Mechanism for the Regulation of the Gene Expression of CYP93A Gene Family Involved in Isoflavone Biosynthesis of Soybean
Hiroyuki OHTA, Hideaki SUEOKA and Ken-ichiro TAKAMIYA
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501
Soy Protein Research, Japan, 3(Serial Vol. 21), 1 - 5, 2000.
A soybean cytochrome P450, CYP93A1 is dihydroxypterocarpan 6a-hydroxylase (D6aH) which is involved in the elicitor-inducible biosynthesis of glyceollin, the isoflavonoid compound and the phytoalexin of soybean. Recently, it has been proposed that isoflavonoids have various good effects on human health. So, it is of interest to clarify the regulation mechanism of the biosynthetic pathway of glyceollins and other flavonoids in soybean plants. Although three genes (CYP93A1 to A3) were isolated as CYP93-subfamily of soybean, the function of CYP93A2 and A3 is unknown. In this study, the genomic DNA fragments containing CYP93A2 and A3 coding region were isolated and CYP93A3 was sequenced. CYP93A2 and A3 mRNAs were specifically induced in soybean cells by an elicitor which was derived from cell wall fraction of fungal pathogen, Phytophthora megasperma f. sp glycinea similarly to CYP93A1. Furthermore, in particular, the CYP93A2 mRNA was induced in accordance with the accumulation of phytoalexin glyceollin in soybean cotyledon. The induction of CYP93A3 mRNA by yeast elicitor was much earlier than those for genes involved in the early stage of glyceollin biosynthesis. Moreover, the induction by methyl jasmonate (MeJA) was not observed in CYP93A3. We observed elicitor-induced several products including glyceollin, whereas MeJA induced no glyceollin in SB-P. These facts suggest that the P450 enzymes are coordinately involved in flavonoid biosynthesis in soybean cells.
Key words : isoflavonoid, cytochrome P450, elicitor, phytoalexin, disease-resistance
Linkage Analysis of the Genes for Deficiency of 7S Globulin in Soybean
Kyuya HARADA1, Masaki HAYASHI1 and Yasutaka TSUBOKURA2
1 Faculty of Horticulture, Chiba University, Matsudo 271-8510
2 Graduate School of Science and Technology, Chiba University, Matsudo 271-8510
Soy Protein Research, Japan, 3(Serial Vol. 21), 6 - 11, 2000.
The 7S globulin (b-conglycinin) is one of the main protein components of soybean seed and is composed of three major subunits a, a', b. Two mutant lines lacking all the major subunits have been reported. The deficiency is controlled by a single recessive gene cgdef and a single dominant gene Scg, respectively. But the cloning of these genes has not yet tried. As a first step to isolate the responsible genes, we analyzed the map position of each gene. Two AFLP markers were identified that co-segregated with Cgdef using the segregants obtained by selfing of a plant heterozygous for cgdef. After cloning and sequencing the two AFLP fragments, they were converted to sequence-characterized amplified regions (SCARs). Linkage analysis of the two AFLP-SCAR markers in F2 plants derived from a cross between Moshidou Gong 503 and the plant heterozygous for cgdef showed that these markers are tightly linked to Cgdef at a distance 1.1 ±} 0.12 cM. Southern hybridization suggested that Scg is located at or near the coding region of a' subunit gene. Because transcription of each subunit gene was observed by RT-PCR even in plants homozygous for Scg, it was estimated that Scg acted at the post-transcriptional level.
Key words : soybean, b-conglycinin, deficient mutant, AFLP-SCAR marker, dominant-negative allele
Investigation of Structure-function Relationships of Soybean b-Conglycinin Using Mutant Soybean Cultivars
Nobuyuki MARUYAMA and Shigeru UTSUMI
Research Institute for Food Science, Kyoto University, Uji 611-0011
Soy Protein Research, Japan, 3(Serial Vol. 21), 12 - 17, 2000.
b-Conglycinin is a trimeric protein consisting of three subunits a, a' and b . All subunits are N-glycosylated and a and a' contain extension regions in addition to the core regions common to all subunits. We prepared homogeneous trimers consisting of only a, a' and b from soybean mutant cultivars lacking one or two subunits : a homotrimers from an a'-lacking mutant, a' homotrimers from an a-lacking mutant and b homotrimers from an aa'-lacking mutant. Structural features (secondary structure, thermal stability) and functional properties (solubility, emulsifying ability, heat-induced association) of the three homotrimers were examined and compared with those of recombinant homotrimers having no carbohydrate moiety (Soy Protein Reserach, Japan 1, 19-24, 1998) in order to elucidate the structure-function relationships and the roles of the carbohydrate moieties of b-conglycinin. The results indicate that 1) the recombinant homotrimer has the same structure as that of the native one, 2) thermal stability is different among subunits (b > a' > a) and the carbohydrate moieties do not contribute to thermal stability, 3) a and a' having the extension regions exhibit better solubility, emulsifying ability and heat-induced association than does b, and 4) the carbohydrate moieties contribute to solubility of three subunits at low ionic strength, emulsifying ability of b and heat-induced association of a and a'.
