The Multivariate Functions of Adipose Tissue and the Metabolic Syndrome:
The Effects of Soybean Protein on Fat Distribution and Adipocytokines
Yuji MATSUZAWA
Osaka University Graduate School of Medicine, Osaka 565-08711
ABSTRACT
The metabolic syndrome, a cluster of insulin resistance, hypertension and
dyslipidemia, is a common basis of Type 2 diabetes and atherosclerotic vascular
diseases in industrialized countries. The most common cause of the metabolic
syndrome is the accumulation of intra-abdominal visceral fat. Adipose tissue secretes
a variety of bioactive substances called adipocytokines. Dysregulation of
adipocytokines by overnutrition may play an important role in the development of the
metabolic syndrome. Soybeans, a part of the Japanese traditional diet, may protect
against the development of the metabolic syndrome through regulation of
adipocytokines. This study was aimed to clarify 1) the role of adipocytokines in the
development of the metabolic syndrome, and 2) the significance of soybean protein, on
the regulation of adipocytokines. Adiponectin is an adipose-specific plasma protein,
which we identified in the Human Adipose cDNA Project. Adiponectin possesses the
activities to suppress the expression of adhesion molecules in vascular endothelial
cells; the expression of scavenger receptor mRNA in macrophages; and growth
factor-induced proliferation of vascular smooth muscle cells. Furthermore
adiponectin promotes glucose uptake in myocytes. Therefore, adiponectin may play a
key role in the development of the metabolic syndrome. In previous studies, we
reported dysregulation of adiponectin in subjects with body fat accumulation. In the
present study, we investigated 1) the role of adiponectin in the metabolic syndrome
using mice lacking the adiponectin gene and mice oveexpressing adiponectin, and also
studied 2) the effect of a soy protein diet on adiponectin concentrations. Adiponectin
knockout mice exhibited severe insulin resistance when fed a high-fat, high-sucrose diet. The knockout mice also showed severe vascular neointimal thickening from
vascular injury. Overexpression of adiponectin in the apoE knockout mice, which is
an animal model for atherosclerosis, significantly reduced fatty streak lesions in
aorta. In obese KK-Ay mice, body fat weights, plasma cholesterol, triglyceride, free
fatty acid, and glucose levels were lower in mice on a calorie-restricted diet
containing soy protein isolate. Among the genes related to glucose and fatty acid
metabolism, adiponectin mRNA levels in adipose tissue and adiponectin plasma
concentrations were elevated in mice on calorie-restricted diets, although there were
no significant differences between soy protein and casein protein groups. Our results
indicate that adiponectin has anti-atherogenic and anti-diabetic activity in vivo, and a
calorie-restricted diet elevates plasma adiponectin concentration in obesity.
Soy Protein Research, Japan6, 1-10, 2003.
Molecular Mechanisms of Genetic Variations in Seed Storage Proteins and
Their Transmission During the Process of Breeding in Soybean
Michiko YOSHINO1, Ken-ichi TSUTSUMI2, Yoshiya SHIMAMOTO1
and Akira KANAZAWA1
1Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589
2Cryobiosystem Research Center, Faculty of Agriculture, Iwate University, Iwate 020-8550
ABSTRACT
The 7S globulin (β-conglycinin), a major component of seed storage proteins in
soybeans, is comprised of three subunits, α, α' and β. In order to reveal genetic
bases for variations in 7S globulin, we analyzed the structures of genomic DNA
around genes for the proteins. We first constructed restriction maps around genes
for each subunit in cultivated soybeans 'Forrest' and 'Keburi'. We next analyzed the
structures around genes in various lines of soybean that show genetic variations
regarding these subunits. As a result, polymorphisms were detected in these soybean
lines with regard to the presence or absence of genes for each subunit as well as the
structures around these genes. Large deletions of gene regions were found to be a
major cause of genetic variations. We previously found that a gene (CG-2), which is
located in the downstream of the α-subunit gene (CG-3) in 'Forrest', shares highsequence
homology with the α-subunit gene. Because CG-3 was deleted while CG-2
was present in soybean lines that express α subunit at a low level, the CG-2 was
regarded as a second gene for the α subunit. In addition, the deletions of gene
regions of the α- and α'-subunit genes were found to be useful genetic variations for
producing soybean strains that accumulate 7S globulin at a low level.
Soy Protein Research, Japan6, 11-17, 2003.
Key words : soybean, β-conglycinin, α subunit, gene expression, deletion
Identification and Linkage Analysis of the Genes Expressed in Soybean Seeds
Kyuya HARADA1 and Toyoaki ANAI2
1Faculty of Horticulture, Chiba University, Matsudo 271-8510
2Faculty of Agriculture, Saga University, Saga 840-8502
ABSTRACT
Map-based cloning is a general method for isolating genes responsible for agriculturally
important traits. The use of cDNA markers makes the method more effective. To
identify the genes for seed traits including seed quality and quantity of seed
constituents, high quality cDNA libraries were made from developing soybean seeds.
The 5' partial sequences of 1,500 randomly selected clones were determined and the 3'
partial sequences of 233 non-redundant clones were also determined to develop locusspecific
cDNA markers by PCR. Ribosomal proteins were most highly expressed and
electron transferring proteins had a relatively high expression in the early stage of
developing seeds, while storage proteins were highly expressed in the middle stage.
