed on a reverse-phase octadecyl silica gel column coated with, S-dioctyl-D-penicillamine, SUMICHIRAL OA-5000 (particle-type), using cupric sulfate as a mobile phase additive. A UV absorbance detector was set to 254 nm. The elution orders were fairly consistent, and the D-enantiomers were retained for a time longer than the L-enantiomers on the column. The calibration graphs were linear in the range of 5-100 mg/mL. The recovery of the compounds added to the beverages at the 50 mg/mL level was >98%, and the limit of quantification values was found to be 5 mg/mL for the nine D-amino acids. Eight commercial beverages were analyzed by the proposed method. Although D-Val was detected in a sample, by using LC-MS, it was proved that the peak was not due to D-Val.
4. Chiral analysis for proline (Determination of praline in fermentation products by liquid chromatography-mass spectrometry)
As for proline, which is optically active, only the L-isomer is permitted as a food additive, whereas the D-isomer has been reported to possess neurotoxicity. Therefore, it is necessary to confirm whether the D-isomer is present in order to secure food safety.
In this study, a sensitive and selective method using liquid chromatography-mass spectrometry (LC/MS) for the determination of proline and hydroxyproline in the fermentation food has been developed. The LC separation was performed on a chiral CYCLOBONDⅠ2000RN (Astec Corp.) column.
This analytical method was applied to fermentation products for proline and hydroxyproline measurement to detect the D-isomer at a concentration of a few percent in several samples.

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Fundamental Safety Assessment of the Registered Existing Food Additives (Natural Source Food Additives)
（From Butyrospermum parkii KOTSCHY. to Hevea brasiliensis MUELL.-ARG.）

From 1995 the natural source food additives can not be used without the permission of Ministry of Health, Labor and Welfare although the previously listed food additives have been continuously supplied. Among them the 450 items of food additives previously used were classified in the following three categories (March 2005).
(1) Food additives of 247 items have been confirmed their fundamental safety by the international evaluation of FAO/WHO Joint Expert Committee on Food Additives (JECFA) and/or can be evaluated as safety by the experimental data.
(2) The safety of food additives 132 items are recognized to be not considered urgently depending on their resources, processing and natures.
(3) The additives of 71 items are recommended to be immediately and effectively confirmed their safety cause of few scientific information and data regarding their safety.
This project carries out the fundamental investigating researches about the above categories (3) [the registered additives which need urgent evaluation of safety] and (1) [the registered additives confirmed their basic safety]. In order to evaluate the safety of registered food additives, the fundamental investigating researches depending upon the reliability of animal and plant resources, the historical survey of human consumption, the non-injurious resources obtained from animal and/or plant, and the injurious chemical components have been carried out and furthermore the standardized drafts of animal and plant resources, and registered food additives have been prepared except minerals and enzymes. The food additives included in (1) and (3) were categorized as except the registered food additives without urgent analysis in this project.