Soy Protein Research(Japan)

25-1

The Multivariate Functions of Adipose Tissue and the Metabolic Syndrome: The Effects of Soybean Protein on Fat Distribution and Adipocytokines (Part III)

Yuji MATSUZAWA

Osaka University Graduate School of Medicine, Osaka 565-0871

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 industrial countries. The most common cause of the metabolic syndrome is 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. The Japanese traditional diet may protect against the development of the metabolic syndrome. 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, one of the major components of Japanese traditional diet, on the regulation of adipocytokines. In the previous reports, we demonstrated that adiponectin, which we discovered in adipose cDNA project, possessed anti-atherosclerotic properties, but its plasma concentration was decreased in visceral fat obesity. Calorie-restricted diet containing soy protein could raise plasma adiponectin in diet-induced obesity. In this final report, we describe a global function of this adipocyte-derived factor and the effect of soy protein diet on the regulation of adipocytokines. In adiponectin knockout mice, we found that CRP and TNFαmRNAs were overexpressed in the adipose tissue. Highly sensitive Plasma CRP level was correlated negatively with plasma adiponectin level in humans. Adiponectin-null mice also showed severe hepatic fibrosis induced by carbon tetrachloride. Overexpression of adiponectin suppressed hepatic fibrosis via inhibition of smad 2 signaling. High plasma adiponectin level was a negative risk for breast cancer. In mouse tumor model, injection of adiponectin inhibited the tumor growth. These data indicate that adiponectin has multivalent functions including anti-inflammatory, anti-fibrosis and anti-tumor activities, and that the reduction of adiponectin may have a causative role in inflammation, hepatic fibrosis and some types of cancer frequently associated with visceral obesity. Soy protein diet suppressed the expression of type 1 plasminogen activator inhibitor mRNA and induced mRNA expression and plasma concentration of adiponectin as compared to the casein diet without affecting the body weight. However, we did not observe any significant change in plasma adiponectin level after the ingestion of the formula containing 20 g soy protein per day for 12 weeks. Our results indicate that adiponectin has multivalent functions and that the reduction of adiponectin may play an important role in the development of the disorders in visceral obesity. Soy protein diet has potential beneficial effect on adiponectin. However, further clinical studies will be necessary.
Soy Protein Research, Japan 7, 1-12, 2004.

Key words : soy protein, adipocytokine, viscera fat, adiponectin, metabolic syndrome
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25-2

Molecular Biomedical Evaluation in Monkeys of Efficacy of Soy Products, Soy Protein and Soy Isoflavone for Treatment of Hypercholesterolemia (Part I)

Shin NAKAMURA1, Fusako MITSUNAGA1, A-Ram JOENG1, Hidenori YAMAUCHI1, Takashi HAYASHI2 and Joseph GABRIEL2

1Primate Research Institute, Kyoto University, Inuyama 484-8506
2
Ina Research /Ina Research Philippines, Nagano 399-4501

ABSTRACT
Soy Products are known to protect against both hypercholesterolemia and hypercholesterolemia-mediated atherosclerosis, which are typical life-style-related diseases in the advanced countries. These diseases are also closely linked with agerelated dysfunction in postmenopausal women. In the current study, we examined protective and/or therapeutic effect of soy protein (β-Conglycinin; β-CG) and the soy isoflavone (Soyaflavone HG; IF), which are expected to be the functional components in soy products, on hypercholesterolemia that was developed in ovariectomized (OVX) crab-eating monkeys with experimentally created postmenopausal condition. The molecular mechanism of protective and/or therapeutic action of the soy products against hypercholesterolemia was investigated by genomic biomedical analyses of plasma, liver, adipose tissue, and stool samples. Plasma levels of cholesterol and other related biochemical markers were monitored weekly. Plasma levels of adipocytokines, leptin and adiponectin, were also assayed by a sandwich ELISA established for monkey samples. Gene expression profiling was performed on thrombotic genes (TF, TFPI), Th1/Th2 cytokine genes (IL-4, INF-γ), cholesterol metabolic genes (LDL-R, VLDL-R, HMG-CoA, SR) and adipocytokine genes (leptin, adiponectin, TF) by means of a Real-Time RT-PCR/SYBRY method with ABI PRISM 7700. (Also, using bacterial DNA prepared from monkey stool, intestinal microflora (Bacteroides, Bifidobacteria, E. coli, and Clostridia) was analyzed by means of PCR. β-Conglycinin revealed significant protective and/or therapeutic effect on hypercholesterolemia in OVX-monkeys, whereas Soyaflavone did not show any effect. Little change was observed in the plasma levels of adipocytokines. β-Conglycinin caused up-regulation of gene expression of TF, LDL-R, and HMG-CoA reductase in both peripheral MNL (monocytes) and the liver (hepatocytes), down-regulation of gene expression of TF and leptin, and up-regulation of adiponectin in adipose tissue (adipocytes). Interestingly β-Conglycinin had significant effect on intestinal microflora. It was effective in reducing the cell numbers of harmful bacteria, E. coli and Clostridia, and increasing those of beneficial bacteria, Bifidobacteria. From these results using a monkey model, it is suggested that β-Conglycinin, but not soyaflavone, is the functionally effective component that protects or cures postmenopausal hypercholesterolemia. These results also suggest that β-Conglycinin- or soy protein-enriched-food is a possible candidate for alternative-complementary medicine for these age-related disorders.
Soy Protein Research, Japan 7, 13-19, 2004.

Key words : β-conglycinin, OVX-hypercholesterolemia, monkey model, adipocytokines, intestinal microflora, alternative medicine


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25-3

Characterization of Seed Storage Proteins of Wild Soybean for Application of Breeding

Nobuyuki MARUYAMA1, Takako FUKUDA1, Yoshiya SHIMAMOTO2, Akira KANAZAWA2 and Shigeru UTSUMI1

1Graduate School of Agriculture, Kyoto University, Uji 611-0011
2
Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589 Japan 7, 20-25, 2004.

