Research Reports No.8 Abstract(2002)


Establishment of Antioxidant Assay System Using Fish Liver Microsome
-Development of a New Experimental Animal Substituted for Rat

Hideo Hatate
Faculty of Agriculture, Miyazaki University

In order to assess the adaptability of fish tissues to antioxidative assay, we performed fatty acid analysis of lipids extracted from the ordinary muscle, brain, liver, and liver microsomes of tilapia Oreochromis niloticus. Although the ordinary muscle was poor in fatty acids, the brain and liver had a large amount of polyunsaturated fatty acids such as arachidonic and docosahexaenoic acids. These fatty acids are susceptible to active oxygen species and are thus essential for the experimental animal tissues used for evaluating antioxidant activity. Furthermore, the fish liver microsomes was prepared successfully from tilapia liver by an ultracentrifugation and they had a large amount of polyunsaturated fatty acids enough to be available as oxidative substrate for antioxidative assay. Tilapia could survive without diet for more than two months. The liver weight decreased quickly during the starvation, along with decrease of significant amount of indwelling a-tocopherol in the liver, of which antioxidative activity would disturb assay of other antioxidant sample. This result indicated that preferable experimental fish lucking confusable a-tocopherol will be prepared only by starvation.


Study on stability of red color component, crocin

Yukihiro Shoyama
Graduate School of Pharmaceutical Sciences, Kyushu University

Crocetin-glucosides are sensitive for the presence of oxygene, light irradiation and moisture. It becomes evident that storage of saffron at -20 ℃ promotes the constant supply of saffron.
Saffron crude extracts and crocin antagonized the inhibitory action of ethanol on the central nervous system in terms of learning behavior in mice and LTP in the dentate gynus of hippocampus in rats.
Although it was well known that saffron extracts have anti-tumor activities, this is the first finding of anti-skin tumor promotion activities of saffron and crocin.
We make clear that crocin suppresses the cell death of neuronally differentiated PC-12 cells induced by tumor necrosis factor.
Crocetin-glucosides are sensitive for the presence of oxygene, light irradiation and moisture. It becomes evident that storage of saffron at -20 ℃ promotes the constant supply of saffron.
Saffron crude extracts and crocin antagonized the inhibitory action of ethanol on the central nervous system in terms of learning behavior in mice and LTP in the dentate gynus of hippocampus in rats.
Although it was well known that saffron extracts have anti-tumor activities, this is the first finding of anti-skin tumor promotion activities of saffron and crocin.
We make clear that crocin suppresses the cell death of neuronally differentiated PC-12 cells induced by tumor necrosis factor.


Antioxidant activity of quinoa and its utility as antioxidant food-stuff

Katsumi Watanabe
Department of Food and Nutritional Science, Faculty of Agriculture, Kinki University

Albumin and globulin consisted of 35.7 wt% and 21.9 wt% of the total protein of quinoa. On the contrary, prolamin and glutelin consisted only of 3.6 wt% and 3.3 wt%. The protein composition of quinoa differed from that of rice and wheat. Quinoa protein contained large amounts of lysine and S-containing amino acids, compared to rice and wheat. On the other hand, quinoa had many antixoidants effective in inhibition of lipid peroxidation and/or radical scavenger. These results suggested that quinoa was available for resources of protein and antioxidant food-stuff.


Studies on genes and enzymes involved in the biosynthesis anthocyanins
in Ipomoea batata cv. Yamagawamurasaki.

Ikuro Abe
School of Pharmaceutical Sciences, University of Shizuoka

The physiological function of chalcone synthase, the key enzyme involved in the biosynthesis anthocyanins in Ipomoea batata cv. Yamagawamurasaki, the formation of naringenin chalcone. The enzyme uses 4-coumaroyl-CoA, performs three condensations with malonyl-CoA, and folds the resulting tetraketide into a new aromatic ring system. We investigated the capacity to build novel and unusual polyketides from alternative substrates. Three types of products were obtained: (i) complete reaction (chlcone-type), (ii) three condensations without formation of aromatic ring (CTAL-type pyrone derailment), (iii) two condensations (BNY-type pyrone derailment). All product types were obtained from 4-fluorocinnamoyl-CoA and analogs in which the coumaroyl moiety was replaced by furan or thiophene. The results show that minor modifications can be used to direct the enzyme reaction to form a variety of different and new products. Manipulation of the biosynthesis of polyketides by synthetic analogs could lead to development of a chemical library of pharmaceutically interesting novel polyketides.


