Research Reports No.11 Abstract（2005）

11-01

Miroencapsulation of PH Sensitive Dye and Application to Sense System of Rottenness and Deterioration of Food

Masato Tanaka
Department of Chemical Engineering, Niigata University

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Microencapsulation of the PH-sensitive dye was investigated in order to apply these microcapsules to the sense system of the rottenness and deterioration of food.
As the material of the microcapsule shell,methylcellulose (MC) and polyvinyl alcohol (PVA) were used.
It was investigated how the operating conditions such as the mixture ratio of MC to PVA and the concentrations of gelation agents affected the functionality of microcapsules.
It was found that microcapsules prepared changed in color according to the PH change.

11-02

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

Yoshiyuki Mizushina
Laboratory of Food & Nutritional Sciences, Department or Nutritiona1 Science,
Kobe-Gakuin University

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Petasipheno1, a bio-antimutagen isolated from a Japanese vegetable, Petasites japanicus, selectively inhibits the activities of mammalian DNA polymerase l (pol l) in vitro. We found here that another phenolic compound, curcumin (diferuloylmethane), which is known as bioactive food material and is structurally quite similar to petasiphenol, was also a potent pol l inhibitor. Curcumin did not influence the activities of replicative pols such as pol a, d and e, but also showed no effect even on the pol b activity, the three-dimensional structure of which is thought to be highly similar to po1 l. The inhibitory effect of curcumin on intact pol l including the BRCA1 C-terminus (BRCT) domain was dose-dependent, and 50 ％ inhibition was observed at a concentration of 7.0 mM. The curcumin-induced inhibition of the pol l activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. Curcumin did not only inhibit the activity or the truncated pol l including the pol b-like core, in which the BRCT motif was deleted in its N-terminal region. BIAcore analysis demonstrated that petasiphenol bound selectively to the N-terminal domain of po1 l, but did not bind to the C-terminal region. Based on these results, the pol l-inhibitory mechanism of curcumin is discussed.

11-03

Developmednt of an animal model of rapid induction of tongue neoplasms
in human c-Ha-ras proto-oncogene transgenic rats by 4-nitroquinoline 1-oxide:
potential use for preclinical chemoprevention study

Takuji Tanaka
Department of Oncologic Pathology, Kanazawa Medical University

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We developed an animal model using the human c-Ha-ras proto-oncogene-carrying transgenic (Tg) rats and the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in the drinking water to produce tongue carcinomas. 4-NQO (20 ppm) was given for 8 weeks by drinking water to Tg and non-Tg rats. During the experimental period of 22 weeks, tongue carcinogenesis was compared by the incidence and multiplicity of preneoplasm and neoplasm. Also, we examined the immunohistochemical expression of some of the biomarkers associated with the process of oral carcinogenesis, such as cyclin D1, glutathione S-transferase placental form, cyclooxygenase (COX)-2, inducible nitric oxide synthase, and -catenin. In addition, using Tg rats, the cancer chemopreventive effects of a specific COX-2 inhibitor, a ligand for peroxisome proliferator-activated receptor (PPAR), and a synthetic geranylated derivative that were reported to be inhibitors of tongue carcinogenesis in rats were examined in Tg rats treated with 4-NQO. During or after treatment with 4-NQO in the drinking water, tongue dysplasia and tumors were observed on the tongues of both Tg and non-Tg rats, with greater incidence and multiplicity in Tg rats. Histopathologically, squamous cell dysplasia, papilloma, and carcinoma with or without invasion were present in the tongue. Immunohistochemistry revealed that expression levels against five biomarkers increase with disease progression. Subsequent chemoprevention study using Tg rats showed that all three different chemicals suppressed the occurrence of tongue carcinomas when fed after 4-NQO-exposure. These results may indicate that our 4-NQO-induced Tg rat tongue carcinogenesis model simulates many aspects of human oral carcinogenesis and can be applied for analysis of oral cancer development and for search candidate cancer chemopreventive agents against oral cancer within 20 weeks.