ABSTRACT
Both soybean (Glycine max) and wild soybean (Glycine soja) storage proteins are composed of two major components, β-conglycinin (7S) and glycinin (11S). We detected electrophoretic variants of the α' subunit of β-conglycinin and the A3 and A4 polypeptides of glycinin of wild soybean storage proteins on SDS-PAGE and designated them as small α', small A3, and large A4. Their N-terminal sequences were identical to normal ones. We isolated cDNAs of smallα', small A3, and large A4 by RT-PCR and sequenced them. Their sequences indicated a deletion of 24 amino acids in smallα', a deletion of 4 amino acids in a small A3 and substitutions of 2 amino acids in large A4 in comparison with the sequences of normal subunits of wild soybean. This deletion and substitution probably causes the difference in the mobility on SDS-PAGE. Furthermore, we analyzed the solubility, emulsifying ability, and thermal stability, of 7S from lines containing small α', and the 11S from lines containing small A3 or large A4. Solubility and emulsifying ability of all samples were very similar to those of normal wild soybean, but the denaturation-starting temperatures of 11S from lines containing small A3 or large A4 were about 4oC lower than that of 11S of normal wild soybean. This characteristic is valuable for lowering the cost of food production from soybean proteins by heating.
Soy Protein Research, Japan 7, 20-25, 2004.

Key words : wild soybean, seed storage protein, β-conglycinin, glycinin
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25-4

Analysis of the Regulatory Mechanisms ofβ-Conglycinin Gene Expression in Modification of Seed Storage Protein Components in Soybean

Akira KANAZAWA

Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589

ABSTRACT
Theβ-conglycinin, a major component of seed storage proteins in soybean, comprises three subunits, α, α' and β. Reporter gene constructs comprising upstream sequences of the gene encodingαsubunit ofβ-conglycinin andβ-glucuronidase (GUS) were made in order to identify regulatory elements of transcription in seeds. The constructs were introduced into Arabidopsis thaliana plants via Agrobacteriummediated gene transfer. GUS gene expression was detected in Arabidopsis seeds when a sequence comprising 545 bp upstream of the transcription start site was fused to the GUS gene, suggesting that regulatory elements for gene expression in seeds are present in this region. Multiple sequence motifs including box I sequence, which are highly conserved among various seed storage protein genes, are present in the upstream region of the α subunit gene. Interactions between these sequences and nuclear factors detected so far suggest that sequences located particularly in the vicinity of the box I are co-operatively involved in the transcriptional control within the DNA region that enabled the expression in Arabidopsis seeds.
Soy Protein Research, Japan 7, 26-31, 2004.

Key words : soybean, β-conglycinin, α subunit, transcription, DNA-protein interaction
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25-5

Studies on Biosynthetic Enzymes of Soybean Saponins for the Breeding of Low Unpleasant taste Soybean Variety

Masakazu SHIRAIWA and Kazumi YASUDA

School of Agriculture, Ibaraki University, Ibaraki 300-0393

ABSTRACT
Group-A and -DDMP saponins exist in the soybean seed as intact saponins. While group-A saponin is the main cause of the unpleasant taste of the soybean, the group- DDMP saponin has various medicinal properties. Therefore, it is thought that the elucidation of characteristics of the enzymes which catalyze biosynthetic reaction of saponins will be useful toward the breeding of the value-added soybean variety in the future. In this research, we have characterized the glucuronosyltransferase and hydroxylase which is the key enzyme of group-A saponin biosynthesis. The substrate specificity of glucuronosyltransferase for each aglycon of group-A and -DDMP saponins changed with metal, reaction temperature and pH. The possibility that by controlling the cultivation conditions of soybeans based on the characteristics of this enzyme, the composition of group-A and -DDMP saponins in an soybean seed might be controlled, is suggested. Furthermore, we searched for the catalytic enzyme in the biosynthetic pathway which could stop only the biosynthesis of group-A saponin. Instead, we found a new hydroxylase, and characterized it. The finding of this hydrohylase has enabled us to depress biosynthesis of group-A saponin and raise biosynthesis of group-DDMP saponin relatively. The result is considered to contribute to development of low unpleasant taste and high functional soybean variety.
Soy Protein Research, Japan 7, 32-41, 2004.

Key words : soybean, saponin biosynthesis, glucuronosyltransferase, hydroxylase
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25-6

The Structure of Tofu for Preventing the Change of Lipid

Tomotada ONO, Takahiro WADA and Atsuko IMAI

Faculty of Agriculture, Iwate University, Morioka 020-8550

ABSTRACT
The tofu (soybean curd), which is a traditional food in Japan & China, includes 38% lipid and 52% protein in dry matter. The lipid in the curd is extremely stable against ooze and oxidation by cooking and storing. Thus the tofu is an important food source for a stable lipid supply. Soy milk was prepared from the soybean ground with water, heated, and filtered. We have already proposed that the soy milk proteins consist of particulate and soluble proteins. The particulate protein is made up of mainly basic peptide (B) of 11S globulin andβsubunit of 7S globulin. The soluble protein is made up of acidic peptide (A) of 11S, α and α' subunits of 7S. The tofu was made from soy milk by the addition of CaSO4 or glucono-δ-lacton (GDL) as coagulants. The protein and lipids from tofu were extracted by the addition of urea or guanidine hydrochloride (GuHCl) as a denaturant with or without 2-mercaptoethanol. The proteins extracted from tofu were up to 40% even by the addition of 8M urea or 5M GuHCl without 2-mercaptoethanol. The yield of lipids was also low. In the presence of 2-mercaptoethanol, yield of both protein and lipid increased to 100% with each denaturant. The composition of extracted proteins was analyzed with SDS-PAGE. Proteins liberated from tofu, in turn, A,αandα', andβand B with increasing denaturants. These results indicate that oil body particle covered with oleosin in tofu is packed with three protein layers, that is, oleosin, protein particles (mainly B and β), and soluble proteins (A, α and α'). Therefore, the lipid of tofu is extremely stable against ooze and oxidation by cooking and storing.
Soy Protein Research, Japan 7, 42-47, 2004.