Studies on effects of food additives on adipocyte differentiation

Masayoshi Imagawa
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences,
Nagoya City University

To elucidate the effects of food additives on adipocyte differentiation, two assay systems were developed; one is a yeast two-hybrid system and another one is a PPARg-expressing cell line. These two systems were applied to eight kinds of food additives, and clear effects were not observed. The preadipocyte cell line was also used for testing the effects of food additives on the multiple steps of adipogenesis, and ascorbic acid was found to be an activator for adipocyte differentiation. Although it is required that other kinds of additives are tested and also effects on adipogenesis should be more characterized in detail, these studies seems to be a new approach to elucidate the relationship between food additives and obesity.


Study for inhibitory effects of citrus essential oils and terpene compounds
on the formation of N-nitrosodimethylamine

Masayoshi Sawamura
Faculty of Agriculture, Kochi University

Citrus essential oils and their components were studied for inhibitory effects on the formation of N-nitrosodimethylamine (NDMA). The reaction mixture consisted of dimethylamine and sodium nitrite adjusted at pH 3. 6, in addition to essential oils and an emulsifying agent. The quantification was determined by high-performance liquid chromatography monitored at 220 nm. All of the essential oils inhibited the formation of NDMA in the range of 22-85%. The oils of ujukitsu (Citrus ujukitsu Hort. ex Shirai), yuzu (C. junos Tanaka), mochiyu (C. inflata Hort. ex Tanaka) and ponkan (C. reticulata Blanco cv. F-2426) inhibited the formation of NDMA much more effectively than other citrus oils. The inhibitory proportion of components of citrus essential oils such as myrcene, a-terpinene and terpinolene was as high as 80%. This suggests that the high activity of citrus essential oils was induced by double bonds in their compounds. Yuzu essential oil was also studied for inhibitory effects on the formation of NDMA in various vegetable foods. Twenty-five kinds of vegetables were examined and the rate of NDMA inhibition was 40% at the highest. The samples of onion, Chinese chive and spinach demonstrated high inhibitory effectiveness. NDMA inhibition effectiveness increased to 75-85% when yuzu essential oil was added to the vegetable samples, especially in the case of bean sprouts, eggplants and sweet peppers.
This result shows that yuzu essential oil enhances the inhibition of the formation of NDMA in vegetables; in other words, yuzu essential oil's inhibitory function is not lost, even in vegetables.


Masami Ishibashi
Graduate School of Pharmaceutical Sciences, Chiba University

Biological activity tests on 77 plants extracts collected mainly in Thailand were carried out against toxicity against a crustacea Artemia salina (brine shrimp) and toxicity against murine lymphocytes, and several plants extracts proved to be active against these toxicity tests.
Chemical constituents of a Zingiberaceous plant Cantimbium speciosum, whose extract was toxic against A. salina, were investigated, and four flavonoids, pinocembrin (CS-1), naringenin (CS-2), 3-methylkaempferol (CS-3), and pinocembrin chalcone (CS-4) were isolated. Particularly pinocembrin (CS-1) exhibited toxicity against A. salina. Three new natural onoceranoid triterpenes, lansionic acid (LD-2), a, g-onoceradiene-21-one-3-ol (LD-3), and a, g-onoceradiene- 3-one-21-ol (LD-5) were isolated from the fruit peel of Lansium domesticum (Meliaceae) together with two known triterpenoids (LD-1 and LD-4), and their structures were elucidated mainly from spectral data. These triterpenoids exhibited toxicity against A. salina at 100 mg/mL.
Chemical constituents of a Euphorbiaceous plant Bridelia siamensis, which was shown to be toxic against murine lymphocytes, were examined to obtain nine compounds including six known and three new compounds. The structures of three new compounds were elucidated mainly from spectral data, and two were found to be new coumarin-type natural products (BS-7 and B-8) and one proved to be a new flavonoid glycoside, myricetin 3-rhamnoside-3'-rhamnoside (BS-12). The major coumarins, BS-1 and BS-5, were found to be toxic against murine lymphocytes.


