Research Reports No.15 Abstract（2009）

15-01

Real-Time Evaluation of Quality for Food Emulsifiers by Terahertz Spectrum

Yasushi Endo

Tokyo University of Technology

We tried to evaluate the quality of food emulsifiers by terahertz spectrum. The terahertz spectra at the range between 10 and 400 cm-1 were measured for sugar esters and egg lecithin. Sugar esters had absorption maxima at 295 cm-1 due to sucrose and the absorbance was correlated with the content of monoester. The absorbance (100 cm-1) of egg lecithin depended on the purity of phospholipids. The terahertz spectrum method seemed effective for nondestructively evaluating the quality of food emulsifiers.

15-02

Microencapsulation of hydrophilic physiologically active substance with hydrophobic shell material and application

Masato Tanaka

Department of Chemical Engineering, Faculty of Engineering, Niigata University

It was tried to microencapsulate hydrophilic physiologically active substance with hydrophobic shell material.
In this study, L-cystein as core material and tripalmitin as hydrophobic shell material were adopted respectively.
Microencapsulation is based on the melting dispersion cooling method.
Core material was added into the shell solution and mixed, and then poured into the continuous water phase dissolving a stabilizer of given concentration.
To obtain the desired droplet size, this dispersion was stirred with the homogenizer, and then cooled to a room temperature to form microcapsules.
In this fundamental operation, the concentration of soy bean lecithin was changed stepwise and a few fatty acids and fatty acid esters were added into shell solution to modify the physical properties of shell material.
Microencapsulation efficiency was increased with the concentration of soy bean lecithin and changed according to additive species to modify shell material.

15-03

Development and Application of Efficient Purification of Main Contents from Natural Food Colorant such as Turmeric and Monascus Yellow

Hisao Oka

School of Pharmacy, Kinjo Gakuin University
Graduate School of Human Ecology, Human Ecology Major, Kinjo Gakuin University

Recent studies reported that natural food colorant's contents have the biological effects such as anti-tumor initiation, antiinflammation and important pharmacological activities. We report the first procedure for the high, efficient, and low-cost purification of the main contents from natural food colorants such as Turmeric and Monascus yellow. Purification involves high-speed countercurrent chromatography (HSCCC) with a simple two-phase solvent system composed of hexane/chloroform/methanol/water (5/10/7.5/2.5, V/V) for Turmeric, and ethyl acetate/n-butanol/water (4/1/5, V/V) for Monascus. The HSCCC fractionated effluent peaks indicated that the peak resolutions (Rs) were 1.7 between curcumin and demethoxycurcumin, and 2.1 between demethoxycurcumin and bisdemethoxycurcumin for 25 mg of loaded Turmeric powder. In addition, the Rs was 1.8 between xanthomonasin A and B for 10 mg of loaded Monascus yellow. These purified main contents were evaluated by liquid chromatography tandem mass spectrometry with scan and daughter scan positive-modes, and the wide absorbance from 200 to 500 nm was monitored by photodiode array detection. The separation yielded 1.1 mg of curcumin, 0.6 mg of demethoxycurcumin, and 0.9 mg of bisdemethoxycurcumin (>98% purity) from Turmeric powder, and 2.7 and 0.6 mg of xanthomonasin A and B (>95% purity) by HSCCC purification. These reference standards of these contents can be used for various evaluations of their pharmacological activities.

15-04

Prevention of metabolic syndrome by regulation of key molecules using natural food color

Takanori Tsuda

College of Bioscience and Biotechnology, Chubu University

Anthocyanins are pigments widespread in the plant kingdom and used for food additives as a pigment. Anthocyanins have the potency of anti-obesity and possibility of regulation of adipocyte function. In this study, we have shown anthocyanins can suppress down-regulation of adiponectin induced by TNF-a and activation of AMP-activated protein kinase which is recognized as an important energy sensor. Cyanidin and delphinidin significantly suppressed down-regulation of adiponectin in 3T3-L1 adipocytes. Cyanidin-3-glucoside significantly activated AMP-activated protein kinase (phosphorylation of AMP-activated protein kinase subunit at Thr172) during 24 h in myotubes. These data have provided an new insight of anthocyanins related with anti-obesity and anti-diabetes that merit further investigation.

15-05

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

Hiroshi Morita

Faculty of Pharmaceutical Sciences, Hoshi University

Anthraquinones are organic compounds whose structures serve as a basic building block for a number of naturally occurring plant pigments. The compounds have been used in the production of dyes and as a catalyst for the production of wood pulp. Madder color is a coloring agent extracted from the roots of Rubia tinctorum, which has been allowed as one of the existing food additives in Japan until recently. However, many positive data have been reported in mutation tests for lucidin and its related anthraquinones. There are many positive data, but most of them are in vitro test system. Therefore, we are interested in the structures of their metabolites and their safety.
On the other hands, we have been isolated some peptides from Rubia species such as R. akane and R. cordifolia. Some of the compounds showed a potent cytotoxicity against various cell lines. In addition, mollugin, a naphtoquinone has been also isolated from the extract of Rubia tinctorum as one of yellow pigments. We found that iNOS induction of LPS-activated macrophages was shown to be suppressed by mollugin in a concentrate depending manner. These results suggested that mollugin inhibited NO production mainly due to its inhibitory activity against iNOS induction in LPS-activated macrophages.
Nitric oxide (NO) plays an important role in various physiological processes. NO is synthesized by a family of NO synthases (NOS). Among the three isoforms of NOS, inducible NOS (iNOS) is closely related to inflammatory and autoimmune diseases. The suppression of excess NO production in participating cells may be helpful in improving disease status.

