Skip to content


  • Research
  • Open Access

Screening of soy protein-derived hypotriglyceridemic di-peptides in vitro and in vivo

  • 1, 4,
  • 1,
  • 1,
  • 1,
  • 1,
  • 1,
  • 2,
  • 3,
  • 3,
  • 3 and
  • 1Email author
Contributed equally
Lipids in Health and Disease201110:85

  • Received: 1 April 2011
  • Accepted: 22 May 2011
  • Published:



Soy protein and soy peptides have attracted considerable attention because of their potentially beneficial biological properties, including antihypertensive, anticarcinogenic, and hypolipidemic effects. Although soy protein isolate contains several bioactive peptides that have distinct physiological activities in lipid metabolism, it is not clear which peptide sequences are responsible for the triglyceride (TG)-lowering effects. In the present study, we investigated the effects of soy protein-derived peptides on lipid metabolism, especially TG metabolism, in HepG2 cells and obese Otsuka Long-Evans Tokushima fatty (OLETF) rats.


In the first experiment, we found that soy crude peptide (SCP)-LD3, which was prepared by hydrolyze of soy protein isolate with endo-type protease, showed hypolipidemic effects in HepG2 cells and OLETF rats. In the second experiment, we found that hydrophilic fraction, separated from SCP-LD3 with hydrophobic synthetic absorbent, revealed lipid-lowering effects in HepG2 cells and OLETF rats. In the third experiment, we found that Fraction-C (Frc-C) peptides, fractionated from hydrophilic peptides by gel permeation chromatography-high performance liquid chromatography, significantly reduced TG synthesis and apolipoprotein B (apoB) secretion in HepG2 cells. In the fourth experiment, we found that the fraction with 0.1% trifluoroacetic acid, isolated from Frc-C peptides by octadecylsilyl column chromatography, showed hypolipidemic effects in HepG2 cells. In the final experiment, we found that 3 di-peptides, Lys-Ala, Val-Lys, and Ser-Tyr, reduced TG synthesis, and Ser-Tyr additionally reduced apoB secretion in HepG2 cells.


Novel active peptides with TG-lowering effects from soy protein have been isolated.


  • HepG2 Cell
  • Hydrophilic Fraction
  • Choline Bitartrate
  • apoB100 Secretion
  • Hypocholesterolemic Peptide


In industrialized countries, lifestyle-related diseases such as hyperlipidemia, arteriosclerosis, diabetes mellitus, and hypertension are widespread and increasingly prevalent, thus contributing to the increases in cardiovascular morbidity and mortality [1, 2]. Accompanied by the rapid increase in the number of elderly people, this increase in lifestyle-related diseases becomes important not only medically, but also socioeconomically. A clustering of metabolic disorders in an individual, defined as metabolic syndrome, is known to increase cardiovascular morbidity and mortality. Although the pathogenesis of metabolic syndrome is complicated and the precise details of its underlying mechanisms are not known, lipid abnormality is now proposed as a feature of metabolic syndrome along with insulin resistance [13]. Many studies have suggested that the quality of dietary proteins can be an important modulator of the risks associated with this syndrome [47].

In general, plant protein intake is inversely related to the risk of hypertension and cardiovascular disease [47]. For example, several studies have shown that dietary soy protein reduces cholesterol and triglyceride (TG) levels and lowers blood pressure in animals and humans [49]. Because it has been reported that peptides or protein hydrolysates show greater bioactivities than intact proteins or amino acid mixtures [10, 11], bioactive peptides have been produced in vitro through chemical or enzymatic hydrolysis of several food proteins, in order to modify and improve the physiological functions of dietary proteins [11, 12]. Bioactive peptides are prepared from both plant and animal sources, with the antihypertensive peptides IAP and VY and the hypocholesterolemic peptide IIAEK derived from wheat gliadin, sardine muscle, and bovine milk α-lactoglobulin, respectively [1317]. For soy protein, several bioactive peptides, including the hypocholesterolemic peptide LPYPR, antihypertensive peptide NWGPLV, antioxidant peptide LLPHH, and anti-obese peptide VRIRLLQRFNKRS, were derived from its major constituents, glycinin and β-conglycinin (BCG) [1821]. Of the limited number of peptides reported to have hypotrigliceridemic activities, VVYP, VYP, and VTL were identified in hydrolyzed globin from animal blood [22].

