Research Reports No21 Abstract (2015)

21-01

Physical and psychological characteristic of children raised by mothers who are sensitive to food additives and the support for the children

TOKUDA Katsumi¹，MIZUNO Tomomi¹，NISHIDATE Arisa²，AJIMI Akiko³，NISHIMURA Miho⁴，
¹University of Tsukuba，²University of Toyama，³Mejiro University，⁴Tokyo Future University

The purpose of this research is to clarify awareness of parents who have children under school age and their behavior relating to food (research 1) and to clarify what kind of influence is given physically and mentally to the children who are raised by parents who are oversensitive about food additives from the perspective of nursery teachers (research 2).
　When we compare the awareness of food additives between parents and nursery teachers without children, we clarified that there is a difference between their ideas. It was also verified that children of parents who are sensitive about food safety and make many restriction to where their life in kindergarten is influenced are easy to develop physical and psychological problems. Restriction of diet, which the parents consider to be for their child, is becoming a cause of their physical and psychological problems.
The role of nursery teachers is to explain the parents that excessive diet restriction for children has a risk of them not enjoying meals and consider making the environment for the children to enjoy meals with the parents.

21-02

Evaluation of constituents in Citrus microcarpa Bunge and C. depressa collected from Okinawa based on the anti-pancreatic cancer activity

Hiroyuki Morita
pision of Natural Products Chemistry, Institute of Natural Medicine, University of Toyama

Citrus microcarpa Bunge is actually a hybrid of mandarin and the Japanese Kumquat (Citrus japonica). It is the source substance of natural fragrance group in the food hygiene law, and its fruit (Shikikan) is frequently used for a drink and food fragrance mainly in Okinawa, instead of C. depressa. Recently, C. microcarpa has been contained in Citrus depressa juice. In our previous study, we identified three Flavonoids, 3,3'-di-O-methylquercetin, 3'-methoxycalycopterin, and 3,3',4',5,6,7,8-heptamethoxyflavone that exhibit preferential cytotoxicity against pancreatic cancer cells under nutrient deprived medium. To further clarify the possibility of C. microcarpa as the health food material useful for the pancreatic cancer prevention, we investigated the constituent of 70% ethanol extracts obtained from C. microcarpa and C. depressa peels by using high performance liquid chromatography (HPLC)－mass spectrometry. The comparison of the constituents of C. microcarpa with those of C. depressa revealed that C. microcarpa peel characteristically contains 3',3'-di-O-methylquercetin as a minor product. Furthermore, C. microcarpa peel characteristically contained phloretin-3',5'-di-C-glucoside, apigenin-8-C-glucosyl-2''-O-rhamnoside, and diosmetin-7-O-rutinoside. Hesperetin-7-O-rutinoside was also found to be a rich constituent in C. micrcarpa peel, as compared with that in C. depressa peel. However, the analysis revealed that C. depressa peel contains the polymethoxyflavones such as nobiletin, as compared with those in C. microcarpa peel.

21-03

Study on infrared spectroscopic method for confirmation test of Saccharin Sodium

Tomoaki Sakamoto¹, Kyoko Sato^2
1pision of Drugs, National Institute of Health Sciences ²pision of Food Additives, National Institute of Health Sciences

The dihydrate and anhydride of Saccharin Sodium are listed in the 8^th Japan's Specification and Standards for Food Additives. Although four confirmation tests are established in the official monograph of Saccharin Sodium, there are no spectroscopic methods such as an infrared (IR) spectroscopy which provide characteristics of chemical structure. In order to propose new confirmation test of Saccharin Sodium using an infrared spectroscopy to Japan's Specification and Standard for Food Additives, we studied test conditions. The sample preparation method including drying condition were examined according to the monographs of the Japanese Pharmacopoeia XVI (JP XVI), the United States Pharmacopoeia 38 and the National Formulary 33 (USP 38-NF33), and European Pharmacopoeia 8^th Edition (EP 8.0) . The drying condition at 105℃ for 16 hours (or to constant weight) provided better spectrum with good reproducibility than that at 120℃ for 4 hours. Since dried saccharin sodium rapidly absorbed moisture during grind and its moisture content affected the IR spectrum, we found that rapid grinding of dried saccharin sodium and compression of fine powder under reduced pressure drying were important to prepare a KBr disc providing reproducible good IR spectrum.