15-06

Improvement of Starchy Foods by Acylated Brabched Oligosaccharide Phosphate with Multi-Functional Properties

Koji Takahashi

Faculty of Agriculture, Tokyo University of Agriculture and Technology

Starchy foods easily retrograde, resulting in the reduced quality, and generally less interact to fat and oil giving good body. Surfactants such as sucrose fatty acid ester (SE) are thus often used. However, SE has some large defects such as low solubility, lost emulsifying ability under the acidic reason and the presence of NaCl, and a limited usage due to unacceptable taste. In this study, oleyl branched oligosaccharide phosphate exhibiting a high solubility, superior emulsifying ability, calcium-binding ability, and controlling ability to gelatinization and retrogradation of starch was thus prepared from phosphate-rich potato starch by limitedly hydrolyzing with -amylase followed by exhaustively hydrolyzing with glucoamylase, and then by acylating with lipase.

15-07

Effects of Phosphatic Additives on Skeletal Development and Maintenance

Toshimi Michigami

Osaka Prefectural Hospital Organization,
Osaka Medical Center and Research Institute for Maternal and Child Health,
Department of Bone and Mineral Research

Phosphates are broadly utilized as food additives such as an emulsifier and a meat sticker. Although phosphates are known to be involved in mineralization of bone, their precise roles in differentiation and function of bone cells remain to be elucidated. In the current study, we investigated the effects of phosphates on differentiation and function of osteoblasts and chondrocytes. Treatment of osteoblastic MC3T3-E1 cells and chondrocytic ATDC5 cells with inorganic phosphate, pyrophosphate or polyphosphate induced the phosphorylation of ERK1/2. Moreover, the treatment with pyrophosphate or polyphosphate of ATDC5 cells markedly facilitated the mineralization, which was accompanied with the increased expression of matrix Gla protein (MGP) gene. Treatment with inorganic phosphate also increased the mineralization. As to the expressions of Col2a1 and aggrecan, the marker genes for early chondrocytes, they were suppressed by the addition of phosphate, pyrophosphate or polyphosphate. These results suggest that phosphates might facilitate the transition from the early stage of chondrocytic differentiation to mineralization, and control the skeletal development through the regulation of intracellular signaling and gene expression.

15-08

Comparative Investigation on the Safety Assessment of Food Additives

Yoichi Konishi¹，and Fumitake Fukutomi²

　International information concerned the safety assessment of food additives was continued to collect based on the findings of previous years. The information of this year which contains the present status of the utilization of TTC concept and the future development of the risk assessment of environmental chemicals including food additives, the changing strategy of human risk assessment of the results of carcinogenicity test in animals, alternative models for classical carcinogenicity test and predictive model of chemical toxicity utilizing computer systems (ToxCast Program). The concise monograph entitled "TTC" originally published by ILSI Europe was published in Japanese translation and available for everybody who wants to understand precisely the TTC concept. It is widely utilized for the risk assessment of food additives, especially flavoring in FDA, JECFA and EFSA but not in Japan. It is proposed that the safety value of TTC is 1.5 mg/day and that of chemical structure classes, and are 1,800, 540, and 90 mg/kg B.W./day, respectively and further, safety value of genotoxicity is 0.15 mg/day. ILSI/HESI initiated to find the possible usefulness of TTC concept for the risk assessment of combine exposure of chemical mixtures. The attention should be paid on the advance of this project. The symposium focused on alternative models for classic carcinogenicity tests in animals will be held U.S. STP in its 2009 annual meeting and the value of evaluation of the various models for risk assessment will be discussed. With regard to the changing strategy of the risk assessment on the results of animal carcinogenicity tests, the importance of MOA for epigenetic agent, dose-response reaction by chemicals, and MOE for genotoxic carcinogens are indicated. The computer program for the prediction of chemical toxicity has been introduced by U.S. EPA with cooperation by HESI. This program will be the important source of information related to chemical toxicity for EPA and contribute to the advance in the field of risk assessment of environmental chemicals.

