Research Reports No.9 Abstract（2003）

9-1

Study on the protein-protein interaction among reguratory factors
involved in anthocyanin production in Perilla frutescens.

Kazuki SAITHO, Yukiko MAKITA and Mami YAMAZAKI
Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba Univesity

A bHLH2 factor gene F3G1 was isolated from Perilla frutescens. This gene expresses specifically in red form but not in green form. The protein-protein interactions between F3G1 and one of other factors, bHLH1, MYB and WD40, were investigated by using yeast two-hybrid system. F3G1 interact with MYC-RP (bHLH1) and PFWD (WD40) but not with pther factors. From these results, it was supposed that F3G1 interacted with MYC-RP and PFWD, and participate in the regulation of anthocyanin biosynthesis.

9-2

Molecular Aggregation of Sugar Chain and Alkyl Chain
Linked Tetraphenyl Porphrins for Photodynamic Therapy

Shigenobu YANO
pision of Functional Material Science, Graduate School of Human Culture, Nara Women's University

Recently much attention has been focussed to photodynamic therapy (PDT) as clean and new modalities of selective treatments of cancer. Porphyrin derivatives have been used as effective photosensitizers. However, the solubility into water, tumor selectivity, dark toxicity and a deep penetration of the light into tissue of these compounds are unsatisfied. Therefore, the development of effective photosensitizers is highly desirable. Porphyrins and their aggregates play an important role in nature and in technological processes such as photochemistry, photobiology and medicine etc. Therefore, it is an interesting subject how to control the aggregation behavior of porphyrins by changing their side chains. In the present study, we have synthesized and characterized a series of water-soluble porphyrins, which contain three maltohexaosyl chains as hydrophilic groups (ethyl, butyl, hexyl, decyl, hexadecyl) and an alkyl chains as hydrophobic unit, and examined their molecular aggregation by means of an electronic absorption and circular dichroism spectroscopy. In DMSO solution, all these compounds showed no Cotton effects in CD spectra and showed fairly sharp Soret bands in electronic absorption spectra. These results indicated that these compounds should exist monomeric form. In contrast, the CD spectra of these compounds in water give a fairly large Cotton effect. The intensity of the Cotton effect decreased with elongation of the alkyl chain and red shift of the Soret bands accompanying by the line broadening has been observed in water. These observations suggested that stable chiral face to face aggregates changed to head-to-tail aggregates with increasing the alkyl chains dependent on the hydrophobic interactions between alkyl chains. Thus, the hydrophobic/hydrophilic balance of maltohexaosyl and alkyl chains is important factors in the control of aggregation.

9-3

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

Hideo HATATE
Faculty of Agriculture, Miyazaki University

To assess antioxidative activity by the animal testing, many researchers frequently use mammals and their tissues. The experimental use of mammals, however, are limited or avoided as the current trend in the world. In this study, we prepared fish liver microsomes from tilapia Oreochromis niroticus by a differential ultracentrifugation method and then studied their adaptability for antioxidative assay by comparison with usual method that employed rat liver microsomes. By using fish liver microsomes, antioxidative activity of some authentic antioxidants such as butylated hydroxyanisole, a-tocopherol, propyl gallate, and sesamol was assayed and it was found that their evaluated antioxidative potencies were very similar to those obtained from rat. Moreover, fish liver microsomes were stable for 30 days at -80ｺC. These results suggest that fish liver microsomes are available for antioxidative assay as substitution of rat.

9-4

Exploration for New Functions of Polyphenol Food Additives and
Investigation on Structures and Safety of Their Metabolites.

Nobutoshi MURAKAMI
Graduate School of Pharmaceutical Sciences, Osaka University

Exploration for new functions of polyphenol food additives disclosed antimalarial activity of flavonol glycosides. Furthermore, structure activity relationship was examined by use of readily available and naturally occurring flavonols. Consequently, flavonol diglycosides were shown to retard the life cycle of malaria parasites. On the other hand, flavonol monoglycosides completely arrested the life cycle at the trophozoite stage to inhibit proliferation of the parasites.