In the present study, we investigated the effects of soy protein-derived peptides on hepatic lipid metabolism, especially TG metabolism, both in vitro and in vivo. We used human hepatoma HepG2 cells, the most suitable and accessible human-derived cells that retain many of the biochemical functions of human liver parenchymal cells [23], for the in vitro study. For the in vivo study, Otsuka Long-Evans Tokushima fatty (OLETF) rats, which develop a syndrome with multiple metabolic and hormonal disorders that shares many features of human obesity, were used. OLETF rats exhibit hyperphagia, owing to the lack of cholecystokinin receptors; as a result, they become obese and develop hyperlipidemia, fatty liver, and diabetes [2426].

Materials and methods

Preparation of soy peptides

Preparation of soy crude peptides (SCP)-LD3 was performed by hydrolyze of soy protein isolate with endo-type protease derived from Bacillus sp. and fractionation with centrifugation [27]. SCP-LD3 was separated into 2 fractions (hydrophobic fraction and hydrophilic fraction) on the basis of hydrophobicity by using hydrophobic synthetic absorbent (DIAION HP21; Mitsubishi Chemical Corporation, Tokyo, Japan). The amino acid composition of SCP-LD3, hydrophobic fraction, and hydrophilic fraction was analyzed with an automatic amino acid analyzer; results are shown in Table 1. Hydrophilic fraction was further fractionated by gel permeation chromatography-high performance liquid chromatography (GPC-HPLC, Bio-Rad Superdex Peptide HR10/30 column, 30% CH3CN/0.1% trifluoroacetic acid (TFA), 0.3 mL/min), and di- or tri-peptide fractions were collected at an elution time ranging from 45 to 75 min (Figure 1; Fraction A, Frc-A, 45-55 min; Fraction B, Frc-B, 55-65 min; Fraction C, Frc-C, 65-75 min). Then, 2 peptide fractions (0% CH3CN/0.1% TFA and 20% CH3CN/0.1% TFA fractions) were obtained by octadecylsilyl (ODS) open-column chromatography (20 × 60 mm) from Frc-C peptides. Finally, 0% CH3CN/0.1% TFA fraction peptides were separated by reversed-phase HPLC (COSMOSIL 5C18-AR-II column; Nakalai tesque, Kyoto, Japan), and 7 di-peptides (Figure 2; Ala-Leu, Ala-Tyr, Ala-Val, Leu-Val, Lys-Ala, Ser-Tyr, and Val-Lys) were identified. These di-peptides, except Leu-Val, could be synthesized using a Fmoc solid-phase synthesis method according to the manufacturer's instructions (Kokusan Chemicals, Osaka, Japan). The sequence of the di-peptides was confirmed on a PPSQ-21 amino acid sequencer (Shimadzu Co., Kyoto, Japan).
Table 1

Amino acid composition of soy peptides

Amino acid

Soy crude peptide-LD3

Hydrophobic peptide

Hydrophilic peptide












































































Tryptophan content could not be determined under the present experimental condition.

Figure 1
Figure 1

Calibration of Superdex Peptide HR10/30 gel permeation chromatography column by using standard peptides and the elution profile of the hydrophilic fraction of soy crude peptide-LD3.

Figure 2
Figure 2

Reversed phase-high performance liquid chromatography elution profile of 0% CH 3 CN/0.1%TFA fraction peptides on COSMOSIL 5C 18 -AR-II column and identification of di-peptides.

Cell culture

HepG2 cells were maintained in Dulbeccos's modified Eagle's medium (DMEM) containing 100 units/mL penicillin and 100 μg/mL streptomycin and supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2. At approximately 70-80% confluence, the medium was preincubated with 1% bovine serum albumin (BSA)-DMEM with 0.5 mM oleic acid for 24 h. The fatty acid-BSA complex was prepared as described by Van Harken et al [28]. The cellular protein concentration was determined using the method of Lowry et al [29] or the bicinchoninic acid method [30]. To measure cytotoxicity, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) activity was determined using a MTT Cell Growth Kit (CHEMICON International, Inc., Temecula, CA, U.S.A.); and after 24-h incubation with 1-10 mg/mL of the soy peptides, the MTT activity of HepG2 cells was unaffected in all experiments (data not shown).