Elucidation of pharmacokinetics of natural plant sterol as emulsifiers for safety

Yoshie Maitani
Fine Drug Targeting Research Laboratory, Institute of Medicinal Chemistry,
Hoshi University

Plant sterols as natural additives including b-sitosterol (Sit) are used as emulsifier of o/w emulsion for improvement of manufactured processes and quality of elaborated food. However, the effect of Sit on the biological membrane in the intestine and the liver is not clarified yet. Therefore, in this study, these effects were examined compared with cholesterol (Chol) forming three kinds of particles such as emulsions, liposomes, and nanoparticles.
Like Chol, the particles of Sit did not affect the intestinal absorption of FITC-dextran 4, 400 (FD-4) used as a model compound following infusion of their particles and FD-4 in the small intestine. These particles at 25 mM concentration of sterols did not affect the integrity of the membrane in HepG2 cells since uptake amount of FD-4 into HepG2 cells was not increased, but 1, 205 mM concentration of Sit and Chol particles decreased the survival of HepG2 cells with a significant increase of FD-4 uptake in HepG2 cells. Sit nanoparticles showed significantly this effect. The internalization of Sit and Chol nanoparticles labeled by fluorescent dye in HepG2 cells was also observed by confocal laser microscopy. From these findings, it was demonstrated that Sit is safety as the natural additives like Chol.


Control and Elucidation of Biomechanism by the Cooperative Effects
of Sugar and Biologically Originated Dye

S. Yano
pision of Material Science, Graduate School of Human Culture,
Nara Women's University

From the viewpoint of quality of life, recently, much attention has been focused to photodynamic therapy (PDT) as clean and new modalities of selective treatments of cancer. PDT treatment, which results in the destruction of the cancer tissue, depends on the presence of molecular oxygen, and hence this is the example of the photodynamic effect. The photosensitiser employed for PDT must have not only (1) good phototoxicity and (2) low dark toxicity, as well as (3) tumor localization tendency. Furthermore, the photosensitizer must have (4) light absorption window at longer wavelength for tissue transmittance, (5) water solubility for administration. In order to reduce the dose, new photosensitizers have been extensively explored, and porphyrin derivatives including chlorin and bacteriochlorin, and phthalocyanine derivatives, and d-aminolevulic acid are focused as a promising photosensitizer.
For the porphyrin derivatives, 5,10,15,20-tetrakis(3-hydroxyphenyl)chlorin (Foscanョ, hereinafter referred as m-THPC) is a potent second generation photosensitizer used for photodynamic treatment of early stage cancer. Currently the drug is clinical trials for head and neck cancer and has also been to treat brain cancer and esophageal cancers. However, the solubility into water and tumor selectivity of Foscanョ is unsatisfied. Therefore, the development of effective photosensitizers is highly desirable. Since sugars have a number of OH groups and exists in important biological molecular recognition site, it can be expected that the introduction of sugar units to porphyrin derivatives increase water solubility and tissue selectivity. From this point of view, we have prepared a series of new tetraphenylchlorin (TPC) derivatives having sugar units in addition to zinc (II) compounds and examined their singlet oxygen producing ability, cell permeability, phototoxicity against the HeLa cell, and cyclic voltammometry (CV). All compounds prepared increase solubility into water and have little or no dark cytotoxicity. The photodynamic efficiency generating singlet oxygen is almost identical in all compounds. From the photocytotoxic results in OH-protected and OH-free chlorin derivatives, water-soluble OH-free compounds exerted higher activity, and sugar-dependent significant differences in phototoxicity have been observed. These information are very important to develop the high functional photosensitizers in PDT. In order to prove the stability of these chlorin derivatives, electrochemical properties of corresponding glycoconjugated porphyrin derivatives was studied by cyclic voltammetry (CV) in DMSO. On the cathodic scan of p-THPC and m-THPC, no reversible reduction wave was found. This trace corresponds to the imcompleteness in the diimide reduction of p-THPC and m-THPC. On the other hand, glycoconjugated porphyrin derivatives show two chemically reversible redox couples. This electrochemical property sustains the formation of pure glycoconjugated chlorins. Even though the mechanism of stabilization is still beyond understanding, hence, the saccharide moiety gives stability to the chlorin. The electrochmical behavior of the starting porphyrin derivatives is in accordance with the formation of the target chlorin derivatives. These observations are very important for the development of new effective glycoconjugated photosensitizers for PDT.