9-5

Screening for the pharmacological activity of foodstuff components and additives
using their membrane effects as an indication

Hironori TSUCHIYA
Co-worker: Motohiko NAGAYAMA
Asahi University School of Dentistry

Various foodstuff components and additives were subjected to the screening for their pharmacological activity based on a potent interaction with bio-membranes. Membrane fluidity changes were comparatively studied by a fluorescence polarization method using liposomal model membranes for tumor cells and platelets. The anti-tumor and anti-platelet activity was evaluated by cell culture and aggregation experiments, respectively. The screening method using the membrane effects as an index was also applied to the inedible brownish scale of onion.
The active components in functional foodstuffs, and preservative, coloring and anti-oxidative agents influenced the membrane fluidity of liposomes with the lipid composition corresponding to tumor cells. At ~10 mM, (-)-epigallocatechin gallate, genistein, apigenin, resveratrol and a reference anti-tumor drug doxorubicin rigidified liposomal membranes by acting on the hydrophobic regions rather than the hydrophilic regions. They were more effective in rigidifying the tumor cell model membranes than the normal cell model membranes. Quercetin specifically showed a biphasic effect on the membrane core to rigidify above 5 mM and fluidize below 2.5 mM. The structure-activity relationship among flavonoidal components was derived as follows: anthocyanidins > flavonols > Flavones > flavanones > isoflavones, flavanols > epicatechins, isoflavones > isoflavanones, and flavones > chalcones. It indicated the importance for membrane rigidification of ring closure, and hydroxylation at the 3-position of C ring, the 5,7-positions of A ring and the 3',4'-positions of B ring. At 10~100 mM, all of the membrane-rigidifying foodstuff components and additives not only inhibited the growth of tumor cells but also rigidified cell membranes. Quercetin with the biphasic membrane effects concentration-dependently showed the cell-growth inhibition and promotion. Two quercetine dimers and quercetin-4'-glucoside were isolated from the outer scale of onion in addition to quercetin. The most membrane-active quercetin dimer of them significantly inhibited the tumor cell growth together with rigidifying cell membranes at 10 mM. It also showed both anti-aggregatory and disaggregatory effects on human platelets by interacting with membrane lipids. Although preservatives such as p-hydroxybenzoic esters and BHT were membrane-active at 0.0002~0.002%, they fluidized the membrane core of liposomes.
From these results, the new screening method using the membrane effects as an index appears to be useful for evaluating the utility and safety of foodstuff components and additives.

9-6

Effect of Meals Containing High and Low Nitrate Content
on Nitrate and Nitrite Level in The Saliva and Urine

Masataka ISHINAGA
Hiroshima Prefectural Women's University
Collaborator: Mihoko MURATA
Suzugamine Women's College

The effect of meals containing high and low nitrate content on nitrate and nitrite level in the saliva and urine was examined. The meal of high content nitrite contains the much vegetable (vegetable meals), and the content was 260.9mg/day. The meal of low content nitrate contains the much processed foods (processed foods meals), and the content was 18.1mg/day. The contents of nitrite in both meals were 1.51mg/day.
The nitrate content of the saliva in eating the vegetable meals were also higher than the case in which the processed foods meals was eaten about 3 time. When the vegetable meal was eaten, excretion quantity of the nitrate to the urinary was 68% of an intake. However, in the case of the processed foods meals, it was 248% of an intake. That is, the excretion quantity greatly exceeded intake.
In both meals, the nitrite detected about 0.10mg in urinary.