Measurement of cellular TG synthesis

To evaluate the effects of the peptides on cellular TG synthesis, HepG2 cells were preincubated with 1% BSA-DMEM with 0.5 mM oleic acid for 24 h and then incubated with experimental medium (Control: 1% BSA-DMEM; Peptides: 1% BSA-DMEM with 1-10 mg/mL peptides) containing 18.5 KBq [1-14C] acetate (American Radiolabeled Chemicals, St. Louis, MO, USA) for 24 h. After incubation, cells were washed once and collected in 2 mL of phosphate-buffered saline using a rubber policeman. Cells were thawed and homogenized with a sonicator (Sonifier 250TM; Branson Ultrasonic Co., CT, USA) before analysis. Total lipids in the cells were extracted and purified by the method of Bligh and Dyer [31], then radioactivities were measured by a liquid scintillation counter (Wallic System 1410; Pharmacia, Uppsala, Sweden). The lipids were fractionated using thin-layer chromatography (TLC) in a solvent mixture of petroleum ether:diethyl ether:acetate (82:18:1, v/v/v). After separation with TLC, the radioactivities of the lipid fractions were quantitated with a bio-imaging analyzer (BAS1000; Fuji Photo Film, Kanagawa, Japan). Activity of TG synthesis was calculated as "cpm per mg protein" and represented as "% of control" in figures.

Measurement of apolipoprotein B100 secretion

To evaluate the effects of the di-peptides on apolipoprotein B100 (apoB100) secretion, HepG2 cells were preincubated with 1% BSA-DMEM with 0.5 mM oleic acid for 24 h and further incubated with experimental medium (Control: 1% BSA-DMEM; Peptides: 1% BSA-DMEM with 5 mg/mL di-peptides) for 24 h. At the end of the experiment, the media were harvested for the measurement of apoB100 levels, and the cells were used for determination of cellular protein levels. ApoB100 levels in the culture media were quantitated using the ApoB Microwell ELISA Assay Kit (AlerCHEK).

Animals and diets

All aspects of the animal experiment were conducted according to the guidelines provided by the ethical committee of experimental animal care at Saga University. Male OLETF rats aged 4 weeks were provided by the Tokushima Research Institute (Otsuka Pharmaceutical, Tokushima, Japan). The rats were housed individually in metal cages in a temperature-controlled room (24°C) under a 12-h light/dark cycle. In the first animal experiment, the rats were assigned to 1 of 2 groups (6 rats each), and were fed 1 of 2 diets: (i) a semi-synthetic diet containing (in weight %) casein, 20; corn oil, 7; cornstarch, 15; vitamin mixture (AIN-76™), 1; mineral mixture (AIN-76™), 3.5; DL-methionine, 0.3; choline bitartrate, 0.2; cellulose, 5; and sucrose, 48 (control diet, Con); (ii) a semi-synthetic diet containing (in weight %) SCP-LD3, 19.1; corn oil, 7; cornstarch, 15; vitamin mixture, 1; mineral mixture, 3.5; DL-methionine, 0.3; choline bitartrate, 0.2; cellulose, 5; and sucrose, 48.9 (soy peptide diet, SoyPep) for 2 weeks. In the second animal experiment, the rats were assigned to 1 of 3 groups (6 rats each) and were fed 1 of the following 3 diets: (i) control diet; (ii) soy peptide diet; and (iii) a semi-synthetic diet containing (in weight %) hydrophilic peptide, 11.85; casein, 10; corn oil, 7; cornstarch, 15; vitamin mixture, 1; mineral mixture, 3.5; DL-methionine, 0.3; choline bitartrate, 0.2; cellulose, 5; and sucrose, 46.15 (hydrophilic peptide diet) for 4 weeks. Basal semisynthetic diets were prepared according to recommendations of the AIN-76 [32].