Key words : soybean, b-conglycinin, carbohydrate moiety, structure-function relationships
Research and Application of Cold Denaturation of Soy Protein
Rikimaru HAYASHI
Graduate School of Agriculture, Kyoto University, Kyoto 606-8502
Soy Protein Research, Japan, 3(Serial Vol. 21), 18 - 21, 2000.
Protein coagulates or gelatinizes by high pressure-treatment as well as by heat-treatment. Pressure-denaturation of protein could be brought about by pressurization even at low temperature. To elucidate the cold denaturation process under high pressure and to apply the process to food protein gelation, we studied the possibility of cold-denaturation with approximately 30 proteins stocked in our laboratory by treating them at 60 to -30 oC under 400 MPa. As a result, we found that carboxypeptidase Y denatured at -20 oC under 200 MPa. The denaturation process was studied by means of fluorescence and absorption spectroscopy and circular dichoroism under high pressure (in situ observation) and after high-pressure treatment (ex situ observation) in reference to its heat-denaturation process. Soy proteins caused pressure-induced coagulation depending on the temperature, but further studies are required to apply the cold denaturation process to forming soy protein gel of high quality.
Key words : soy protein, cold denaturation, pressure denaturation, carboxypeptidase
Y
Proteolytic Digestion of Soy Protein Isolates and the Coagulation of the Digests
Kuniyo INOUYE, Teisuke TAKITA and Kohhei NAGAI
Graduate School of Agriculture, Kyoto University, Kyoto 606-8502
Soy Protein Research, Japan, 3(Serial Vol. 21), 22 - 28, 2000.
We investigated the deodorization of soy protein isolates (SPI) with various solid absorbents and reported that some of them removed free or bound hexanal on the surface of proteins but not at the inside of proteins. Since the chymotryptic digestion of SPI did not promote the removal of hexanal by the absorbents, it was concluded that the inside hexanal might exist in the similar state even in the digests. For more efficient deodorization of SPI with absorbents, the proteolytic digestion of SPI must be improved. In this study, the digestions of SPI by subtilisin Carlsberg, subtilisin BPN', chymotrypsin, papain, and trypsin were investigated. The measurement of the absorbance at 280 nm of acid-soluble fraction and SDS-PAGE of the digested SPI indicated that subtilisins Carlsberg and BPN' showed similar digesting activity against SPI and their activity was significantly larger than that of the other proteases. Precipitation of the coagulum was observed in the digestion of SPI by subtilisin Carlsberg, subtilisin BPN', and papain, but not by chymotrypsin and trypsin. The time needed for the precipitation by subtilisins Carlsberg and BPN' was considerably shorter than that by papain, indicating that the fragmentation of SPI significantly causes the coagulation. However, the differences in the clotting time and in the time-course of the turbidity between two subtilisins albeit the comparable digesting activity indicated that the property of the digested fragments also plays an important role in the coagulation. These results suggest that the states of SPI digests significantly depend on the digesting activity and the substrate specificity of the proteases.
Key words : protease, digestion, coagulation, soybean, soy protein isolate
Search of Plasticizers for Soy Protein Isolate-Films
Hironori YAMAZOE, Shuhei SOGOYA and Kiyoshi YAMAUCHI
Faculty of Engineering, Osaka City University, Osaka 558-8585
Soy Protein Research, Japan, 3(Serial Vol. 21), 29 - 33, 2000.
About 80 chemicals were examined in order to search the plasticizers for a soy protein isolate-film. The film was prepared by casting a mixture of SPI, plasticizer and formaldehyde (weight ratio ; 1 : 0.1-0.4 : 0.01-0.1). The resulting films were evaluated on the basis of ultimate elongation strength, elongation extent, Young's modulus, swelling extent (in water) and tolerancy to bending (by 90 degree). Although structural factor of plasticizers for SPI was not evaluated well, it appeared that (i) the most compounds, which showed the plasticizer-effect, had the molecular-weight less than about 1000, (ii) the compounds having plural OH, SH, NH2, COOH, S=O groups often played a role of a plasticizer, (iii) cyclopropane and cyclohexane groups are more effective than the similar member-aromatic rings, (iv) a combinational use of plasticizers is more effective than the use of single plasticizer.
Key words : chemical modification, plasticizer, protein film, soy protein isolate
Development of Novel Antioxidants Originated from Soy Protein by Conjugation with Galactomannan
Soichiro NAKAMURA
Faculty of Education, Shimane University, Matsue 690-8504
Soy Protein Research, Japan, 3(Serial Vol. 21), 34 - 39, 2000.
Phytic acid associated proteins (pAPP) and polypeptides (SPP) were prepared from acid precipitated proteins from SPI (APP) and tryptic digests of SPI, respectively, by using ion-exchange chromatography with DEAE-Sepharose Fast Flow. A polysaccharide, galactomannan was prepared from soybean's brawn. APP, pAAP and SPP were conjugated with galactomannan via a naturally occurring Maillard reaction at 60 oC in 79% relative humidity for 2 weeks. Chelating activities of pAAP and SPP were not significantly lowered by the polyglycosyl modification. On the other hand, emulsifying properties of pAPP and SPP were dramatically improved by the controlled Maillard-type conjugation. Besides, radical scavenging power was substantially elevated by the conjugation, thus resulting in high antioxidant effects in pAPP- and SPP-galactomannan conjugates. This study suggests that the phytic acid associated proteins and polypeptides could be used as a new food antioxidant having good emulsifying properties.
