Purification and Characterization of Glucuronosyltransferase for the Elucidation of Physiological Role of Saponin in Soybean Plant and the Breeding of a Value-added Soybean Variety
Masakazu SHIRAIWA and Yasunori KUROSAWA
School of Agriculture, Ibaraki University, Ami-machi 300-0393
ABSTRACT
We solubilized and purified microsomal glucuronosyltransferase from soybean, and elucidated its enzymatic properties. A microsome fraction was isolated from germinating soybean seed and treated with various detergents to solubilize the enzyme. The enzyme activity was monitored throughout purification using [14C]-UDP GlcA and soyasapogenol B as substrates. Purification of glucuronosyltransferase was achieved by HiTrap Q, Superdex 200, HiTrap Blue chromatography procedures. This resulted in an enrichment >130-fold relative to the starting homogenate. Purified enzyme was found to require cation for activity. Studies of the substrate specificity of the purified enzyme demonstrated that the specificity for the sugar residue transferred was very high, as no activity was found when UDP-GlcA was replased by other UDP sugars: UDP-Glc and UDP-Gal. Soyasapogenols which are aglycone of soybean saponin are usable acceptors but glycyrrhetinic acid or flavonone is not. These findings suggest that this glucuronosyltransferase was a specific enzyme for UDP-GlcA as donor and soyasapogenols as acceptor, and it was related to biosynthesis of the sugar chain in soybean saponin. This study provides a basis for molecular characterization of key enzyme in saponin biosynthesis in soybean. The isolation of the gene may enable its use in the elucidation of the biosynthesis and physiological role of saponins in soybean.
Soy Protein Research, Japan 4, 1-10, 2001.
Key words : soybean, saponin biosynthesis, glucuronosyltransferase
Analysis of the Regulation of Gene Expression and Genetic Variations in Seed Storage Proteins in Soybean
Michiko YOSHINO1, Ken-ichi TSUTSUMI2, Jun ABE1, Yoshiya SHIMAMOTO1 and Akira KANAZAWA1
1 Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589
2 Cryobiosystem Research Center, Faculty of Agriculture, Iwate University, Iwate 020-8550
ABSTRACT
The 7S globulin ( b-conglycinin), a major component of seed storage proteins in soybean, comprises three subunits, a, a' and b. The nucleotide sequence of a 7.6-kb EcoRI-digested fragment of genomic DNA, which contains a gene, CG-3, of the b-conglycinin gene family, was analyzed. By comparing the sequence with cDNAs of b-conglycinin, we found that CG-3 encodes the a-subunit. The gene contained 6 exons and 5 introns, of which positions were conserved among genes for a-, a'- and b-subunits. A major transcription start site of the a-subunit gene was located at 56 bp upstream of the initiation codon by a primer extension analysis. Binding of proteins in the DNA region upstream of the a-subunit gene was examined by DNase I footprint analysis. Binding of proteins was found at 4 sites in the region -163 to +7. We focused our analysis on the DNA-protein interactions on a 39-bp sequence designated as Box I, which is conserved among upstream regions of genes for 7S-type seed storage proteins. Gel mobility shift assay showed that at least three different protein factors bind to three distinct elements located within the Box I sequence.
Soy Protein Research, Japan 4, 11-18, 2001.
Key words : soybean, b-conglycinin, a-subunit, cis-acting element, DNA-protein, interaction
Elucidation of Structure-physicochemical Function Relationships at Subunit Level of Soybean b-Conglycinin by Using Mutant Cultivars
Nobuyuki MARUYAMA and Shigeru UTSUMI
Graduate School of Agriculture, Kyoto University, Uji 611-0011
ABSTRACT
We purified four single molecular species of b-conglycinin heterotrimers consisting of the a and b subunits or the a' and b subunits from mutant soybean cultivars lacking the a or a' subunit, respectively, and examined their structural features and physicochemical functions. The presence of more a or a' subunits in heterotrimer makes it more hydrophobic. The thermal stabilities of heterotrimers composed of two kinds of subunit are mainly influenced by that of the subunit which has lower thermal stability. Solubilities at low ionic strength ( m= 0.08) of the heterotrimers containing the a or a' subunits are very similar to those of the a or a' homotrimers (Soy Protein Research, Japan 3, 12-17, 2000), respectively. Emulsifying abilities and heat-induced associations of the heterotrimers containing one b subunit are similar to those of the a or a' homotrimer, while those of the heterotrimers containing two b subunits are similar to those of the b homotrimer. Furthermore, it was suggested that the number of the extension region, the surface hydrophobicity and the structural stability relate to the emulsifying abilities of the individual trimers of b-conglycinin.
Soy Protein Research, Japan 4, 19-26, 2001.