9-7

Effects of food additives mostly consumed in childhood
on atopic dermatitis-like experimental dermatitis in NC/Jic mice

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

We demonstrated here the effects of food additives on contact hypersensitivity induced by 2,4,6-trinitrochlorobenzene (TNCB) in BALB/c mice. Anato, sunset yellow and benzoic acid did not show any effects on contact hypersensitivity, however, tartrazine and L-tartaric acid worsened contact hypersensitivity. So that, we studied the effects of tartrazine and L-tartaric acid on chronic dermatitis induced by repeated application of TNCB in NC/Jic mice, which is the experimental model of human atopic dermatitis. Surprisingly, both food additives enhanced the ear swelling, AD-like symptoms and increase of serum total IgE level. These results suggested the possibility that tartrazine and L-tartaric acid might affect the on-set and symptoms of AD in human.

9-8

Changes of intestinal flora by chemicals.

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

Host-intestinal flora interaction alters absorption, metabolism, and toxicity of chemicals. Major part of intestinal flora is composed bacteria which are uncultureable or difficult to culture. Analytical methods to detect uncultureable bacteria by 16s rDNA-based RFLP, FISH and DGGE were developed and already applied in the field of environmental microbiology. We tried to apply PCR-DGGE to analyze the change of intestinal flora. Various parameter were evaluated and defined. Under the defined condition, penicillin G- and metronidazole-induced changes in DGGE profile of intestinal flora were clearly observed in gel electrophoresis and two dimensional scaling.

9-9

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. Using PPARg-expressing cell line, we also isolated the PPARg-target genes by two-dimension gel electrophoresis and mass spectrometry. Moreover, we determined the new DNA binding sequences of PPARg. 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.

9-10

Pigment production by intensifying the enzyme genes of anthocyanin biosynthesis

Takashi YAMAKAWA
Department of Global Agricultural Sciences, The 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. However, the enzymatic activity was not observed. A cDNA of anthocyanin synthase (ANS) from Ipomoea nil was introduced into sweet potato(Ayamurasaki) using Agrobacterium rhizogenes. Hariy roots were induced and anthocyanin production was observed by the investigation of culture condition.

9-11

The research on opening to the public and accumulation of the infrared absorption spectrum and the ultraviolet and visible absorption spectrum of the food additive

Tadashi EZAWA
Faculty of Engineering, University of East Asia
Akio NAKANO and Kunitoshi YOSHIHIRA
pision of Food Sciences, Graduate School of Integrated Science and Art, University of East Asia

Infrared absorption (IR) spectra of 229 items, and ultraviolet (UV) and visible absorption (VIS) spectra of 197 items were measured to evaluate the safety of food additives. The results including IR absorption spectra, wave number, and intensity (transmittance) of the additives were opened to the public on the Web. In addition, an on-line system for whole qualitative analysis of food additives included in a food was designed and constructed using the database of the absorption wave numbers we measured. Lastly, to examine the efficiency of this system, the data of food additive analysis by the online analyzer was compared with those of NMR spectrum already shown on the same website since 2002.

9-12

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 and its derivertives 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 receptora tranfected HepG2 cells, the ApoA-I gene transcription is significantly increased by genistein compared to control.The other side,In estrogen receptora tranfected HepG2 cells, The secreted ApoA-I level is no changed by Genistein's derivertives (4'-O-methyl Genistein, 7-O-methyl Genistein) compared to control in HepG2 cells. In estrogen receptora tranfected HepG2 cells, The mRNA level of ApoA-I is no changed by genistein's derivertives compared to control in HepG2 cells. In estrogen receptora tranfected HepG2 cells, the ApoA-I gene transcription no changed is by genistein's derivertives compared to control.
Genistein increased ApoA-I and its mRNA levels, and transactivated the ApoA-I gene expression via estrogen receptora in Hep G2 cells. We suggest that the effects of genistein and its derivertives on the level of ApoA-I and its mRNA and the mechanism of genistein action on the ApoA-I gene expression is related to genistein's C4' or C7 hydroxyl group.