15-09

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

Masayuki Taniguchi

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

In manufacture of Kamaboko, several endogenous proteases autohydrolyse partially proteins in surimi (fish meat paste); the proteolysis by endogenous proteases in surimi causes Modori (softening) and therefore lowering of quality of Kamaboko. In this study, we examined prevention of the softening of Kamaboko using rice protein (oryzacystatin) with protease inhibitory activity.
Our previous study showed that polished rice contains rice protein (oryzacystatin) with protease inhibitory activity. The 10-20% of total protease activity in extract of surimi (pollack and lizardfish) was decreased by adding the extract of rice protein, independent of NaCl concentration in the extract of surimi. The total protease activity in extract of surimi(lizardfish) was about three times higher than that in extract of surimi (pollack).
The kinds of protease in surimi were examined by using protease inhibitor specific to each protease such as cysteine protease, metal protease, and serine protease. The extract of surimi (pollack and lizardfish) contained not only cysteine protease but also metal protease and serine protease. We found that the activities of proteases were affected by NaCl concentration in the extract.
On the basis of these results, we prepared model Kamaboko using surimi (pollack) as a starting material. When the rice protein was added to surimi paste with 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 (pollack and lizardfish) was effective to prevent Kamaboko from Modori (softening) in the presence of NaCl. We also found that the improvement of the breaking force of Kamaboko was responsible for both the partial inhibition of protease activity in surimi and the enhancement of water holding activity by adding rice protein to surimi.

15-10

Development of quality assessment method for optically active food flavor compounds and its biological behavior

Koichi Saito

Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University

A quality assessment method for optically active food flavor compounds, such as borneol, menthol, menthyl acetate, perillaldehyde, and 1,8-cineole, was developed, which utilizes GC-FID for the chemical purity test and GC/MS for the optical purity test. Of the 25 standard samples subjected to the chemical purity test, six were found to have lower purity than that indicated on the reagent labels. When the same samples were subjected to the optical purity test, 13 were found to have lower purity than that indicated on the reagent labels. The results suggest that not only the chemical purity test but also the optical purity test is necessary for the quality assessment of food flavor standards.

15-11

Antioxidant enzymatically modified isoquercitrin or melatonin supplementation reduces oxidative stress-mediated hepatocellular tumor-promotion of oxfendazole in rats

Kunitoshi Mitsumori

Graduate School, Institute of Symbiotic Science and Technology,
Tokyo University of Agriculture and Technology

To clarify whether enzymatically modified isoquercitrin (EMIQ) or melatonin (MLT) supplementation reduces oxidative stress-mediated hepatocellular tumor- promoting effect of oxfendazole (OX), a benzimidazole anthelmintic, male rats were administered a single intraperitoneal injection of DEN and were fed a diet containing OX (500 ppm) for 10 weeks with or without EMIQ (2,000 ppm) or MLT (100 ppm) in the drinking water after DEN initiation. One week after the commencement of the administration of OX, rats were subjected to two-thirds partial hepatectomy. The number of GST-P-positive foci promoted by OX was significantly inhibited by the combined antioxidant EMIQ or MLT administration, and the area of GST-P-positive foci was inhibited by the administration of MLT. Real-time RT-PCR analysis revealed decreases in mRNA expression levels of Cyp2b2 and Me1 in the DEN-OX-EMIQ and DEN-OX-MLT groups and decreases in mRNA expression levels of Cyp1a1 and Akr7a3 in the DEN-OX-MLT group compared to those in the DEN-OX group. In in vitro ROS production assay, inhibited production of NADPH-dependent ROS was observed by the treatment with EMIQ or MLT. These results suggest that coadministration of EMIQ or MLT suppresses the hepatocellular tumor-promoting activity of OX in rats through the decrease in ROS production by hepatic microsomal activation.

15-12

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

Mamoru Totsuka

Department of Applied Biological Chemistry, The University of Tokyo.

Intestinal epithelial cells (IECs) maintain an intestinal barrier function, and also play an important role in regulation of the intestinal immune system. In the present study, we aimed to evaluate the safety and physiological functionality of dietary factors by analyzing their effects on immune-related functions of intestinal epithelial cells. For this objective, we established a novel evaluation system using mouse small intestinal epithelial cell (sIEC) lines we established recently. In previous studies, we found that melatonin suppressed IL-6 secretion from a mouse sIEC line stimulated with bacterial components or proinflammatory cytokines. We further investigated the effect of melatonin on cytokine response of IECs by a DNA microarray analysis. Melatonin was found to down-regulate mRNA expression of IL-1a, IL-11, LIF (Leukaemia Inhibitory Factor), G-CSF, GM-CSF, CXCL2/MIP-2, and CCL20/MIP-3疝och by a mouse sIEC cell line iMoS4 stimulated with bacterial components. Next, we examined anti-inflammatory effect of melatonin in vivo using dextran-sodium sulfate (DSS)-induced colitis model. Oral administration of melatonin down-regulated fecal occult blood score and serum IL-6 concentration significantly. These results indicate that melatonin modulates cytokine response of IEC and suppresses intestinal inflammation.

15-13

A Survey of Literatures Concerning Chemical Sterilization and Preservation of Food and the Construction of Antimicrobials Database

Tetsuaki Tsuchido¹ and Ichiro Nakamura²

We built a database ChemoKill Database R0305b of chemical sterilization and preservation for sanitation and sterilization of environments of food manufacturing and also for food preservation, based upon the data extracted and collected from papers published in two major academic journals from 2003 to 2005. It contains data concerning bibliography, cell death results, treatment conditions, and food preparation conditions on 85 items as a total and, in addition, it has a lookup function. Since chemicals have also been used in heat sterilization and pasteurization, we extracted the data of microbial death caused by a combination treatment with heat from the thermal death database, ThermoKill Database R0305, and designated as ThermoChemoKill Database R0305 on the basis of papers published from the same period as the above. For the analysis of data statistics, the database R0105, which contained not only R0305 but also R0102, was used. These databases are expected as useful tools for not only food manufacturing but also medical and environmental treatments for microbial decontamination.