Key words : phytic acid, galactomannan, Maillard-type conjugation, emulsifying
properties, radical scavenging power, antioxidant activity
Research for an Antioxidative Pathway Induced by Soybean Components
Asako TAKENAKA
Faculty of Agriculture, Yamagata University, Tsuruoka 997-8555
Soy Protein Research, Japan, 3(Serial Vol. 21), 40 - 44, 2000.
The preventive effects of soy protein on paraquat-induced oxidative stress were determined in rats. Male Wistar rats of 4 weeks old were fed diets with 20% casein, 20% soy protein isolate or 20% casein supplemented with saponin and isoflavone. Decreased body weight gain and increased lung weight by feeding the rats diets containing paraquat were clearly suppressed in rats fed a diet containing soy protein isolate. Supplementation of saponin and isoflavon of soy protein did not have the preventive effect. These results suggested that soy protein isolate acted preventively against the oxidative stress in vivo induced by active oxygen species formed through the action of paraquat.
Key words : soy protein, paraquat, oxidative stress, rat
Relationship between Soy-derived Isoflavones and Endocrine Disruptors in Cultured Chinese Hamster Lung Fibroblast (CHL/IU) Cells
Junko YAMASHITA1 , Yoshiko SEKI2 , Eisuke SHIMIZU2
1 Faculty of Nursing, Kure University, Kure 737-0004
2 Jikei Universtiy School of Medicine, Tokyo 105-8461
Soy Protein Research, Japan, 3(Serial Vol. 21), 45 - 47, 2000.
Isoflavones in soybeans are estrogenic compounds, and are categorized as phytoestrogens. The aim of the present experiment was to examine the effects of isoflavone complex on the growth and induction of micronuclei in cultured Chinese hamster lung fibroblast (CHL/IU) cells. The complex was extracted from soybean hypocotyls, containing about 40 mg of at least 12 types of isoflavones, along with 20.4 mg of daizin, 11.1 mg of glycitin, 4.6 mg of genistin and small amounts of their aglycons per 100 mg. The growth-inhibitory and micronuclei induction effects of the complex were tested using the protocol in the guidelines "Research for Factors in Mutagenicity Test" issued by the Ministry of Labor in 1996. When the cells were treated with the complex for 24 h at the concentration of around 2 mg/mL in the culture medium, the dose-dependent induction of micronuclei was observed. When the treatment with the complex in the medium along with S9 for 6 h was followed by a post-treatment period of 18 h in fresh medium, an increased number of micronuclei was observed. Our results suggest that a large isoflavone intake may cause adverse health effects such as induction of mutagenicity. Further investigation is necessary to establish concrete guidelines for the intake of isoflavones.
Key words : isoflavones, phytoestrogen, micronuclei, mutagenicity, CHL/IU
Effect of Ethanol-extracted Soy Protein Isolate on Cytokine Productivity in Macrophages from Hepatoma-bearing and Nephritic Rats
Kazumi YAGASAKI, Tatsuhiko YAMADA, Masaharu KANEKO and Yutaka MIURA
Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu 183-8509
Soy Protein Research, Japan, 3(Serial Vol. 21), 48 - 53, 2000.
The effect of ethanol-extracted soy protein isolate (SPI-EE) on cytokine productivity in macrophages was studied using tumor-bearing and nephritic rats. As model cancer cells, a rat ascites hepatoma cell line of AH109A was employed. The hepatoma cells were transplanted in the back of rats. Nephritis was induced in rats by injecting nephrotoxic serum into tail vein. The animals were given either a 20% casein diet (20C), 20% soy protein isolate diet (20S), or 20% SPI-EE diet (20SE) for 14 days (hepatoma-bearing rats) or 10 days (nephritic rats). In the hepatoma bearing state, productivity of tumor necrosis factor-a (TNF-a) in resident peritoneal macrophages was found to be significantly lower in 20S-fed rats than in 20C-fed ones, and tended to be higher in 20SE-fed rats than in 20S-fed ones. Likewise, in the nephritic state, productivity of TNF-a in macrophages tended to be lower in 20S-fed rats than in 20C-fed ones, and was significantly higher in 20SE-fed rats than in 20S-fed ones. These results suggest that suppressive effect of SPI on TNF-a productivity in macrophages from both hepatoma-bearing and nephritic rats is reduced by ethanol extraction and that some factor(s) suppressing TNF-a productivity must exist in the ethanol extract.
Key words : hepatoma, macrophage, nephritis, soy protein isolate, tumor necrosis factor-a
Effect of Isoflavones on Proliferation of Human Mammary Cancer MCF-7 Cells
Koji YAMADA1 , Dal-Ho HAN1 , Yoshiyuki MIYAZAKI1 , Michihiro SUGANO2 and Hirofumi TACHIBANA1
1 Division of Bioresource and Bioenvironmental Sciences, Graduate School of
Kyushu University, Fukuoka 812-8581
2Faculty of Environmental and Symbiotic Sciences, Prefectural University of
Kumamoto, Kumamoto 862-0920
Soy Protein Research, Japan, 3(Serial Vol. 21), 54 - 58, 2000.