9-13

Basic analysis on active structures and preparative expression of new taste-modifying proteins
occurring in a tropical plant

Keiko ABE
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo

We conducted purification of taste-modifying protein from Curculigo latifolia seeds, which can convert sourness to sweetness. Several kinds of HPLC were used for the purification. The use of a phenyl-hydrophobic column resulted in a good degree of separation, affording an active fraction with a high yield. N-terminal amino acid sequence analysis made it possible to identify more than two curculin-like proteins. The result indicates that the principles the taste-modifying activity are not only curculin itself but also some heteropolymers constituted with these curculin-like analogues.

9-14

Study on biological functions of glycosaminoglycans including chondroitin sulfate as a food additive.

Toshio IMANARI
Graduate School of Pharmaceutical Sciences, Chiba University

Chondroitin sulfate (CS) is one of glycosaminoglycans widely distributed in animal tissues, which has an anti-inflammatory property. We demonstrate here that a specific sulfation pattern of the CS polysaccharide is required for the Th1 promoted activity, as other polysaccharides such as dextran and dextran sulfate do not significantly induce this activity. While the presence of some O-sulfo groups are essential for activity, CS-A and synthetically prepared, partially O-sulfonated CS induced higher Th1 promoted activity than did synthetically prepared, fully O-sulfonated CS. In addition, chondroitin sulfate E induced greater activity than CS-A or chondroitin sulfate D. These results suggest that the GlcA?1-3GalNAc (4,6-O-disulfo) sequence in CS is critical for Th1 promoted activity. Furthermore, rat anti-mouse CD62L antibody, an antibody to L-selectin, inhibits the Th1 promotes activity of CS. These results suggest that the Th1 promoted activity is mediated through L-selectin on lymphocytes. These findings provide a new mechanism for the anti-inflammatory and chondroprotective properties of CS and may be useful for designing new therapeutic applications for CS for the treatment of immediate-type hypersensitivity.

9-15

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 colorants, which are used for food pigments, have phenolic structure. Our research project in the last year revealed that some of these colorants showed potent antioxidant activity in micell system. Food grade lac pigment is one of the potent active colorants, therefore, the antioxidant property and antioxidant mechanism of the pigment were examined in this year's project. First, laccaic acid A, which is the main color compound of lac pigment, was purified and isolated as a pure form from food grade lac. The antioxidant activity of the pure laccaic acid A was measured in a micell system, showing potent antioxidant activity comparable to that of tocopherol.
However, laccaic acid A did not show antioxidant activity in homogeneous system. Further examination revealed that the antioxidant activity of laccaic acid A was affected by pH conditions of the measuring system. Especially, below pH 6.3, the antioxidant activity of laccaic acid A was remarkably reduced. The antioxidant reaction of laccaic acid A was analyzed by HPLC technique, revealing that two products were detected in the chromatogram. Although isolation of the products was failed for the instability of the products, the psudo-molecular ion peak for the main product could be detected in LC-MS analysis using negative ESI mode. A possible chemical structure of the main compound was estimated from the consideration of the reactivity of laccaic acid A and the molecular weight obtained by the MS.

9-16

Preparation and DPPH Radical-scavenging Activity of the Anthocyanin-soyprotein Complexes

Kazutaka NISHIKAWA
Faculty of Health and Living Sciences Education, Naruto University of Education

Anthocyanin-soyprotein complexes were prepared from the mixture of autoclaved defatted soybean extraxt and methanol extract of various plants containing anthocyanin. All anthocyanin-soyprotein complexes contained anthocyanin and showed reddish or purple coloration. The maximum concentration of anthocyanin (0.23% dry weight as cyanidin equivalent) was observed in the complex prepared from black soybean. Anthocyanin-soyprotein complexes showed large radical-scavenging activities which levels were higher than that observed in soyprotein precipitate containing some isoflavones. The largest radical-scavenging activity was observed in the complex prepared from the extract from red leaves of Sapium sebiferum. The highest radical-scavenging activity was presumed to be originated from tannin constituents (geraniin and chebulagic acid) as well as anthocyanin. Anthocyanin-soyprotein complexes also have good potential to be as a new functional material valuable in food industries.