Proteinase inhibitors showed relatively high expression in both stages.
Phosphoenolpyruvate carboxylase (PEPCase) and acetyl-CoA carboxylase (ACCase) were
selected as putative candidate enzymes for protein and lipid contents respectively.
Their loci were determined based on RFLP and PCR-RFLP using recombinant inbred
lines and compared with the quantitative traits loci (QTLs) for protein and lipid
contents, but there was no correspondence among the enzyme loci and the QTLs.
Further analyses are necessary to identify QTLs for protein and lipid contents by cDNA
mapping and map-based cloning.
Soy Protein Research, Japan6, 18-21, 2003.
Key words : soybean, seed, quantitative trait locus (QTL), EST, cDNA marker
Chemical Treatment of Plants May Activate Defense Mechanisms and
Increase Allergenicity
Tadashi OGAWA1, Tatsuya MORIYAMA1, Noriko NOMURA2
and Koji TAKAHASHI3
1Graduate School of Agriculture, Kyoto University, Kyoto 611-0011
2Taiyo Chemical Co., Mie 510-0844 3National Agriculture Research Center, Ibaraki 305-8517
ABSTRACT
Many plant allergenic proteins have been isolated and identified as pathogenesisrelated
proteins. They are produced when biological, chemical, and physical stresses
activate defense mechanisms of plants. These defense proteins are thought to play an
important role in pathogen resistance, and some defense-related proteins are
significant cross-reactive allergens. Accordingly, the allergenicity of plant foods may
depend on the growth conditions of plants or given stress activating defense
mechanisms. Using turnips and soybeans, we measured the allergenicity of these
model food plants after treatments activating their defense mechanisms to determine
whether the allergen content of these plants increase. On immunoblotting profiles,
IgE-binding protein bands were found to increase in both turnip and soybean treated
chemically, and the allergenicity given as total ELISA values were also increased in
the stress-given plant samples. These results demonstrated that activating defense
mechanism of plants may increase their allergenicity.
Soy Protein Research, Japan6, 22-28, 2003.
Key words : plant, soybean, allergen, allergenicity, pathogenesis-related proteins,chemical stress
Development of a New Emulsifier with the Conjugation Between Soy Protein and
Pectin from Food Waste
Hidefumi YOSHII and Takeshi FURUTA
Faculty of Engineering, Tottori University, Tottori 680-8552
ABSTRACT
Depolymerized citrus pectin has emulsifying properties. It is thought that the
presence of a protein moiety in the depolymerized pectin plays a major functional
role. In this study, the kinetic characterization of acid hydrolysis was investigated
with high-methoxy (HM) pectin and soy protein to obtain water-soluble
polysaccharides (WSP). A 6% by weight solution of HM-pectin and soy protein (5:1
w/w) at pH3-5 was incubated at a constant temperature (80oC-120oC) in an autoclave.
Acid degradation kinetics of HM-pectin indicated a first-order degradation rate as
the viscosity change of WSP. The change of mean emulsion diameter of d-limonene
with WSP from HM-pectin and soy protein also showed a first-order rate. Both
kinetic activation energies (42 kJ/mol) were obtained at pH 3.0.
Soy Protein Research, Japan6, 29-33, 2003.
Key words : pectin, hydrolysis, conjugates, emulsifier
Antifreeze Protein Improves the Quality of
Soy Protein Processed Frozen Food
Hidehisa KAWAHARA, Runa TANAKA and Hitoshi OBATA
Faculty of Engineering, Kansai University, Osaka 564-8680
ABSTRACT
We examined the effect of antifreeze protein (AFP) on the solubilization of soy
protein, which is used as food material, under freezing conditions. The soy protein
suspension (8 mg protein/mL) was prepared and mixed with AFP to yield its final
concentration of 0-100μg/mL. This mixture was frozen at -20oC for 24 h, and then
thawed at room temperature. When AFP was added at 5 and 100μg/mL, their
solubilization rates were 45% and 48%, respectively. Also, this high level of
solubilizing effect was achieved with only one freezing treatment. Furthermore, this
solubilizing effect could improve the manufacture of bean curd with different protein
contents.
Soy Protein Research, Japan6, 34-37, 2003.
Key words : antifreeze protein, bean curd, soyprotein
Functions of Lecithin in Dough and Bread Supplemented with β-Conglycinin
Reiko URADE
Graduate School of Agriculture, Kyoto University, Uji 611-0011
ABSTRACT
The soy protein, β-conglycinin is known to decrease plasma triacylglycerol and
cholesterol levels in humans. Because these effects of β-conglycinin are transient, it is
necessary to take it daily to maintain such effects. Bread is a convenient vehicle for
introducing β-conglycinin into the daily diet. However, because of its high waterabsorption
properties, β-conglycinin increases the water-absorption capacity of flour,
alters the rheological properties of dough, and decreases the loaf volume of bread.
Studies show the addition of soybean protein isolate or β-conglycinin decrease loaf
volume. Strategies to overcome these adverse effects should be developed. We added
phosphatidylcholine (PC) to the bread ingredients, in addition to protein isolate and β-
conglycinin, and the adverse effect of the proteins on loaf volume was counteracted.