Application of commercial paprika color to edible oil

Hiroshi Matsufuji
College of Bioresource Sciences, Nihon University

The antioxidant properties of paprika color on oxidation of linoleic acid and the decrease of the color during the storage were investigated. The addition of paprika color suppressed the oxidation of linoleic acid at 37コC under the air exposed to light for 8 hr per a day. However, the antioxidant effect was slight in comparison with that in the dark. Furthermore, the addition of a-tocopherol to linoleic acid with paprika color added improved slightly the effect. On the other hand, the destruction of the paprika pigments occurred with a gradual decrease in its concentration throughout the antioxidant action, and in the vicinity the decoloration, the drastic oxidation of linoleic acid was observed. Thus, it was implied that paprika color might be a good antioxidant and be utilized as an indicator, which could judge oxidative deterioration of edible oils visually by a panel of observation.


Two cytochrome P450s involved in the biosynthesis of flavones and anthocyanins:
molecular cloning and characterization from Perilla frutescens

Kazuki Saito, Chika Kitada and Mami Yamazaki
Department of Molecular Biology and Biotechnology,
Graduate School of Pharmaceutical Sciences, Chiba University

In Perilla frutescens, there are two varietal forms on anthocyanin accumulation, i.e., red and green forms. The cDNA clones encoding flavone synthase II (FSII) and flavonoid 3'-hydroxylase (F3'H), two cytochrome P450s that are involved in the biosynthesis of flavones and anthocyanins, were isolated from P. frutescens. The FSII cDNA encoded 57.1 kDa protein designated as CYP93B6, and the F3'H cDNA did 57.5 kDa protein designated as CYP75B4. Both proteins exhibit several amino acid sequence motifs characteristic of cytochrome P450 proteins. Recombinant CYP93B6 expressed in yeast converted flavanones, i.e., naringenin and eriodictyol, to flavones, i.e., apigenin and luteolin, with Km values of 8.8mM and 11.9mM, respectively. Recombinant CYP75B4 catalyzed 3'-hydroxylation of flavanones, i.e., naringenin, apigenin and dihydrokaempferol, to the corresponding compounds, i.e., eriodictyol, luteolin and dihydroquercetin, with Km values of 18-20mM. The CYP93B6 transcript accumulated to an equal level in leaves of both red and green forms of P. frutescens, in agreement with the accumulation pattern of flavones in the leaves. However, the CYP75B4 transcript was predominantly expressed in the red form of P. frutescens, and its expression was induced by light in conjunction with other transcripts of biosynthetic enzymes of anthocyanin. These results indicate that gene expression of a set of anthocyanin biosynthetic enzymes including F3'H is regulated coordinately only in the red form of P. frutescens but not in the green form, whilst FSII gene expression is controlled in a similar manner in red and green forms of P. frutescens.


Determination of the mean intake per day with an inpidual Japanese
in the detailed item of food

Aiko Yamauchi
Graduate School of Pharmaceutical Sciences
The University of Tokushima

The average daily intake of nutrient composition, additives or pollutant in the food with an inpidual Japanese is calculated using the numerical value of "a nutrient intake according to the food group" based on the national nutrition survey by the Ministry of Health and Welfare. However, the national nutrition survey is carried out in only one day in November. There seems to be some problems as follows, 1) small extraction populations, 2) the seasonal variation, 3) no data of the detailed food item. In this study, the mean food intake per day with an inpidual Japanese was determined in the detailed item of foods. With respect to the perishables, several agriculture, forestry and fishery statistics in 1995-1999 were used for compensation and reevaluation of the numerical value. The amount of supply food per year was calculated using the amount of production, export, import and loss under distribution, and disposal rate etc. The intake of season food was averaged by the monthly purchase amount reported in the Prime Minister's Office investigation of family income annual report. The rate of eating-out and the post-supply disposal rate were estimated by variously comparing the statistic


Study on absorption, metabolism and function of chondroitin sulfates
as a food additive and other glycosaminoglycans.