11-04

Inhibition of hUGT1A6 activity by Xanthene food dyes

Takaharu Mizutani
Department of Drug Metabolism and Disposition,
Graduate School of Pharmaceutical Sciences, Nagoya City University

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Synthetic food dyes are used for coloring of foods, pills, and cosmetics. We previously studied the influence of synthetic food dyes on the activities of UGT1A6 and CYP2A6. Erythrosine (ET) among those dyes inhibited UGT1A6 activity. ET is one of Xanthene dyes and has some halogens on its. Thus, we studied the influence of other Xanthene dyes, such as Acid Red (AR), Rose Bengal (RB) and Phloxine (PL), and some drugs containing high halogens such as Ioxaglic acid, Iodixanol, Iotalamate Meglumine, and Diatrizoate sodium. ET, PL, and RB made of Xanthene with halogens well inhibited UGT1A6 activity at IC50 value＝50 ■ochM, 40■ochM, and 15■ochM, respectively. AR of Xanthene without halogen did not inhibit the activity. Thus, these results suggest that the inhibition might be dependent upon Xanthene structure and halogens on it.

11-05

Food chemical Studies on polyphenols of seed coat of adzuki bean, Vigna angularis.

Kumi Yoshida
Graduate School of Information Science, Nagoya University

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The polyphenol composition of the water extract of the seed coats of the red adzuki bean, Vigna angularis, was analyzed. 3D-detection HPLC analysis revealed that the water extract contains several catechin derivatives, procyanidins and glycosylflavonols. Major components were isolated by various chromatographies and the structure was determined by MS spectrometry and 1D and 2D-NMR experiments. We clarified the existence of (+)-catechin, 7-O-b-D-glucopyranosyl-catechin, 2-O-((E)-p-coumaroyl)-meso-tartaric acid and its (Z)-p-coumaroyl isomer, 3-O-((6-O-a-L-rhamnopyranosyl)-b-D-galactopyranosyl)-7-O-a-L-rhamnopyranosylquercetin, 3-O-((6-O-a-L-rhamnopyranosyl)-b-D-glucopyranosyl)-7-O-a-L-rhamnopyranosylquercetin, 3-O-((6-O-a-L-rhamnopyranosyl)-b-D-galactopyranosyl) quercetin, 3-O-((6-O-a-L-rhamnopyranosyl)-b-D-glucopyranosyl)quercetin, procyanidin B1 (epicatechin-4-b-8-catechin), and epicatechin-4-b-8-epicatechin-4-b-8-catechin.

11-06

Studies for physiological functionality of anthocyanins using DNA microarray analysis

Takanori Tsuda
Office for Research Initiatives and Development, Doshisha University

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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 first time the gene expression profile in human adipocytes treated with anthocyanins (cyanidin 3-glucoside; C3G or cyanidin; Cy). The human adipocytes were treated with 100 M 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 lipid metabolism related genes by the treatment of adipocytes with C3G or Cy. These data have provided an overview of the gene expression profiles in adipocytes treated with anthocyanins and identified new responsive genes with potentially important functions in adipocytes related with obesity and diabetes that merit further investigation.

11-07

Development of the sensitive and high-throughput analysis of metabolite by FT-ICR MS
and its application to evaluate effects of food ingredients.

Shigeo Takenaka
Department of Veterinary Science, Graduate School of Life and Environmental Sciences,
Osaka Prefecture University

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Metabolic profiling is an emerging discipline under system biology. The goal of metabolomics in toxicological assessment of food ingredient is to know the changes of metabolites induced by them and find the surrogate markers to assess its toxicity. Here we examined the availability of urinary metabolic profiling using FT-ICR MS for toxicological assessment in amiodarone-induced phospholipidosis (PLD) rat as a model.
Spectra of the amiodarone-treated and non-treated urine were obtained and the resulting profiles were examined by multivariate data analysis. The urinary metabolite profiles of the drug-treated were separated from those of the control on the PCA plot. A number of potential markers were observed and some were identical to the markers reported in 1H-NMR based metabonomics analysis. These results indicated that FT-ICR MS based metabolic fingerprinting can be applicable for toxicological assessment of food ingredient.

11-08

Breeding study for increasing anthocyanin content of red radish

Koji Tsuji
Graduate School of Pharmaceutical Sciences, Osaka University

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In spite of characteristic properties of anthocyanins derived from red radishes, breeding studies of red radishes aiming at increase in anthocyanin content were little conducted. This circumstance prompted us to examine gamma ray irradiation against seeds and crossing between inpiduals with high anthocyanin content selected by HPLC quantitation in the first instance. In the selection of inpiduals with high anthocyanin content, aliquots of roots cut off from center to surface were subjected to HPLC quantitation because of disproportion for distribution of anthocyanins in roots. Consequently, both gamma ray irradiation by 200 Gy and crossing between inpiduals with high anthocyanin content were revealed to bring about increase in anthocyanin content.