21-04

Study on safety and efficacy of antioxidants using electron spin resonance

Yusuke IWASAKI
Department of Analytical Chemistry, Hoshi University

Food additives, such as preservatives, sweeteners, coloring agents, and flavoring agents, are commonly used in food manufacturing. However, their combined effects on the human body are not known. The purpose of this study was to determine the effects of the simultaneous intake of antioxidant and metal ion on reactive oxygen species (ROS) generation. We investigated the interaction of antioxidant with metal ion under in vitro conditions using electron spin resonance. Moreover, we examined sample stability of antioxidants under several conditions, and assessed membrane permeability using PAMPA assay. In the presence of copper ion, substantial amounts of ROS were generated from Asc and Car in artificial intestinal juice. However, antioxidants are decomposed under artificial intestinal juice, and not to be absorbed by passive transport. It was concluded that there was low effect of oxidative stress in human body on combined reaction of antioxidants and metal ion.

21-05

Development of physical-chemical information retrieval system for food additives

Naoki Sugimoto
pision of food additives, National Institute of Health Sciences

About two decades ago, Dr. Yoshihira research group in University of East Asia had measured NMR, IR UV/Vis spectra of over 200 food additives and then built up the retrieval database to evaluate the safety of them. The retrieval database was opened on website to the public, but the website had been closed in 2006 because the aging server PC was no longer able to maintain the service. Since any researchers had used the retrieval database through internet, they lamented the end of service. Moreover, at present, there is no specialized retrieval database for food additives in Japan. The situation led us to rebuild up a system to offer the retrieval database service to those in need. From the hard disk stored database on an aging server PC, we extracted over 200 datasets of NMR, IR and UV/Vis spectra of food additives. And then by using the datasets, new retrieval database system was built up on our server PC. The new retrieval database system of spectra of food additives was generated as a dynamic page on our web site by PHP: Hypertext Preprocessor script. To improve the retrieval database system, we also measured about 150 of quantitative NMR (qNMR) spectra. As a next project, we just started building up other database of specification of food additives that will be connected to the retrieval database system.

21-06

Effects of pharmaceutical agents on fatty liver disease and antipsychotic drug and preventive effects of
anti-oxidant through analyzing oxidative stress, senescence, and autophagy

Toshinori Yoshida
Tokyo University of Agriculture and Technology, Graduate School, pision of Animal Life Science, Institute of Agricultural Science

To determine effects of enzymatically modified isoquercitrin (EMIQ) on blood lipids and intrahepatic oxidative stress, we examined blood and/or liver samples in rats subjected to a two-stage hepatocarcinogenesis model. Rats were fed with high fat diet (HFD) containing a fatty liver modified substance malachite green (MG) and co-administered with EMIQ or a NADPH oxidase (NOX) inhibitor apocynine (APO). The HFD increased total cholesterol (T.Chol), triglyceride, and alkaline phosphatase in blood samples from the control HFD-fed rats. MG treatment decreased T.Chol, which was further reduced by EMIQ supplement. EMIQ supplement decreased TG as well. EMIQ or APO supplement reduced expression of a NOX component p22phox in preneoplastic lesion, glutathione S-transferase placental form (GST-P)⁺ foci; EMIQ also increased apoptotic hepatocytes. We further examined the effect of EMIQ on intrahepatic oxidative stress in a drug metabolizing enzyme inducer piperonyl butoxide (PBO)-promoted rats. Co-administration of EMIQ or bilberry extracts (BBE) suppressed PBO-induced GST-P+ foci, the effects was associated with decreases in phosphorylated PTEN- or smad4- foci and Ki-67 labeling index in GST-P+ foci. EMIQ also reduced PBO-mediated thiobarbituric acid reactive substances as a marker of lipid peroxidation in liver tissues. The results indicated that EMIQ had a potential of suppressing hyperlipidemia and preneoplastic liver lesions through suppression of intrahepatic oxidative stress and suggested that the antioxidant might prevent obesity-related diseases in rats.