Key words : tofu, oil-body, proteins of tofu, structure of tofu
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25-7

A Novel and Convenient Assay Method for Proteinase Activity by Measuring Turbidity Change in Proteolytic Digestion of Soy Protein Isolates

Kuniyo INOUYE and Kouhei NAGAI

Graduate School of Agriculture, Kyoto University, Kyoto 606-8502

ABSTRACT
A novel, convenient and accurate assay method for proteinase activity was developed. This method is based on measuring the turbidity change directly in the proteolytic digestion of soy protein isolates (SPI). For all proteases examined, bromelain (Bro),β- chymotrypsim (Chy), ficin (Fic), subtilisin BPN' (SB), subtilisin Carlsberg (SC), thermolysin (TLN), and pronase (Pro), the initial velocity (vo) of the turbidity decrease changed depending on the enzyme and substrate concentrations following the Michaelis-Menten equation. Values of the catalytic constant (kcat) and Michaelis constant (Km) were evaluated for the respective proteinases: The kcat values ((OD units/min)/(g/L)) were 11 ± 0 for Bro, 3.9 ± 0.1 for Chy, 29 ± 0 for Fic, 77 ± 1 for SB, 66 ± 1 for SC, 13 ± 0 for Pro and 277 ± 3 for TLN; the Km values (mg/mL) were 0.83 ± 0.07 for Bro, 0.97 ± 0.08 for Chy, 0.60 ± 0.06 for Fic, 0.58 ± 0.06 for SB, 0.93 ± 0.05 for SC, 1.3 ± 0.1 for Pro and 1.3 ± 0.1 for TLN. The kcat values obtained with turbidity measurement showed high correlation (R=0.995) with those evaluated in the SPI digestion accompanied with acid treatment, which was the general assay method for the proteinase activity. It was demonstrated that the turbidity measurement in the proteolytic digestion of SPI could provide us with a convenient and accurate method for evaluating proteolytic activity without pre- and post-treatment of the samples and reaction solutions.
Soy Protein Research, Japan 7, 48-51, 2004.

Key words : soy protein isolates, proteinase, proteolytic activity, turbidity
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25-8

Interactions of β-Conglycinin and Phosphatidylcholine with Dough Components

Reiko URADE

Graduate School of Agriculture, Kyoto University, Uji 611-0011

ABSTRACT
Soy protein, β-conglycinin is known to decrease plasma triacylglycerol and cholesterol levels in humans. Since these effects of β-conglycinin are transient, it is necessary to take it daily to keep in good health. Bread is a convenient vehicle for introducing β-conglycinin into the daily diet. However, β-conglycinin decreases the loaf volume of bread. Therefore, strategies to overcome this adverse effect should be developed. When soy phosphatidylcholine (PC) was added to the ingredients including β-conglycinin, the adverse effect of this protein on the loaf volume was shown to be counteracted. This study was carried out to elucidate the mechanism of this improving effect of PC on bread-making. By confocal microscopy, PC was shown to associate with gluten in dough. The effects of PC on the volume of dough prepared with de-lipidated wheat flour were determined. The addition of PC showed improving effect on the volume of dough with or without β-conglycinin. This effect reached a plateau when soy PC was supplemented at 4% of wheat flour weight. However, when PC was supplemented in combination with wheat lipids, a synergistic increase in dough volume was obtained. These results suggest that PC acts in two ways, dependent or independent, on wheat lipids in dough. PC showed different effects depending on the PC molecular species.
Soy Protein Research, Japan 7, 52-56, 2004.

Key words : β-conglycinin, bread, dough, phosphatidylcholine, molecular species
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25-9

Masking of Soyprotein-derived Astringency and Its Astringency-sensitive Neuron Profile in Chorda Tympani Nerve Fibers

Michio KOMAI1, Mayuko FUKUNARI1, Hitoshi SUZUKI2 and Tsutomu SAITO3

1Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555,
2School of Science and Engineering, Ishinomaki-Senshu University, Ishinomaki 986-8580,
3
Fuji Oil Co., Ltd., Izumi-Sano 598-8540

ABSTRACT
Astringency perceived in the mouth upon ingestion of tannic acid and other substances is generally regarded as a dry, puckering sensation, which seems more closely allied to the tactile than to the gustatory sense. On the other hand, according to some studies perceptual assessments of astringency may be closely linked to bitterness in human taste, and some electrophysiological studies showed that the chorda tympani and glossopharyngeal nerves transmit the astringency signal. We attempted to find tactile sensitive nerve bundles from the lingual trigeminal nerves by applying 0.6 M tannic acid, but found instead 18 lingual trigeminal nerve bundles that did not respond to this at all. This indicates that the lingual trigeminal nerves do not respond to astringent stimuli. On the other hand, many chorda tympani nerve fibers responded to the tannic acid solution. Therefore, we analyzed the response profiles of potential astringentsensitive neurons in the chorda tympani nerves in adult female Wistar rats. Astringent compounds, tannic acid, and acidified soy protein isolate [glycinin] were applied to the tongue of the rat. According to the cluster analysis of 80 chorda tympani nerve fibers, the responses to glycinin (3% soln, pH 3.5) that cause astringency were most closely related to the response to sour taste (0.01 M HCl, r=0.721) and tannic acid (0.01 M, r=0.693). They were less related to bitter taste (0.02 M quinine-HCl, r=0.559) and umami taste (0.035 M MSG, r=0.483), and not correlated with other basic taste stimuli. Several candidate substances that reduce astringency derived from acidified glycinin are proposed.
Soy Protein Research, Japan 7, 57-62, 2004.