Key words : b-conglycinin, structure-physicochemical function relationship, solubility, thermal stability, soybean
Structural Relationship between Proteolipid-apoproteins and Oleosins of Soybean
Miyoko SUZUKI, Tomoyuki NAKAMURA, Naoko OKAI, Tomohisa OGAWA and Koji MURAMOTO
Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555
ABSTRACT
A proteolipid was preapared from defatted soybean meals by successive extraction with chloroform-methanol (2:1) and 0.73% NaCl. The lipid moiety was removed from the proteolipid by ethanol-diethyl ether (3:1) to give apoproteins. SDS-PAGE of the apoproteins showed a major band with an apparent molecular mass of 18 k. The hydrophobic apoprotein 18 k was purified by preparative SDS-PAGE. Oleosins of different molecular masses, ranging from 18 to 34 kDa, were prepared from soybean oil bodies by successive extraction and centrifugation, and then purified by preparative SDS-PAGE. Structural comparison of apoprotein 18 k and oleosin 18 k showed that they were distinct protein molecules. A new procedure for the separation of hydrophobic proteins was also proposed by using gel filtration and reversed-phase HPLC under denatured and reduced conditions.
Soy Protein Research, Japan 4, 27-32, 2001.
Key words : soybean proteolipid, oleosin, apoprotein, hydrophobic protein
Utilization of Phytase in Koji natto : Hydrolysis of Phytate in Soy Foods and Improvement of the Utilities of Micro amount Minerals
Kozo OHTSUKI, Kenji SATO and Yasushi NAKAMURA
Faculty of Human Environment, Kyoto Prefectural University, Kyoto 606-8522
ABSTRACT
Phytic acid tightly combines with the minerals, such as Ca, Mg, Fe, Cu and Zn, which are essential for human nutrition. Less phytic acid content in food is better for the child nutrition, whereas soybean food contains much phytic acid. Some fermentation processings hydrolyze phytic acid. Koji-natto is one of the fermented foods of soybean. Koji-natto includes Shio-natto, Hama-natto and Daitokuji-natto. We analyzed the phytic acid in Koji-natto and found it lower quantity compared with that in Kinako. Shio-natto was made of Itohiki-natto (50%), Koji (Asp. oryzae, 45%) and NaCl (5%) in our laboratory, and aged at 4oC for 5 weeks. The content of phytic acid in the Shio-natto decreased to 50% in 2 weeks and to 10% in 4 weeks. The ratio of free mineral to total mineral in the Shio-natto was higher than that in Kinako. Koji-natto is a good process food of soybean for the mineral nutrition.
Soy Protein Research, Japan 4, 33-38, 2001.
Key words : soybean, Shio-natto , koji, phytic acid, phytase
Allergenicity of a Soybean Allergen, Gly m Bd 28K
Hideaki TSUJI1, Miki HIEMORI1, Masumi KIMOTO1, Hiromi YAMASHITA1, Tadashi OGAWA2 and Shigeru UTSUMI2
1 Faculty of Health and Welfare Science, Okayama Prefectural University, Soja 719-1197
2 Graduate School of Agriculture, Kyoto University, Uji 611-0011
ABSTRACT
A soybean allergen, Gly m Bd 28K has been purified by various chromatographic techniques. The purified allergen was shown to be an Asn-linked glycoprotein with a molecular mass of 26 kDa. A cDNA encoding the allergen has been cloned using a lZAP cDNA library prepared from mRNA in developing soybean cotyledons. The cDNA contained 1567 bp with an open reading frame encoding 473-amino acid sequence. Homology analysis shows that the product for the cDNA exhibits high homology with MP27/MP32 in pumpkin seeds and a carrot globulin-like protein and that the product may be a preproprotein. The allergen was shown to locate in the former half part of the preproprotein and might be biosynthesized in the same manner as pumpkin MP27/MP32 is. Interestingly, the recombinant allergen expressed in E. coli using a pGEX vector showed weak responses in reactions with the sera of several soybean-sensitive patients, whereas the native allergen showed strong responses. The glycopeptide isolated reacted strongly with the above sera and the deglycosylated peptide showed no reactivity. These observations demonstrate that the sugar moiety of the allergen binds to IgE antibodies in the sera
of the patients.
Soy Protein Research, Japan 4, 39-44, 2001.