To establish an assay system for evaluation of estrogenic activity of soybean isoflavones, effects of medium components on proliferation of human mammary cancer MCF-7 cells were examined. Proliferation of the cells was much slower in phenol red (PR)-free DMEM medium than in PR containing normal medium, and the addition of PR to PR-free medium enhanced the proliferation of the cells. In addition, the cells proliferated well in charcoal-treated fetal bovine serum (cFBS), but not in charcoal-treated calf serum. Thus, the cells were cultured in the PR-free DMEM medium supplemented with 5% cFBS to assay estrogenic activity of various environmental estrogens. Among flavonoids, daidzein and genistein exerted weak estrogenic activity at around 10-9 M. Among environmental estrogens, tamoxifen and mestranol exerted strong estrogenic activity at around 10-9 M, while 17a-ethynylestradiol and bisphenol A at around 10-5 M.
Key words : MCF-7 cells, isoflavone, anti-estrogenic activity
Study on Cancer Preventive Substances in Soybeans
Hoyoku NISHINO
Kyoto Prefectural University of Medicine, Kyoto 602-8566
Soy Protein Research, Japan, 3(Serial Vol. 21), 59 - 62, 2000.
In the previous study, we showed that genistein, one of the isoflavonoids found in soybean, inhibited the proliferation of prostate cancer DU145 cells. In addition to prostate cancer cells, various tumor cell lines were also proven to be sensitive to genistein. In the present study, we confirmed that genistein has anti-proliferative activity on human tumor cells, including gastric cancer cell line and lung cancer cell lines. Thus, genistein seems to be useful for the cancer control in a wide range spectrum. Further analysis of action mechanism of genistein is important before starting new clinical intervention trials, because of the development of novel methods, such as DNA array technology and proteomics technology, has recently been achieved. In this context, we evaluated the potency of genistein on expression of wide variety of genes using DNA macroarray, and found that the treatment of DU145 cells with genistein resulted in early induction of cell cycle related genes, such as p53, p53-dependent cell growth regulator CGR19, MDM2-like p53-binding protein, RBQ-3 and so on. We are now extending this kind of study by means of DNA microarray. And introduction of proteomics is now in planning. Since various substances co-exist with isoflavonoids in soybean, studies on these soybean constituents seem to be also valuable. Thus, we have started to assess biological activities of these substances, including soyasaponins, tocotrienol, and phytic acid. In the present study, we confirmed anti-tumor promoter activity of soyasaponin I and II.
Key words : anti-carcinogenic activity, anti-tumor promoter activity, genistein,
soyasaponins, DNA array
Effect of Soybean Peptide Fractions on the Growth of Helicobacter pylori
Ah-Mee PARK and Masayasu INOUE
Osaka City University Medical School, Osaka 545-8585
Soy Protein Research, Japan, 3(Serial Vol. 21), 63 - 66, 2000.
H. pylori, a gram-negative and microaerobic bacterium plays important roles in the pathogenesis of gastritis, peptic ulcer and gastric cancer. To test the possibility that dietary consumption of soybean proteins might beneficial for the improvement of the related risk factors, we studied their effects on the metabolism and growth of H. pylori. The three insoluble fractions of soybean peptide, DL, D1 and S, strongly inhibited the growth of H. pylori, but not of E. coli. Some of their fractions, R and D3, accelerated the respiration and ATP generation of H. pylori, while Fujipro -R had no appreciable effects. The understanding of the mechanism by which some soybean peptide fractions show bacteriostatic action against H. pylori might permits establishment of the method for minimizing H. pylori-dependent gastric injury and cancer.
Key words : H. pylori, soybean peptide, metabolism, antibacterial
Immunochemical Characterization of Soybean Allergen, Gly m Bd 30K as a Syringolide Receptor
Tadashi OGAWA, Tatsuya MORIYAMA, Chizuko FUJITA and Taro UESUGI
Research Institute for Food Science , Kyoto University, Uji 611-0011
Soy Protein Research, Japan, 3(Serial Vol. 21), 67 - 72, 2000.
A major soybean allergen, Gly m Bd 30K is known as a protein classified into a member of papain (thiol protease) super family, a homologue of a house dust mite allergen, Der p (f) 1, and a receptor of an elicitor, syringolide, produced by Pseudomonas syringae. Gly m Bd 30K was shown to occur in the vegetative plant (leaves) by immunoblotting with anti-Gly m Bd 30K monoclonal antibody, F5. Isolated native Gly m Bd 30K showed no hydrolytic activity like papain and bromelain. Syringolide was prepared from the culture medium of the plasmid (avrD)-transformed E. coli. Syringolide treatment elicited the hypersensitive response on leaves of the strain with disease resistance gene Rpg4 (+). Both the allergen and the syringolide-treated allergen gave the same molecular mass by a TOF-MASS analysis. The syringolide-treated allergen was dot-blotted on the nitrocellulose membrane and immuno-stained with F5 mAb and patient's serum. The syringolide treatment of the allergen enhanced the binding activity between immunoglobulins and the allergen, indicating that syringolide binding site (receptor site) seems to be different from the epitope site.