21-07

Study on Efficient Purification of the Components of Gardenia Blue

Sayuri Matsuyama
School of pharmacy, Kinjo Gakuin University

In the present study, a thin-layer chromatographic (TLC) method for the analysis of gardenia blue was described. Gardenia blue is obtained from the fruits of Gardenia augusta Merrill or Gardenia jasminoides Ellis. It is produced by adding -glucosidase to a mixture of iridoid glycosides from gardenia fruits and protein degradation products. Recently, gardenia blue has frequently been used in various foods as a natural coloring in Japan. However, structural characterization of gardenia blue components has not been yet clarified and even chromatographic separation of the components has not been reported. Synthetic colors in foods are generally analyzed by TLC. Therefore, we investigated the analysis of gardenia blue in foods with TLC and separation of gardenia blue components using high-speed countercurrent chromatography (HSCCC). We established two TLC conditions; reversed phase C18 TLC using a solvent system of aqueous 0.2 % TFA-acetonitrile-ethanol (1:2:3) and TLC on cellulose plates using a solvent system consisting of acetone-3-methyl-1-butanol-water (6:5:5). Both conditions yielded three well-delineated spots with good separation. We applied these separations to the analyses of gardenia blue in coloring matter preparations from different manufacturers and in foods on the market. After the gardenia blue was extracted from the samples with water, the extract was evaporated and the residue was dissolved in water-methanol (1:1). Aliquots of the dissolved solutions were then applied to TLC and their chromatographic behaviors were observed. Each preparation showed characteristic spot patterns depending on the manufacturers, so it is possible to specify the manufacturers of the preparations used in foods. For food samples, the Rf values were slightly different between each color extracted from foods and gardenia blue, and we were thus able to identify the manufacturers using the spot pattern of gardenia blue. Next, we attempted isolation of gardenia blue components by HSCCC. We investigated HSCCC separations using 3 two-phase solvent systems, however, the systems did not give sufficient separation. Further investigation is necessary on the optimization of two-phase solvent system for HSCCC separation. The present study is considered to be useful for the establishment of a method of analysis for gardenia blue in coloring preparations and foods.

21-08

Prevention against zinc deficiency by the use of food additives increasing zinc absorption in the intestinal epithelial cells

Taiho Kambe
Graduate School of Biostudies, Kyoto University

Shortage of dietary zinc puts human health at risk, because zinc is required for multiple physiological functions. Hence, a food-based strategy enabling efficient zinc absorption should be extensively explored. In the small intestine, the zinc transporter ZIP4/Slc39a4 functions as an essential component for zinc absorption, and thus food components with the activity to increase .ZIP4 expression may be a potential enhancer of zinc absorption. We have found several food additives to increase ZIP4 expression and increase cellular zinc contents. However, these results have been obtained in studies using mouse cell lines, because of lacks of cultivated human cell lines expressing endogenous ZIP4. In this study, we showed the positive effect of soyasaponin on human ZIP4 expression using human pancreatic carcinoma AsPC1 cells, which we found to express ZIP4 endogenously. Since food additives that increases human ZIP4 expression are promising potential enhancers of zinc absorption, we are now examining the effects of other food additives, which have the activity to increase mouse Zip4 expression.