11-09

Development of useful food additives by chemical modification
and enzymatically combining oligosaccharides to flavons

Taku Chiba
Department of Molecular Health Science,
Graduate School of Pharmaceutical Science, Nagoya City University

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In order to increase solubility to water, D-glucose or maltose was combined with an aglycone of flavons by chemical modification and coupling. Elongation of the sugar part of the aglycone was enzymatically effected with cyclodextrin glucanotransferase and dextrin. As a model compound, quercetin was usud and synthetic route was established.

11-10

Effects of the Complex of Tea Catechins and Soybean Protein on Lipid Metabolism in Rats

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The effects of the addition on the complex of tea catechins and soybean protein (the complex), a new food material, on lipid metabolism in the plasma and liver of rats with or without cholesterol diets were studied. Body weight gain, food intake, food efficiency, liver weight, and lipid concentrations were not influenced by the addition of the complex in rats without cholesterol diets. In rats with cholesterol diets, the complex also showed no remarkable influence on growth factors, liver weight, or phospholipid levels, and unfavorable symptoms were not observed at the ingestion of the complex. The addition of soybean protein decreased plasma triglyceride levels, but the addition of the complex decreased these levels more than did soybean protein. The tea catechin and soybean protein in the complex would collaboratively contribute to decrease the plasma and liver triglyceride levels in rats with cholesterol diets. The complex also decreased total plasma cholesterol levels but showed no effect on the liver. Furthermore the complex suppressed thiobarbituric acid reactive substances (TBARS) levels in the plasma and liver. Thus, some part of the tea catechin, especially (-)-epigallocatechin 3-O-gallate (EGCG) and (-)-epicatechin 3-O-gallate (ECG), in the complex was metabolized and showed antioxidant activity. In addition, the complex showed no significant effect on the fecal excretion of neutral steroids and bile acid. These results indicate that accumulation of plasma triglyceride, cholesterol, and TBARS, and liver triglyceride and TBARS are prevented and amended by the ingestion of the complex under a cholesterol-loading diet.

11-11

Changes of intestinal flora by chemicals: PCR-DGGE based approach

Norio ITOH
Department of Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University

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Host-intestinal flora interaction alters absorption, metabolism, and toxicity of chemicals. Major part of intestinal flora is composed bacteria which are uncultureable or difficult to culture in vitro under one defined condition. Analytical methods to detect uncultureable bacteria by 16S rDNA-based RFLP, FISH and DGGE were developed and already applied in the field of environmental microbiology. We have tried to apply PCR-DGGE to analyze the change of intestinal flora and have newly developed a statistic test to evaluate PCR-DGGE images. Various fungicides were evaluated by the newly developed methods. Risk assessment was performed under our experimental conditions.

11-12

Molecular authentication of Myricae Cortex based on
the nucleotide sequence of a chloroplast trnK gene

Hajime Mizukami
Graduate School of Pharmaceutical Sciences, Nagoya City University

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Fruits, leaves and bark of Myrica rubra (Myricaceae) are used to prepare 斗ochMyrica Extract派ich, a natural food additive. The dried bark of M. rubra has also been used as a crude drug, together with baurks of M. esculenta and M. adenophora. To establish an efficient protocol to identify and /or discriminate the dried bark materials of these three species, the chloroplast trnK gene encoding tRNALys(UUU) was amplified by PCR from fresh leaves of M. rubra as well as the dried burk specimens, and sequenced. Based on the aligned sequences thus obtained, we could authenticate the dried bark materials originating from M. rubra, M. esculenta and M. adenophora.

11-13

Exploitation of antibiotic browning-inhibitors from marine algae

Tadahiko Kajiwara
Department of Biological Chemistry, Faculty of Agriculture,
Yamaguchi University

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An oxo-C9-acid, 9-oxo-nonadienoic acid, which will be formed during the formation of C6 and C9-aldehydes such as (3Z)-hexenal, (2E)-hexenal and (2E)-nonenal from C18- and C20-polunsaturated fatty acids in marine algae, and a methanol extract from Hijiki fusiform, showed strong antifungal activities against Botorytis cinerea. Noticeable activities against gram-negative and gram-positive bacteria such as Escherichia coli TG-1, Erwinia carotovora , and Staphylococcus aureus were not observed in the both. Antimicrobial activities of essential oils from fresh fronds including green, brown and red algae such as Ulva pertusa, Sargassum thubergii, Laminaria japonica, Dictyopteris prolifera, and Undaria pinnatifida by using SDE (simultaneous distillation and extraction) apparatus were much weaker than those of hexenals and nonenals.