15-14

Behavior of microorganisms from decontaminated food additives within foods

Masakazu Furuta¹ and Masao Kishida²

The purpose of the study is to investigate the microbial growth recovery in response to killing stresses such as heating and 60Co-gamma irradiation especially within the processed meat products. We employed flow cytometry, MTT assay, pulse field gel electrophoresis to monitor cell damage of Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis, and Bacillus megaterium after the irradiation stress. These assay revealed that the irradiation stress caused the delay of the time point of growth recovery accompanied with nucleic acid synthesis according to increasing doses. Irradiation also primarily broke the double strand of genome DNA of S. cerevisiae as reported previously while heating caused damage on their respiration systems first, then membrane transport system.
Black pepper powder treated by each process in which less than 1000 microbial loads was enumerated by aerobic plate counts was mixed with minced sausage and incubated at 30oC for several days. Outgrowth of microorganism was monitored according to the procedure described by Japanese Food Hygiene Law.
Sausage samples containing the treated black pepper powder showed the similar microbial growth from less than 1000 CFU/g of the sample to approximately the order of 109 CPU/g of the sample during the incubation at 30oC, irrespective of gamma-irradiation or superheated-steam treatment. There were no significant differences between gamma-irradiation and superheated-steam treatment in outgrowth of the surviving microorganisms when the treated black pepper are added to sausage.

15-15

Studies on the elicitation of sweetness of thaumatin

Tetsuya Masuda and Naofumi Kitabatake

Thaumatin is a sweet-tasting plant protein extracted from the aril of the fruit of Thaumatococcus daniellii Benth. In order to clarify the mechanism responsible for elicitation of the sweetness of thaumatin, we prepared mutant thaumatin proteins, using yeast Pichia pastoris. The results indicated that K67 and R82 were particularly important for eliciting the sweetness of thaumatin. To clarify the structure-sweetness relationships in thaumatin, we performed high resolution crystal structure analysis of the mutants on K67 and R82. The overall structures of these mutants are identical to that of plant thaumatin as well as recombinant thaumatin. However the surface electric potential on the cleft containing side of the thaumatin was dramatically changed by the mutation at R82. These results demonstrated that the strict surface electrostatic potentials at the specific site formed by R82, K67 are required for thaumatin sweetness and not the e-amino group but the guanidino group at R82 would be important for sweetness of thaumatin.

15-16

Analysis of anti-noroviral effects of persimmon tannin

Tadashi Shimamoto

Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science, Hiroshima University

Noroviruses cause nonbacterial gastroenteritis worldwide. Although sodium hypochlorite is commonly used for disinfection of noroviruses, it is a hazardous chemical and requires careful handling. Alternative disinfectants for noroviruses need to be developed. However, due to the absence of an in vitro cell culture system or small animal model, detection of infectious noroviruses has been impossible. Evaluation of disinfectants for noroviruses has been very difficult.
We recently discovered that a formulation of persimmon tannin in ethanol could inactivate noroviruses. This discovery was confirmed by using the real-time reverse transcription (RT)-PCR base detection system. A new disinfectant was created to disinfect more than 99% of noroviruses in 30 seconds. This disinfectant contains only natural persimmon tannin and other food additives and can be applied safely in areas where cookware, dishes, and even food are exposed.

15-17

Identification analysis of major allergen proteins in cochineal dye

Hiroshi Akiyama

pision of Novel Foods and Immnochemistry, National Institute of Health Sciences

Carmine is a natural red pigment obtained from the dried gravid female cochineal insects (Dactilopius coccus or Coccus cacti). There have been several reports of allergies to carmine, but the major allergens responsible have not been identified. To identify the major allergenic proteins in cochineal. Immunoblots of purified cochineal extract were probed with sera from three allergic patients. Partial amino acid sequences were determined for the proteins bound by IgE and the corresponding cDNA, containing a complete coding region, was cloned by 5'- and 3'- rapid cDNA extension (RACE) and PCR. The recombinant protein was expressed in yeast and subjected to immunoblotting. We identified a full-length cDNA encoding a protein, which we named CC38K, with 335 amino acids and a molecular mass calculated as 38 kDa. This amino acid sequence included all the partial amino acid sequences obtained from the purified proteins identified by IgE from allergic patients. Recombinant CC38K protein was recognized by patients' sera indicating that this is a major allergen present in carmine. The CC38K sequence showed homology to phospholipases. We have, for the first time, identified the major allergen in cochineal extract. This protein may be a phospholipase or related enzyme, which in other insects are known to be allergens.