21-09

Development of discrimination test for plant origin of starches by quantification of color vision of iodine-starch reaction

Tamaki Miyazaki
National Institute of Health Sciences

Starches for food products and food ingredients are originated from various plants such as maize, wheat, potato and rice. Microscopic observation is the most general method to discriminate their plant origin, however, it requires highly proficient skills and experience. And the method cannot be applied to modified starches and starches in food products. It is also well known that the color of iodine-starch reaction is different depending on the plant origin. However, color perception is one of organoleptic properties and the current color reaction method has disadvantage in the ambiguity of expressing color. Recently, convenient and sophisticated spectrophotometers have become popular, and it is applied in nature products as well as industrial products for quality control with digital color information. In this study, the color of iodine-starch reaction of 30 starch products from maize, wheat, potato and rice were measured with a spectrophotometer. It was suggested that the plant origin of the starches could be discriminated from each other, since the values of (a^*, b^*) in L^*a^*b^* color space varied according to their plant origin. The difference in the values of (a^*, b^*) might be due to the difference in amylose/amylopectin ratio, arrangement and degree of polymerization of amylose chains; they can vary depending on species of plant.

21-10

Effects of non-caloric sweeteners consumption in early stage of life on microbiome and systemic metabolism

Takashi Uebanso
Department of Preventive Environment and Nutrition,
Institute of Biomedical Sciences, Tokushima University Graduate School

Sucralose is one of the non-caloric artificial sweeteners (NAS) which is not absorbed in mammalian gut, so it can affect non mammalian cells, including gut bacterium. Sucralose containing sweetener (Splenda) consumption reduce intestinal bacterium in rats but there is no direct evidence in which pure sucralose affect microbiota or not. Recently, Suez J et al. indicated the possibility that NAS consumption induces glucose intolerance through the gut dysbiosis. Because they used much sucralose (1,000 fold as acceptable daily intake (ADI)) for the experiments, it is not clear the effect of ADI levels (15 mg/kg body weight) of sucralose on gut microbiota. In the present study, we examined effect of ADI levels of sucralose consumption on in mice. We evaluated changes in enterobacterium by PCR and body composition. Sucralose consumption for 70 days but not 7 days reduces Clostridium cluster 14a levels in feces. In contrast to Splenda reports, there is no differences in bacterial number in feces between sucralose and control group in 70 days treatment. There are no difference is observed in liver, epididymal fat and cecum weights between sucralose and control group. These results suggest that daily sucralose consumption is less affected on microbiota than it have been reported so far.

21-11

Preservation of Enzymes during Freeze-Drying by Sugar Surfactant

Koreyoshi Imamura
pision of Chemistry and Biochemistry, Graduate School of Natural Science and Technology, Okayama University

Enzymes are indispensable in the food manufacturing industries and, however, is apt to lose its native conformation and consequently bioactivity under non-physiological conditions. Hence, to date, the methodologies to protect enzymes against denaturation have been investigated, and various substances including sugars, amino acids, and certain types of polymers have been known to serve to maintain the enzyme conformation under different conditions. On the other hand, the sugar surfactants that are frequently used as food additives have been found to stabilize protein in various situations. Herein, the effectiveness of various sugar surfactants in the enzyme preservation against denaturation in freezing and freeze-drying was investigated. The influences on the enzyme stabilizing characteristics by alkyl chain length, sugar head group structure, and ester/ether bondings of sugar surfactant are discussed. It was also indicated that, when a decent amount of sugar was also added, the enzyme stabilizing effect of a small amount of sugar ester through the freeze-drying process could be enhanced.

21-12

Consumer's Attitudes toward the Safety of Food Additives

Masakazu Horie, Miki Iwabori, Marina Kudo, Mari Kumaki, Natsumi Takano, Chie Tanaka, Mizuki Hayashi, Kana Funakoshi
Department of Food Science, Faculty of Home Economics, Otsuma Women's University

In representing consumers, an opinion survey pertaining to the safety of food additives was conducted of people in charge of school lunches and students belonging to the dietitian or registered dietician curriculum. Of the people in charge of school lunches, more than 70% had feelings of concern, citing food additives as the primary factor and expressed concern for the carcinogenicity of the additives. Further, as for the people in charge of school lunches, more than 50% responded that they carefully check the additives label when purchasing food and avoid food using food additives as much as possible. On the other hand, students have a vague feeling of concern for food additives, but only about 15% carefully read the food label when purchasing food and less than 30% avoid food that use additives. It can be said that the feeling of concern that the students have toward food additives is vague and does not actually impact their consumption activities.