Key words : soybean allergen, Gly m Bd 28K, cDNA, pumpkin MP27/MP32, carrot globulin-like protein
Occurrence of IgE Antibody Recognizing Asparagine N-linked Glycan Moiety of Soybean Glyco-proteins and Its Cross-reactivity
Tadashi OGAWA, Tatsuya MORIYAMA, Chizuko FUJITA and Kyoko TOMITA
Graduate School of Agriculture, Kyoto University, Uji 611-0011
ABSTRACT
A major soybean allergen, Gly m Bd 30K is known as a glycoprotein having a typical oligo-mannose type glycan moiety of Man3GlcNAc2 backbone with the b1-2 xylose branch, and/or b1-2 xylose and a1-3 fucose branches, which is located on Asn-170 residue. Patient's serum containing the allergen-specific IgE antibodies was fractionated by an affinity chromatography using horseradish peroxidase-immobilized column. The fraction absorbed on the column was found to contain the glycan specific IgE antibodies and the flow-through fraction from the column contained the IgE specific to peptide backbone. When the inhibition-blotting experiment was carried out using the small peptides bearing glycan moiety prepared from bromelain hydrolyzate, the dose-dependent inhibition of the binding between IgE and the allergen was observed. A contribution of glycan-specific IgE antibody on the RAST values was estimated to be about 70%, which is assumed to be one of the possible factors to give the false positive diagnostic results as to the causative foods, because the common glycan moiety will behave as a pan-allergen among the plant food stuffs.
Soy Protein Research, Japan 4, 45-51, 2001.
Key words : food allergy, soybean allergen, glycoprotein, glycan moiety, radioallergosorbent test, cross-reactivity
Establishment of Immunoassay for Soybean Trypsin Inhibitor (KSTI) Based on Its Inhibitory Activity and Its Application for the Detection of KSTI Absorbed in Portal Vein
Wakako YANO, Chikako YAMADA and Tsukasa MATSUDA
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-0601
ABSTRACT
The absorption of soybean trypsin inhibitor (KSTI) from digestive tract to portal vein was investigated. To detect KSTI retaining its trypsin inhibitory activity two assay methods based on the specific binding of KSTI to trypsin were developed. An ELISA-plate was coated with bovine trypsin and incubated with KSTI added to mouse serum at varied concentrations. KSTI bound to the trypsin on the plate was detected by ELISA using anti-KSTI and POD-labeled secondary antibodies. The KSTI in serum was successfully detected at a level of 10 ng/mL, though mouse serum competitively interfered the KSTI-binding to trypsin probably due to its endogenous trypsin inhibitors. As an alternative assay method an affinity precipitation method using trypsin-coupled Sepharose was applied to precipitate KSTI from serum. The precipitate was subjected to SDS-PAGE and immunoblot using the anti-KSTI and POD-labeled secondary antibodies. KSTI was also detected by this method, and the detection limit was about a level of 50 ng/mL serum. These assay methods were applied for the detection of KSTI absorbed from intestine into portal vein. KSTI was injected into duodenum of anesthetized mice, and blood was collected from the portal vein 10-30 min later. In the serum of portal vein, KSTI retaining its trypsin inhibitory activity was detected at a level of 100-300 ng/mL. The anti-KSTI antibody detected a band with a molecular mass of about 20 kDa, corresponding to the intact protein.
Soy Protein Research, Japan 4, 52-57, 2001.
Key words : protein absorption, soybean trypsin inhibitor (KSTI), immunoassay, portal vein
Molecular Recognition of Bile Acids by Soy Protein and Modeling of Its Mimetics
Kimikazu IWAMI, Hideharu MATSUDA, Takeo KOMORI and Ryuhei KANAMOTO
Faculty of Agriculture, Kyoto Prefectural University, Kyoto 606-8522
ABSTRACT
Resistant protein has some properties beneficial for health. Soybean resistant protein was prepared from HMF by a combination of pepsin and pancreatin digestions (hereinafter, referred to as HMF-RP). HMF-RP was superior to HMF in bile acid-capturing capacity. As for fecal nitrogen and bile acid excretions, they did not differ between both groups of rats fed 10% HMF-RP and 20% HMF diets. The feces were gathered from the rats fed the 10% HMF-RP diet and washed with water, 70% ethanol and 8 M urea. The fecal leavings thus obtained and HMF-RP itself after acid hydrolysis were quite similar in amino acid composition to each other. This implies that HMF-RP is scarcely impaired throughout the digestive tract and excreted, as it is, into the feces. HMF or HMF-RP was characteristic of masking bile acids. In this connection, several soy protein products were examined for bile acid-capturing capacities by equilibrium dialysis. These products were bovine serum albumin (BSA) > 7S-G > HMF > soy flour > 11S-G > SPI in order of deoxy cholate-capturing capacity. Interestingly the capacity of BSA decreased with increasing concentrations of coexistent fatty acid, while the presence of high concentrations of fatty acid did not affect those of other soy protein products at all. It was assumed from this observation that soy protein might have the structural specificity for the skeleton moiety of steroids.
Soy Protein Research, Japan 4, 58-64, 2001.