Toshio Imanari
Graduate School of Pharmaceutical Sciences, Chiba University

The pharmacological interest in glycosaminoglycans (GAGs) has markedly heightened with the increased knowledge on their biological role. In addition to the anticoagulant effect of heparin, some other functions are now recognized such as the chodroprotection of chondroitin sulfate and the use of hyaluronan in microsurgery. On the other hand, there is no method for normalization of chondroitin sulfate as functional food or a food additive. In this study, the instrumental and chemical analytical methods for normalization of chondroitin sulfate have been investigated. As the results of the trial, NMR spectroscopy and some chromatographic approaches show determination of molecular weight, sulfation degree and sulfation pattern of chondroitin sulfate. Furthermore, the micropretreatment procedure using centrifugal micro devices for analysis of chondroitin sulfate in biological samples such as blood plasma and urine has bee newly established. Furthermore, therapeutic effect of orally administrated chondroitin sulfate on experimental inflammation of joints (arthrosis) was observed. The mechanism of this phenomenon and the metabolic fate of chondroitin sulfate will be investigated in the near future.


Effect of endocrine disruptors and food additives on the development of mouse embryos in vitro

Masaharu Miyake and Yuichi Kawai
Faculty of Pharmaceutical Sciences, Kobe-Gakuin University

In recent years, a lot of reports concerned with endocrine disruptors (EDs) have been done. It was suspected the responsibility that the chemicals might induce a reproductive abnormality. In addition, we take daily many food additive and EDs in the food. It is uncertain on the risk of a simultaneous intake of these chemicals, though the ADI values of each food additive have been established.
In this report, we assayed the effects of EDs and food additives on the development of mouse two-cell embryos in vitro. Bisphenol A, DOP nonylphenol and b-estradiol inhibit the development of mouse embryos in a dose- dependent manner. The 50 % inhibition concentrations of those EDs were about 100mM. The food additives (such as food red, food yellow and food blue) did not show any inhibitory effect in ADI value. While they showed the suppressive effects at an extremely higher concentration in the comparison to ADI value of each food additive, but not the synergistic effect in the co-existence of EDs.


Characterization of Kaoliang-induced lymphocyte proliferation

Tsuyoshi Nakanishi
Department of Toxicology, Graduate School of Pharmaceutical Sciences,
Osaka University

We previously reported that food coloring agent, Kaoliang, stimulated B-lymphocyte proliferation and enhanced in vitro proliferative response of mouse T-lymphocytes mediated by anti-CD3 monoclonal antibody, but not concanavalin A (Con A). In the present study, we characterized Kaoliang-induced lymphoccyte proliferation in vitro. We fractionated Kaoliang by HPLC and evaluated effects of fractionated Kaoliang on proliferative response of mouse splenic lymphocytes. The hydrophilic fractions were stimulated B-lymphocyte proliferation, while the hydrophobic fractions were enhanced proliferative response of T-lymphocytes mediated by anti-CD3 monoclonal antibody. Kaoliang also stimulated proliferative response and enhanced anti-CD3 monoclonal antibody induced-proliferation of mouse Peyer's patch cells. Additionally, The proliferative response of human peripheral blood lymphocytes were stimulated and enhanced by Kaoliang. These results indicate that Kaoliang acts as a immunomodulator of systemic and mucosal immue systems.