11-14

Effects of food ingredients on lipid metabolism through the activities of nuclear receptors

Ryuichiro Sato
Department of Applied Biological Chemistry,
Graduate School of Agricultural and Life Sciences, The University of Tokyo

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In this study we examined the ligand activity of food ingredients to several nuclear receptors including FXR, LXR, PPAR, RXR and HNF-4, which are deeply involved in regulation of lipid metabolism. For this purpose we established a new assay system to evaluate the ligand activity using 96-well plates. The ligand-binding domain of each nuclear receptor was fused to GST, and this fusion protein was expressed in E. coli and purified. The coactivator protein fused to alkaline phosphatase was also prepared. This system takes advantage of the recruitment of the coactivator protein to the ligand-binding domain in the presence of the ligand. Using this assay system we examined the ligand-binding activity of several isoflavones to these nuclear receptors. Some isoflavones were found to be able to bind to LXR weekly. By using other nuclear receptor ligand-binding domains this new assay will enable us to reveal novel functions of food ingredients.

11-15

Effects of Erythorbate Sodium and Sodium Nitrite on the Vascular Endothelial Functions:
A Study on the Safety Evaluation Methods for Food Additives.

Hiroshi Okamoto and Katsutoshi Yayama
Department of Pharmacology, Faculty of Pharmaceutical Sciences,
Kobe Gakuin University

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It is well-documented that nitric oxide (NO) from the vascular endothelium is believed to be implicated in the development and progression of atherosclerosis and hypertension, and the impaired endothelial function has been demonstrated in vessels exposed to the oxidative stress, such as hypercholesterolemia and atherosclerosis. The present studies have been undertaken to develop novel methods targeting the vascular endothelial functions for the safety evaluation of food additives. We studied two food additives, erythorbate sodium and sodium nitrite, because erythorbate sodium is one of potent antioxidatives, and sodium nitrite is a product of the endothelium-derived relaxation factor, NO. Male ddY mice received erythorbate sodium or sodium nitrite at a dose of 500 mg/kg/day or 20 mg/kg/day corresponding to 100-times higher dose of ADI, respectively, via drinking water for 4-weeks. Systolic blood pressures (SBP) measured by the tail-cuff method were not significantly different in mice received either additives from mice without receiving additives. Relaxation response to acetylcholine of the aortic rings prepared from thoracic aortas was not significantly different between mice with and without receiving additives. Administration of these additives for 4-weeks to mice having hypertension which had been induced by administration of a NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), did not influence the elevated SBP in L-NAME-treated mice. Acetylcholine-induced relaxation was significantly decreased in aortic rings from L-NAME-treated mice, and this decreased endothelium-dependent relaxation was also found in aortic rings from mice received erythorbate sodium and sodium nitrite. The increased SBP by L-NAME was significantly decreased by the administration of enalapril (1 mg/kg/day, i.p.), an angiotensin-converting enzyme inhibitor, and this hypotensive effect of enalapril was not influenced by administration of either additive. The decreased acetylcholine response of aortic rings from L-NAME-treated mice was restored by the administration of enalapril, and a restoration of the endothelium function by enalapril was not influenced by the administration of either additive. These results suggest that two food additives, erythorbate sodium and sodium nitrite, does not influence not only the vascular dysfunction by decreased NO production but also an ameliorative effect of antihypertensive drug, such as an angiotensin-converting enzyme inhibitor.