15-18

Thermal Inactivation of a Sweet Protein Thaumatin and Its Protection by Phosvitin

Naotoshi Matsudomi

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University

The sweet protein thaumatin became insoluble on heating in a phosphate buffer (pH 7), and the sweetness disappeared. The thermal inactivation and aggregation of thaumatin were examined by heating at 80ｰC for 15 min in pH 7 under various anions. The thermal aggregation of thaumatin was enhanced strongly in phosphate and citrate. In the presence of phosvitin, however, the thaumatin insoubilization was suppressed completely, the sweetness was retained by the addition of 0.01% phosvitin to 0.1% thaumatin. The mechanism for phosvitin inhibition against thermal inactivation of thaumatin was investigated from CD and tryptophan fluorescence spectra analyses. The results indicate that thaumatin was less stable to heat in the presence of phosvitin, while the thermal aggregation of thaumatin was prevented by interaction of a denatured thaumatin with phosvitin. Such an interaction may be possible to assist refolding of the denatured thaumatin.

15-19

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

Hideyuki Ito

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

Interactions between food components and drugs have recently come into focus of interest. The interactions often occur as a result of inhibiting or inducing of the metabolic enzymes, cytochrome P450 (CYP). It is well known that concomitant intake with grapefruit juice increases the concentration of calcium antagonists in human by suppression of CYP3A4 activation. We have investigated the inhibitory effects of 30 natural food additives on CYP3A4 or CYP2C9 activities. In our previous report, Turmeric oleoresin and Kaoliang color among tested additives exhibited potent inhibition on CYP3A4 and CYP2C9 activations. Here we studied the characterization of the inhibitor of CYP3A4 or CYP2C9 in Kaoliang color. Kaoliang color was extracted with n-hexane, ethyl acetate and water saturated n-butanol, successively. n-Hexane extract that was shown inhibitory activity against CYP3A4 and CYP2C9 activations, was analyzed by reversed-phase HPLC, to identify apigenin as a CYP inhibitor. The eluate from MeOH with Diaion column chromatography of Kaoliang color extract was analyzed by gel permeation chromatography. A fraction composed of the large molecular weight compounds was characterized as potent CYP inhibition. These results suggested that constituents of a part of natural food additives may have the potential to inhibit the metabolism of drugs, so that further in vivo study should be investigated to clarify the clinical relevance.

15-20

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

Nobuyuki Sakai

Department of Human Sciences, Kobe Shoin Women's University

In our everyday life, we are exposed to many stressors that induce mental stress, and consequently many people may suffer mental illness, such as depression, bipolar mood disorders, and so on. Although there are many empirical studies showing that smelling an aroma or eating/drinking something is useful for easing mental stress, those studies did not use standardized methods for evaluation of stress and/or for easing stress. Preceding studies also used simple and boring tasks, such as Kraepelin test requiring mental subtraction of a number sequence. In this study, the strategy, which university students use to ease stresses in their daily lives, were surveyed, and first investigate their effect on easing stress. Then convenience task using SUDOKU puzzles for evoking mental stress were introduced. At last, convenient but effective methods for easing stress were investigated. The third study revealed that the aroma of coffee had a suppressive effect on stress, but the aromas of grapefruit, orange and hinoki did not suppress mental stress. Also drinking canned coffee, bottled water, cola drink, or herb drink did not have a suppressive effect on mental stress. This study suggests that using coffee aroma as ambient odor suppresses mental stress. At the last study, anticipation effect was investigated. Participants presented herb drink with a description that the beverage has an easing effect on mental stresses showed a significant suppression of stress after drinking the beverage.

15-21

Pharmacokinetis and mechanisms for intestinal absorption of several flavonoid glycosides and the suppressive effects of these compounds on blood glucose increase

Toshiaki Makino

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

Quercetin, a flavonol contained in various vegetables and herbal medicines, has various biological activities including anti-cancer, anti-allergic and anti-oxidative activities. However, its low oral bioavailability due to insolubility in water has limited its use as a food additive or dietary supplement. Since the water solubility is enhanced by glycosyl conjugation, in the present study, we evaluated the bioavailability of several quercetin glycosides with different sugar moieties in rats. Quercetin, quercetin-3-O-rutinoside (rutin), and quercetin-3-O-glucoside (isoquercitrin, IQC) in suspension, and quercetin-3-O-maltoside (Q3M), quercetin-3-O-gentiobioside (Q3G), a-monoglucosyl rutin (aMR), a-oligoglucosyl rutin (aOR), and enzymatically modified isoquercitrin (a-oligoglucosyl isoquercitrin, EMIQ) dissolved in water, were orally administered to rats under anesthesia. Bioavailability (F value) was calculated from the concentrations of total quercetin in plasma from 0 to 12 hr after the administration. F value of quercetin was 2.0 %, and those of IQC, Q3M and EMIQ were 12%, 30%, and 35%, respectively, suggesting that elongation of a-linkage of glucose moiety in IQC enhances the bioavailability of quercetin. Although Q3G, MR and OR have high water solubility, their F values were low (3.0%, 4.1% and 1.8%, respectively). In the in vitro study, the homogenate of rat intestinal epithelium rapidly hydrolyzed IQC, Q3M and EMIQ to quercetin, and aMR and aOR to rutin. However, it could not hydrolyze Q3G or rutin to quercetin. These results suggest that intestinal epithelial enzymes such as lactase phrolizin hydrolase or mucosal maltase-glucoamylase play important roles in the hydrolysis and absorption of flavonol glycosides.