Screening of chemopreventive agents in herb-related extracts against colon carcinogenesis

Yoshihiro Yoshimura
Department of Analytical Chemistry, Hoshi University

This study was designed to investigate the chemopreventive effects of three natural products, cyanidine-3-glycoside (Cy), acetoside (Ac) and rosemaric acid (Ro) on 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP)-induced colon carcinogenesis in rats. F344 male rats were maintained on powdered basal diet containing 0.03% PhIP alone, PhIP together with 1% or 5% Cy, Ac or Ro, 1% or 5% Cy, Ac and Ro alone or basal diet for 2 weeks. Immunohistochemically, PhIP-DNA adduct positive cells as well as BrdU positive cells induced by PhIP treatment were significantly reduced by the combined treatment with 5% cyanidine in the proximal colon and pancreatic acinar cells. Combined treatment with 5% rosemaric acid also reduced these parameters in the proximal colon, distal colon and ventral prostate. These results indicate that cyanidine and rosemaric acid prevent PhIP-induced colon, prostate and pancreas.


Pigment production by genetic improvement of anthocyanin biosynthesis

Takashi Yamakawa
Graduate School of Agricultural and Life Sciences, University of Tokyo

A dihydroflavonol reductase gene (DFR), coding one of the enzyme on biosynthetic pathway of anthocyanin pigments in purple-fleshed sweet potato, was expressed in Escherichia coli and the expressed protein was characterized for gene manipulation into a culture variety of purple-freshed sweet potato, "Ayamuarasaki" with high anthcocyanin productivity. The cheracetrizeion of the enzyme of the expressed protein in E. coli is in progress. A DFR gene was introduced into Ayamurasaki using Agrobacterium rhizogenes by inducing hairy roots and 16 lines of hairy roots were obtained which were thought to be carrying DFR gene.


Evaluation of safety and physiological function of polyphenol
utilizing gene engineering and cell technology

Satoshi Nagaoka
Department of Agriculture, Gifu University

As the structure of the isoflavonoids is similar to that of estrogen and isoflavonoids has a estrogenic activity, the beneficial effects of dietary soy isoflavonoids on atherosclerosis, cancer, osteoporosis has been extensively reported. As it is reported that the serum level of the antiatherogenic factor, apolipoprotein A-I (ApoA-I) changes by hormone such as estrogen, isoflavonoids is expected to have its effects. There is few reports that the effects of isoflavonoids on the ApoA-I gene expression. Thus, in the present study, we investigate that the effects of soy isoflavonoids, genistein on the level of ApoA-I and its mRNA, the mechanism of genistein action on the ApoA-I gene expression. The secreted ApoA-I level is significantly increased by genistein compared to control in HepG2 cells. The mRNA level of ApoA-I is significantly increased by genistein compared to control in HepG2 cells. In estrogen receptor αtranfected HepG2 cells, the ApoA-I gene transcription is significantly increased by genistein compared to control. We found for the first time that genistein increased ApoA-I and its mRNA levels, and transactivated the ApoA-I gene expression via estrogen receptor α in Hep G2 cells.


Studies for Analysis and Control of the Oxidative Change of Natural Food Colorants

Toshiya Masuda
Faculty of Integrated Arts and Sciences, University of Tokushima

Many natural food colorants have phenolic moiety, which shows potent antioxidant activity. The antioxidant activity and color change of these colorants were examined. Curcumin, a main yellow pigment of the turmeric colorant which showed potent antioxidant activity and color stability during antioxidation, have been chosen for the purpose of this study. From the quantitative HPLC analysis of antioxidation products of curcumin and following structural analysis of them, an antioxidant mechanism of curcumin against lipid oxidation has been proposed. And the stability of color of curcumin or turmeric pigment can be explained by the contribution of the antioxidaion products to the yellow color display.


Binding of Porphyran from Died Nori to Lipid

Koji Takahshi
Faculty of Agriculture, Tokyo University of Agriculture and Technology

A suspension of low-quality dried nori processed from Porphyra yezoensis, a red alga, was autoclaved at 120コC for 30 min, and from the supernatant, the purified porphyran without protein was obtained by precipitation with ethanol at stepwise increasing concentrations of 50 and 67% followed by size-exclusion chromatography. The purified porphyran had about 82% total saccharide content, 9% sulfate content, 11% 3,6-anhydrogalactose content, and Mr 40 KDa. The porphyran content of the supernatant separated from the emulsion prepared with porphyran exhibited that some porphyran in the aqueous phase was adsorbed on the surface of the oil droplets. The absorption spectrum of the aqueous phase containing toluidine blue exhibited a decrease in g peak assigned to porphyran toluidine blue complex, supporting the adsorption of porphyran on the surface of the oil droplets.