Key words : resistant protein, HMF-RP, bile acid-capturing capacity, equilibrium dialysis
Effect of Soy Protein Isolate on Cytokine Productivity and Abnormal Lipid Metabolism in Rats with Carrageenan-induced Inflammation
Kazumi YAGASAKI, Masaharu KANEKO and Yutaka MIURA
Department of Applied Biological Science, Tokyo Noko University, Fuchu 183-8509
ABSTRACT
Effect of soy protein isolate (SPI) on tumor necrosis factor-a (TNF-a) productivity and abnormal lipid metabolism was studied in rats with carrageenan-induced inflammation. Two days after carrageenan injection, serum triglyceride (TG) and albumin concentrations decreased, while liver TG content and TNF-a productivity in macrophages increased. Feeding previously for 2 weeks a 20% SPI diet, compared with feeding a 20% casein diet, decreased pouch fluid volume and carrageenan-induced rises in TNF-a production in macrophages 2 days after carrageenan injection. The SPI diet remedied carrageenan-induced hypoalbuminemia, but failed to suppress carrageenan-induced, temporary accumulation of liver TG. These results suggest a potent anti-inflammatory action of SPI.
Soy Protein Research, Japan 4, 65-69, 2001.
Key words : soy protein isolate, carrageenan, inflammation, albumin, triglyceride, cytokine
Effect of Amino Acid Mixture Simulating the Composition of Soy Protein Isolate on Muscle Atrophy by Suspension Hypokinesia/Hypodynamia in Rats
Osamu TADA and Hidehiko YOKOGOSHI
Graduate School of Nutritional and Environmental Sciences, The University of Shizuoka, Shizuoka 422-8526
ABSTRACT
Under the microgravity condition like the universe, it is known that various nutritional and physiological changes in the body are induced. Especially in the aspect of nutrition, muscle atrophy is a characteristic phenomenon accompanying the weightlessness. This study was conducted to investigate the ameliorating effect of muscle atrophy caused by suspension hypokinesia, using the amino acid mixtures simulating the composition of soy protein isolate (SPI) as the protein source as compared with simulating the composition of casein. Male Wistar strain rats (190 g) were divided into two groups, suspended with suspension harness, and were fed on a 20% SPI pattern amino acid mixture or a 20% casein pattern amino acid mixture for 10 days. The body weight of the suspended rats fed casein pattern diet and SPI pattern diet decreased similarly. The weights of the gastrocnemius and soleus muscles were decreased by suspension hypokinesia, however the degree of the decrease of the weight of muscles, especially soleus muscle, of rats fed SPI pattern diet was smaller than that of rats fed casein pattern diet. Serum Nt-methylhistidine concentration was significantly lower in the rats fed SPI pattern diet than that in the rats fed casein pattern diet. Similarly, the activities of muscle protein-degrading enzymes such as calpain and proteasome were significantly lower in the rats fed SPI pattern diet than those in the rats fed casein pattern diet. Therefore, it is suggested that SPI pattern amino acid mixture caused to reduce the proteolysis of skeletal muscles through the reduction of the activities of calpain and proteasome, in consequence to ameliorate the muscle atrophy.
Soy Protein Research, Japan 4, 70-76, 2001.
Key words : hypokinesia/hypodynamia, muscle atrophy, calpain, proteasome, soy protein isolate
Effect of Soybean Peptide Fractions on the Helicobacter pylori-infected Mice
Ah.Mee PARK and Masayasu INOUE
Osaka City University Medical School, Osaka 545-8585
ABSTRACT
Helicobacter pylori (H. pylori), a gram-negative and microaerobic bacterium, plays important roles in the pathogenesis of gastritis, peptic ulcer and gastric cancer. We previously reported that some fractions of soy bean peptides have bacteriostatic effect on H. pylori in vitro. The present work studied the effect of soy bean peptide fractions on the growth of H. pylori infected in mice. Effect of oral administration of fraction S peptides on the growth of H. pylori infected in c57BL/6j mice was analyzed in vitro. H. pylori infectivity and antioxidants were assayed. Plasma levels of antibody against H. pylori and reactive oxygen species generated by blood neutrophils increased in H. pylori-infected mice. Administration of S faction of soy bean enhanced these changes induced by H. pylori. Plasma levels of ascorbic acid were increased by the conformation of H. pylori infection and administration of fraction S peptides. These results suggest that, although the fraction S peptides of soy bean exhibited the appearance actions against H. pylori, such effect is not achieved in infected mice. In vivo effects of other peptides fractions of soy bean on the infected H. pylori should be studied further.
Soy Protein Research, Japan 4, 77-82, 2001.
Key words : Helicobacter pylori, soybean peptide, antioxidant, antibacterial
Effects of Soybean Peptide on the Damaged Muscle : Immunological Research
Ken-ichi MASUDA, Taroh TOMURA, Takako MATSUO and Yasuyuki UCHIDA
Kansai College of Oriental Medicine, Osaka 590-0482
ABSTRACT
We investigated effects of soybean peptide on the muscle damage in order to examine the immunological influence of soybean protein. Thirty-two Wistar male rats about 150 g weight were meal-fed in two groups, 16 rats each, 1) with a 20% casein, and 2) 20% soybean peptide as their protein source for two weeks. Each group of 16 rats was further divided into two, a) with no weight-bearing by the tail suspension method, and b) with exercise of treadmill until reaching all out. We especially focused on the damages of the skeletal muscle through b) the hard exercise from an immunological point of view. We compared those groups on parameters of the blood samples, and the histological specimens. Clear effects of soybean peptide were resulted from the lower value of creatine phosphokinase and IL-6, specifically in the no weight-bearing groups. In terms of histology, the type I fiber of the soleus muscle increased in the exercise group.
