Research Reports No.13 Abstract（2007）

13-01

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

Kanji Ishimaru

Faculty of Agriculture, Saga University

A new kaempferol trioside (kaempferol 3-O-rutinoside 7-O-b-D-apiofuranoside, 1) was isolated together with nine phenolic compounds, trans-tiliroside (2), caffeic acid (3), chlorogenic acid (4), 3-O-caffeoylquinic acid (5), 4-O-caffeoylquinic acid (6), 3-O-coumaroylquinic acid (7), 4-O-coumaroylquinic acid (8), 3,5-di-O-caffeoylquinic acid (9) and 4,5-dicaffeoylquinic acid (10) from the leaves of Solidago altissima L. The structure elucidations of the compounds were based on the analyses of spectroscopic data.
Shoot, adventitious root and hairy root cultures of S. altissima L. were established. Chlorogenic acid and 3, 5-dicaffeoylquinic acid were isolated from the hairy root cultures. 3, 5-dicaffeoylquinic acid was the major secondary metabolites in various tissue cultures of this plant. These polyphenol compounds were easily isolated as the polyphenol-soybean protein complex from the extract of this plant. S. altissima L., producing polyphenol compounds with the high concentrations, seemed to have a potential to be as a new resource for functional polyphenols.

13-02

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

Toshihiko Toida and Shinobu Sakai

Graduate School of Pharmaceutical Sciences, Chiba University

Chondroitin sulfate shows various biological activities and high affinity to the CD62L (L-selectin). We have already reported that 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 unfractionated chondroitin sulfate has been fractionated by gel filtration chromatography and enzymatically depolymerized to investigate the possibility of absorption of chondroitin sulfate through oral administration, and the effects of the fractionated chondroitin sulfate on the immune response have also been investigated. The fractionated chondroitin sulfate, which average molecular weight is 21 kDa, showed the strongest binding to CD62L among the fractionated chondroitin sulfate which average molecular weights were from12 kDa to 40 kDa, and the binding constants were related to the productions of cytokines of murine splenocytes when they were co-cultured with each fractionated chondroitin sulfate for 48 h. On the other hand, all of these fractionated chondroitin sulfate samples (12, 15, 18, 21, 25, 28, 35 and 40 kDa) could not be detected in blood serum after oral administration to mice. The effects of the fractionated chondroitin sulfate on intestinal immunosystem are going on the investigation.

13-03

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.
Western blot analysis using antibody against a recombinant oryzacystatin, which was prepared and purified in this study, showed that polished rice contains rice protein (oryzacystatin) with protease inhibitory activity. Moreover, 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 (lizardfish) showed papain like protease. On the basis of these results, we prepared model Kamaboko using surimi (lizardfish) as a starting material. When the rice protein was added to surimi paste with 3% 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 (lizardfish) was effective to prevent Kamaboko from Modori (softening) in the presence of 2-3% NaCl. Further study on clarification of relationship between degree of inhibition of protease activity in surimi and physical properties of Kamaboko is necessary.

13-04

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 in 2005 (See the annual report No.12).
In 2006, we additionally analyzed 7 kinds of crude drugs (5 companies, 33 herbal materials) purchased from the Japanese market. By using modified Rankine method, more than 300 mg/kg of sulfur dioxides was detected only from Moutan Bark.
Next, be aimed for development of a new simple method for the measurement of sulfur dioxides, we investigated correlation between the color value obtained by a hand-held spectrometer and the sulfur dioxides content in 19 kinds of crude drugs. As the results, the good correlation between the color index L* value and the sulfur dioxide content and the good inverse correlation between the color index C* value and the content were observed in four powdered crude drugs (Pueraria Root, Gastrodia Tuber, Lilium Bulb and Moutan Bark). Therefore, the color index L* and C* values may be suitable as the screening index of sulfur dioxide content in these powdered crude drugs.

13-05

Comparative Investigation on the Safety Assessment of Food Additives

Yoichi Konishi¹ ，and　Fumitake Fukutomi²

1 International Federation of Societies of Toxicologic Pathologists　(IFSTP)
2 Ochanomizu University・ Life World Watch Center

Recent international information concerned the safety assessment of food additives is presented. The risk assessments are conducting based on the results of 2-year carcinogenicity tests in rats and mice, TTC and NOAEL values. However, the carcinogenicity tests need long period and high expenses, the alternative methods such as genetically modified mice and gene expression profil using microarray etc. are developing, but those are useful for hazard identification but not being alternative method.
TTC is accepted for the safety assessment of low dose exposure of chemicals such as flavor in US and various countries of European regulatory agencies. The advantage of TTC to be used for the safety assessment of food additives including the flavor should be paid attention in Japan since many those substances have not been evaluated their safety. The importance of NOAEL principle is recognized world-wide. However, it contains unclear factors such as no standard definition, differentiation between adverse and non-adverse effects and setting reasonable safety factor to calculate ADI. The solution of these uncertain factors is the matter of great urgency.
Finally, the safety assessment of food additive is possible to be proposed as following: The components of substances should be analyzed exactly and chemical structure of the components to be used for TTC determination. Negative TTC substances are not needed for further tests and positive substances are needed the tests of the genotoxicity and 90 days toxicity test. Based on the evaluation results, the substances needed to test the carcinogenicity are possible to be selected.