Key words : allergen, syringolide , Gly m Bd 30K, receptor, Pseudomonas syringae, elicitor
Effect of the Intestinal Protease Inactivation by Soybean Trypsin Inhibitor on the Immune Response to Oral Protein Antigens
Chikako YAMADA, Noriko NINOMIYA and Tsukasa MATSUDA
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601
Soy Protein Research, Japan, 3(Serial Vol. 21), 73 - 78, 2000.
The previous reports (Ninomiya et al., Soy Protein Research, 1, 75-80, 1998; 2, 70-75, 1999) showed that the oral administration of Kunitz soybean trypsin inhibitor (KSTI) inhibited not only trypsin activity but also chymotrypsin and elastase activities in the small intestine of B10.A mice. In the present study, the effect of such decrease in intestinal protease activities on digestibility and oral immunogenicity of proteins co-administered with the inhibitor was investigated. The soybean trypsin inhibitor (KSTI) and chicken egg trypsin inhibitor (ovomucoid) were used as protease inhibitors, and chicken egg lysozyme as a co-administered protein. Both trypsin inhibitors, especially KSTI, were more resistant to the gastrointestinal digestion than lysozyme. KSTI inhibited intestinal trypsin activity almost completely, whereas ovomucoid did not. Western blot analysis using anti-lysozyme antibody demonstrated that intact lysozyme remained in small intestine when lysozyme was co-administered with KSTI but not when it was done with ovomucoid. The responses of serum IgG1 and IgE antibodies agaisnt the orally administered lysozyme were analyzed for B10.A mice by ELISA. The oral co-administration of KSTI, or ovomucoid with lysozyme did not enhance the antibody response to the lysozyme as an oral antigen. Some mice in the KSTI/lysozyme-administration group showed rather lower response than those of the lysozyme administration. These results suggested that inhibition of the intestinal protease activity did increase residual intact food proteins in small intestine, but did not enhance the antibody response to the proteins.
Key words : soybean trypsin inhibitor (KSTI), ovomucoid, digestibility, antibody
response, food allergy
Effects of Soybean Isoflavones on Bone Mineral Density and Bone Metabolism in Human
Yusuke ARAI, Mariko UEHARA, Kikue OSHIMA, Noriko TAKADA, Mitsuru KIMIRA and Shaw WATANABE
Faculty of Applied Bio-science, Tokyo University of Agriculture, Tokyo 156-8502
Soy Protein Research, Japan, 3(Serial Vol. 21), 79 - 86, 2000.
The similar structure between soy isoflavone and estrogen and the findings that isoflavones possess weak estrogenic properties as shown by various experiments provided the initial basis for speculation that isoflavones may effect bone metabolism. In human, however, epidemiological data are insufficient to show the interaction between isoflavones and osteoporosis by the prospective cohort studies. To clarify the beneficial effects of soy isoflavones for bone health, we estimated the interaction among dietary intakes of isoflavones, plasma concentrations and urinary excretions, bone mineral density (stiffness), and bone formation and resorption biomarkers. The subjects were 41 female volunteers (40-69 years old; premenopause, 7; postmenopause, 34), who gave their written informed consent to participate to the study. Each subject completed a 3-day dietary record and the health check-up. Their plasma and 24 h-urine samples were collected for measurements of isoflavonoid concentration by the time-resolved fluoroimmunoassay and HPLC. Osteocalcin (OC) and deoxypyridinoline (DPY) were measured by enzyme linked immunosorbent assay. Stiffness value of bone was used for the assessment of subjects' bone mineral density. After multiple regression analysis to assess the relationship of bone health, dietary factor and life style, eliminating confounding factors (age, BMI, regular exercise, number of delivery and intake of carbohydrate and vitamin B2), significant negative correlation between plasma genistein and urinary DPY was observed (r = 0.300, n = 41, P < 0.05). Urinary equol, a metabolite of daidzein, showed significantly positive correlation with plasma OC (r = 0.461, n = 22, P < 0.05). In conclusions, it is suggested that genistein is useful for preventing bone loss, and equol may have a beneficial role on bone formation in this cross-sectional study. Since the plasma and urinary isoflavones are useful as biomarkers for the dietary intake, it is hypothesized that high intake (not excess) of isoflavones reduces the risk of osteoporosis.
Key words : genistein, daidzein, equol, osteocalcin, deoxypyridinoline
Effects of Dietary Isoflavone-extract from Soybean Hypocotyl on Bone Metabolism in Ovariectomized Young and Old Female Rats
Hidemichi EBISAWA and Yasuko KOSHIHARA
Department of Nutrition, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015
Soy Protein Research, Japan, 3(Serial Vol. 21), 87 - 94, 2000.