15-22

Preventive functions against disease-germs of outer skin chitinase prepared from citrus fruits

Yukio Takii

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

Chitinase activities as for preventive functions against the disease germs were surveyed among the outer skin extracts obtained from 54 fruits and vegetables. One of the citrus fruits exhibiting highly amounts of chitinase was selected to be chitin destructive materials. The enzyme was purified further to electrophoretically homogeneous state and its molecular weight was determined to be 30,000.

15-23

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

Kaeko Murota, Yoshichika Kawai, and Junji Terao

Institute for Health Biosciences, the University of Tokushima Graduate School

Quercetin is a major flavonol-type flavonoid distributed in a wide variety of vegetables and other plant foods. Numerous epidemiological studies and animal experiments suggest the protective effect of quercetin against various diseases, and it may be associated with the antioxidative property. Quercerin mostly exists as its metabolites in the body. Thus, the estimation of its bioavailability is crucial for understanding the efficacy of this flavonoid intake against oxidative stress and related disorders. Sugar moiety plays a key role on modifying flavonoid bioavailability, although the previous human studies are not enough to verify the effect in humans' cases. In this study, human bioavailabilities of naturally occurred quercetin glucosides were compared with that of enzymatically modified isoquercitrin (EMIQ). EMIQ was shown to be absorbed more efficiently than other glucosides when taken up with water, as well as with cooked onion. Plasma concentration of quercetin metabolites after EMIQ intake reached the maximum level at 1.5 h after intake and subsequently decreased, indicating that EMIQ could be absorbed in the proximal intestine. EMIQ intake could not enhance the antioxidative capacity of plasma lipoproteins ex vivo.

15-24

Analysis of molecular behavior of glycyrrhizin, the main active compound in licorice, by natural compound-specific monoclonal antibodies

Takuhiro Uto, Osamu Morinaga, and Yukihiro Shoyama

Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagasaki International University

Licorice (Glycyrrhiza spp.) is worldwide food additive and important ingredient used in 70 % of traditional Chinese medicine (TCM). Glycyrrhizin (GC), a main saponin component of licorice has been reported to possess numerous pharmacological effects like anti-inflammation, anti-ulcer, anti-tumor, anti-allergy, and hepatoprotective activities. However, the molecular mechanisms underlying its effects are not completely understood. In this study, we aimed to elucidate the molecular behavior and intracellular localization of GC and its metabolite, glycyrrhetinic acid (GA), by using anti-GC and anti-GA specific monoclonal antibodies (MAb).
GC inhibited pro-inflammatory mediators such as prostaglandin E2 (PGE2) and nitric oxide (NO), and cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264 cells. ELISA by anti-GC MAb showed that intracellular GC was observed immediately after treatment and reached a plateau at 6 h. Moreover, immunocytochemistry demonstrated that GC localized in cytoplasm. The analysis of GC-free licorice extract (GC knock-out extract) by immunoaffinity column conjugated with anti-GC MAb showed that GC may exert synergistic suppression of NO production with the other constituents contained in licorice extract. GA, but not GC, strongly induced apoptotic cell death of human colon cancer Caco-2 cells. ELISA by anti-GC and anti-GA MAb indicated the cellular uptake of GA was much higher than GC.

15-25

Blood pressure depressing effects of polyphenols with acid-amide bonding, contained in raw food materials, and its relation to chemical structures and action mechanism

Kiharu Igarashi

Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University

To determine physiological functions of polyphenols with acid-amide bonding such as caffeoyltyrosine and p-coumaroyltyrosine, which are found in cacao beans, inhibitory effects of these compounds on angiotensin I converting enzyme (ACE) were determined in vitro. Effects of oral administration and feeding of these compounds on systolic blood pressure and its related markers were also determined on spontaneously hypertensive rats (SHR). Although ACE inhibitory activity in vitro was found in both caffeoyltyrosine and p-coumaroyltyrosine, the activity was stronger in the former than in the latter. On the other hand, ACE-inhibitory activity of tyrosine was weaker than that of these two compounds, indicating that cinnamic acid moiety of these two compounds was important for stronger ACE-inhibitory activity. The blood pressure-depressing effect of caffeoyltyrosine by its oral administration tended to be stronger than that of p-coumaroyltyrosine.
Feeding of caffeoyltyrosine and p-coumaroyltyrosine to SHR over 22 days suppressed increases in blood pressure observed in the control, but there were no differences in the suppressing effects between these two compounds. Although ACE activity in the kidneys in SHR did not differ between the control groups and groups fed caffeoyltyrosine and p-coumaroyltyrosine, the serum concentration of NO-related compounds tended to be higher in groups fed these compounds, suggesting that these two compounds showed anti-hypertensive effects via ACE-inhibitory activity and enhancement activity of NO production.