Soy Protein Research, Japan 4, 83-86, 2001.
Key words : soybean, muscle, creatine phosphokinase, IL-6, histology
Effect of Dietary Soybean Protein Isolate Hydrolysate on Oxidation of Food Components : Measurements of Substrate Oxidation Using Stable Isotope
Kengo ISHIHARA1, Shinnichi OYAIZU2, Yoshiko FUKUCHI1, Wataru MIZUNOYA2, Tohru FUSHIKI2 and Kyoden YASUMOTO1
1 School of Life Studies, Sugiyama Jogakuen University, Nagoya 464-8662
2 Graduate School of Agriculture, Kyoto University, Kyoto 606-8502
ABSTRACT
The effects of a long-term feeding of soy protein isolate hydrolysate (SPI-H) on the expiration of 13CO2 from dietary 13C-glucose or 13C-triolein in a type II diabetic mice, KK-Ay strain were investigated. The mice of 6 wk old were provided free access to a high fat diet (30% of energy from fat) for the initial 4 wk to induce diabetes. The animals were divided into two groups and during the subsequent 4 wk each group of animals was fed a diet containing 35% SPI-H or casein, 10% sucrose, and 5% soybean oil on energy intake restricted regimen to 60% of that given during the preceding high fat diet. For the last 3 days on the restricted dietary regimen, the animals were housed in the respiratory metabolic chambers. For the middle 24 h of the metabolic measurement, half of animals in each group fed a diet that sucrose was replaced by 13C-glucose and the remaining half fed another diet that soybean oil was replaced by 13C-triolein. The expiration rate of 13CO2 from 13C-glucose during the
period of initial 12 h was higher in the animals fed the SPI-H diet than those fed the casein diet. To the contrary, the expiration from 13C-triolein was lower in SPI-H group during the period of 7 to 22 h after the diets. Diabetic renal hypertrophy was alleviated in the SPI-H diet group compared with the casein diet group. These results are consistent with the notion that the mice on the SPI-H diet can utilize exogenous glucose more readily than those on the casein diet. It thus appears that SPI-H feeding for a prolonged period ameliorates glucose tolerance in type II diabetic mce.
Soy Protein Research, Japan 4, 87-96, 2001.
Key words : KK-Ay, 13C, glucose, diabetes, soybean
Influence of Early Postnatal Exposure to Isoflavone on the Reproductive Organ in Mice
Junko YAMASHITA1, Tsukasa MINETA2 and Kohshi NORIKOSHI2
1 Faculty of Nursing, Kure University, Kure 737-0004
2 Life Science Institute, Sophia University, Tokyo 102-8554
ABSTRACT
Isoflavones, classified as a phytoestrogen, are known to have estrogen-like effects. Since early postnatal treatment with estrogen induces persistent estrus in mice, we wanted to examine how isoflavones affect mice in this regard. Newborn female mice were injected subcutaneously with either isoflavone, daizein or genistein, or estradiol-17 b for 5 consecutive days from the day of birth. When the mice were between 4 and 7 weeks of age, vaginal smears were examined, and the vagina from each mouse was removed for histological study at 7 - 8 weeks of age. The vaginal smear test revealed that all the estradiol-treated mice had cornifying cells of the vaginal epithelia, indicating persistent estrus. Similar manifestations were observed in some mice treated with isoflavone, especially when the dose was higher than that of estradiol-17 b. However, the injection of equimolar isoflavone together with estradiol-17 b tended to arrest the harmful effect of estradiol. It seems necessary to establish concrete guidelines regarding intake of isoflavones for pregnant women and babies.
Soy Protein Research, Japan 4, 97-101, 2001.
Key words : isoflavone, daizein, genistein, persistent estrus, vaginal epithelia
Absorption and Transport of Soy Isoflavones and Their Estrogenic Effect
Shinichiro TAKASHIMA, Hiroyoshi SATO, Shoichiro INOUE, Hiroko TOMOYORI,Masao SATO and Katsumi IMAIZUMI
Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581
ABSTRACT
The purpose of this study was to investigate if the type of isoflavones influences their absorption, transport and estrogenic effect in rats and cultured cells. Isoflavone-rich fractions were isolated from soy protein isolate (SPI-I) and Soyaflavone E (Soya-I) preparations. Concentration of the serum isoflavones, in particular daizein, was higher in rats fed a diet containing Soya-I in relation to SPI-I for 2 weeks. These isoflavones affected, in a tissue-dependent manner, the mRNA abundance of cytochrome c oxidase subunit VII related protein (COX7RP) that contains a gene responsive to estrogen. Female rats were sham-operated (Sham) or ovariectomized (OVX) and fed a control diet or a diet containing Soya-I for 3 weeks. The OVX group fed the control diet increased their food consumption, body weight gain and the liver triglyceride concentration. Feeding the diet containing Soya-I improved these parameters. There was no prominent effect of ovariectomy on the COX7RP mRNA abundance in tissues. In cultured COS-7 cells or Hela cells transfected with estrogen receptor (ER) gene and ER responsive element gene, the transactivation effect by genistein was higher than by daizein or glycitein. These results indicate that dietary isoflavones can ameliorate the endocrine derangement, but the active principle involved to be determined.