Isoflavones, such as genistein and daidzein were accepted as phytoestrogen that has similar structure to estrogen and functions with little effects on reproductive organs. Isoflavone extracted from soybean hypocotyl (IF) that contains daidzein and genistein in abundance is expected as dietary modulator for bone metabolism without side-effects. But, there are very few investigations that relate to dietary isoflavone and bone metabolism, especially in old animals. The purpose of this study was to elucidate the effects of dietary isoflavone on bone metabolism. Female Wistar rats aged 2 and 24 months old were received ovariectomy, and then the rats fed 0.5% IF (OVX-IFL) or 2.5% IF (OVX-IFH) isoflavone supplemented diet. In young rats, OVX-IFH group showed significantly higher bone mineral density (BMD) and trabeculae in femur than that of control group, although no changes were observed in uterine weight in both groups. In old rats, IF did not increase bone mineral density and trabeculae in femur, although relatively lower bone resorption and relatively higher bone formation were observed in biochemical markers. These results suggest that dietary isoflavone has beneficial effects on bone metabolism in ovariectomized young rats, but not in old rat. Further experiments, with longer experimental period and increased number of rats were required in old rat.
Key words : isoflavone, bone metabolism, BMD, old rat, OVX
Effect of Soy Protein on Hepatic Autophagy
Motoni KADOWAKI, Fumihiro FURUYA, Makiko WATANABE and Shinobu FUJIMURA
Faculty of Agriculture, Niigata University, Niigata 950-2181
Soy Protein Research, Japan, 3(Serial Vol. 21)
The possibility that soy protein may have a specific effect on hepatic protein turnover, especially autophagic proteolysis, was investigated. Wistar strain male rats weighing about 200 g were meal-fed with a 20% casein or soy protein diet from 1000 h to 1800 h for 2 to 3 weeks. One day after last meal, the liver was perfused to measure in vivo proteolytic rates, or separated into hepatocytes to measure in vitro proteolytic rates and their responses to amino acids and glucagon. The in vivo proteolysis, which was measured from valine release for initial 15 min perfusion, was 20% lower in a soy protein group than a casein group. Since the liver weight was not different between the two groups, protein synthesis, an another part of protein turnover, must also have been lower in the soy protein group, which strongly suggests a lower protein turnover with this protein. In the isolated hepatocytes, there were no differences in proteolytic responsiveness to amino acids between the two groups, but the rsponsiveness to glucagon was more sensitive with soy protein. Measurement of amino acid patterns in the portal blood proved that the effect of soy protein was not due to a direct effect of amino acid composition of the diet. It is concluded for the first time that hepatic autophagy can be controlled by dietary protein.
Key words : amino acid, autophagy, glucagon, proteolysis, soy protein
Effect of Isoflavone on DES-induced Cell Proliferation and Genetic Hypertension
Katsumi IKEDA, Yu SEKINE, Hiroko MACHIDA and Yukio YAMORI
Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606-8501
Soy Protein Research, Japan, 3(Serial Vol. 21), 100 - 104, 2000.
Genistein (10 mM) inhibited the cultured MCF7 cell proliferation induced by 500 pM diethyl-stilbesterol (DES). In vivo study using the immature rat uterotrophic assay, genistein attenuated the increase in uterine weights induced by DES (10-3 mg/kg/day). Genistein may antagonize the estrogen-agonist action of DES in MCF7 cell and the rat uterus. Eighteen week-old stroke-prone spontaneously hypertensive rats (SHRSP), 33 in total, were divided into 3 groups, 11 in each ; ovariectomized SHRSP (Ovx), ovariectomized SHRSP receiving a diet containing 0.067% of daidzin (Dai) and sham operated SHRSP (sham). Tail blood pressure was checked once a week. Soy isoflavone, daidzin, significantly attenuated blood pressure rise in Ovx. Endothelium-dependent relaxation to acetylcholine was augmented in aortic rings from Dai in comparison with Ovx. Daidzin may attenuate menopausal blood pressure rise, supposedly due to increased nitric oxide production.
Key words : genistein, daidzin, MCF, SHRSP, hypertension, DES
Study on Lipid Metabolism Regulation by a Low Molecular Weight Peptide Derived from Soybean Protein
Yasuyuki TAKENAKA, Futoshi NAKAMURA and Masaaki YOSHIKAWA
Research Institute for Food Science, Kyoto University, Uji, 611-0011
Soy Protein Research, Japan, 3(Serial Vol. 21), 105 - 109, 2000.
Leu-Pro-Tyr-Pro-Arg, a fragment peptide derived from soybean glycinin A5A4B3 subunit, has structual homology to enterostatin (Val-Pro-Asp-Pro-Arg) which is known as an anorexigenic peptide derived from procoplipase. We found that both Leu-Pro-Tyr-Pro-Arg and enterostatin had hypocholesterolemic and anorexigenic activities. So, we compared the activities of these two peptides in detail. Leu-Pro-Tyr-Pro-Arg increased excretion of bile acids in feces while enterostatin did not. Furthermore, we found that enterostatin had anti-analgesic and anti-amnesic activities, while Leu-Pro-Tyr-Pro-Arg did not. Thus, it is suggested that Leu-Pro-Tyr-Pro-Arg and enterostatin might act by different mechanisms.
Key words : soybean, enterostatin, hypocholesterolemic, anti-analgesia, anti-amnesia
Hypocholesterolemic Effect of Ethanol Extract of Soy Protein Isolate
Shinichiro TAKASHIMA, Hiroyoshi SATO, Yasuyuki TSUDA, Masao SATO and Katsumi IMAIZUMI
Division of Bioresource and Bioenvironmental Sciences, Graduate School of Kyushu University, Fukuoka 812-8581
Soy Protein Research, Japan, 3(Serial Vol. 21), 110 - 114, 2000.