Measurement of TG levels in serum and liver

All rats were killed by aortic exsanguination under diethyl ether anesthesia, and the liver was excised for analysis. The serum was separated from the blood, and serum TG levels were measured using a commercial enzyme assay kit (Wako Pure Chemicals, Tokyo, Japan). Hepatic lipid was extracted according to the method of Folch et al. [33], and the TG concentration was measured by the method of Fletcher [34].

Preparation of hepatic subcellular fractions

A piece of liver from each rat was homogenized in 6 volumes of a 0.25 M sucrose solution containing 1 mM ethylenediaminetetraacetic acid in a 10 mM Tris-HCl buffer (pH 7.4). After the nuclear fraction was precipitated, the supernatant was centrifuged at 10,000 × g for 10 min at 4°C to obtain mitochondrial fractions. The resulting supernatant was recentrifuged at 125,000 × g for 60 min to precipitate microsomes, and the remaining supernatant constituted the cytosol fraction. The protein concentration was determined according to the method of Lowry et al. [29], with BSA used as the standard.

Assays of hepatic enzyme activity

The enzyme activity of phosphatidate phosphohydrolase (PAP) [35], fatty acid synthase (FAS) [36], malic enzyme [37], glucose 6-phosphate dehydrogenase (G6PDH) [38], and carnitine palmitoyltransferase (CPT) [39] was determined as described elsewhere.

Statistical analysis

All values are expressed as mean ± SE. Data were analyzed by student t-test to assess differences between 2 groups. To assess differences between 3 groups, data were analyzed by 1-way ANOVA, and all differences were analyzed by the Tukey-Kramer post-hoc test (KaleidaGraph, Synergy Software, Reading, PA). Differences were considered significant at P < 0.05.

Results and discussion

The liver is the pivotal organ concerned with lipid metabolism. Nonalcoholic fatty liver disease (NAFLD) is often associated with features of the metabolic syndrome and is emerging as the most common liver disease worldwide [4042]. NAFLD is the preferred term used to describe the spectrum of liver damage, ranging from hepatic steatosis to steatohepatitis, liver fibrosis, and cirrhosis. Most liver-related morbidity and mortality events are associated with the development of cirrhosis. Cirrhosis is most likely to occur in individuals who have progressed from hepatic steatosis to steatohepatitis. Although the processes through which steatohepatitis evolves from hepatic steatosis are not fully understood, it is necessary to develop effective therapies for the treatment of NAFLD, and the discovery of nutrients that reduce the risk of NAFLD would be useful. We previously discovered novel hypolipidemic dietary components such as functional lipids and phytochemicals by in vitro (HepG2 cells) and in vivo (OLETF rats) evaluation [4347].

Effects of SCP-LD3 on lipid metabolism in HepG2 cells and OLETF rats

In the first part of the current study, we evaluated the effects of SCP-LD3 treatment on TG synthesis in HepG2 cells. As shown in Figure 3A, incorporation of [1-14C] acetate into the cellular TG fraction was significantly lowered by SCP-LD3 treatment. These results suggest that SCP-LD3 has the ability to normalize lipid abnormalities possibly through the suppression of TG synthesis in hepatocytes. Next, we evaluated the effects of SCP-LD3 on lipid metabolism in OLETF rats. As shown in Figure 3B, 2-week feeding of SCP-LD3 alleviated the obesity-induced TG accumulation in the liver of OLETF rats without significant alterations in other growth parameters such as food intake (control, 29.2 ± 0.6 g/day; SCP-LD3, 29.0 ± 0.6 g/day) and final body weight (control, 539 ± 12 g; SCP-LD3, 531 ± 17 g). Additionally, serum TG level was also lowered by SCP-LD3 feeding in OLETF rats (control, 251 ± 40 mg/dL; SCP-LD3, 143 ± 21 mg/dL, P < 0.05). These results indicate that SCP-LD3 has TG-lowering properties, which led us to perform further isolation and identification of hypolipidemic peptide sequences from this peptide during subsequent experiments.
Figure 3
Figure 3

Effects of soy crude peptide (SCP)-LD3 on triglyceride (TG) synthesis in HepG2 cells (1 mg/mL) and hepatic TG level in Otsuka Long-Evans Tokushima fatty (OLETF) rats. Values are expressed as mean ± standard error of 5 samples in vitro and 6 rats in vivo. Asterisks indicate a significant difference at P < 0.05.