21-13

Evaluation of immunotoxicity of nanomaterials in foods

Yasuo Yoshioka
Laboratory of Toxicology and Safety Science, Graduate School of Pharmaceutical Sciences, Osaka University

In considering the safety of ingested nanomaterials, quantity of transfer across and effects on intestinal cells are important points to evaluate. However, little information exists about how quantity of　transfer across intestinal cells and cellular effects of nanomaterials change between apical and basolateral exposure. Here, we examined quantity of cellular internalization and transcellular transport, and cellular effects of nanomaterials on Caco-2 cell monolayer after apical and basolateral exposure by using Ag and Au nanoparticles. When treated in apical side, smaller Ag nanoparticles were more easily internalized and transfer to basolateral side. However, larger Au nanoparticles were more easily internalized, though the transfer rate of Au nanoparticles was the same for 10 nm, 50 nm, and 90 nm ones. On the other hand, when treated in basolateral side, larger Ag nanoparticles were more easily internalized, whereas smaller Ag nanoparticles more easily transfer to apical side. After basolateral treatment, larger Au nanoparticles were more easily internalized, but smaller Au nanoparticles more easily transfer to apical side. In addition, Ag nanoparticles whose diameter are less than 10 nm temporarily increased permeability of Caco-2 monolayer when treated in basolateral side, whereas they did not when treated in apical side. These results suggest that quantity of cellular internalization, transfer rate, and cellular effects on epithelial cell monolayer of nanomaterials change depend on size, composition, and exposure side of nanomaterials. Our findings are thought to be of importance as base information for absorption of ingested nanomaterials.

21-14

Basic study on an effect of polysaccharides on absorption, distribution, and excretion of radionuclides

Shuichi Enomoto
Department of Pharmaceutical Analytical Chemistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University

After Fukushima nuclear accident occurred, radionuclides such as ¹³⁷Cs, ¹³¹I, and ⁹⁰Sr, were released into the environment. After the accident, radioprotection became a subject of social concern. Therefore, effective and easily obtained radioprotector is urgently needed. Although it has been shown that pectin promotes the excretion of ¹³⁷Cs, the mechanism remains unclear. In the present study, we analyzed distribution of radionuclides in order to reveal the effects of pectin intakes to the dynamics and excretion of radionuclides.
Seven pectin samples (A - G) which have different molecular weight (50-360 kDa) and character were mixed with a cocktail solution of ¹³⁷Cs, ¹³¹I, ⁸⁵Sr, ⁵⁴Mn, ⁵⁹Fe and ⁶⁵Zn. The solution was pided into macromolecular fraction (>3 kDa) and small-molecule fraction (<3 kDa) by ultrafiltration. Subsequently, the radioactivity of each fraction was measured by using a Ge detector to evaluate the binding property of pectin with radionuclides. In addition, ¹³⁷Cs, ¹³¹I and ⁸⁵Sr were orally administrated to BALB/c mice and 1 hour later, pectin was orally treated into the mice. Then, we noninvasively evaluated biodistribution of each radionuclide by using GREI (Gamma-Ray Emission Imaging), which is a multiple nuclide imaging modality.
The radioactivity of ⁸⁵Sr in the macromolecular fraction of samples D, E, and F increased and that in the small-molecule fraction of the identical samples decreased. These results suggest that samples D, E, and F adsorb ⁸⁵Sr. In addition, on noninvasive imaging experiments with GREI, we succeeded to visualize sample D accelerates the excretion of ⁸⁵Sr from the body. These results suggest that pectin sample D accelerates ⁸⁵Sr excretion from the body by interaction with ⁸⁵Sr. Therefore, pectin sample D has potential to reduce the internal radiation exposure caused by ⁸⁵Sr.