Improvement and the Mechanism for Gel Formation of Food Protein
using Food Emulsifier

Naoko Ohta
Junior College at Mishima, Nihon University
Collaborator; Mikio Sugiki*, Yuuichi Maekawa*, Akihiro Horiuchi
*: Department of Agriculture and Biological Chemistry,
College of Bioresource Sciences, Nihon University

We have studied on the effects of fatty acid salts on the gelation properties of some kinds of food proteins such as sesame 13S globulin and b-lactoglobulin with heat-treatment. We have found that sesame 13S globulin can form a soft and high-water holding gel by the addition of sodium caprate or sodium dodecyl sulfate with heat-treatment. On the other hand, sesame 13S globulin and b-lactoglobulin formed gels similar to each control gel by the addition of sucrose monolaurate or DK-ester F160; (marketing sucrose fatty acid ester). These results suggest that the water-holding ability of the gels was increased by the addition of ionic fatty acid derivatives. In the case of b-lactoglobulin, it formed the gel softer than the control gel by the addition of Carrot powder (less than 0. 1%) which consists of 10% b-carotene and 30% gum with heat-treatment. The gels with different amount of Carrot powder showed different texture and FT-IR spectra. It seemed that the difference of FT-IR spectrum of gels reflected difference in inpidual gel hardness. Furthermore, we found a new phenomenon that sesame 13S globulin or rice g-globulin can form aggregates by the incubation with sodium caprate for more than 24 hr at room temperature.


Development of efficient production system on bioactive compounds
with radical scavenging and antioxidative activities

Tadashi Fujii
Faculty of Life Sciences, Toyo University

Beniazuma is the kind of sweet potato showing red skin accumulated anthocyanin and light cream yellow in color inside. The skin of the other, Tanegashima-murasaki, shows light cream close to white in color, but inside dark red. Radical scavenging activity of the raw and steamed sweet potato skin and edible part (inside) were investigated. The high radical scavenging activity (34.7 % - raw potato, 26.1% - steamed potato) was observed in the peel of Tanegashima-murasaki, and pigment formation on sweet potato is not related to radical scavenging activity. Two transformed root clones of Murasakiimo showed pink were established by infection with Agrobacterium rhizogenes MAFF03-01724.
 Ethylene was detected by GC-MS analysis in petri dish where the shoots were cultured for 4 weeks. Therefore, the possible involvement of ethylene on shikonin production was investigated on the shoot cultures. Treatment of ethylene increased shikonin derivatives contents in cultured shoots. In contrast any changes in levels of shikonin derivatives were observed by exposure of the ethylene precursor, 1-aminocyclopropane-1carboxylic acid (ACC). Silver ion, an ethylene-response inhibitor or aminoethoxyvinylglycine (AVG), an ethylene biosynthesis decreased shikonin derivatives contents in cultured shoots.


Developments and applications of the analytical method for hydrogen peroxide using HPLC

Shigenori Kumazawa and Tsutomu Nakayama
School of Food and Nutritional Sciences, University of Shizuoka

 We developed a method for separating hydrogen peroxide (H2O2) by HPLC with ECD. We applied the method for the determination of H2O2 during the oxidation of tea catechins such as epicatechin (EC), epicatechin gallate (ECg), epigallocatechin (EGC), and epigallocatechin gallate (EGCg). The amount of H2O2 formed depended on their structures, pH, temperature, and incubation time. The gallyl moiety in the B-ring of the gallocatechins, namely EGC and EGCg, mainly contributed to the H2O2 formation. Superoxide dismutase (SOD) inhibited oxidation of the gallocatechins and, consequently, the H2O2 formation. Cytotoxic effects were investigated by a colony formation assay with Chinese hamster V79 cells, and the effects of the gallocatechins were stronger than those of EC and ECg. The cytotoxic effects of the gallocatechins were inhibited completely by catalase and partially SOD. These results indicate that the gallocatechins were oxidized by superoxide, accompanied by the formation of H2O2, and their cytotoxic effects were ascribed to the H2O2 formation.