Additionally, adding PC showed little effect on the rheological properties of dough,
determined with a Reolograph Gel, a Rheoner, and a Microfarinograph. The function
of PC could not be replaced by phosphatidylethanolamine, phospatidic acid, or
lysophospatidylcholine. The ultrastructure of bread was examined by scanning
electron microscopy. The gas cells in the bread containing proteins from soybean
were smaller than those in the control bread without soy proteins. In the bread
supplemented with soy proteins and PC, the size of the gas cells was similar to those in
the control bread. These results may suggest that PC made the cell walls resistant to
breakage by gas pressure, thereby maintaining the gas-retaining ability and
maintaining the usual loaf volume of the bread.
Soy
Protein Research, Japan6, 38-42, 2003.
Key words : β-conglycinin, soybean protein isolate, bread, dough, phosphatidylcholine
1Faculty of Agriculture, University of the Ryukyus, Okinawa 903-0213
2Faculty of Biotechnology, Fukui Prefectural University, Fukui 910-1195
Bacillus subtilis
ABSTRACT
Soybean-milk-coagulating enzyme (SMCE) is important for coagulating soybean
protein, and the technology for its effective production is needed. In this study, we
investigated the expression of SMCE gene in genetically engineered Bacillus subtilis.
The SMCE structural gene containing the putative promoter and terminator regions
were isolated from B. pumilusby PCR using the primers, each of which contains the
BamHI or XbaI site. The unique amplified band was digested with BamHI and XbaI,
and then ligated to the BamHI and XbaI sites of plasmid pHY300PLK. The resultant
plasmid (pNK) was introduced into Bacillus subtilis DB 403 that was a proteasedeficient
strain by electroporation, and three transformants containing pNK were
obtained. When the recombinant strains ( B. subtilisDB 403 (pNK)) were cultured on a
LB agar plate containing skimmed milk, all strains were shown to form haloes. The
band corresponding to SMCE from B. pumilusand SMCE activities were observed in
the culture fluid from the recombinant B. subtilisDB403 (pNK). These results showed
that the SMCE gene was expressed and the active enzyme was produced in B.
subtilis. Soy Protein Research, Japan6, 43-47, 2003.
Enhanced Function of Calcium Absorption
by Phytate-Removed and Deamidated Soybean Proteins
Hitomi KUMAGAI
College of Bioresource Sciences, Nihon University, Fujisawa 252-8510
ABSTRACT
Calcium is an important mineral for the formation of bones and teeth. Currently,
calcium intake of Japanese is nearly half that of Westerners, and does not meet the
recommended level. The amount of calcium absorbed in the intestine is about 20-50%
of intake. Therefore, increasing calcium absorption as well as intake is important for
achieving optimal calcium status. Casein phosphopeptide (CPP) and calcium citrate
malate (CCM) enhance calcium absorption, and are used in foods for specified health
purposes. However, the use of these substances in foods is limited because of their
low molecular weight, and CPP has a milky flavor, and CCM a sour taste. Soybeans
are a traditional part of the Japanese diet and the isoflavones are known to inhibit
calcium resorption in bones. However, phytate in soybeans renders calcium insoluble,
and inhibits calcium absorption by the body. If one could enhance calcium absorption
from soybeans, it would expand their use and decrease the calcium inadequacy in
Japanese. We showed previously that the removal of phytate and the deamidation of
acid amide from soybean proteins improves their calcium binding, and that about 70%
of the total acid amide can be deamidated by ion-exchange resins without any
detectable peptide-bond hydrolysis. In this study, we evaluated the calcium-binding
properties of phytate-removed and deamidated soybean proteins (PrDS) during
hydrolysis by digestive enzymes, and examined the enhancement effect of calcium
absorption by PrDS on bone formation. The results indicate that PrDS retained the
high calcium binding properties even after hydrolysis by digestive enzymes, and
enhanced calcium absorption from the intestines. Soy Protein Research, Japan6, 48-52, 2003.
Comprehensive Evaluation of Soy Protein Functions
by DNA Microarray Analysis
Keiko ABE and Ichiro MATSUMOTO
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657
ABSTRACT
Feeding rats with soy protein isolate (SPI) for a short term (2 weeks) decreased serum
cholesterol and neutral lipid to a similar degree as feeding for a longer term (8 weeks).
To better understand the molecular mechanism behind this phenomenon, we carried
out a comprehensive analysis of the resulting gene expression profiles, with the
expectation that it would contribute to our in-depth understanding of SPI
functionalities. Male Wistar rats aged 6 and 12 weeks were dichotomized and fed an
SPI or casein diet (control) for 2 weeks. Liver samples were obtained from rats in
each diet group and a cRNA sample was prepared for use in DNA microarray
analysis. The SPI feeding significantly up-regulated 138 and down-regulated 218
genes in the 6-week old rats, and significantly up-regulated 87 and down-regulated
248 genes in the 12-week old rats. Down-regulated genes included those for enzymes
involved in fatty acid anabolism, the same genes down-regulated in longer term SPI
feeding (mentioned above), as well as genes for enzymes involved in cholesterol
catabolism, which were not down-regulated in the longer-term feeding studies. The
present study suggests the possibility that the use of DNA microarray analysis is
effective as a method for evaluating physiological functions of food proteins in general
as well as those of SPI in particular.
Soy Protein Research, Japan6, 53-56, 2003.