Key words : astringency, glycinin, taste nerve, nerve fiber profile, rat
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25-10

High Efficiency Dye-adsorbent Produced from Okara, a By-product of Soy Foods Processing
-Adsorption Characteristics and the Elucidation of the Mechanism-

Norie NISHIKAWA, Akihiko MASUI, Yoshihiro YOBIKO and Nobuaki FUJIWARA

Technology Research Institute of Osaka Prefecture, Izumi 594-1157

ABSTRACT
Electrophoresis is a preparative and analytical technique broadly used in biological sciences, particularly in protein research. Avoiding background raised by dyestuff, gel staining and destaining are crucial steps in detection of protein bands in the gel electrophoresis. Particularly important is fast and efficient gel destaining. Okara, a byproduct obtained during the production of tofu (bean curd) and soymilk, quickly adsorbed Coomassie Brilliant Blue (CBB) stain. Compared to activated carbon and/or carbon fiber adsorption, Okara efficiently adsorbs significantly higher amount of dye. In this research, the characteristics and mechanism of adsorption were examined with the aim to develop an efficient dyestuff adsorption system using Okara. The amount of the saturation dye adsorption, Qm (mg-dye/g-okara) obtained from the Langmuir adsorption isotherm (25oC) after 24 h at pH 3 was 197. It was found that Okara's adsorption ability is approximately 66 times higher than the ability of activated carbon. There was no significant difference in the adsorption of the dyestuff between defatted sample with n-hexane and intact sample. It was shown that the dyestuff was not adsorbed on the oil body in Okara. The adsorption efficiency of dye increased proportionally to the content of remaining protein in Okara and was influenced by pH. These results show that dye was attached to the residual protein in Okara. The decrease in adsorption capacity, which was influenced by the addition of salt (NaClO4), was only 30%, however the addition of methanol decreased the adsorption capacity approximately 70%. These results showed that dye adsorption was enhanced by hydrophobic interactions.
Soy Protein Research, Japan 7,63-68, 2004.

Key words : okara, dye, coomassie brilliant blue R-250, adsorption, ionic bond, hydrophobic interactions
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25-11

Effects of Soy Protein or Its Derived Peptides on Muscle Protein Degradation

Takeshi NIKAWA, Katsuya HIRASAKA, Harumi FUROUCHI, Yuko OHNISHI and Kyoichi KISHI

Institute of Health Bioscience, The University of Tokushima Graduate School, Tokushima 770-8503

ABSTRACT
We have previously demonstrated that muscle proteases and the related molecules play important roles in disorders of muscle function. For example, rats subjected to an acute running exercise have significantly high calpain activity in gastrocnemius muscle. Rat gastrocnemius muscle atrophied by spaceflight or tail-suspension is specifically sensitive to the ubiquitin-proteasome proteolytic pathway. In this study, we examined the effects of soy protein isolate (SPI) or soy protein-derived peptides on these proteolytic pathways. SPI diet prevented exercise-induced protein degradation in skeletal muscle possibly through inhibition of calpain-mediated proteolysis, as compared with the casein diet. Soy protein-derived peptides inhibited proteinubiquitination caused by oxidative stress in mouse myoblastic cells. Our results suggest that soy protein may contain bioactive factors for regulating proteolysis in skeletal muscle.
Soy Protein Research, Japan 7, 69-74, 2004.

Key words : soy protein isolate, soy protein-derived peptides, calpain, ubiquitin, skeletal muscle
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25-12

Studies on Interaction between Soy Constituents and Central Nervous System

Masaaki YOSHIKAWA and Soushi SONODA

Graduate School of Agriculture, Kyoto University, Uji 611-0011

ABSTRACT
Effect of a pentapeptide Leu-Pro-Tyr-Pro-Arg (LPYPR), which corresponds to residues No. 63-67 of soy bean glycinin A5A4B3 subunit, on central nervous system was investigated in mice. LPYPR showed analgesic activity after intracerebroventricular (icv) administration at a dose of 30 nmol/mouse. The analgesic activity was nonopiodic since it was not blocked by naloxone. In passive avoidance test, LPYPR improved memory retention at a dose of 20 nmol/mouse (icv) or 100 mg/kg (per os).
Soy Protein Research, Japan 7, 75-78, 2004.

Key words : soy, glycinin, peptide, central nervous system, analgesia, memory, learning
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25-13

Isolation and Application of Physiologically Active Peptides from Soybean Whey and Okara Proteins

Masami YONEKURA and Ayako YAMAMOTO

School of Agriculture, Ibaraki University, Ibaraki 300-0393

ABSTRACT
In this study, the antihypertensive effect of a peptide, Leu-Ala-Pro (LAP), which has angiotensin I-converting enzyme (ACE) inhibitory activity and was isolated from thermolysin digests of soybean whey proteins, was investigated in spontaneously hypertensive rat (SHR). Oral administration of soybean whey peptide preparation containing 0.1% LAP at a dose of 1 g/kg of body weight/day resulted in a significant reduction of systolic and diastolic blood pressures in SHR. Single oral administration of synthesized LAP at a dose of 10 mg/kg had ability to lower blood pressure in 21- week-old SHR. The ACE inhibitory peptides from whey protein digested with protease S were also isolated by 60% ethanol fractionation, gel filtration on Sephadex G-25, and reverse phase HPLC, and their amino acid sequences were determined. Seven kinds of the ACE inhibitory peptides were identified as LN, LHP, LKP, VTY, LYQA, YEAP, and YQAP. These results suggest that the soybean whey peptide preparations containing LAP prepared by thermolysin digestion and the protease S digests of soybean whey proteins may be promising materials for the development of a functional food with antihypertensive activity.
Soy Protein Research, Japan 7, 79-84, 2004.