9-17

Binding of Porphyran from Died Nori to Lipid

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

Binding of the purified porphyran from dried Nori to lipids was investigated by measurig zeta-potential of the oil droplets of the O/W emultion prepared with porphyran, and by evaluating the release of calsein from the porphyran-coated liposomes by digestion with phospholipase D. Porphyran remarkably reduced zeta-potential of the emulsion, and the release of calsein incorporated in liposomes. These results showed that porphyran could effectively bind to lipids to form stable emulsions.

9-18

Enzyme-catalyzed Direct and Regioselective Acylation of Flavonoid Glucosides
for Stabilization and Functionarization of Plant Pigments

Kohji ISHIHARA
Department of Chemistry, Kyoto University of Education
Co-worker: Nobuyoshi Nakajima
Department of Nutritional Science, Okayama Prefectural University

Regioselective acylation of flavonoid glucosides was achieved by lipase-catalyzed transesterification in dry organic solvent. The participation of the acyl group in flanovoid glucoside molecules resulted in increasing of the physiological function of the acylated flavonoid glucosides.

9-19

Natural Antimicrobial Peptides from Egg White Lysozyme

Hisham R. IBRAHIM
Department of Biochemistry & Biotechnology, Faculty of Agriculture, Kagoshima University.

Lysozyme is an antibacterial enzyme widely distributed in nature and is found in various biological fluids including avian eggs, plants, bacteria, insects, mammalian tissues and secretions, tears, saliva, milk, respiratory secretions, and secreted by polymorphonuclear leukocytes. We have recently discovered a potent bactericidal peptide within the sequence of lysozyme with a unique structural motif similar to many natural bactericidal peptides. Suggesting that partial proteolysis might be a biological process to trigger the specific antimicrobial activity of lysozyme by which its natural antimicrobial peptide could be isolated. Here, we studied the antimicrobial activity of lysozyme as a function of proteolysis by specific chemical and enzymatic means. Partial proteolysis at Asp-X sites of the molecule remarkably promoted the bactericidal potency of lysozyme against Gram-negative and Gram-positive bacteria. Cleavage of disulfide bonds of the proteolysed lysozyme, by food grade reducing agents, completely librated the peptides and further enhanced the antimicrobial activity. The results introduce an interesting structure-antimicrobial relationship that the antimicrobial conformation of lysozyme can be triggered by proteolysis at specific sites (Asp). This specific proteolysis represents a simple method to switch the antimicrobial specificity of lysozyme to include the food-borne pathogens and heralding fascinating opportunities for application of lysozyme peptides in formulated food and drug systems.

9-20

Structure and Application of highly Water Adsorbent Polysaccharide
on the surface of seeds of Artemisia sphaerocephala Krasch

Masahiro TADA
Laboratory of Bio-organic Chemistry, Tokyo University of Agriculture and Technology

Desert wormwood, Artemisia sphaerocephala Krasch is distributed over dry desert of yellow soil in northwest China, Ningxia and Neimenggu Zizhigu. The surface of seeds is coated with highly water adsorbent polysaccharide. When it is rain on the desert, the polysaccharide on the surface of the seeds becomes a gum by adsorbing water to spread over the yellow soil and the seeds can germinate. The seeds of A. sphaerocephala Krasch are used for additives of noodles to improve the quality in the northwest China. We found efficient method to separate the polysaccharide from the seeds and found that the polysaccharide contains water-soluble substances in it on the seeds surface. The isolated polysaccharide by the known procedures is partially crystallized and difficult to adsorb water again. The water adsorbing polysaccharide could be prepared by adding some hydrophilic substance before drying it. The polysaccharide consists of glucose, mannose (in a ratio of 3:1) and small amount of unknown carbohydrates. These informations give new possibility for application of the seeds of A. sphaerocephala Krasch as foods additives.