Key words : soy protein isolate, functions of food proteins, DNA microarray,
comprehensive analysis
Phytomedical Effect of Soy Protein and Soy Isoflavone
on Hypercholesterolemia and/or Atheroscelerosis :
Its Molecular Pathophysiological Mechanism in Monkeys
Shin NAKAMURA1, Fusako MITSUNAGA1, Maki OKADA1,
Aram JOENG1, Takashi HAYASHI2, 3, Rosario M. PEREZ3, Romualdo L. CARRASCO3,
and Hiroshi NAKAGAWA2, 3.
1Primate Research Institute, Kyoto University, Inuyama 484-8506
2Ina Research Inc., Nagano 399-4501
3INA Research Philippines, Philippines.
Research, Japan 6, 57-62, 2003.
ABSTRACT
Soy protein (β-Conglycinine) and soy isoflavone (Soyaflavone HG) are known to
regulate hypercholesterolemia and its mediated atherosclerosis. In the current study
we examined the phytomedical effect of β-Conglycinine and Soyaflavone using an
established monkey hypercholesterolemia/atherosclerosis model, in normal male,
female and ovariectomized (OVX)-female monkeys. The molecular mechanism of the
phyto-medicial function of the soy products was investigated by analyses of blood lipid
markers (total Chol, LDL-C, HDL-C, GOT, GPT, Glu, CPK, TG, Apo-Al, Apo-B, and
Apo-E). To elucidate the genetic mechanism, gene expression profiling analyses of an
inflammatory and thrombotic gene (TF) and lipid metabolic genes (LDL-R, VLDL-R,
HMG-CoA, SR) were performed by RT-PCR. β-Conglycinine and Soyaflavone both
suppressed upregulated TF gene expression in hypercholesterolemia, suggesting a new
mechanism of these soy products in hypercholesterolemia/atherosclerosis.
Soy Protein Research, Japan6, 57-62, 2003.
Studies on Soy Proteins Stimulating Cholesterol Efflux Driven by the Intestinal
ABCA1 Transporter
Ryuichiro SATO
Graduate School of Agricultural and Life Sciences. The University of Tokyo, Tokyo 113-8657
ABSTRACT
Cholesterol efflux from human cultured intestinal (Caco-2) cells was studied. ABCA1,
which stimulates cholesterol efflux in a manner similar to HDL from various cells,
was expressed in differentiated Caco-2 cells. A nuclear receptor, Liver X
Receptor(LXR), was also expressed in these cells. ABCA1 gene expression was
augmented by treatment of cells with an LXR agonist. [3H]cholesterol efflux to the
apical or basolateral side of Caco-2 cells was measured after induction of ABCA1
expression by the LXR agonist. Cholesterol efflux to the basolateral side was
increased by the agonist or the addition of apo A-1, which is involved in formation of
HDL by ABCA1, to the culture medium, suggesting that ABCA1 particpates in HDL
production on this side. Cholesterol efflux to the apical side was increased by the
agonist or the addition of bile acid, not apo A-1. This indicates that certain
transporters including ABCG5 and G8 induced by LXR stimulate cholesterol efflux to
the apical side in a bile acid-dependent manner. We speculate that some peptide
molecules digested from soy proteins as well as bile acids might stimulate cholesterol
efflux from the intestinal epithelial cells. Alternatively, it is possible that some
peptides in the small intestinal tract directly enhance the transport activity of
transporters involved in cholesterol efflux. To find out functional soy peptides
reducing cholesterol absorption (increasing cholesterol efflux), several kinds of
screening are now being carried out.
Soy Protein Research, Japan6, 63-66, 2003.
Key words : ABCA1, ABCG5, ABCG8, cholesterol efflux, LXR
Studies on Suppressive Effect of Soy Protein on LPS-induced Hepatitis
Kimio SUGIYAMA, Ena ATSUMI, Yasuhiko SHIMADA and Tatsuya MORITA
Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529
ABSTRACT
We investigated the effect of dietary protein on lipopolysaccharide (LPS) +Dgalactosamine
(GalN)-induced hepatitis by measuring plasma alanine aminotransferase
and aspartate aminotransferase activities as hepatitis markers in rats. When rats
were fed diets containing 25% casein or an equivalent amount of other proteins
(purified whole egg protein, soy protein isolate (SPI) or wheat gluten) for 10 days and
then injected with LPS+GalN, hepatitis was most suppressed in rats fed an SPI diet.
The hepatitis-suppressive effect of SPI could not solely be ascribed to impurities
included in SPI. When L-Methionine was added to the SPI diet to make it comparable
to that of the casein diet, hepatitis was significantly intensified, suggesting that the
hepatitis-suppressing effect of SPI might be attributable, at least in part, to the low
methionine content of the protein. LPS is thought to induce apoptosis of liver cells
through the action of TNF-α, which is produced by macrophages in response to LPS,
and leading to hepatitis. Thus, we investigated the effects of an SPI diet on plasma
TNF-α concentration and apoptosis of liver cells. The SPI diet did not suppress
LPS+GalN-induced enhancement of plasma TNF-α concentration as compared with
the casein diet, but it significantly suppressed apoptosis of liver cells. On the other
hand, the SPI diet significantly suppressed TNF-α+GalN-induced hepatitis compared
to the casein diet, suggesting that SPI might elicit its protective effect against
LPS+GalN-induced hepatitis by suppressing the action of TNF-α. Our results suggest
that SPI might be effective in preventing certain types of hepatitis that are mediated
by TNF-α.