21-15

Studies on the chemical structure and the mechanism for production of gardenia red

Yusai Ito
Faculty of Home Economics, Kyoritsu Women's University

Gardenia red is hydrophilic colorant produced by the addition of an iridoid glucoside, geniposidic acid, prepared from geniposide of gardenia fruits by alkali hydrolysis to a mixture of -glucosidase and hydrolyzate of proteins. The colorant is a complex of high-molecular compounds, therefore the chemical structure of red pigments and the mechanism for producing the pigments have not been elucidated. The production of gardenia red generally needs the bubbling of argon to nitrogen gas to keep anaerobic condition and the addition of citric acid. Geniposidic acid prepared from gardenia fruits hydrolysed by -glucosidase and then were dissolved in distilled water with Gly and ascorbic acid and heated at 90℃. The mixture turned to red and had a maximum absorption at 520 nm. While, the mixture without ascorbic acid tuned to blue, which showed the indispensable of a reductive condition in the reaction mixture for the production of red pigments. Furthermore, the addition of five times more than equivalent of グリシン suppressed the production of precipitate in the reaction mixture. While the addition of Ala, Gln, Glu and Lys as well as Gly also produced red pigments in the mixture containing ascorbic acid, the addition of Lys and Cys did not produce red pigments but dark brown precipitate.

21-16

Elucidation of carcinogenic mechanism of the nongenotoxic liver carcinogen dammar resin in rats

Hideki Wanibuchi
Osaka city university graduate school of medicine, Department of molecular pathology

Dammar resin is a food additive primarily used as a thickening agent. We previously reported that dammar resin induced liver tumors in a two-year rat carcinogenicity study and showed that it is negative for mutagenicity in gpt delta rats. The purpose of this study is to clarify the mechanisms underlying the liver carcinogenesis induced by dammar resin in rats. 6 weeks old F344 rats were pided into 2 groups and were fed diet supplemented with dammar resin at doses of 0, 2% (carcinogenic dose), for 4, 13, 32 or 52 weeks. CYP1A1 was increased in livers of rats treated with dammar resin compared to respective controls and showed a time-dependent manner. These results suggest that activation of aryl hydrocarbon receptor may involve in dammar resin-induced rat hepatocarcinogenesis. Line-1 methylation analysis revealed that dammar resin induced global hypomethylation in rat livers. Moreover, we also found that low expression of de novo DNA methyltransferases DNMT3b might attribute to the global hypomethylation. Our findings suggest that activation of nuclear transcription factor and aberrant DNA methylation are involved in the dammar resin-induced rat hepatocarcinogenesis.

21-17

Genetic and breeding study for increase and stabilization of yield of food additive color from safflower

Tsuneo Sasanuma
Faculty of Agriculture, Yamagata University

In order to increase the amount and improve the stability of yield of safflower petals as a food color material, basic morphological and agronomical traits and petal harvest were investigated, and isolation of genes involved in safflower petal color synthesis was carried out. For investigation of the traits, 14 accessions of safflower genetic resources were grown in the experimental field of Faculty of Agriculture, Yamagata University, and 20 basic traits such as flowering date, plant height, number of capitula, diameter of capitulum, number of petals, maximum length of petals, etc. were measured. In addition, the by hand harvest of petals from densely grown safflower like farmers' field were investigated. The results revealed that number of capitula varied greatly among years and that Mogami-benibana that is an accession of Yamagata Prefecture cultivated as dye materials had long petals and showed high stability and yield in spite of change of environmental conditions, indicating that it is a superior accession as dye plant. It is also revealed that several accessions among genetic resources showed high scores in each of the traits and some accessions such as KKB27 showed a high petal yield as well as Mogami-benibana with different characters. These results clarified the potential of safflower genetic resources as future breeding materials. As for the isolation of color synthesis related genes, comparative expressional analysis between red and white flowers using PCR-based cDNA subtraction method was conducted and obtained a candidate gene that showed much higher expression in red flower petals.

21-18

Investigation of the classification and chemical composition of ginger varieties using metabolomics

Daigo Wakana
Department of organic chemistry、Hoshi University

Ginger (Zingiber officinale) is well known spice and cultured on a temperate region. We attempted to analysis of the samples of ginger extracted by hexane, acetone and ethanol using ¹H-NMR metabolomics. The samples of ginger extracted by each solvent were possible to classify by solvent using toextract.