Key words : soybean, peptide, angiotensin I-converting enzyme inhibitor, whey protein
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25-14

Analysis of Soybean Protein for Producing Root Hair Promoting Peptide

Motoki KUBO, Youhei YAMAJI, Takuma MATSUKURA, Sayoko SUMIYOSHI and Satoko HIRANO

Faculty of Science and Technology, Ritsumeikan University, Kusatsu 525-8577

ABSTRACT
The growth of plants was promoted by the degradation of soybean meal products (DSP). The root hair number of Brassica campestris was increased when DSP was added. The whey protein, SPI protein, and the insoluble protein were separated, and their degraded products obtained by digestion with protease from Bacillus circulans HA12, were assayed for analyzing the root hair promoting activity. Degradation products from the whey protein had the root hair promoting activity. The whey protein was further separated by chromatography, and the degradation products from each peak were analyzed by the root hair assay. Degradation products from 20 kDa protein showed the root hair promoting activity and the protein was identified as Kunitz trypsin inhibitor.
Soy Protein Research, Japan 7, 85-89, 2004.

Key words : soybean, whey protein, trypsin inhibitor, Bacillus circulans, alkaline protease, root hair
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25-15

Studies on Liver Triglyceride-lowering Action of Soyapeptide Prepared by Using Hydrophobic Absorbent Resin Column

Mayumi MINAMI, Masanobu SAKONO and Nobuhiro FUKUDA

Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192

ABSTRACT
In previous studies, we have demonstrated a potent hypolipidemic effect of both LD3 (hydrolyzate of soy protein isolate obtained by digestion with fungus protease) and F5 (prepared by using hydrophobic adsorbent resin column). In the present studies, we have attempted to clarify the active components occurring in the LD3 and F5 and to elucidate the mechanism responsible for the observed reduction, using primary rat hepatocytes. When the hydrolyzates of casein, LD3 and F5 were incubated with primary hepatocytes, followed by addition of [1-14C]oleate as an exogenous fatty acid substrate (experiment 1), the incorporation of oleate into the medium lipids, but not into cellular lipids, was significantly lowered in the groups of LD3-D (enzyme hydrolyzate of LD3) and F5-D (enzyme hydrolyzate of F5) in a dose-dependent manner; the extent of lowering activity being greater in F5-D than in LD3-D. This lowering activity was not true for casein hydrolyzate. Although the effects on lipid synthesis of addition of three types of di-peptides, which may be absorbable from the intestine into the blood stream, were not consistent in experiment 2 and 4, it nevertheless appeared to have some beneficial effects on lipid synthesis. In the third experiment, we examined the effects of isoflavone and saponin on the incorporation of radiolabelled oleate into lipids. The results showed that there was a dose-dependent reduction in incorporation of oleate into the cell and/or medium lipids, especially the triglycerides, in the cells incubated with isoflavone and saponin, respectively. In the fourth experiment, we further examined the effects of combinations of di-peptide with isoflavone or saponin on the incorporation of [14C]oleate into lipids, and found that under certain conditions there was an additional effect of this combination of di-peptide with the isoflavone or saponin in terms of the reduced synthesis of lipids, especially triglycerides, as compared to those of either di-peptide, isoflavone or saponin alone. On the other hand, there was an increased incorporation of radiolabelled oleate into water soluble fraction (ketone body). These results suggest that serum and liver lipid-lowering effect of LD3 or F5 is in part due to the combinational effect of di-peptide and/or other components such as isoflavone, flavone and saponin the mechanism responsible for the observed lowering effect is attributed to an altered metabolism of fatty acid between oxidation and esterification in the liver.
Soy Protein Research, Japan 7, 90-97, 2004.

Key words : liver triglyceride-lowering action, soypeptide, rat, hepatocyte
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25-16

Soy Proteins Affect Activities of Small Intestinal Transporters and Improve Lipid Metabolism

Ryuichiro SATO

Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657

ABSTRACT
Cholesterol efflux from human cultured intestinal cells, Caco-2, was studied. [3H]cholesterol efflux to the apical side of Caco-2 cells was measured after induction of ABC transporter gene expression by the LXR agonist. Cholesterol efflux to the apical side was enhanced by the agonist and/or the addition of bile acid, but 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. Among several bile acid molecules, chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) are potent acceptors. Crude peptide molecules digested from soy proteins as well as bile acids, stimulated cholesterol efflux from the intestinal epithelial cells. We also developed a novel assay system for evaluating bile acid absorption through the intestinal bile acid transporter (IBAT). Some soy peptides did not affect bile acid absorption, but appeared to stimulate the farnesoid X receptor (FXR) activity in cells after being absorbed. This stimulating effect of soy peptides on the FXR activity might partly explain the molecular mechanism by which dietary soy proteins reduce serum triacylglycerol and cholesterol levels.
Soy Protein Research, Japan 7, 98-101, 2004.

Key words : cholesterol, bile acid, IBAT, FXR, ABCG5, ABCG8
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25-17

Beneficial Effects of a Soy Protein Diet and a Soybean Trypsin Inhibitor in Genetically Obese and Diabetic Rats Lacking Cholecystokinin-A Receptor

Kyoko MIYASAKA, Setsuko KANAI, Minoru OHTA and Ayako SEKIME

Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015

ABSTRACT
Otsuka Long-Evans Tokushima Fatty (OLETF) rats were developed as a model for noninsulin-dependent diabetes mellitus (NIDDM). OLETF rats are characterized by late onset of hyperglycemia at 18 weeks of age, followed by insulin-dependent diabetes mellitus at 65 weeks of age. Soybeans and their products are classical Japanese food and are generally believed to be good for health. In this study, we compared the effect of a special diet, consisting of soy protein, on energy metabolism of normal LETO rats and genetically diabetic OLETF rats. Twelve-week-old male rats of LETO and OLETF strains were fed this experimental diet in parallel with CRF-1 (control) and examined for respiratory quotient (RQ) and the blood or plasma parameters associated with diabetes mellitus. We also examined whether abnormal intestinal triglyceride metabolism in OLETF rats with NIDDM could be improved by soy protein and/or soy peptides diet. A mesenteric lyphphnode fistula was prepared. Administration of a soy protein diet increased daily food intake but did not change daily energy expenditure in LETO rats, resulting in increased body weight. In diabetic OLETF rats, administration of a soy protein diet increased daily energy expenditure, but daily food intake did not change therefore, the body weight slightly decreased. Moreover, glucose tolerance test was slightly improved by administration of a soy protein diet. The intestinal fat absorption was not influenced by long-term administration of a soy protein diet. However, simultaneous infusion of a soy peptide with fat into the small intestinal lumen, decreased the intestinal fat absorption. Thus, ingestion of soy protein and/or peptide might prevent fat absorption and hyperlipidemia as well as obesity.
Soy Protein Research, Japan 7, 102-107, 2004.