Soy Protein Research, Japan 4, 102-106, 2001.
Key words : isoflavones, absorption, ovariectomy, estrogen receptor
Effect of Isoflavone Aglycone on Thrombogenicity Assessed by the Mouse Carotid Artery Thrombosis Method Using apoE-/-ÅELDLR-/- Double Knockout Mice
Yoshinobu IJIRI1,2, Mayuko MIURA1,2, Masaru HASHIMOTO1,2, Tomomi TAKA1,2, Akira KUBOTA3, Sadahiro WATANABE4, Kazuhiro OIWA5, Chizuru FUKUNAGA6, Toyoko OKUDA7 and Junichiro YAMAMOTO1,2
1 Faculty of Nutrition, Kobe Gakuin University, Kobe 651-2180
2 High Technology Research Center, Kobe Gakuin University, Kobe 651-2180
3 Department of Surgical Pathology, Hyogo College of Medicine, Nishinomiya 663-8501
4 Division of Basic Medical Science, Kobe City College of Nursing, Kobe 651-2103
5 Communications Research Laboratory, Kansai Advanced Research Center, Kobe 651-2492
6 Kakogawa Synthetic Public Health Center, Kakogawa, Japan 675-0101
7 Department of Ars and Sciences, Osaka Kyoiku University, Kashiwara 582-8582
ABSTRACT
Thrombosis is deeply involved in life style-related diseases and prevention of these diseases by diet is very important. However, suitable animal models to demonstrate epidemiological results by experiments have been poorly established. The aim of the present study was to establish the animal model, which can evaluate the effect of diet on thrombogenicity, and to demonstrate antithrombotic effect of isoflavone aglycone using the animal model. Congenital atherogenic mice, apolipoprotein E and LDL receptor double knockout mice (apoE-/- and LDLR-/-), were used. Helium-neon laser-induced carotid artery thrombosis method and the cross section method of carotid artery were used to evaluate thrombotic tendency and atherogenicity, respectively. Diet models were prepared on the basis of Western and Japanese diets in 1970s and AIN93G. These were given to mice for 4 or 8 weeks. Western diet feeding with high fat (40% in energy) resulted in significant enhancement of thrombotic tendency and atherogenicity. Isoflavone aglycone supplemented in Western diet suppressed enhanced thrombotic tendency.
Soy Protein Research, Japan 4, 107-116, 2001.
Key words : thrombosis, animal model, atherogenic mice, diet, isoflavone
Preventive Effect of Soy Isoflavones on DMBA-induced Mammary Tumor Enhanced by Endocrine-disrupting Chemicals in the Rat
Kunitaka NASHIKI, Miho WATAKABE, Taro KISHIDA and Kiyoshi EBIHARA
Faculty of Agriculture, Ehime University, Matsuyama 790-8566
ABSTRACT
It has been reported that 3, 3', 4, 4' -tetrachlorobiphenyl (TCB), a polychlorinated biphenyl congener, can enhance the development of 7, 12-dimethylbenz (a) anthracene (DMBA) -induced mammary tumors in the rats (Nesaretnam et al. : Eur J Cancer, 34, 389-393, 1998). In this study, the ability of TCB to enhance the development of DMBA-induced mammary tumors and the possible role of soy isoflavone aglycone, daizein and genistein, in preventing tumor progression were studied in Sprague-Dawley (SD) virgin female rats. Mammary tumors were initiated in 80 rats at 50 days of age by intragastric administration of a single dose of 10 mg of DMBA. Sixty rats were also given 1.6 mg of TCB by intragastric administration at the same time as the DMBA and were subsequently also fed for one week a TCB (100 mg/kg diet) containing high-fat (20% w/w corn oil) diet. Of the 60 rats given TCB, 20 rats were then fed for 9 weeks a high-fat diet, 20 rats were then fed for 9 weeks a daidzein (200 mg/kg diet) containing high-fat diet and 20 rats were then fed for 9 weeks a genistein (200 mg/kg diet) containing high-fat diet. TCB had no effects on the development of DMBA-induced mammary tumors. Daidzein and genistein could not retard the first appearance of palpable tumors and reduce the weight and volume of tumors.