11-16

Establishment of agricultural plants producing high bioactive compounds

Koichiro Shimomura
Faculty of Life Sciences, Toyo University

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To induce callus of red cabbage (Brassica oleracea L. var. capitata DC), leaf or petiole segments of red cabbage shoots were cultured on LS solid medium supplemented with 1mg/L NAA and 2mg/L BA for 8 weeks. Calli were induced on both of segments, and adventitious buds and roots generated on callus induced on the leaf segments. An adventitious bud transferred onto phytohormone-free LS solid medium grew well and was able to be subcultured on the same medium. For production of mutant, callus induced on LS solid medium containing 4 mg/L IBA and 0.2 mg/L BA for 2 weeks was exposed to the heavy ion beams (0 - 200 Gy). Although adventitious roots were formed on calli exposed to 0 - 10 Gy of heavy ion beams, adventitious bud did not formed. Leaf and petiole segments cultured on LS solid medium supplemented with 1mg/L NAA and 2mg/L BA for 1 day were irradiated heavy ion beam. Both segments exposed to 0 - 10 Gy of heavy ion beams formed adventitious roots. Only one adventitious bud generated on the leaf segment exposed to 0.5 Gy of heavy ion beam, which could be maintained as shoot. A new variety of blueberry was produced in Agricultural Technology Center (Gunma), the new variety of blueberry fruits had higher antioxdative activity when measured with superoxide anion-scavenging activity (SOSA) than those of parent strain. Among of 12 different variety of blueberries tested, the new variety of blueberries established through tissue culture had the highest SOS activity.

11-17

Anti-atherogenic effect of enzyme-modified isoquercitrin in apo E-deficient atherogenic mice.

Yoshiki Nishizawa, M.D., Ph.D.
Department of Metabolism, Endocrinology and Molecular Medicine,
Osaka City University Graduate School of Medicine

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Anti-atherogenic effect of enzyme-modified isoquercitrin (EMIQ), an antioxidant flavonoid, was examined using apo E-deficient atherogenic mice. Male apo E -/- mice (7 week-old), were treated with or without EMIQ (0.026% in total diet) for 14 weeks. Mice were fed with high fat diet through the experiment. At 20 week-old, mice were scarified and atherosclerotic legions in the aorta were measured by Oil-red O staining and morphometry. Throughout the experiment, there were no significant differences in body weight between the groups. In EMIQ-treated group, aortic atherosclerotic legion area was significantly less than control group (5.91ｱ2.46% (n=9) vs 9.49ｱ4.11% (n=10), meanｱSD, p=0.037, Student's t-test). There were no significant differences in plasma total cholesterol (1405ｱ253 vs. 1399ｱ282 mg/dl), triglyceride (105ｱ43 vs. 103ｱ38 mg/dl) and HDL cholesterol (22.4ｱ5.0 vs. 17.9ｱ5.0 mg/dl). Thus, EMIQ was shown to have anti-atherogenic effect.

11-18

Effects of BEETRED on Diabetes in ALS mice

Katsunori SATO and Tasuku YAMASHITA
pision of Animal Science, Graduate School of National Science and Technology (Faculty of Agriculture), Okayama University

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BEETRED extracted from a kind of beet (Beta vulgaris LINNE) is a natural pigment and contains betacyanin-like compounds. Recent years, it has been reported that this pigment demonstrated strong antioxidant activity in vitro. Therefore, BEETRED is expected to prevent us from lifestyle-related disease. Then, to uncover the physiological effect of this pigment, BEETRED-ingested ALS mice were investigated by administrating alloxan, inducing pancreatic b-cells to damage specifically. After 30 days of drinking BEETRED solution, 36, 3.6 and 1.8ppm, from 4 weeks of age, ALS mice were treated with alloxan. Female mice which ingested 36ppm-solution, didn't show alloxan-induced diabetes (ALDM) and degenerative changes including b-cell degranulation in many pancreatic islets. Furthermore, this effect of inhibition against ALDM had a dependence on the concentration of ingested BEETRED solution. Then, ALS mice induced to be obese and diabetes by neonatal treatment with monosodium-L-aspartate (MSA), which was an agent impairing hypothalamic ventromedial nucleus, were investigated by ingesting 36ppm-BEETRED solution from 3 to 18 weeks of age. Surprisingly, the onset of diabetes in these mice was delayed for 2 or 3 days. In this study, it was suggested that the ingesting BEETRED was effective in preventing alloxan-induced diabetes.

11-19

Studies for safety assessment of additives used to foods and pharmaceuticals

Kunikazu Teshima
School of Nursing and Rehabilitation Sciences, SHOWA University

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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 made a manual and decided to be based on that.
As a result, 44 ingredients of safety information have been collected in 2004.

11-20

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

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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. For the qualification and quantification by PCR method described in Japanese official method, the DNA sequences for target genes should not be not broken or degradated. However, the degradation of DNA sequences during food processing has not been studied in details. Here, the degree of DNA degradation during processing was investigated to soybean and corn. It was confirmed that current Japanese official method was applicable to soybean under the heating conditions below boiling temperature and to corn by fry-processing at 180℃ shorter than 1 min. In contrast, it was observed that simultaneous treatment of heat and pressure caused to be significant degradation of genomic DNA in both soybean and corn during food processing.