Key words : soy-protein diet, energy expenditure, non-insulin dependent diabetes mellitus, aging, health
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25-18

Mechanism of Inducing Satiety Effects by Peptides Derived from Soybean β-Conglycinin

Hiroshi HARA, Toshihiro MAEKAWA and Tohru HIRA

Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589

ABSTRACT
Soybean β-conglycinin peptone (β-con) enhances satiety via cholecystokinin (CCK) secretion, and the β-conglycinin β-subunit 51-63 (β51-63, VRIRLLQRFNKRS) is responsible for this effect. The aim of the present study is to identify signaling pathway induced with β-con by using the enteroendocrine cell line, STC-1. We found that β-conglycinin peptone induced Ca signaling in the STC-1 cells, which correlated with the binding activity of peptides derived from β-conglycinin to the rat jejunal brush-border membrane. The Ca signaling induced by β-con was dependent on Ca outside of cells passing through L-type Ca channel. Adenylate cyclase was partly involved in the induction of Ca signaling byβ-con in STC-1 cells.
Soy Protein Research, Japan 7, 108-113, 2004

Key words : soybean β-conglycinin, satiety effect, cholecystokinin, calcium signaling, STC-1 cells
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25-19

Effect of Dietary Soybean Protein Isolate-hydrolysate on Oxidation of Dietary Carbohydrate and Diabetic Disorders in Diabetic Obese Mice

Kengo ISHIHARA1, Wataru MIZUNOYA2, Tomono UCHIDA1, Tetsuro SHIBAKUSA2, Yukiko MITA1, Tohru FUSHIKI2 and Kyoden YASUMOTO1

1School of Life Studies, Sugiyama Jogakuen University, Nagoya 464-8662
2
Graduate School of Agriculture, Kyoto University, Kyoto 611-0011

ABSTRACT
The aim of this study was to investigate the effects of chronic feeding with dietary protein, on insulin resistance during weight loss, in type II diabetic mice. Obesity was induced in KK-Ay mice by high fat feeding, followed by weight loss induced through restrictive isoenergetic and isonitrogenous diets (35% of energy as protein and 5% as fat) for five weeks. Restrictive diets were based on casein, wheat gluten hydrolysate (GLU-H), or soy protein isolate- hydrolysate (SPI-H). The postprandial RQ and generation of 13CO2 from ingested 13C-glucose were higher in nondiabetic mice than in diabetic mice. The postprandial RQ was higher in GLU-H-fed and SPI-H-fed mice than in casein-fed mice after 3 weeks. We also observed the elevated postprandial RQ in GLU-H and SPI-H groups after all the mice had single feedings of the casein diet. In an oral glucose tolerance test, the average oxidation of 13C-glucose derived from orally administered glucose solution was higher in GLU-H-fed than in casein-fed mice. The average plasma insulin level after 30 min. of glucose administration was lower in GLU-H-fed than in casein-fed mice. In fasted mice, the RQ and levels of plasma glucose and insulin did not differ significantly between dietary groups. These results indicate that chronic feeding of GLU-H increases postprandial carbohydrate oxidation and prevents development of insulin resistance in type II diabetic mice.
Soy Protein Research, Japan 7, 114-120, 2004.

Key words : KK-Ay, 13C, glucose uptake, diabetes, soybean
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25-20

A Novel Hybrid-type Artificial Bone Prepared from Plant Protein

Toshiki MIYAZAKI

Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Fukuoka 808-0196

ABSTRACT
The so-called bioactive ceramics such as Bioglass®, sintered hydroxyapatite, and apatite- and wollastonite-containing glass-ceramics (glass-ceramics A-W), bond to living bone when implanted in bony defect. They have been already subjected to clinical applications as bone substitutes in orthopedic surgery, neurosurgery, dentistry, and so on. However, their higher brittleness and lower flexibility than natural bone, limits their clinical application to low loaded conditions. Materials exhibiting both high bioactivity and high flexibility are required for novel bone substitutes. Natural bone is a kind of organic-inorganic hybrid, which is composed of organic collagen and inorganic apatite nanocrystals. Based on this we think that apatite-polymer hybrid produces novel bone-repairing materials, which exhibit both bioactivity and flexibility. In living body, the bone tissue is produced by site-selective precipitation of apatite on collagen fibers abundant in carboxyl group. In the present study, we attempted to synthesize organic-inorganic hybrids from various plant proteins such as wheat-derived gluten and corn-derived zein. This kind of plant protein is free from diseases of virus and prion origin. Apatite deposition was observed on the surfaces of these proteins in aqueous solution supersaturated with respect to the apatite, when their surfaces were previously modified with calcium ions. This type of organic-inorganic hybrid is quite attractive as a novel bone substitutes in orthopedic and dental fields.
Soy Protein Research, Japan 7, 121-124, 2004.