It has been reported that isoflavones in soy protein isolate (SPI) have hypocholesterolemic effect, but the mechanisms remain unclear. The structure of isoflavones is similar to that of estrogen, so they were called as phytoestrogen. In this study, we have investigated the hypocholesterolemic effect of dietary isoflavones in exogenously hypercholesterolemic (ExHC) rats. When isoflavones were fed to ExHC rat, serum cholesterol level was decreased. The mRNA abundance of cholesterol 7a-hydroxylase and apolipoprotein E (Apo E) were decreased and of cytocrome c oxidase subunit VII related protein (COX7RP), which is estrogen responsive gene because estrogen response element (ERE) is present in promoter region of the gene, was increased. It has been also reported that binding of the estrogen or phytoestrogen, isoflavones, to the nuclear estrogen receptor, ERa or ERb, initiates interaction with ERE and activation of gene transcription. These results suggest that the hypocholesterolemic effect of dietary isoflavones is the estrogen like effect and isoflavones can contribute to the regulation of gene expression via estrogenic action, which is involved in lipid metabolisms.
Key words : isoflavones, serum cholesterol, mRNA abundance, cytochrome c oxidase subunit VII related protein, exsogenously hypercholesterolemic rat
Effect of Soybean Protein Intake on Energy Metabolism :
Measurement of Respiratory Quotient Using Small Laboratory Animals
Kengo ISHIHARA, Shinnichi OYAIZU and Tohru FUSHIKI
Graduate School of Agriculture, Kyoto University, Kyoto 606-8502
Soy Protein Research, Japan, 3(Serial Vol. 21), 110 - 121, 2000.
The effect of amino acid mixture simulating soybean peptide on oxygen consumption and respiratory quotient of Std ddY mice was investigated for the purpose of examining the anti-obesity effect of soybean protein. The author developed an apparatus to measure the respiratory gas of 6 mice, independently. A gas sampler adjusted the flow line and, in turn, led the respiratory gas from chambers into a mass spectrometry gas analyzer. When flow meters were installed in each line of the sampler, the errors of measured flow rate by each flow meter were nearly 50%. In case a shared flow meter was installed in a shared part of each line, the errors were less than 10%, and there were no effect of the line number on measured oxygen consumption and respiratory quotient. Std ddY male mice fasted for 18 h and were orally administered 500 mL of 5% amino acid solution simulating soybean peptide. Oxygen consumption was temporarily increased in all groups after administration, and rapidly declined to the value before administration in control, but the acute increase tended to last longer in mice administered soybean amino acid mixture.
Key words : oxygen consumption, respiratory quotient, energy expenditure
Mechanism of Elevation in Plasma Glucagon Concentration by Soybean Protein Intake, with Reference to Changes in the Concentration of Plasma Glucagon-like Peptide-1
Shin HASEGAWA, Hironao KATO, Sanae IBA, Aya YAMAMOTO and Tohru MOTOKI
Graduate School of Science and Technology, Kobe University, Kobe 657-8501
Soy Protein Research, Japan, 3(Serial Vol. 21), 122 - 127, 2000.
The present study was conducted to elucidate the mechanism of elevation in plasma glucagon concentration by soybean protein intake in chicks, with reference to changes in the concentration of plasma glucagon-like peptide-1 (GLP-1). Firstly, chicks were fed purified diets containing two types of protein (casein and soy protein isolate), and the effects of dietary protein types on growth performance, liver triglyceride content, and the concentrations of plasma triglyceride, free fatty acid, glucose, glucagon, insulin and GLP-1 were examined. Soybean protein intake significantly reduced adipose tissue weight, liver weight, liver triglyceride content and plasma triglyceride concentration, and had tendencies to reduce the concentrations of free fatty acid and glucose, although it had tendencies to raise the concentrations of plasma glucagon and GLP-1, and glucagon/insulin ratio. Secondly, chicks were administered intravenously with GLP-1 (7-36) amide and its fragments, namely GLP-1 (7-16), GLP-1 (17-26) and GLP-1 (27-36) amide. All the fragments had no effect on the concentrations of plasma free fatty acid, glucose, glucagon and insulin, although GLP-1 (7-36) amide had tendencies to raise the concentrations of plasma free fatty acid, glucose and glucagon, and glucagon/insulin ratio. From the results obtained above, the mechanisms by which soybean protein intake affects the plasma glucagon concentration were discussed.
Key words : soybean protein diet, plasma glucagon concentration, GLP-1 (7-36)
amide, glucagon/ insulin ratio, body fat accumulation, liver triglyceride content
Effects of Soy Protein Isolate on TG-rich Lipoproteins and Safety among Healthy Men
Kenji HIGASHI1, Shojiro SAWADA1 , Tetsuya HISADA1 , Noriyuki IWAMOTO1 , Toshimitsu ITO1, Kei NAKAJIMA1 , Koji TOMIYASU1 , Takeshi YAMASHITA1 , Emiko MIYAJIMA2 , Fumitaka OHSUZU1 and Haruo NAKAMURA2
1National Defense Medical College,Tokorozawa 359-0042
2 Mitsukoshi Health and Welfare Foundation, Tokyo 160-0023
Soy Protein Research, Japan, 3(Serial Vol. 21), 128 - 132, 2000.