Soy Protein Research, Japan6, 67-72, 2003.
Key words : lipopolysaccharide, hepatitis, soy protein, tumor necrosis factor-α
Production of Hypotensive Peptide, SVY, from 7S Globulin of Soybean Protein
and Its Physiological Functions
Toshiro MATSUI
Faculty of Agriculture, Kyushu University Fukuoka 812-8581
ABSTRACT
To clarify the latent hypotensive effect of soybean proteins, we measured production
of angiotensin I-converting enzyme (ACE) inhibitors from 7S globulin, and
characterized peptides responsible for an in vivo depressor effect. Soybean protein
was hydrolyzed by pepsin, chymotrypsin, and trypsin, and the prepared hydrolysate
(F-1) with a peptide length of 7.2 showed an ACE inhibitory activity with an IC50 value
of 0.286 mg/mL. The F-1 also contained 44.2% di- & tri-peptides (IC50: 0.358 mg/mL),
which could be absorbed intact through the intestinal tract. From the di-/tri- peptide
fraction, Val-Tyr (VY), Ile-Val-Tyr (IVY), and Ser-Val-Tyr (SVY) (IC50: 1.7 mM) were
isolated and identified. A single oral administration rat study of the fraction revealed
that it had an ability to lower blood pressure, in which a 0.3 g/kg dose in an 18 wk old
spontaneously hypertensive rat resulted in a significant reduction of systolic blood
pressure of 19.7 mmHg at 6 h. Useful information on the hypotensive effect of
soybean proteins was obtained by an intestinal membrane transporting study using
the Ussing system. Results from the rat small intestinal transport experiment
indicate that F-1 and 18 di- and tri-peptides, including SVY and IVY with ACE
inhibitory activity, were absorbed intact through the membrane. Phe-Ala (FA) was
the most absorbable di-peptide with a permeation rate of 164.9 nmol/cm2/30 min/mL.
Soy Protein Research, Japan6, 73-77, 2003.
Graduate School of Agriculture, Kyoto University, Uji 611-0011
ABSTRACT
Yield of soymetide-13 (MITLAIPVNKPGR), an immunostimulating peptide, from a
recombinant β-conglycinin α' subunit after trypsin digestion was 15%. Yield of
soymetide-13 from a recombinant β-conglycinin α' subunit fortified with 4 mol/mol
of RPLKPW, a potent hypotensive peptide, by site-directed mutagenesis was 17%.
Yields of soymetide-13 and DPR, a hypocholesterolemic/bile-secreting peptide, from a
recombinant β-conglycinin α' subunit fortified with 3 mol/mol of DPR were 14% and
40%, respectively. Thus, it was concluded that RPLKPW and DPR sequences
introduced into the β-conglycinin α' subunit did not interfere with release of
soymetide-13 by the action of trypsin.
Soy Protein Research, Japan6, 78-82, 2003.
Effect of Water-Soluble Soybean Peptide Fractions
on Liver and Serum Triglyceride Concentrations in Rats
Takayuki KURAYAMA, Masanobu SAKONO and Nobuhiro FUKUDA
Faculty of Agriculture, Miyazaki University, Miyazaki 889-2192
ABSTRACT
Effects of various peptides prepared by enzymatic hydrolysis of soy protein isolate
(SPI) on liver and serum triglyceride (TG) were compared with those of SPI alone or
casein in Sprague-Dawley rats fed diets with and without cholesterol. In experiment
1, the liver triglyceride-lowering effect of SPI, HD1 (a low molecular peptide fraction
with residual high molecular peptides), and LD3 (a low molecular peptide fraction)
was evident, compared to casein. In addition, SPI and LD3 exhibited a potent
hypotriglyceridemic effect in rats fed diets with and without cholesterol; the extent of
this lowering effect being greater in LD3 than in SPI, suggesting the effectiveness of
LD3 in lowering serum and liver triglyceride concentrations. In experiment 2 with
no dietary cholesterol, the effect of increasing amounts of LD3 on liver and serum
triglyceride was examined, and we found that there was a dose-dependent reduction
in the concentrations of triglyceride with increasing amounts of LD3 in the diet. In
experiment 3, also with no added cholesterol in the diet, other fractions such as HMF
(a high molecular peptide fraction in HD1) and D5 (an acid-soluble low molecular
peptide fraction) were compared with SPI, HD1 and LD3; the D5 fraction exhibited a
potent serum and liver cholesterol lowering activity; the extent of reduction appeared
to be greater in D3 than in LD3. HMF had no lowering effect. In experiment 4, the
effect of F5 (a highly water soluble low molecular peptide) was examined in rats fed
diets free of cholesterol, and we found that there is a greater reduction in the
concentration of serum and liver triglycerides compared to SPI and LD3. The results
of these four experiments suggest that the water-soluble low molecular fraction
prepared from SPI is a useful dietary component to prevent and/or ameliorate hypertriglyceridemia and fatty liver.
Soy Protein Research, Japan6, 83-87, 2003.