Anti-Salmonella and anti-rotavirus actions of phosphopeptides
from egg-yolk phosvitin, acid precipitated soy-protein and milk casein,
and the possibility of application as new functional food additives

Soichiro Nakamura
Faculty of Education, Shimane University

Anti-Salmonella and anti-rotavirus effects of phosphopeptides from egg-yolk phosvitin, acid precipitated soy-protein and milk casein were investigated using Salmonella enteritidis and rotavirus-Wa strain as target microorganisms. All phosphopeptides tested showed strong abilities to prevent amplification of S. enteritidis in CV-1 cells, compared to the control system. Rotavirus infections to the host MA 104 cells were also prevented by co-existing with the phosphopeptides. These effects were significantly improved by the polyglycosylation with galactomannan. In addition, food safeties of the phosphopeptides and polyglycosylated phosphopeptides were confirmed by bacterial mutagenesis tests, rec-assay and Ames test. Thus, it is revealed that these food-derived phosphopeptides and their polyglycosylated forms gain great potential of the application as food aids. This study also shows that the polyglycosylation of polypeptides is a new approach to enhance their functional properties.


Effect of arginine on growth of lactic acid bacteria for fermented sausage
under high concentration of salt

Mitsuo Sekikawa
Laboratory of Meat Science,
Obihiro University of Agriculture and Veterinary Medicine

In this study, the effect of Arginine (Arg) of the NaCl induced growth inhibition was examined for Sta. carnosus and P. acidilactici which isolated from the commercial starter culture of fermented sausage. When Arg and/or NaCl were added to MRS broth and cultured with shaking, the differences were not recorded in the broth pH, the nitrate reduction activity, and the protein degradation activity. However, the viable count of both strains had decreased by adding NaCl. The addition of Arg has increased the number of recovered cell and it was remarkable in Sta. carnosus. Arg added broth was metabolized by both strain, the content of Arg in broth was decreased, especially it was disappeared in cultured broth of Sta. carnosus. SEM photographs showed an enlarged and cohesive morphology of both spheroidal strains by adding NaCl to the broth. Addition of Arg, this tendency was decreased in P. acidilactici, whereas a small wrinkle on the surface of Sta. carnosus. It was thought that, the two bacterial strains used in this study, the growth inhibitory effect caused by high concentration of NaCl was partly recovered by the addition of Arg to the broth.


Enzymatic Glucosylation of Naturally Occurring Spices by Plant Cell Cultures

Hiroki Hamada
Department of Applied Science, Okayama University of Science

The effective glucosylation of naturally occurring spices by plant cell cultures was investigated. Thymol and carvacrol were glucosylated by the cultured cells of Eucalyptus perriniana to their corresponding glucosides, respectively.


Preparing Model System of the Rational and Qualified Application
of Resistant Polysaccharides

Masaru Ohtsuru
Department of Food and Nutrition, Mukogawa Women's University

The effects of 17 kinds of polysaccharide(carrageenan(3 types), alginic acid, locust bean gum, guar gum, arabic gum, tragacanth gum, karaya gum, pectin, xanthan gum, gellan gum, pullulan, ghatti gum, soybean polysaccharide, cellulose, glucomannan), on plasma lipids and triglyceride, total cholesterol and bile acid in feces were studied in rats. Rats were fed control diet and 5% of each polysaccharide diets for a week.
 The plasma total cholesterol was not significantly difference in every polysaccharide diets, but the triglyceride levels in plasma were lowered in rats fed seed gums(locust bean gum, guar gum), pectin and xanthangum diets compared to rats fed the control diet. Neutral sterol and bile acids excreted in feces were increased in rats fed the diets of seaweed gums(carrageenan and alginic acid. Levels of minerals excreted in feces, especially Na+ and K+ were higher in rats fed seaweed gums diets.

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