Soy protein has been reported to possess a hypocholesterolemic action and an estrogen-like action as well. However, it has not been clear about the effect on triglyceride (TG) rich lipoproteins, and safety to men. To elucidate the effects of soy protein on TG rich lipoproteins and safety, 14 healthy male volunteers were either given 20 g per day of soy protein isolate (SPI) for 4 weeks or not, in a cross over study. We compared the changes in lipids, in particular TG and remnant-like particles (RLP) cholesterol, transaminases, iron, calcium, testosterone, and vitamin E in plasma between SPI and control groups. Hypocholesterolemic effect was most apparent 3 weeks after SPI intake, and total cholesterol and low-density lipoprotein (LDL) cholesterol were insignificantly reduced by 4.3% and 4.5%, respectively. TG and RLP cholesterol were significantly reduced by 13.4% and 9.8%, respectively. Lecithin cholesterol acyltransferase (LCAT), cholesteryl estser transport protein (CETP), lipoprotein lipase (LPL), and hepatic lipase (HL) activity were not significantly changed. Transaminases, iron, calcium, testosterone and vitamin E levels did not significantly differ between SPI and control groups during the experiment. However, vitamin E were reduced significantly by 9.7% from baseline after 3 weeks by SPI intake. These results show that intake of soy protein in men exerts a beneficial effect on TG rich lipoproteins, however reduces sex hormone and vitamin E levels. Further study should be needed about the association of soy protein with reduction in sex hormone and vitamin E.
Key words : soy protein, remnant like particles (RLP), testosterone, vitamin
E, transaminase, cholesterol
Effects of Soybean Yogurt Intake on Serum Lipids and Eating Behaviors
Masako WAKI, Yoshimi SHINMURA and Kiyomi YASUHARA
Shimada Municipal Hospital, Shimada 427-8502
Soy Protein Research, Japan, 3(Serial Vol. 21), 133 - 138, 2000.
The effect of soybean yogurt containing 3.5% of soybean globulin on serum lipids and apo lipoproteins was studied in 6 normo- and 15 hypercholesterolemic subjects. Four-weeks intake of the yogurt either 180 g or 270 g a day reduced LDL-cholesterol levels in both the normocholesterolemics (0 wk, 133.2±12.8 ; 2 wk, 121.7±14.5 ; 4 wk, 125.0±3.5 mg/100 mL : P < 0.01 by ANOVA) and the hypercholesterolemics (0 wk, 165.7±23.8 ; 2 wk, 150.3±27.7 ; 4 wk, 159.9±28.4 mg/100 mL ; P < 0.01 by ANOVA). LDL-C to HDL-C ratio and apo E levels were also reduced significanly in the hypercholesterolemics. These reductions were prominent especially in 10 subjects with elevated cholesterol level and normotriglyceridemia (< 150 mg/100 mL) at baseline. Through the study, day-to-day variations in energy intake was reduced, and more than a half of the subjects became aware of the problems on their own diet composition and dietary habits. The result of the study suggests that soybean yogurt may be useful to improve atherogenic hypercholesterolemia and eating behavior.
Key words : soybean yogurt, hypercholesterolemia, apolipoprotein, eating behavior
Effect of Energy Restricted Diets Supplemented with Soy Protein Formula Product on Body Weight Reduction and Basal Energy Expenditure in Adult Obese Patients
Yutaka MORI1, Hideaki KOMIYA2, Yoshiko INABA2 and Yoshio IKEDA3
1National Higashi Utsunomiya Hospital, Utsunomiya 329-1104
2Faculty of Education, Utsunomiya University, Utsunomiya 321-8505
3Tanita Scientific Institute for Best Weight, Tokyo 174-8630
Soy Protein Research, Japan, 3(Serial Vol. 21), 139 - 144, 2000.
Effect of soy protein formula product named "Koresukkiri" (6 pieces of biscuit and a cup of soup) on the body weight reduction by energy restriction was studied in obese patients. Nineteen patients were divided into 2 groups, the energy-restricted diet supplemented with soy protein formula product (soy protein isolate ; SPI) group (1,060 kcal, protein 59.5 g) and the energy-restricted diet (control) group (1,000 kcal, protein 57.0 g), and followed for 4 weeks. Although body weights siginificantly decreased in both groups, body weight reduction was greater in SPI group compared with that in control group. With body weight reduction, basal energy expenditure significantly decreased in control group, whereas there was no significant change in SPI group. The decrease in basal energy expenditure was greater in control group than that in SPI group. Fasting plasma glucose and serum insulin levels, and HOMA-r significantly decreased in both group. Although serum total cholesterol (TC) and triglyceride (TG) levels significantly decreased in both groups, the decrease in TC and TG levels were greater in SPI group than those in control group. Serum HDL-C levels were unchanged in both groups. In the clinical study of body weight reduction by energy restriction, the supplement with soy protein formula product was suggested to have benefical effect on the amelioration of obesity and preventive effect on increase in body weight after body weight reduction.
Key words : obesity, body weight reduction, basal energy expenditure, soy protein
isolate