9-21

Availability of Anthocyanin Pigments in Vitis Coignetiae Grape Berry Skin
as a Coloring Agent and Healthy Food

Goro OKAMOTO
Faculty of Agriculture, Okayama University

Vitis coignetiae grape berries are usually harvested for wine making during September at Hiruzen vineyards. It is known that the berries contain abundant anthocyanin in their skins. However, the wine quality is not excellent due to low sugar levels and high acidity. The present study, using 30 vines cultivated in Hiruzen, revealed that juice TSS (total soluble solids) contents reached to about 20 Brix, whereas anthocyanin levels continued to increase as the harvest date is delayed to late October. Juice amino acid contents were considerably low in most coignetiae vines compared to usual vinifera grapes for red wine making. Results of RAPD analyses using 26 primers showed wide genetic differences among tested coignetiae vines, causing various compositional deviations in juice amino acids and skin anthocyamins. Total polyphenol contents were 2 to 4 times higher than those in Cabernet Sauvignon (CS) berries, a representative red wine grape cultivar. Anti-oxidative activity, as indicated by DPPH scavenging activity, of coignetiae berry juice was 1.8 times higher than that of CS berries. The present study indicated that Vitis coignetiae grapes, produced in Hiruzen, contain sufficiently high levels of sugars and higher levels of anthocyanin and polyphenol than other usual red wine grapes when fully ripen, although the levels and composition are depending on vines. Selection and propagation of vines producing berries with higher levels of such useful constituents will enable us to produce more excellent grape berries as red wine materials.

9-22

Enhancement of taste for cereal mix (Glanola) by adding oligosaccharides to the mix prepared from koji

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

Amylolytic enzyme was purified to electrophoretically homogeneous state from culture broth of Aspergillus oryzae MIBA316. This enzyme hydrolyzed preferentially maltotriose, maltoteraose, amylose, amylopectin and starch. The genes coding for amylase were cloned within a 2.0 kb fragment and the primary structure of amylase was deduced from its molecular sequences The amino acid sequence of the enzyme consisted of 534 residues with a molecular weight of 54,000.
Approaches to complete breakdown of starch to its components and their utilization in food processing were discussed.

9-23

Exploitation of antibiotic browning-inhibitors from marine algae

Tadahiko KAJIWARA
Department of biological chemistry, Faculty of Agriculture, Yamaguchi University

Generally, polyphenol oxidase (PPO) occurs in most vegetables and fruits concomitant with polyphenols. Polyphenols and PPO react under the presence of oxygen when the tissues of vegetables and fruits are damaged. Tyrosinase is one of PPO, which is a copper-containing enzyme and is widely distributed in microorganisms, animals and plants. The aim of this study was to find the efficient and novel inhibitors of PPO. Efficient inhibition of the browning at the cut surface would be useful to process cut vegetables.
L-3,4-Dihydroxyphenylalanine (L-DOPA) is an intermediate in a pigment-forming process, which is produced from tyrosine with tyrosinase. Subsequently, dopaquinone is oxidized to form melanin pigments through polymerization. Thus, inhibition of the browning can be achieved at either one step of the two, namely, oxidation reaction by tyrosinase or subsequent nonenzymatic polymerization. Tyrosinase activity was measured with monitoring absorbance at 475 nm originated from dopachrome formed from L-DOPA.
Many kinds of aliphatic carboxylic acids and aldehydes and alcohols, saturated or unsaturated, are used as inhibitors for PPO activity. Some of them resulted in efficient inhibition of the oxidation of L-DOPA catalyzed by tyrosinase. The results obtained indicated that the a,b-unsaturated carbonyl compounds inhibit tyrosinase activity.
In preliminary bioactive experiments, the a,b-unsaturated aldehydes such as (2E)-nonenal and (2E)-hexenal showed antibiotic activities to Fusarium oxysporum f sp. raphani and Botrytis cinerea. When marine algae were damaged or macerated, the a,b-unsaturated aldehydes were first found to be generated from C20-polyunsaturated fatty acids. These facts suggested that a,b-oxo-acids, the counter parts of a,b-unsaturated aldehydes in the biosynthesis, might be found as antibiotic browning inhibitors in marine algae.