Key words : artificial bone, organic-inorganic hybrid, gluten, zein, apatite
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25-21

Regulation of Heme Oxygenase-1 and Ligand-binding Transcription Factors by Soybean Isoflavones

Kazuhiro OGAWA1, Koichi NAGATA1, Katsuya SUZUKI1, Toshihiro KONNO1, 2, Shigeki SHIBAHARA1 and Teruyuki YANAGISAWA1

1Tohoku University School of Medicine, Sendai 980-8575
2
Tohoku University Graduate School of Dentistry, Sendai 980-8575

ABSTRACT
Genistein and daidzein, soybean isoflavones, have been described to bind to estrogen receptors and to exert estrogenic and/or anti-estrogenic activity. We have shown that heme binds to Bach1, a transcriptional repressor, and that this binding abrogates the DNA-binding activity of Bach1 to enhance the transcription of genes carrying Bach1-binding sequences. This mechanism is involved in heme oxygenase (HO)-1 induction by heme. HOs degrade heme, a pro-oxidant, to produce bilirubin, an anti-oxidant, and HO-1 is an inducible isozyme of HOs. In this study, we investigated the effects of the isoflavones on gene regulation of HO-1 and its related transcription factors, including Bach1, in human lung carcinoma cells. Both genistein and daidzein inhibited the induction of HO-1 by sodium nitroprusside (SNP), a nitric oxide donor, while genistein but not daidzein inhibited the hememediated HO-1 induction. Genistein and daidzein had little effect on the mRNA levels of Bach1 and Nrf2, the transcription factors which regulate transcription of the HO-1 gene. Further study is under way to elucidate the regulatory mechanism of the HO-1 expression by the isoflavones. As the HO-1 induction is an important protective response against excessive free heme and other oxidative stresses, daidzein might be, in some cases, a useful phytoestrogen in that it does not inhibit the heme-mediated HO-1 induction.
Soy Protein Research, Japan 7, 125-129, 2004.

Key words : isoflavone, genistein, daidzein, heme, heme oxygenase-1, phytoestrogen
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25-22

Effect of Genistein on Immune Responses in NC/Nga Mice Used as an Animal Model for Atopic Dermatitis

Tohru SAKAI, Mari KOGISO, Kaori MITSUYA and Shigeru YAMAMOTO

1Department of Clinical Nutrition, Osaka Prefecture College of Nursing, Habikino 583-8555
2
Institute of Health Bioscience, The University of Tokushima Graduate School, Tokushima 770-8503

ABSTRACT
The isoflavone genistein is a phytoestrogen found in high levels in soy products that has been associated with decreased incidence of breast and prostate cancers. In addition, a recent clinical trial suggests that supplementation with the dietary genistein may be as effective as hormone replacement therapy in attenuating menopause-related bone loss without causing the associated side effects. In this study, we examined the effect of genistein on the immune responses in NC/Nga mice that are used as an animal model for atopic dermatitis. Groups of mice were exposed to vehicle or the genistein by gavage. The doses of genistein used were 4 and 20 mg/kg body weight/day. Treatment with picrylchroride in the ear of NC/Nga mice developed dermatitis. Mice that received genistein showed mild dermatitis when compared to the control mice. We also determined serum IgE level, but significant difference was not observed between the groups. Although genistein did not affect the in vitro lymphocyte proliferation, it suppressed cytokine production by spleen cells. These results showed that dietary genistein modulates an inflammatory response resulting in amelioration of dermatitis in the skin of NC/Nga mice.
Soy Protein Research, Japan 7, 130-136, 2004.

Key words : genistein, NC/Nga mice, IgE, cytokine, dermatitis
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25-23

A Soybean Kunitz Trypsin Inhibitor Suppresses Ovarian Cancer Cell Invasion by Blocking Urokinase Upregulation

Hiroshi KOBAYASHI and Mika SUZUKI

Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Shizuoka 431-3192

ABSTRACT
We have previously reported in a series of papers that a Kunitz-type protease inhibitor, bikunin, suppresses upregulation of urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) expression, phosphorylation of ERK1/2 and cancer cell invasion in vitro and the peritoneal disseminated metastasis in vivo. In the present study, we investigated the effects of soy bean trypsin inhibitor (SBTI) on the net enzymatic activity of secreted extracellular uPA, the signal transduction involved in the expression of uPA and the invasion in HRA human ovarian cancer cells. SBTI contains a Kunitz trypsin inhibitor (KTI) and a Bowman-Birk inhibitor (BBI). Here, we show 1) that KTI and BBI were purified separately from soybeans, 2) that neither KTI nor BBI effectively inhibits enzymatic activity of uPA, 3) that uPA upregulation observed in HRA cells was inhibited by preincubation of the cells with KTI with an IC50 of ~2μM, whereas BBI failed to repress uPA upregulation, as measured by enzyme-linked immunosorbent assay, 4) that the cell invasiveness was inhibited by treatment of the cells with KTI with an IC50 of ~3μM, whereas BBI failed to suppress cell invasion, as measured by an in vitro invasion assay, 5) KTI suppresses HRA cell invasion by blocking uPA up-regulation which may be mediated by a binding proteins other than a bikunin-binding protein and/or its receptor, and 6) that the transforming growth factor-beta 1 (TGF-β1)-mediated activation of ERK1/2 was significantly reduced by preincubation of the cells with KTI. In conclusion, KTI, but not BBI, could inhibit cell invasiveness at least through suppression of uPA signaling cascade, although the mechanisms of KTI may be different from those of bikunin.
Soy Protein Research, Japan 7, 137-144, 2004.

Key words : bikunin, urokinase, cancer metastasis, ovarian cancer, soybean protein
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25-24

Anti-platelets Effects of Genistein, an Isoflavonoid from Soybean

Atsushi ODA

Department of Preventive Medicine, Hokkaido University School of Medicine, Sapporo 060-8638

ABSTRACT
It has been reported that genistein inhibits platelet functions in vitro. The mechanisms of the inhibitory effects are poorly understood. Genistein is a wellknown inhibitor of protein tyrosine kinases. However, I show here that genistein inhibits platelet activating factor (PAF)-induced elevation of ionized calcium concentrations and IP3 production in platelets, induced by PAF, in the presence of staurosporin, which abolishes increased protein tyrosine phosphorylation. Thus, it appears that genistein inhibits activation of phospholipase C in stimulated platelets, apparently independent of its effects on tyrosine kinases. I also show that catechins, which might be consumed along with genistein in the Japanese diet, had markedly different effects on platelets in comparison with genistein. As people take different flavonoids simultaneously, it is highly desirable that the combined effects of flavonoids are elucidated in different systems. In this sense, platelets may be useful in these settings.
Soy Protein Research, Japan 7, 145-148, 2004.