Effects of hydrophobic and hydrophilic fractions on lipid metabolism in HepG2 cells and OLETF rats

Several reports have suggested that the amino acid composition of dietary proteins and peptides influences their bioactivities [11]. It has been reported that high amounts of histidine and hydrophobic amino acids contribute to antioxidant potency and that hydrophobic peptides can bind bile acids, thereby enhancing fecal steroid excretion [20, 4850]. In the second part of the current study, we evaluated the effects of hydrophobic and hydrophilic fractions, separated from SCP-LD3 with hydrophobic synthetic absorbent, on TG synthesis in HepG2 cells. As shown in Table 1, the hydrophobic fraction was found to contain a higher amount of phenylalanine, whereas the hydrophilic fraction contained a higher amount of glutamine compared to SCP-LD3. In HepG2 cells, incorporation of [1-14C] acetate into the cellular TG fraction was significantly and dose-dependently lowered by hydrophilic fraction treatment (Figure 4A). In the next in vivo experiment, we evaluated the effects of the hydrophilic fraction on lipid metabolism in OLETF rats. Though there was no significant alteration in growth parameters (Table 2), feeding of the hydrophilic fraction alleviated hepatomegaly and hepatic TG accumulation in OLETF rats (Table 2, Figure 4B). Moreover, despite the fact that the degree of supplementation with the hydrophilic fraction in the diet (substituted for 10% casein) was half that of SCP-LD3 (substituted for 20% casein), the TG-lowering effects of these 2 diets were almost the same. In agreement with the in vitro study, the TG-lowering effects were attributable to the suppression of fatty acid synthesis (represented by lowered activities of FAS, G6PDH, and malic enzyme) and TG synthesis (represented by lowered PAP activity) in the liver of OLETF rats.
Figure 4
Figure 4

Effects of soy peptides on triglyceride (TG) synthesis in HepG2 cells (1 or 10 mg/mL) and hepatic TG level in Otsuka Long-Evans Tokushima fatty (OLETF) rats. Values are expressed as mean ± standard error of 5 samples in vitro and 6 rats in vivo. Different letters indicate a significant difference at P < 0.05.

Table 2

Effects of dietary soy peptides on growth parameters and activities of hepatic triglyceride metabolism-related enzymes in Otsuka Long-Evans Tokushima fatty rats



Soy crude peptide-LD3

Hydrophilic peptide

Initial body weight (g)

465 ± 11

467 ± 7

463 ± 9

Final body weight (g)

558 ± 10

554 ± 7

550 ± 8

Food intake (g)

743 ± 3

740 ± 8

738 ± 10

Liver weight (g/100 g b.w.)

3.45 ± 0.05a

3.14 ± 0.05b

3.16 ± 0.06b

Hepatic enzyme activity (nmol/min/mg protein)



17.0 ± 0.7a

13.6 ± 0.8b

13.3 ± 1.3b


127 ± 7a

82.7 ± 4.2b

77.0 ± 5.2b

   Malic enzyme

115 ± 5a

79.0 ± 3.7b

86.5 ± 2.1b


17.3 ± 1.1a

14.5 ± 0.5b

14.8 ± 0.5b


5.16 ± 0.31

5.66 ± 0.25

5.42 ± 0.10

FAS, fatty acid synthase; G6PDH, glucose 6-phosphate dehydrogenase; PAP, phosphatidate phosphohydrolase; CPT, carnitine palmitoyltransferase.

Values are expressed as mean ± standard error of 6 rats.

Different superscript letters indicate a significant difference at P < 0.05.