13-06

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 third generations of red radish by way of successive selective-crossing among the first generations were cultivated and their anthocyanin contents were quantitated by HPLC. Consequently, the third generation was shown to maintain character to produce anthocyanin in higher content than the original population. In addition, several inpiduals with remarkably high anthocyanin content were disclosed among the third generations.
On the other hand, effect of prolonged cultivation from autumn to spring for content and composition of anthocyanin was also examined. The red radishes harvested in spring were shown to contain anthocyanin with higher content and different composition as compared with those harvested in winter.

13-07

Basic Study of Dissolution Behavior of Silicates in Liquid Foods

Teruhisa Fujimaki

Kanagawa Prefectural Institute of Public Health

Imported foods currently comprise over 60% of the foods consumed in Japan on a calorie basis. Many imported foods contain additives that are banned in Japan despite the fact that the safety of such additives has been guaranteed on the basis of international standards. In considering sanitary measures for such foods containing undesignated additives, the Ministry of Health, Labour and Welfare is reviewing regulations on additives with a view towards international conformity for food additives whose safety has been validated internationally and which are in wide use. As part of this review, in 2005, a request for evaluation of the health effects of four types of silicates (sodium aluminosilicate, calcium silicate, aluminum calcium silicate, magnesium silicate) was made to the Food Safety Commission in accordance with the Food Safety Basic Law. In North America and Europe, silicates are mainly used as an anti-caking agent in table salt and substitute salts. Therefore, it can be assumed that table salt and other foods that contain silicates as anti-caking agent are extensively used in food preparation processes together with other ingredients, adulterating vinegar and other liquid foods. It is assumed that JECFA does not specify the ADI of these four types of silicates based on the premise that they are insoluble in water.In this regard, insoluble minerals (activated acid clay, acid clay, green tuff, granite porphyry, talc, perlite, and diatomaceous earth) and the four types of silicates were subjected to fluorescence X-ray analysis. In addition, the dissolution behavior of each element including silicon from the insoluble minerals and silicates under various conditions was explored using food-simulating solvents by ICP optical emission spectrometry.

13-08

Mechanisms underlying changes in metabolic profiles seen
in diabetes by means of analysis of metabolome

Yuka Fujimoto

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

Impaired suppression of hepatic glucose production and a defect in hepatic glucose uptake in response to the increased plasma glucose and insulin levels are common feature in obesity and diabetes. In these subjects, postprandial hyperglycemia, hyperinsulinemia, and hyperlipidemia create a vicious cycle. Our research is interested in the relationship between hepatic glucose and lipid metabolism to break the cycle seen in obesity and diabetes, using catheterized diabetic and/or obese animals. At first, the present study represents that 3 week high-fat diet-fed rats and also combination of high-fat diet-fed and low-dose streptozotocin-treated rats are available for an animal model for type 2 diabetes. Secondly, to examine a role for dietary glycaemic index (GI) in treatment of diabetes, healthy normal rats were given low-GI or high-GI diet for 4 weeks or 8 weeks. We found that rats given low-GI diet for 8 weeks were likely to show higher plasma glucose and lower plasma insulin concentrations in response to the rise of plasma glucose, compared to rats given high-GI for 8 weeks. And also there are different plasma lipid metabolic profiles in rats given low-GI food compared to high-GI food.

13-09

An efficient glycosylation of useful secondary metabolites using plant glycosyltransferase

Kazuyoshi Terasaka

Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Nagoya City University

Glycosyl conjugation of lipophilic low molecular weight compounds is an efficient tool to enhance water solubility, to improve stability, and thereby to increase bioavailability and modify biological activity. Curcumin glucosyltransferase (CaUGT2) isolated from cell cultures of Catharanthus roseus exhibits unique substrate specificity. We identified an amino acid involved in substrate recognition and catalytic activity of CaUGT2, using a combination of domain swapping and site-directed mutagenesis. The cDNA encoding UDP-glucose: curcumin glucoside glucosyltransferases (UCGGT) was isolated from a cDNA library of cultured C. roseus cells, using a PCR method directed at the conserved UDP-binding domain of plant glycosyltransferases. Additionally, we developed an one-pot system linking recombinant sucrose synthase and plant glucosyltransferases. This system provides us with an efficient method for synthesis of glucosides of lipophilic small molecules, by removing GTase-inhibiting UDP from the reaction mixture and at the same time regenerating the relatively costly UDP-glucose substrate.