11-21

Heat Stability of Sweet Protein Thaumatin by Anionic-Polysaccharides

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

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The effects of anionic-polysaccharides on heat-induced insolubility of sweet protein thaumatin were investigated on heating at pH 7.0 above 80℃. Thaumatin became insoluble on heating at 80℃ for 15 min at pH 7.0, and the sweetness disappeared. In the presence of dextran sulfate and carrageenans, the heat-induced insolubility was suppressed, and the sweetness remained even after heating at 95℃. This indicated that the sweet protein thaumatin is more stable to heat in the presence of the polysaccharides. The ability of dextran sulfate and l-carrageenan to suppress the heat-induced insolubility of thaumatin was maintained even in the presence of food additives such as ascorbic acid and sodium bicarbonate, and the sweetness of thaumatin remained. The addition of anionic-polysaccharides was an effective method to expand further applications of sweet protein thaumatin to heat-processed foods.

11-22

Effects of spices on gastrointestinal and respiratory systems through capsaicin receptor TRPV1

Syunji Horie
Department of Pharmacology, Faculty of Pharmaceutical Sciences,
Josai International University

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Spices such as red pepper and ginger are added into foods as food additives. In some cases, the spices are healthy, and in other cases, they are noxious. My project is undertaken to clarify how we take spices for our better health. In the present study, effects of spices on the functions of gastrointestinal and respiratory systems were studied in basic research experiments using cultured neuronal cells, isolated animal organs and whole animals. The red pepper constituent capsaicin acts on capsaicin receptor TRPV1 (transient receptor potential vanilloid-1) which is recently discovered in sensory nerves. The distribution of TRPV1 in rats was also investigated in order to support the pharmacological effects of spices. As a result of this project, I got the following findings.
1. Peripheral neuronal cells: Capsaicin, a major pungent constituent of red pepper, is found to induce transmitter release from PC12 cells, a rat pheochromocytoma cell line, through TRPV1.
2. Gastric motility: Capsaicin induced an increase in gastric motility in isolated guinea-pig and mouse intestines through TRPV1.
3. Gastroprotection: Capsaicin protected gastric mucosa through activation of TRPV1 in rat gastric corpus lesion model and rat antral ulcer model.
4. Gastric acid secretion: In isolated mouse stomach, capsaicin inhibited the stimulated acid secretion through peripheral TRPV1. In the contrary, centrally-administered capsaicin induced an increase in acid secretion in anesthetized rats through central TRPV1.
5. Distribution of TRPV1 in stomach and lung: In rat stomach, numerous TRPV1-immunoreactive axons were found around arterioles in the submucosal layer. A large number of nerve fibers were seen in the smooth muscle layer and in the myenteric nerve plexus. In the guinea-pig airway, TRPV1-immunoreactive axons were found within the epithelium and subepithelial regions of airway including smooth muscle and blood vessels and within the lower airway, found in the vicinity of bronchi and bronchioles, and around alveolar tissue.
6. Gingerol, a major constituent of ginger, acts on TRPV1, leading to the enhancement of gastric function.
In the present study, we provided the scientific evidences for the pharmacological effects of spices.

11-23

Rapid Synthesis of Kahweofuran and its Derivatives,
the Coffee Aroma Components, and their Functional Studies as Food Ingredients

Shigeo Katsumura
Department of Chemistry, School of Science and Technology,
Kwansei Gakuin University

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Kahweofuran, as an impact flavor component of roasted coffee or cooked meat, possesses a novel 6-methyl-2, 3-dihydrothieno [2,3-c] furan structure. Although some methods on the synthesis of kahweofuran were reported, all of them were not satisfactory on efficiency and yield. In particular, the total yield of the previous syntheses was less than 10%. We now describe the very efficient syntheses of kahweofuran and its derivatives. Thus, starting from thiophene-3-methanol, we obtained 2-acetyl-3-hydroxymethylthiophene 2 and succeeded in a rapid synthesis of kahweofuran by the formal reductive cyclization of 2 with Wilkinson catalyst under hydrogen atmosphere in 30% overall yield. We also achieved the syntheses of the 4-methyl regioisomer, 6-ethyl, and 4,6-dimethyl derivatives in addition to 6-methyl-4,6-dihydrothieno[2,3-c]furan in favorable yield.
Kahweofuran and ethylkahweofuran have smoky and roasted odor, respectively. Homokahweofuran has green and vegetable like odor, and 6-methyl-4,6-dihydrothieno [2,3-c] furan has sweet, fruity, green, and coffee odor.