Key words : soy protein, soy peptide, triglyceride, water soluble, low molecular
Isolation and Application of Physiologically Active Peptides from Soybean Whey
and Okara Proteins
Masami YONEKURA and Ayako TANAKA
School of Agriculture, Ibaraki University, Ibaraki 300-0393
ABSTRACT
Peptides with inhibitory effects on angiotensin I-converting enzyme (ACE) were
isolated from thermolysin digests of soybean whey proteins; basic 7S globulin; and the
"Hinute DC5" soybean peptide preparation. The amino acid sequence of the inhibitory
factor from the whey protein digest was Leu-Ala-Pro and the IC50 value of the peptide
for ACE from rabbit lung was 0.13μM. Isolation of the inhibitory peptide from the
basic 7S globulin digest was attempted, however the sequence of the peptides has not
been clarified. Thirteen kinds of ACE inhibitory peptides from "Hinute DC5" were
isolated. Among these peptides, dipeptides such as Lys-Tyr and Ala-Phe showed
comparatively high inhibitory activity for ACE. and their IC50 values were 25μM and
97μM, respectively. Our results suggest that the thermolysin digests of soybean
whey proteins, basic 7S globulin and "Hinute DC5" may be suitable materials for the
development of a functional food with antihypertensive activity. Soy Protein Research, Japan6, 88-93, 2003.
Identification of Amino Acid Sequence Responsible for the Satiety
Effect of Soybean β-Conglycinin
Hiroshi HARA and Takashi NISHI
Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589
ABSTRACT
Peptides derived from dietary protein promote cholecystokinin (CCK) secretion via a
putative receptor on the mucosal cells of the small intestine, and soybean β-
conglycinin peptone suppresses food intake in rats via CCK secretion. The aim of the
present study was to identify the amino acid sequence (peptide fragment) responsible
for CCK secretion. We found that the β-conglycinin β-subunit 51-63 (β51-63,
VRIRLLQRFNKRS) has the strongest binding activity to the brush-border membrane
proteins of the rat jejunum. Duodenal infusion of the synthetic peptide β51-63
reduced food intake for 1 hr (satiety effect), which was completely abolished by
devazepide, a CCKA-receptor antagonist. Also, duodenal infusion of β51-63 induced
marked increase in portal plasma CCK concentration. We conclude that soybean β-
conglycinin includes the sequence that strongly stimulates CCK secretion by binding
to the brush-border membrane of the small intestine.
Soy Protein Research, Japan6, 94-99, 2003.
Regeneration of Neural Circuit: Role of Soy-Protein
Tatsuhiro HISATSUNE
Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562
ABSTRACT
We studied the effect of dietary soy protein on brain circuit regeneration in nestinenhanced
green fluorescent protein (nestin-GFP) mice. The mice were fed either a
control (MF) or a soy protein-rich (SPR) diet for 14 days, and were supplied with
bromodeoxyuridine (BrdU) during the last 10 days. The mice were killed and their
brains dissected. Cerebral slices (40 micron) were cut by cryostat, and each slice was
stained with both anti-GFP and anti-BrdU. The numbers of nestin-positive cells per
1 mm3 in the cerebral cortices of the MF and SP groups were 503 ± 70 and 569 ± 90,
respectively. In addition, the frequencies for nestin-positive dividing cells per total
dividing cells were 50% and 66% in the MF and SP groups, respectively. These
results suggest that soy protein promotes the generation of neural precursor cells in
the cerebral neocortex, thought to be the center of intelligence, and enhances the
regeneration of neural circuits in the murine cerebral cortex.
Soy Protein Research, Japan6, 100-103, 2003.
Key words : soyprotein, regeneration, neural circuit, cerebral cortex
Immunological Modulative Activity of Soybean Saponins: Suppressive Effect of
Soybean Saponins on High Affinity IgE Receptor Expression
Hirofumi TACHIBANA, Yoshinori FUJIMURA, Satomi YANO and Koji YAMADA
Faculty of Agriculture, Kyushu University, Fukuoka 812-8581
ABSTRACT
We examined the effect of soybean saponins on the cell surface expression of high
affinity IgE receptor (FcεRI) in human basophilic KU812 cells. Flow cytometric analysis
showed that soybean saponin decreased the cell surface expression of FcεRI. FcεRI is
a tetrameric structure comprising of one α chain, one β chain, and two γ chains. The
level of mRNA of each subunit in KU812 cells was not reduced by treatment with the
saponin. Furthermore, immunoblot analysis revealed that total cellular expression of
the FcεRI α and γ chains were not affected by treatment with the saponin. However,
the saponin treatment inhibited the FcεRI cross-linking-induced histamine release.
The cross-linkage of FcεRI causes the activation of basophils, which leads to the
secretion of inflammatory mediators including histamine. These results suggest that
soybean saponins can negatively regulate basophil activation through the suppression of
FcεRI expression.
Soy Protein Research, Japan6, 104-107, 2003.
Key words : soyasaponin, FcεRI, histamine, basophils
Function of Lipid Metabolism and Antioxidative Activity of Dietary Soybean
Isoflavone Aglycone: Differences in Regulative Function between
Male and Female Rats
Kyoichi OSADA, Yuki KAWAKAMI and Tomoko KIYOSAWA
Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561
ABSTRACT
The regulative effects of aglycone (a soy isoflavone) on lipid metabolism and
antioxidative function in male vs. female rats were examined in young and old
Sprague-Dawley rats. Immature (4 wk) and mature (8 mo) male and female rats were
fed either a diet containing a 0.1% isoflavone-aglycone rich powder or an isoflavonefree
diet for 24 days. Hepatic antioxidative activities were higher in rats fed
isoflavone vs. isoflavone-free diets in both sexes. Feeding isoflavones resulted in
lower levels of lipid peroxide in immature male rats fed isoflavones, and decreased
liver and serum peroxide concentrations in mature rats of both sexes. Liver and
serum triglyceride concentrations were lower in all rats fed isoflavones, especially in
the mature females. Tissue cholesterol concentrations were also lower in all rats fed
isoflavones. Thus, dietary aglycone may enhance the biological antioxidative system
when fed over the long-term, regardless of age or sex. Based on our observations, the
regulation of lipid metabolism by dietary isoflavone may be more effective in female
than in male rats. The residence time of isoflavone-metabolites derived from dietary
soy isoflavones may be a factor in our observations.