15-26

Stabilization of plant polyphenols by interaction with food proteins and cyclic polysaccharides

Tsutomu Nakayama, Takeshi Ishii, and Shigenori Kumazawa

School of Food and Nutritional Sciences, University of Shizuoka

Several lines of evidence indicate that plant polyphenols have poor stability in neutral or alkaline solutions. (－)-Epigallocatechin gallate (EGCg) is the major component of green tea and is known to show strong biological activity, although these can be easily oxidized under physiological conditions. In this study, we indicate that EGCg is stable in aqueous solution with milk protein and that casein stabilizes EGCg. In addition, EGCg is also stable in aqueous solution with cyclodextrin (-CD). Under these conditions, EGCg binds to both non-covalently. However, (－)-Epigallocatechin (EGC) lacking the galloyl group, is unstable in solution with both casein and -CD, and binding affinity with these was lower than that of EGCg. These results suggest that the binding capacity of food proteins and cyclic polysaccharides contribute to the stabilization of plant polyphenols in aqueous solution.

15-27

Screening of essential oils and their components which have relaxation effect.

Hitoshi Aoshima

Applied Molecular Bioscience, Graduate School of medicine, Yamaguchi University

Inonotropic g-aminobutyric acid (GABA) receptors (GABAA receptors) are main inhibitory neurotransmitter receptors and target of tranquillizers, sleeping drugs, anesthetics and ethanol. In our previous research, many aromas have induced similar effect on GABAA receptors. In this study, we measured the effect of essential oils of trees and herbs, and alcohol extracts of Bangladeshi medicinal plants and fruits on the electrical response of GABAA receptors expressed in Xenopus oocytes by injecting their cRNAs and found that several samples potentiate the response. Their effects on the pentobarbital-induced sleeping time of mice were also examined by intraperitoneal, oral and/or inhalational administration prior to intraperitoneal administration of pentobarbital, which is known to act on GABAA receptors. Co-administration of several essential oils prolonged the sleeping time. These essential oils and the extracts may modulate our mood through acting on GABAA receptors and induce relaxed feeling, and can be used for the development of foods, food additives and supplements which induce the mental relaxation.

15-28

Quality evaluation of essential oils by a taste-sensing system

Nobuo Kawahara

Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation

Recently, it has been recognized effectiveness and functionality of aromatherapy, a natural holistic approach to therapy using essential oils and other plant extracts. Many common essential oils have medicinal properties that have been applied in folk medicine since ancient times and are still widely used today.
Essential oils are registered in "The Japan's Specifications and Standards for Food Additives" mainly used as bitter substances and anti-oxidants, and also 7 essential oils (Orange oil, Turpentine oil, Eucalyptus oil, Cinnamon oil, Clove oil, Fennel oil and Mentha oil) are registered in "The Japanese Pharmacopoeia Fifteenth Edition".
In the course of our study for development of a new method for the quality evaluation of essential oils, we investigated the profile analysis of 17 kinds of essential oils by a taste-sensing system. As the results, 17 kinds of essential oils were classified into 7 types by the taste distributions. Furthermore, the taste pattern of clove oil was almost same as eugenol, a main component of clove oil and good correlation between the taste intensity of anionic bitterness and the content of eugenol was observed in clove oil. Therefore, the taste-sensing system may be suitable as the quality evaluation of essential oils.

15-29
Chemical Analyses of the volatile components from lavender (Lavandula angustifolia Mill) leaves and stems

Five samples of lavender (Lavandula angustifolia Mill) including four different varieties were collected in Hokkaido Furano area at five different sites. The leaves and the stems of the samples were soaked into dichloromethane and the extracts were analyzed using GC/MS. Eighty peaks were observed and 58 of them were identified or their reasonable structures were estimated. Most abundant components common to the all samples were coumarin (4) and 7-methyl coumarin (6), which accounted for about 80% of the observed peaks. Statistical analyses (a clustering analysis, a principal component analysis) were performed on the abundance of the 80 components. The results were as follows: (I) chemical profiles of the samples which belong to the same variety collected at the same site were essentially the same regardless their collecting time of the day, (II) the different varieties of lavender showed different chemical profiles.

15-30

Identification of volatile aroma compounds in the original citrus fruits in Yamaguchi Prefecture

Yoshihiko Akakabe

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University

The citrus fruit Setomi is a hybrid of Yoshiura ponkan (Citrus reticulate Blanco) and Kiyomi (Citrus unshiu Mrcov. x Citrus sinensis Osbeck) and is evaluated that Setomi fruit has a mellow and a suitable balance of sweet and sour. However, there are no reports concerning the aroma volatiles in Setomi fruits. The aim of this study was to assess the aroma volatiles in the peel of Setomi and its parent cultivars. Furthermore, the author attempted to evaluate the aroma of Setomi by an electronic nose analysis.
The essential oils from the peel of Setomi and its parent cultivars were obtained by a simultaneous distillation extraction technique. Comparing the essential oils of Setomi and its parent cultivars, it was found that volatile components from both parent cultivars were present in the peel of Setomi. However, it was consisted not only inherited compounds from parent cultivar's components such as monoterpenes and aldehydes, but also composed of the original constituents and quantities such as p-cymene, dehydro-p-cymene, d-elemene, (E)-b-farnesene, germacrene D, carvone, (E,E)-a-farnesene, germacrene B, and a- and b-sinensal. Principal component analysis of data obtained with an electronic nose indicated that the odor of Setomi showed displacement in a positive direction and a downward displacement as compared with those of Yoshiura ponkan and Kiyomi in PC1. Furthermore, the oils of Setomi showed displacement in a negative direction and an upward one in PC2. By the principal component analysis, it was found that the odor quality of Setomi was different from those of the parent cultivars.