13-10

Studies for migration into foods and risk assessment of chemical materials
related to food container packaging

Hiroyuki Kataoka¹, Kurie Mitani¹, Nobumitsu Hanioka², Shizuo Narimatsu²

1 School of Pharmacy, Shujitsu University
2 Faculty of Pharmaceutical Sciences, Okayama University

We investigated about an estimation model of food pollution of abietic acid and dehydroabietic acid through paper container and packaging and their safety evaluation by cytotoxic effect. Abietic acids were determined by automated on-line in-tube solid-phase microextraction/liquid chromatography-mass spectrometry. Contents of abietic acids in paper containers and packagings were ranged from mg/g to several hundred mg/g, and below 1% of these levels were easily migrated into food simulants. Abietic acids were also easy to elute into fat soluble solvent at higher temperature. On the other hand, we examined the expression of cytochrome P450 and UDP-glucuronosyltransferases mRNAs in human intestinal epithelial Caco-2 cells, to evaluate the toxicological activities of abietic acid and dehydroabietic acid. Caco-2 cells were cultured in medium containing abietic acids (0-200 mM) for 48 h. The cell viabilities were dose-dependently decreased by exposure of abietic acids. The EC₅₀ values for abietic acids were 26 and 235 mM, respectively. The results obtained in this study should provide useful information for the prediction of toxicological assessment of abietic acids.

13-11

Evaluation of anti-oxidative vitamin-containing vegetables

Hirobumi Yamamoto¹, Koichiro Shimomura¹, Kaori Touno²

1 Faculty of Life Sciences, Toyo University
2 Plant Regulation Research Center, Toyo University

For the supply of safe and tasty functional vegetables to consumers, comprehensive evaluation system including superoxide anion-scavenging activity, HPLC analyses of lipophilic anti-oxidative vitamins a-tocopherol and b-carotene and carotenoids, those of hydrophilic anti-oxidative vitamin ascorbic acid, and the scientific assessment of taste of vegetables were established. Analytical methods for adverse compounds in vegetables such as oxalic acid and nitrogen ion by use of HPLC were also established. Using this evaluation system, it was demonstrated that "Komatsuna" (Brassica campestris L. var. Komatsuna Makino) grown with farmyard manure were more tasty and less bitter than those grown with complex fertilizers, whereas their growth, anti-oxidative activity or the contents of anti-oxidative compounds were not changed. We also found that the content of lipophilic anti-oxidative compounds such as b-carotene in vegetables increases during the preservation at low temperatures or the heating treatment, presumably owing to the induction of biosynthetic enzymes by such treatments.

13-12

Nutrinogenomic Studies on Safety and Biomedical Effect of Food Additive and Artificial Sweetener
Using Monkey Model: Preliminary Genomics Study in OVX-Menopause Monkeys

Shin Nakamura

Primate Research Institute, Kyoto University

As a preliminary genomics study on biomedical effect of oligofructose, a ifunctional sweetener material, nutrinogenomic analyses using the menopause monkey model were performed. The effect of oligofructose to several function genes associating with sugar/lipid metabolism and steroid hormone generation in liver and subcutaneous adipose was clarified. In addition, the effect of oligofructose on the intestinal bacterial flora was also elucidated through genomic analyses of the change in the number of 11 intestinal bacteria species.

13-13

Stimulation of polyhalogenated aromatic hydrocarbons excretion induced by natural color 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. Here, we examined two natural color additives, annatto and lac, to enhance the excretion of storage PHAHs in male mice by using 3H-TCDD as a model PHAH.