Soy Protein Research, Japan6, 108-115, 2003.
Key words : isoflavone, aglycon, lipid metabolism, antioxidation, sex, rat
Study of Daidzein on Bone Metabolism: Individual Variability in Aging Rats
Hidemichi EBISAWA1, 2 and Yasuko KOSHIHARA1
1Bone research group, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015
2School of Human Life Sciences, Showa Women's University, Tokyo 154-8533
ABSTRACT
Soy isoflavones have received increasing attention because of their potential role in
the prevention of osteoporosis. However, we previously observed no response in bone
mineral density (BMD) in some aging rats fed isoflavones. Thus, in the present study,
we investigated individual variability in bone metabolism of rats in response to
dietary daidzein. Ovariectomized (OVX) 4-mo and 24-mo old rats were fed 13 g of a
diet supplemented with no (OVX-CA), 0.44% (OVX-DL) or 0.88% (OVX-DH) daidzein
for 12 wk. Rats fed OVX-DH had lower femoral BMD and higher bone metabolic
markers than OVX-CA fed rats, although wide individual variation in BMD was
observed in the 24-mo old rats. ER-alpha, ER-beta, HMG-CoA reductase, BMP-2 and
FT-alpha mRNA expressions in the femurs of the 24-mo old rats were measured by
the RT-PCR method. Lower mRNA expression of HMG-CoA reductase was observed
in the OVX-DH group compared to the OVX-CA group. When comparing
characteristics of the 24-mo old rats fed OVX-DH, we found that rats with the highest
BMD (BMD-H) had lower mRNA expression for HMG-CoA reductase and ER-alpha
than rats with the lowest BMD (BMD-L). This suggests that dietary daidzein may
inhibit HMG-CoA reductase mRNA expression. HMG-CoA reductase and ER-alpha
expressions may partially explain the individual variability in response to dietary
daidzein on bone metabolism.
Soy Protein Research, Japan6, 116-121, 2003.
Key words : daidzein, bone metabolism, gene expression, ovariectomized rat
Effects of Soybean Isoflavones on the Proliferation and Invasion of
Hepatoma Cells
Kazumi YAGASAKI, Maki TAKABATAKE and Yutaka MIURA
Department of Applied Biological Science, Tokyo Noko University, Tokyo 183-8509
ABSTRACT
Effects of soybean isoflavones, genistein and daidzein, on the proliferation and
invasion of a rat ascites hepatoma cell line of AH109A were studied in vitro and ex
vivo. Genistein and daidzein dose-dependently suppressed the proliferation and
invasion of AH109A in culture, although the suppressive effect of genistein was more
prominent than that of daidzein. Sera from rats orally given genistein (5 mg/100 g
body weight) 2-3 hours before blood collection most strongly suppressed the
proliferation and invasion of hepatoma cells. Genistein resulted in cell cycle arrest at
the G2/M phase in AH109A cells, suggesting cell cycle arrest may be one mechanism
for the inhibition of AH109A proliferation by genistein. Hepatoma cells pre-cultured
with hypoxanthine (HX) and xanthine oxidase (XO) showed a highly invasive capacity.
Genistein suppressed the reactive oxygen species (ROS)-potentiated invasive capacity
by simultaneously treating AH109A cells with HX, XO and genistein, suggesting that
the antioxidative property of genistein may be involved in its anti-invasive action.
Soy Protein Research, Japan6, 122-127, 2003.
Key words : soybean isoflavones, genistein, hepatoma, proliferation, invasion
Osteogenic Effect of Genistein on Cultured Bone Marrow Cells
Noriko OKUMURA1, Takafumi YOSHIKAWA*1, Jin IIDA2,
Kunio ICHIJIMA1and Yoshinori TAKAKURA2
Departments of Pathology1 and Orthopaedic Surgery2, Nara Medical University, Nara 634-8521
ABSTRACT
The effect of genistein, a soybean isoflavone, on new bone formation by bone marrow
cells from mature rats and humans was examined. Bone marrow cells were
collected from the femoral diaphysis of 7-week-old Fisher rats, cultured in MEM
containing fetal calf serum and then cultured with or without the addition of
dexamethasone to the bone-forming medium. Genistein was added at concentrations
of 10-5, 10-6, 10-7 or 10-8 M. Bone formation was examined 2 weeks after culture.
After informed consent was obtained from a 55-year-old woman with lumbar
spondylosis deformans, bone marrow cells were collected from her ilium for culture
by the same process, and bone formation investigated. In both rats and humans,
when dexamethasone was added to the bone-forming medium, genistein (10-7 M and
10-8 M) caused a significant increase in the levels of calcium, alkaline phosphatase,
and DNA compared with cells not cultured in genistein. In conclusion, genistein was
found to promote bone formation at physiological concentrations across species, and
thus may be useful as a bone formation-promoting factor.