11-24

Making Meat and Bone Meal Harmless and Production of Useful Substances by Sub-Critical Water

Hiroyuki Yoshida
Department of Chemical Engineering, Osaka Prefecture University

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Recently, most countries have banned the use of the meat and bone meal for the feed of cattle. However, the large amount has just been preserved in freeze. The immediate processing them, therefore, is eagerly hoped.
In this study, making the meat and bone meal harmless and production of useful substances were performed by sub-critical water hydrolysis. They were hydrolyzed under the reaction temperature range of 473-563 K and the reaction time of 1-30 minutes. The prion was destroyed by sub-critical water in 30min. After the reaction, oil was extracted. The yield of oil was maximum to 0.13 kg/kg-dry sample. In the aqueous phase, many useful substances were produced, that included organic acids, amino acids and so on. Bone remained to be solid, but to be powdery.

11-25

Reduction in manufacture and cooking of kannsui of Chinese-style noodles, and the coagulant of tofu

Motohiro Nishijima
Jissen Wemen's University, Department of Food and Health Science

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Chinese-style noodles were produced using potassium carbonate and sodium carbonate typical as kannsui (60:40). It turned out that kannsui does not decrease by a manufacturing process.
Moreover, when Chinese-style noodles were boiled between parts with the hot water of quantity 10 times, it turned out that about 20% remains as kannsui, sodium and potassium boil, about 50% remains to noodles, and about 50% shifts to hot water used for boiling.
On the other hand, since there were many examples for which crude salt-ized magnesium is used as a tofu coagulant, the action at the time of tofu manufacture and cooking was examined centering on magnesium.
The quantity exceeding the magnesium with which momen tofu and kinugosi tofu added the magnesium of tofu was detected. The reason is because the amount of magnesium of soy milk was added.
Moreover, the quantity in tofu increased more than the total amount of the added magnesium and the magnesium in soy milk. If magnesium is added to soy milk, the cause will be because the portion to solidify and the portion which is not solidified are made, and will be considered for magnesium to shift to the portion to solidify.
It turned out that the calcium and magnesium in tofu are eluted during cooking, and it decreases.

11-26

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

*: Chairman

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Several materials prepared from cooled lava as origins are useful matters for the production of some foods. In the list of existing food additives of Japan, such materials as perlite, green tuff, bentonite and diatomaceous earth are described in the group of insoluble mineral substances. And they are utilized as a filter aid or a precipitation aid in the process of the production for some limited foods.
In a preliminary study concerning the estimation of the safety of these food additives, we evaluated the methodological relevance for the analyses for these additives, and we also analyzed quantitatively the heavy metals eluted from insoluble mineral substances last year. It was noticeable that we detected arsenic and lead in the eluates, especially considerable level of arsenic from diatomaceous earth.
In the present study, firstly it was shown to quantitate 28 elements in the 8 different insoluble mineral substances in the list of the existing food additives, on the bases of the results of the above study, and further investigation for the improvement of the analyses could be continued.
Next we analyzed the amounts of arsenic contained in commercially available liquid foods for which diatomaceous earth was used as a filter aid in the manufacturing. Both other toxic metals as mercury, cadmium, lead and chrome, and non toxic metals as magnesium, iron, silicon, copper and aluminum were quantitatively determined similarly. Neither arsenic nor the other metals were shown to be contained in the liquid food samples beyond levels causing human health problems.
Finally we examined whether the origin of arsenic detected in the available fluid food was diatomaceous earth used as a filter aid in manufacturing or the raw material of the food. Chemical-morphological analysis of arsenic in an apple juice before and after the filtration by diatomaceous earth indicated that the metal was possibly eluted one from the filtrating material.

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Fundamental Safety Assessment of the Registered Existing Food Additives (Natural Source Food Additives)
(2. From Saccharum officinarum LINN (Sugar cane) to Dipteryx odorata WILLD (Tonka bean))

*: Chairman

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In 1995, the 489 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: 214 items.
2) The additives recognized the verification would not be urgently executed from the nature of the source, process etc: 150 items.
3) The additives recognized the safety verification should be urgently executed, since the scientific results have not been sufficiently corrected: 125 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.