13-14

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

Curcumin is a plant-derived diferuloylmethane compound extracted from Curcuma longa, possessing antioxidative and anticarcinogenic properties. Antioxidants and oxidative stress are known to induce the expression of certain classes of detoxification enzymes. Since the upregulation of detoxifying enzymes affects the drug metabolism and cell defense system, it is important to understand the gene regulation by such agents. In this study, we demonstrated that curcumin could induce the expression of human glutathione S-transferase P1 (GSTP1), In HepG2 cells treated with 20 mM curcumin, the level of GSTP1 mRNA was signincantly increased. In luciferase reporter assays, curcumin augmented the promoter activity of a reporter construct carrying 336bp upstream of the 5'-flanking region of the GSTP1 gene. Mutation analyses revealed that the region including antioxidant response element (ARE), which overlaps AP1 in sequence, was essential to the response to curcumin. While the introduction of a wild-type Nrf2 expression construct augmented the promoter activity of the GSTP1 gene, co-expression of a dominant-negative Nrf2 abolished the responsiveness to curcumin. In addition, curcumin activated the expression of the luciferase gene from a reporter construct carrying multiple ARE consensus sequences but not one with multiple AP1 sites. In a gel mobility shin assay with an oligonucleotide with GSTP1 ARE, an increase in the amount of the binding complex was observed in the nuclear extracts of curcumin-treated HepG2 cells. These results suggested that ARE is the primary sequence for the curcumin-induced transactivation of the GSTP1 gene. The induction of GSTP1 may be one of the mechanisms underlying the multiple actions of curcumin. We postulate here, there is a possibility to establish the evaluation system of antioxidant-responsibility in the mammalian cells through the antioxidant enzyme gene transactivation.

13-15

Global gene expression analysis in the liver of mice administered carcinogens and non-carcinogens
---For the development of a new rapid and reliable carcinogenicity test---

Toshikazu Yoshikawa

Inflammation and Immunology, Kyoto Prefectural University of Medicine

Although much has been elucidated about the mechanism of cellular carcinogenesis, evaluation of the carcinogenicity of substances is still difficult. Since a number of new synthetic materials for drugs and food have been developed, it is necessary to sufficiently evaluate their carcinogenicities. In carcinogenicity tests performed at present, the test substance is administered to rodents for 2 years as their life span, and its carcinogenicity in each organ is evaluated by macroscopic and pathological examinations. Such carcinogenicity tests require considerable time and resources. In this study, we attempted to develop a new carcinogenicity test by comprehensive gene expression analysis, which is independent of target organ-specific activity of carcinogens. All food and chemicals are primarily transported to liver via portal vein, which are independent of the routes of intake. Therefore, we selected liver as the best organ with which to assess carcinogenicity in comprehensive gene expression analysis. The present report indicate that global gene analysis by using DNA microarray in liver at 3 hours after administration of chemicals could make it possible to establish a rapid bioassay to identify carcinogens for various organs.

13-16

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

In 1995, the 450 items of existing food additives (natural source food additives) were registered by the Ministry of Health, Labor and Welfare. These food additives were assessed the safety by existing research data and classified in following three categories.
1) The food additives verified internationally the safety by the several qualified scientific data such as the JECFA(FAO/WHO Joint Expert Committee on Food Additives) etc. : 247 items.
2) The food additives recognized the verification would not be urgently executed from the nature of the source, process etc : 132 items.
3) The food 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 the first and the third categories.
The procedure of assessment was carried on the following properties of the food additives.
1) The identification of the original natural sources (animal and plant) of the food additives.
2) The historical survey of human consumption as food of the natural sources.
3) The investigation of the chemical components in the natural sources and the scientific safety data obtained from the RTECS.
The purpose of this report would be to establish the documents to identify the food additives registered by the Ministry of Health, Labor and Welfare and the original natural sources (plant and animal) corresponded to the food additives.

13-17

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 aspartame
Aspartame (ASP), a synthetic sweetener, was chosen for analysis among optically active food additives. CHIROBIOTIC TAG (Teicoplanin Aglycone) in which silica gel is chemically bound to a macrocyclic glycopeptide, was used as a separation column for detection with MS and UV detectors. This method sharply separated L-a-L-ASP and L-a-D-ASP even when the D-isomer content was 0.1%.
2. Optical isomer analysis for amino acids
An analytical method for amino acids was studied that takes measurements with an ultraviolet absorption detector (1) and a fluorescent detector (2) after postcolumn derivatization with a fluorescent absorption detector and ortho-phthal aldehyde (OPA). (1) In the column, octadecyl silica was coated with N,S-dioctyl-D-penicillamin and a reversed phase mobile phase containing copper (II) ion was used to coordinate the ion and separate amino acids; it was a ligand exchange type. Because a complex made up of an amino acid and the copper ion absorbs light in a range from around 300 nm to that of short wave, this method allows quantification with an ultraviolet absorption detector. On the other hand, (2) CHIROBIOTIC T used as a chiral column sharply separated D- and L-isomers. The analytical results of commercially available soft drinks by this method revealed that all the amino acids detected were L-isomers.
3. Chiral analysis for proline
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, OPA and 9-fluorenylmethyl chloroformate (FMOC-Cl) were used for selective pretreatment to develop a sensitive and selective method for the separation and quantification of the chirals by LC/MS. This analytical method was applied to beverages for proline measurement to detect the D-isomer at a concentration of a few percent in several samples.