Key words : flavonoid, phospholipase C, platelet aggregation, tyrosine kinase, catechin
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25-25

Soy Protein and Isoflavone Intake and the Risk of Colon Cancer

Chisato NAGATA

Gifu University Graduate School of Medicine, Gifu 501-1194

ABSTRACT
This study examined the relationship between soy protein and isoflavone intake and the subsequent risk of colon cancer in a population-based cohort of Japanese men and women. In 1992, the usual diet including soy intake was determined in 13,893 men and 16,331 women in a city of Gifu Prefecture, with the use of a validated foodfrequency questionnaire. The length of follow-up was about 8 y. In men, there were no significant associations between risks of colon cancer and intakes of soy products, soy protein, and soy isoflavones. In women, the highest compared to the lowest tertile of soy product intake was significantly associated with a decreased risk of colon cancer after controlling for covariates. However, the associations of colon cancer risks with soy protein and soy isoflavone intakes were not statistically significant.
Soy Protein Research, Japan 7, 149-151, 2004.

Key words : soy protein, isoflavones, colon cancer, prospective study
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25-26

Clinical Usefulness of β-Conglycinin Containing Snack for the Diet Therapy of Hyperlipidemia and Obesity

Tomomi ADACHI1, Junko NABIKI1, Takeshi MIYAWAKI1, Nayuta MORI1, Tetsuhiko OKAJIMA2, Motohiko HIROTSUKA2, Aki HARANO3 and Yutaka HARANO1

1Koshien University College of Nutrition, Takarazuka 665-0006
2
Fuji Oil Co.,LTD., Izumisano 598-8540
3
Harano hospital, Minoh 562-0005

ABSTRACT
Protein cookie comprising β-Conglycinin (β-CG), one of the major components of soy bean protein that has been reported to reduce blood TG and cholesterol level in animal study, was used as a supplement for the diet therapy of hyperlipidemia and obesity. The protein cookie (9 g β-CG) was ingested by 5 healthy subjects, and their glycemic and insulin responses were compared with those of control subjects who ingested starch cookie in which soy bean protein was replaced with flour starch. Thirty subjects with life- style related diseases, who were obese or having dyslipidemia, were encouraged to take this snack (6 g/day), replacing or reducing regular snack or carbohydrate source). Significant reduction in body weight (0.7 kg), TG (19 mg/100mL) and chol (11 mg/100mL) was observed after 1-3 months. Hamburger (500 kcal containing 37 g fat) was fed daily for a week to each of a total of 25 healthy subjects who were divided into those with or without intake of β-CG cookie. Cookie test was performed before and after the burger-feeding. By PAGE analyses of serum collected at 0, 1, 2 hr, presence or appearance of atherogenic lipoprotein (VLDL, mid-band, decrease of HDL, small-dense LDL) was scored as 1, and judged in total scores. Disappearance of dyslipidemia was counted as -1. In those havingβ-CG no rise in score was noted, whereas in placebo the points increased from 0.5 to 2.5, suggesting an improving effect of β-CG cookie on VLDL metabolism. Hepatic TG lipase when measured after heparin injection (30 U/kg body weight), tended to be activated, but no change was noted for LPL. β-CG cookie was effective in weight reduction or dyslipidemia when used as a supplement in diet therapy. Subjects were encouraged to replace or reduce regular snack, rich in fat or carbohydrate or meal starch, with the newly developed soy bean cookie. The hypolipidemic effect seemed to be attributable to the additive effect of improved insulin sensitivity due to weight reduction and direct effect of β-CG. Antiatherogenic effect on lipoprotein metabolism was mainly observed in TG rather than cholesterol metabolism. Activation of hepatic TG lipase was suggested as one of the mechanisms involved.
Soy Protein Research, Japan 7, 152-156, 2004.

Key words : dyslipidemia, β-conglycinin, soy-bean protein, obesity, life-style related diseases
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25-27

Effect of Soy Isoflavones on Premenstrual Syndrome (Part 2)

Naoko ISHIWATA1, Sayo UESUGI2, Mariko UEHARA2 and Shaw WATANABE2

1Department of Domestic Science, Atomi Junior College, Tokyo 112-8687
2
Faculty of Applied Bio-Science, Tokyo University of Agriculture, Tokyo 156-8502

ABSTRACT
Fifty-four young women with premenstrual syndrome participated in a randomized, double-blind, placebo-controlled intervention trial. They were divided into two groups: Twenty-eight women took a 20 mg/day isoflavone supplement (20 mg/day IF group); 26 women took a 40 mg/day isoflavone supplement (40 mg/day IF group). During the three periods of baseline, IF supplement, and placebo (PL), blood and urine samples were obtained from women at times corresponding to peak estrogen and progesterone in each woman's menstrual cycle. There was a trend toward a decrease in the estradiol/progesterone ratio among women with moderate to serious symptoms, and a decrease in prostaglandin E2 (PGE2) among women with serious symptoms. Independent of symptom severity, the serotonin tended to increase among individuals in the 40 mg/day IF group. Prolactin tended to increase as symptom severity increased, and was significantly lower in the 40 mg/day IF group compared to the placebo among those with severe symptoms. Sex-hormone binding globulin levels tended to increase among the 40 mg/day IF group when symptoms were severe. Only twenty percent of subjects were equol producers, similar to the level observed among Westerners.
Soy Protein Research, Japan 7, 157-161, 2004.

Key words : premenstrual syndrome, soy isoflavones, sex steroid hormone, prostaglandin, equol
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