Soy Protein Research, Japan6, 128-134, 2003.
Key words : genistein, osteogenesis, marrow, dexamethasone
Effect of Soy Isoflavones on Premenstrual Syndrome
Naoko ISHIWATA*1, Sayo UESUGI2 and Mariko UEHARA2
1Department of Domestic Science, Atomi Junior College, Tokyo 112-8687
2Faculty of Applied Bio-Science, Tokyo University of Agriculture, Tokyo 156-8502
ABSTRACT
The purpose of this study was to evaluate the efficacy of soybean isoflavones (IF) in
reducing of premenstrual syndrome (PMS). Two hundred forty-two young women
aged 18-21 yrs, voluntarily participated in the intervention study. Premenstrual
symptoms affected daily life activities in 86.0% of all subjects. Major symptoms were
sleepiness, abdominal pain, backache, breast fullness and mental irritability. The
severity of PMS was related to previous experience with dietary restriction for losing
weight and menstrual pain. Fifty six women with premenstrual symptoms were
identified by questionnaire and participated in the randomized, double-blind,
crossover intervention trial with IF and placebo for 8-menstrual cycles. Physical
symptoms of PMS were significantly reduced in the 40 mg IF/day group compared
with the placebo group ( P< 0.05). Headache was reduced the most. Psychological
symptoms were not improved as much as physical symptoms. Individual personality
influenced the severity of premenstrual symptoms. Significant associations were
observed between openness and mental irritability; agreeableness and acne and
psychological symptoms; and conscientiousness and headache (all P< 0.05). Our
results suggest IF supplements may be an effective treatment for the reduction of
physical symptoms of PMS.
Soy Protein Research, Japan6, 135-139, 2003.
1Research Laboratory, Tsuji Academy of Nutrition, Osaka 530-0021 2Osaka University Graduate School of Dental Medicine, Osaka 565-0871
ABSTRACT
The effects of daily intake of soy and soy products on bone mineral density, body
composition and self-reported fatigue after 16 weeks of physical training were
examined in Japanese collegiate American football players. Forty-six football players
aged 19-25 years in a national university and 105 male students aged 18-27 years in a
nutrition academy (as controls) were recruited. Additionally, football players were
sub-divided into "almond" and "no supplement" groups, where the "almond" subjects
were supplemented with 60 g of almonds per day during the 16 weeks of training prior
to the fatigue testing. Almonds were selected because of their rich vitamin E
content. Bone mineral densities (BMD) and body composition were measured by dual
energy X-ray absorptiometry (DEX). Self-reported fatigue was measured through
Profile of Mood State (POMS) and "subjective fatigue symptoms" questionnaires
established by the Japan Society for Occupational Health. BMDs of total body,
Lumbar Spine (L2-L4) and proximal femur were significantly higher in the football
players than in the control group. BMDs of the proximal femurs of the football
players were significantly higher in those with high (≥3 times/wk) intakes of soy and
soy products than in those with low intakes. "Subjective fatigue symptoms" during 16
wk of physical training were significantly lower in the football players with high (≥3
times/wk) intakes of soy and soy products who were supplemented with almonds,
compared to unsupplemented players. Our data suggest that higher intakes of soy
and soy products may promote higher accumulation of bone mineral density and, with almond consumption, may decrease fatigue after heavy training in collegiate
football players.
Soy Protein Research, Japan6, 140-146, 2003.
Key words : bone mineral density, vitamin E, fatigue, soy and soy products, almond,
antioxidants
Modulating Effects of Soy Protein Isolate and Soy Protein Hydrolysate
on Human Brain Function
Eiko HATAKEYAMA1, Masato YAMAGUCHI1, Koji MURAMOTO2, Go ITO2,
Yutaka MOTOHASHI3 and Shigekazu HIGUCHI3
1Kansei Fukushi Research Center, Tohoku Fukushi University, Sendai 981-8522
2Graduate School of Life Science, Tohoku University, Sendai 981-8555
3Akita University School of Medicine, Akita 010-8543
ABSTRACT
We evaluated modulating effects of soy protein isolate and soy protein hydrolysate on
human brain functions by cerebral blood flow and electroencephalography, both noninvasive
measurements. Oxy-Hb concentration and cerebral blood flow at the
forehead increased significantly while performing assigned tasks and decreased while
listening to music when the subjects ingested soy protein and peptides. In the
electroencephalogram, a significant increase in amplitude of all the frequency bands
was observed in the peptide group. Specifically, there were significant increases in
the amplitudes of theta, alpha-2 and beta-1 frequency bands after the ingestion of
peptides ( P<0.05). These results suggest ingestion of soy protein and peptides has
effects on brain activity. Additionally, salivary cortisol, a stress hormone, was
significantly lower after performing the tasks in the soy protein ( P<0.01) and peptide
( P<0.05) groups compared with the placebo group. This suggests soy protein and
peptides may decrease stress. Further research is required to clarify these points as
well as to identify the active components contained in soy proteins.
Soy Protein Research, Japan6, 147-152, 2003.
Key words : soy protein and peptide, brain function, task, cerebral blood flow,
electroencephalogram