21-19

Analysis of pharmacokinetics variation factors by menthol

Nobutomo Ikarashi
Department of Clinical Pharmacokinetics

Recently, it was reported that the anticoagulant effect of warfarin was reduced when patients receiving warfarin also took menthol. The purpose of this study is to reveal the mechanism of this reduced anticoagulant effect of warfarin from the pharmacokinetic point of view. Moreover, this study evaluated the effects of menthol on the pharmacokinetics of the cytochrome P450 3A (CYP3A) substrate triazolam and the CYP2C substrate phenytoin. Warfarin was orally administered to mice 24 h after the administration of menthol for 2 days, and the plasma warfarin concentration was measured. In the menthol-treated group, the area under the blood concentration time curve of warfarin was decreased by approximately 25%, while total clearance was increased to 1.3-fold compared to the control group. The hepatic CYP2C and CYP3A protein expression levels in the menthol-treated group were significantly increased compared to those in the control group. In the menthol-treated group, the plasma triazolam and phenytoin level were lower and its clearance was higher compared with the control group. The results of this study revealed that menthol causes an increase in CYP2C and CYP3A expression levels in the liver, which leads to an enhancement of warfarin metabolism, resulting in a decreased anticoagulant effect of warfarin. In addition, menthol lowered the plasma concentrations of triazolam and phenytoin when concurrently administered. The results of this study suggested the necessity of exercising caution when taking menthol during treatment with drugs that are substrates of CYP3A or CYP2C.

21-20

thetic effect of eugenol as a natural food additive

Takao Koeduka
Faculty of Agriculture, Yamaguchi University

Eugenol, consisting of a benzene ring (C₆) and propenyl (C₃)-side chain as a basic structure, is an active ingredient of spices and herbs, and it also has been used as food preservatives because of their pleasant aroma and antimicrobial properties. Furthermore, since eugenol exhibits the anesthetic effect to various shrimps and fishes, human has generally used as a natural anesthetizer for the transportation and vaccination of expensive fish such as a globefish. However, despite many previous studies on the anesthetic effect of eugenol, there are few reports regarding the molecular mechanism how eugenol is effective on the motor neural circuit systems in vivo. In this report, the anesthetic effects of eugenol were studied in the zebrafish (Danio rerio). The results suggested that eugenol could be an effective anesthetic for the downstream of muscle constrictive process via calcium channels.

21-21

Study on pharmacokinetics of sulfated polyphenols and the molecular mechanism for their functions

Yoshichika Kawai
Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan

It has been reported that dietary quercetin, a major polyphenol in daily diet, has a variety of biological activity, whereas less attention has been paid on the functions and the molecular actions of quercetin sulfates, major metabolites in human plasma after oral intake of quercetin. In this study, we chemically synthesized quercetin sulfates and examined the anti-inflammatory activity using a macrophage cell line. We found that quercetin sulfates inhibited lipopolysaccharide-induced NO production without deconjugation, in contrast to the glucuronides which could be deconjugated by macrophages. This study suggests that quercetin sulfates, even after conjugation, could be anti-inflammatory agents in vivo.

21-22

Intestinal absorption of curcumin was increased by glycosylation and its mechanism

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

I tried to augment the intestinal absorption of curcumin, the bioactive compound contained in termeric, by glycosylation. Curcumin diglucoside was synthesized using glycosyltransferase isolated from Catharanthus roseus. When curcumin or curcumin diglucoside was orally administered at the same dose as curcumin, the maximum concentration of curcumin and the area under the blood concentration of curcumin-time profile in curcumin diglucoside-treated rat were about 8*- and 7- fold higher than those of curcumin-treated rat, respectively. Curcumin diglucoside was hydrolyzed by the homogenate of rat intestinal epithelium, suggesting that lactose-phlorizin-hydrolase (LPH) existing in intestinal epithelium might digest curcumin diglucoside. Then, I transfected human LPH or rat LPH into human embryonic kidney cells (HEK293 cells), and confirmed that transfected cells can digest phlorizin to make the experimental system to find the substrate of LPH. I will conduct the experiment whether curcumin diglucoside can be digested LPH or not.