9-24

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 17kinds of polysaccharide (*carrageenan (3types), *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.
*: polysaccharides were used to determine the viscosity in the study. The mixtures of karaya gum - xanthan gum and carrageenan-carrageenan K2 which increased the viscosity to a high value than expected from simple addition of both polysaccharide viscosities were tested.
The plasma total cholesterol were decreased in the diet of karaya gum - xanthan gum and carrageenan - carrageenan k2 mixtures. Triglyceride, neutral sterol and bile acids excreted in feces were increased in the diets of karaya gum - xanthan gum mixture. Levels of minerals excreted in feces, especially Na+ and K+ , were higher in the diets of carrageenan - carrageenan K2 mixture, but the diets of karaya gum - xanthan gum mixture showed lower effects than each alone.

9-25

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

Tadashi FUJII
Faculty of Life Sciences, Toyo University

The main anthocyanidin in root skin of Beniazuma and the root skin and edible root without the skin of Ayamurasaki was peonidin. In contrast, the main anthocyanidin with root skin of Tanegashima-murasaki was cyanidin.
Adventitious root cultures of Beniazuma, Ayamurasaki and Tanegashima-murasaki were established. However those adventitious roots did not form pigments. Interestingly the high radical scavenging activity (ca. 60 %) was detectied in the Beniazuma adventitious root (1 mg/L IBA).
The light-pink pigments were partly formed on the roots of Beniazuma, Ayamurasaki and Tanegashima-murasaki plants cultured on Vermiculite (MS liquid medium) at 25 ｰC in the light.
Treatment of ethylene or ACC did not affect the production of caffeic acid derivatives in shoots cultured in the dark. The contents of caffeic acid derivatives in shoots treated with ethylene inhibitors were almost the same level as those in the untreated shoots. The shoots (in the light) treated with 10 L / petri dish ethylene showed about 1.5-fold higher caffeic acid derivatives (RA) content compared with the untreated shoots.

9-26

Functional Evaluation as Food Additive Agents Concerning
the Stereoisomers of Carotenoid Oxidative Metabolites

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

Most of the oxidative metabolites of the carotenoids possess an eleven, thirteen, or fifteen carbon skeleton. Among them, loliolide1 and xanthoxin2 in addition to abscisic acid, have been paid much attention because of their interesting biological activities. (-)-Loliolide, which has been detected in many higher plants and marine mollusks, is well-known to have immunosuppressive, germination inhibitory, and repellent activities. (-)-Xanthoxin has been proposed as a biogenetic precursor of (+)-abscisic acid, which is the primary plant hormone controlling many biological plant processes such as acceleration of abscission, induction of dormancy, and inhibition of rooting, and xanthoxin shows similar biological activities to abscisic acid.
We achieved the stereocontrolled syntheses of (-)-loliolide, (-)-xanthoxin, and their every stereoisomers from (+)- or (-)-3-alkoxy-6-hydroxymethyl-1,1,5-trimethylcyclohexene through the corresponding syn and anti-epoxides, respectively, which were obtained by utilizing the highly diastereoselective Sharpless asymmetric epoxidation or mCPBA oxidation.
(-)-Loliolide and (+)-epiloliolide thus synthesized showed poor germination inhibitory activity, meanwhile, they found to show the clear suppressive activity toward the growth of rice roots. Our further interest is the relationship between the stereochemistry of our synthesized carotenoid metabolites and their repellent or anti-fungi activities.