15-31

Fundamental Study for Safety Evaluation of Food Additives with Optical Activities

Food additives include optically active compounds, such as amino acids, sugars, and organic acids. Some optical isomers of the optically active compounds exert clearly different effects on the body. However, the present component standards of food additives do not include tests for the accurate determination of the ratios of optical isomers (optical purity). Hence, an attempt was made to develop test methods for the accurate determination of optical purity.
1. Optical isomer analysis for amino acids in foods by liquid chromatography-mass spectrometry
　　A sensitive and selective method for the determination of amino acids in foods such as the fermentation foods and soft drink has been developed by liquid chromatography-mass spectrometry (LC/MS). The LC separation was performed on a Chirobiotic TAG column (2.1 mm x 25 cm) with 0.05% acetic acid-ethanol (60:40) as the mobile phase. L-leucine and L-isoleucine was analyzed by LC/MS with gradient elution. This analytical method was applied to fermentation foods for 20 kinds of amino acids measurement. This method is useful as an optical isomer analysis for amino acids in food.
2. Enantiomeric determination of proline in honey by LC-UV
For the chiral separation and quantification of L-proline used as food additive, a selective pretreatment method named "two-step derivatization," which uses o-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC-Cl), was examined, and LC-UV was developed. Most of the impurities that were converted into OPA derivatives were removed by solid-phase extraction, and L-proline and D-proline were converted into FMOC derivatives. Sufficient cleanup of honey and royal jelly samples was achieved with this two-step derivatization method, and selective pretreatment of secondary amino acids was possible. The average recovery rates of L-proline and D-proline from honey and royal jelly were as high as 81.3 % to 98.6 % (RSD: <2.7%). Using this method, L-proline and D-proline in honey and royal jelly can be separated optically and determined.
3. Optical isomer analysis for aspartame using amino acid analyzer
Analysis for aspartame (APM) isomers using amino acid analyzer was investigated. The conditions were as follow : column, LCR-6(4mm i.d.ﾗ120mm) ; precolumn, LCR-7(4mm i.d.ﾗ70mm) ; mobile phase, sodium citrate buffer pH4.25 : pH9.70 (6:4,v/v) ; column temperature,70℃ ; flow rate, 0.35mL/min ; detection, 570nm ; injection volume, 50ylochL. This method could detect 0.04% D-APM in L-APM.

4. An analysis of racemic organic acid food additives utilizing high performance liquid chromatography with UV detection
We have developed a method for determining racemic organic acids, malic acid, tartaric acid and lactic acid, utilizing high performance liquid chromatography (HPLC). These compounds were separated on a reverse-phase octadecyl silica gel column coated with N,S-dioctyl-d-penicillamine (SUMICHIRAL OA-5000, monolithic-type, 4.6mmﾗ100mm). As a mobile phase, 1mM cupric acetate-0.05M ammonium acetate (pH4.5)/isopropyl alcohol (IPA) (85:15) for d,l-malic acid and d,l,meso-tartaric acid, and 2mM CuSO4-IPA (85:15) for d,l-lactic acid. The column was heated at 40ｰC and a UV absorption detector was set at 254nm. The caliblation graphs were linear in the range of 10-100mg/mL for malic acid and tartaric acid, and 10-500mg/mL for lactic acid. The present method is to be useful for the analysis of racemic organic acids.

5. Simultaneous liquid chromatographic determination of racemic aspartame, a food additive, and their degradation products.
A simple and efficient method has been developed for determining of racemic aspartame (APM) and their degradation products, diketopiperazine (DKP) and D,L-phenylalanine (Phe), utilizing high performance liquid chromatography (HPLC). These compounds were separated on a reverse-phase octadecyl silica gel column coated with N,S-dioctyl-D-penicillamine (SUMICHIRAL OA-5000, monolithic-type, 4.6mm x 100mm), using 2mM CuSO4-acetonitrile-isopropyl alcohol (85:10:5) as a mobile phase. The column was heated at 30ｰC and a UV absorption detector was set at 254nm. The detection limits of L-APM, D-APM, DKP and D,L-Phe were 10mg/mL, 1mg/mL, 10mg/mL and 0.1mg/mL, respectively. The concentrations of D-APM and DKP contained in L-APM are prescrived below 0.04% and 1.5% in the Japan's specifications and standards for food additive. The mixture of L-APM 2500mg/mL, D-APM 1mg/mL (0.04% for L-APM) and 37.5mg/mL(1.5% for L-APM) was analyzed using this method.

6. Chiral separation and quantification of amino acids in foods
For the chiral separation and quantification of amino acids in foods, 16 D,L-Amino acids are derivatized with chiral reagent, N-(2,4-dinitro-5-fluorophenyl) -L-alaninamide (FDAA), and then separated via reversed phase high performance chromatography followed by electrospray ionization tandem mass spectrometry detection. Using this method, D,L-amino acids in fish sauce and vinegar can be separated and quantified.

15-32

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

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