Soy Protein Research, Japan 4, 117-122, 2001.
Key words : soy isoflavones, DMBA-induced mammary tunor, TCB, endocrine-disrupting chemicals, rat
Suppressive Effect of Soy Isoflavone on Estrogenic Activity Expression
Koji YAMADA1, Dal-Ho Han1, Michihiro SUGANO2 and Hirofumi TACHIBANA1
1 Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581
2 Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, Kumamoto 862-0920
ABSTRACT
To compare estrogen receptor-binding activity of environmental estrogens, their 50%-inhibiting concentrations (IC50) on the binding of 17 b-estradiol to human recombinant estrogen receptor b were examined. In addition, estrogenic activity of these compounds was examined using human mammary cancer MCF-7 cells. Among soy isoflavones, IC50 of daidzein was 10-7 M, and its maximal proliferation-enhancing effect was observed at 10-8 M. On the other hand, IC50 of genistein was 5 x10-8 M, and its maximal proliferation-enhancing effect was observed at 10-6 M, though it exerted strong estrogenic activity at 10-8 M. In general, there was a positive relationship between IC50 and the concentration giving optimal proliferation-enhancing effect, with some exceptions. In addition, we found that daidzein, genistein and luteolin not only inhibit the expression of proliferation-enhancing activity of environmental estrogens, but also strongly suppressed proliferation of the cells in combinational uses. These results suggest that studies on combinational effect of environmental estrogens are important for the clarification of biological meanings of the compounds.
Soy Protein Research, Japan 4, 123-128, 2001.
Key words : MCF-7 cells, isoflavone, anti-estrogenic activity
Inhibitory Effects of Soy-isoflavones on Osteoclastogenesis in Human Bone Marrow Cell Culture
Hidemichi EBISAWA and Yasuko KOSHIHARA
Department of Nutrition, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015
ABSTRACT
Genistein and daidzein were accepted as phytoestrogen that has estrogen-like effects on bone metabolism with little side-effects on reproductive organs. But, there are very few experimental reports that determine the effects of genistein and daidzein on human bone metabolism. The purpose of the present study is to elucidate the direct effects of genistein and daidzein on bone resorption, such as osteoclast formation and osteoclastogenesis-related cytokine production. Monocyte-rich fraction was collected from human bone marrow and the cells were seeded at 5 x 105 cells/well in 8-well chamber slide. The cells were cultured for 2 weeks in a medium containing either vehicle, genistein, daidzein or 17 b-estradiol at a concentration of 10-7 M to 10-5 M. Genistein, daidzein or 17 b-estradiol caused a significant decrease in a formation of osteoclast-like cell. Daidzein (10-5 M) suppressed IL-6 and M-CSF production in bone marrow cell culture, but genistein had no effect on these cytokine productions. Genistein, daidzein and 17 b-estradiol had no effect on osteoclastogenesis inhibitory factor (OCIF) production. These results suggested that daidzein has suppressive effects on osteoclast-like cell formation through decrease in IL-6 and M-CSF production, and genistein has different mechanisms to decrease osteoclast-like cell formation in human bone marrow cell culture.
Soy Protein Research, Japan 4, 129-134, 2001.
Key words : genistein, daidzein, cell culture, human bone marrow, osteoclastogenesis
Effects of Soybean Isoflavones on Bone Mineral Density and Bone Metabolism in Women : Correlation among Isoflavone Concentrations in Biological Fluids, Sex-hormone Binding Globulin and Bone Biomarkers
Yusuke ARAI, Mariko UEHARA, Mitsuru KIMIRA and Shaw WATANABE
Faculty of Applied Bio-science, Tokyo University of Agriculture, Tokyo 156-8502
ABSTRACT
In the previous study, we found that a significant negative correlation between plasma genistein concentration and urinary deoxypyridinoline (D-Pyr), a marker of bone resorption, and a positive correlation between urinary equol excretion and plasma osteocalcin (OC) were present in women (n = 41) living in an area of Tohoku, Japan. In the present study, to assess the correlation among isoflavone concentrations in biological fluids, sex-hormone binding globulin (SHBG) and bone biomarkers, we analyzed plasma and urinary isoflavonoid concentrations, bone mineral density (BMD), plasma OC and SHBG, and urinary D-Pyr levels. The subjects were 93 female volunteers (33-84 years old, premenopause 19, postmenopause 74), who gave their written informed consent to participate to the study during 1997-2000. Each subject completed a 3-day dietary record and the health check-up. Their plasma and 24 h-urine samples were collected for measurement of isoflavonoid concentrations by the time-resolved fluoroimmunoassay and HPLC. OC and D-Pyr were measured by the enzyme linked immunosorbent assay. Stiffness value of bone was used for the assessment of subjects' BMD. After multiple regression analysis to assess the relationship of bone health, dietary factor and life style, eliminating confounding factors (age, BMI, intake of carbohydrate, calcium, phosphorus, magnesium, vitamin C and vitamin K), a significant positive correlation between plasma daidzein and BMD was observed in equol excreters (r = 0.290, n = 50, P<0.05). Additionally, we measured plasma 17 b-estradiol and SHBG concentrations in pre- (n = 15) and postmenopausal (n = 44) women from the study 2000. In premenopausal women, there was a significant positive correlation between plasma genistein concentration and BMD (r = 0.724, P< 0.001), and between plasma daidzein concentration and SHBG concentration (r = 0.309, P<0.05), respectively.