21-23

Development of novel control method for preventing food-poisoning using food additives derived from polyphenols

Yuko Shimamura
School of Food and Nutritional Sciences, University of Shizuoka

The aims of the present study are first to investigate the binding of the food additives derived from polyphenols, Applephenon (AP) and (-)-epigallocatechin gallate (EGCG) which is a main ingredient in Teavigo, to the active site of staphylococcal enterotoxin A (SEA), and second to examine the inhibitory effects of them on the toxic activity of SEA. The binding affinity of AP and EGCG to toxin active sites was analyzed using prepared 4 different synthetic peptides and rabbit antibodies to their corresponding peptides. As a result, AP and EGCG inhibited the binding affinity of the anti-peptide antibodies to SEA active sites. Especially, samples showed the strong inhibitory effect on the binding affinity to A-6 (corresponding to amino acid residues 81-100). These samples also inactivated the proliferation and interferon production induced by SEA in spleen cells. Next, we examined the effect of pH on the binding of samples to SEA. After treatment at acidic and alkaline pH (pH2.4-8.0) as intragastric conditions, AP and EGCG maintained the binding to SEA. These results suggest that edible and safe plant-derived polyphenols can be used to inactivate biological activity of SEA.

21-24

Study on functions of natural fragrance ingredients exerting anti-obesity and anti-hyperglycemic effects

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

Bile acids exert anti-obesity and anti-hyperglycemic effects through its receptor TGR5. Activation of TGR5 on L cells localized in the small and large intestine increases incretin GLP-1 secretion, thereby improving insulin sensitivity and lowering blood glucose levels. In the skeletal muscle or brown adipose tissues TGR5 induces increased expression of the genes related to heat production, thereby stimulating energy expenditure and exerting anti-obesity effects. Based on these findings, we established an assay system evaluating the TGR5 agonist activity using natural fragrance ingredients. We succeeded to identify a couple of potent agonists among approximately 300 fragrance compounds.

21-25

Digestion characteristics of oil-containing foods for aged persons using Gastric Digestion Simulator equipped with peristalsis

Ichikawa Sosaku¹、Kobayashi Isao)²、Kozu Hiroyuki^1,2
1Faculty of Life and Environmental Sciences, University of Tsukuba，²Food Engineering pision, National Food Research Institute, NARO

Controlling nutrient release in human gut has been more and more important in an aging society. Designing new food products of which digestibility is controlled is necessary for elderly persons whose digestion levels decrease. This study aims to understand breakdown and nutrient release phenomena of solid foods in human stomach using in vitro gastric digestion device, i.e. Gastric Digestion Simulator (GDS) developed in our research group. GDS simulates gastric peristalsis which breaks down solid foods and mixing gastric contents in the stomach. As a model food containing nutrient, O/W-gel emulsion (emulsion gel) was used in this study. Soybean oil was used as dispersed phase as a model nutrient. Considering the situation of nutrient embedded in the solid material, O/W emulsion made of soybean oil was gelatinized using agar alone or mixture of agar and gellan gam. By the direct monitoring of the digestion processes of emulsion gel using GDS, breakdown of gel and release of oil droplets was observed. The type of gelatinizer affected the gel particle size after GDS digestion experiment. Emulsion gel using the mixture of agar and gellan gam reduced the degree of gel breakdown compared to that of emulsion gel prepared with agar alone. In addition, the amount of rereleased oil from emulsion gel after GDS digestion experiment also decreased in the case of agar-gellan gam mixture compared to that of agar alone. These results indicate that the amount of nutrient released from gel to gut lumen can be controlled by changing gelatinizer. Our results provide interesting knowledge about controlling nutrient release in human stomach, which is expected to contribute designing new food products for elderly persons.