Soy Protein Research, Japan 4, 135-141, 2001.
Key words : genistein, daidzein, equol, sex-hormone binding globulin, bone mineral density
Effects of Soy Protein Isolate with or without Vitamin E on Serum Lipids and Safety among Healthy Men
Kenji HIGASHI1, Shinji TABATA1, Tatsuo OBATA1, Tetsuya HISADA1, Shojiro SAWADA1, Masatsune OGURA1, Takeshi YAMASHITA2, Emiko MIYAJIMA2, Fumitaka OHSUZU1 and Haruo NAKAMURA2
1 First Department of Internal Medicine, National Defense Medical College, Tokorozawa 359-0042
2 Mitsukoshi Health and Welfare Foundation, Tokyo 160-0023
ABSTRACT
Soy protein has been reported to improve hypercholesterolemia, however, we previously reported that intake of 20 g soy protein isolate (SPI) for 3 weeks in healthy men might reduce plasma vitamin E and testosterone by 10% and 5%, respectively. The aims of this study were to evaluate the effects of SPI intake on sex hormones and vitamin E levels, and to determine the effect of vitamin E supplement with SPI on vitamin E level. Twelve healthy men were given 20 g per day of soy protein isolate (SPI) either with or without 200 mg per day of vitamin E for each 3-week, in a randomized crossover design. Total cholesterol and triglyceride concentrations were slightly and insignificantly decreased. Testosterone and estrogen concentrations were not significantly changed by 3 weeks SPI intake. However, vitamin E concentration was significantly reduced by 9.2% after SPI intake for 3 weeks. Vitamin E supplement with SPI intake increased vitamin E concentration. These results demonstrate that SPI intake reduces plasma vitamin E, although vitamin E supplement compensates for this reduction. Therefore, vitamin E supplement may be required in subjects with long-term intake of soy protein.
Soy Protein Research, Japan 4, 142-145, 2001.
Key words : soy protein, testosterone, estrogen, vitamin E, cholesterol
Association of the A-204C Polymorphism in the Cholesterol 7a-hydroxylase Gene with Cholesterol Lowering Effect of Soy Protein Isolate
Shinji TABATA1, Kenji HIGASHI1, Masatsune OGURA1, Tatsuo OBATA1, Tetsuya HISADA1, Syoujiro SAWADA1, Makoto AYAORI1, Takeshi YAMASHITA2, Toshitsugu ISHIKAWA3, Fumitaka OHSUZU1 and Haruo NAKAMURA2
1 First Department of Internal Medicine, National Defense Medical College, Tokorozawa 359-8513
2 Mitsukoshi Health and Welfare Foundation, Tokyo 160-0023
3 Sony Corporation Wellness Center, Tokyo 141-0001
ABSTRACT
Soy protein reportedly has a lowering effect on serum cholesterol. However, there were considerable differences in individual response during its supplementation. Cholesterol 7a-hydroxylase (CYP7A1) is one of the important enzymes for cholesterol metabolism and A-204C polymorphism in CYP7A1 gene was reported to be associated with serum cholesterol levels. We investigated the association between the polymorphism of CYP7A1 and the individual responses in serum lipids after soy protein isolate (SPI) supplementation. Sixteen healthy males aged 30.8 ± 2.6 years took 20 g of SPI per day for 4 weeks. They were divided into AA (n = 4), AC (n = 8), and CC (n = 4) group based on A-204C polymorphism of CYP7A1. Serum total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C) was in the order CC > AC > AA group (TC ; 222, 195, 178 mg/100 mL, respectively, P = 0.033 : LDL-C ; 146, 111, 106 mg/100 mL, respectively, P = 0.032), but triglycerides (TG) and high density lipoprotein-cholesterol (HDL-C) did not differ in each group. After SPI supplementation, TC significantly decreased in CC and AC group (5.5 and 4.5% from baseline, respectively), but did not change in AA group. LDL-C appeared to decrease in CC group, but did not reach statistical significance. There were no changes in TG and HDL-C levels in each group. These results indicate that A-204C polymorphism in CYP7A1 affects differential response in lowering serum cholesterol levels by soy protein supplementation.
Soy Protein Research, Japan 4, 146-150 2001.
Key words : soy protein, CYP7A1, cholesterol polymorphism