Effects of peptides fractionated by GPC-HPLC on TG synthesis and apoB100 secretion in HepG2 cells

It has been shown that short peptides, mostly di-, tri-, and tetra-peptides, are absorbed more rapidly than free amino acids and that short peptides are absorbed intact by specific peptide transporters such as PepT1 and PepT2 into the blood circulation and transported to target organs [5154]. Intact absorption of the hypotensive dipeptide Val-Tyr, for example, was confirmed in rats and humans [5557]. In the third part of the current study, we evaluated the effects of di- or tri-peptide fractions, separated from the hydrophilic fraction with GPC-HPLC (Figure 1), on TG synthesis in HepG2 cells. As shown in Figure 5A, all 3 fractions lowered the incorporation of [1-14C] acetate into the cellular TG fraction significantly and dose-dependently. It is known that blood apoB100 level is positively correlated with the incidence of coronary heart disease and that enhanced secretion of apoB100 by the liver is a biomarker of hepatic lipid abnormality [5860]. Therefore, dietary components that control the rate of apoB100 secretion by the liver are of great interest [6163]. In the current study, we evaluated the effects of Frc-B and Frc-C treatment on apoB100 secretion from HepG2 cells. Our results show that Frc-C treatment, but not Frc-B treatment, induced a 33% reduction of apoB100 secretion compared with the control medium (Figure 5B).
Figure 5
Figure 5

Effects of fractionated soy peptides (1 or 5 mg/mL) on triglyceride (TG) synthesis and apolipoprotein (Apo) B100 secretion in HepG2 cells. Values are expressed as mean ± standard error of 5 samples. Different letters indicate a significant difference at P < 0.05.

Effects of peptides fractionated by ODS column chromatography on TG synthesis and apoB100 secretion in HepG2 cells

In the fourth part of the current study, we evaluated the effects of peptide fractions, separated from the Frc-C peptide with ODS column chromatography, on TG synthesis and apoB100 secretion in HepG2 cells. Our results show that 0% CH3CHCN/0.1% TFA fraction lowered both TG synthesis and apoB100 secretion, but 20% CH3CHCN/0.1% TFA fraction lowered only TG synthesis, in HepG2 cells (Figure 6A, B).
Figure 6
Figure 6

Effects of fractionated soy peptides (5 mg/mL) on triglyceride (TG) synthesis and apolipoprotein (Apo) B100 secretion in HepG2 cells. Values are expressed as mean ± standard error of 5 samples. Different letters indicate a significant difference at P < 0.05.

Effects of synthesized di-peptides on TG synthesis and apoB100 secretion in HepG2 cells

Finally, 0% CH3CN/0.1% TFA fraction peptides were separated by reverse-phase HPLC, where by 7 di-peptides were identified (Figure 2). Six di-peptides (Ala-Leu, Ala-Tyr, Ala-Val, Lys-Ala, Ser-Tyr, and Val-Lys), but not Leu-Val, could be synthesized, and we evaluated the effects of these di-peptides on TG synthesis and apoB100 secretion in HepG2 cells. We found that 3 di-peptides, Ser-Tyr, Val-Lys, and Lys-Ala, reduced TG synthesis (Figure 7A), and Ser-Tyr additionally reduced apoB secretion (Figure 7B) in HepG2 cells. The presence of 3 di-peptide sequences in the amino acid sequence of soybean components such as glycinin, the BCG alpha subunit (BCG-α), the BCG alpha prime subunit (BCG-α'), the BCG beta subunit (BCG-β), the trypsin inhibitor, and lipoxygenase is shown in Table 3. The presence of the Ser-Tyr sequence is recognized in the amino acid sequence of glycinin, BCG-α, BCG-α', BCG-β, and lipoxygenase. The presence of the Val-Lys sequence is recognized in the amino acid sequence of glycinin, the trypsin inhibitor, and lipoxygenase. The presence of the Lys-Ala sequence is recognized in the amino acid sequence of glycinin, BCG-α, BCG-α', BCG-β, the trypsin inhibitor, and lipoxygenase. Because of the suppressive effects on both lipogenesis and apoB100 secretion in HepG2 cells and the frequent presence in the amino acid sequence of major soybean components, we speculate that Ser-Tyr is the most effective TG-lowering constituent of soy protein isolate-derived peptides.
Figure 7
Figure 7

Effects of synthesized di-peptides (5 mg/mL) on triglyceride (TG) synthesis and apolipoprotein (Apo) B100 secretion in HepG2 cells. Values are expressed as mean ± standard error of 5 samples. Different letters indicate a significant difference at P < 0.05.

Table 3

Amino acid sequence of major components in soy protein

Glycinin [Glycine max (soybean)]/CAA37044


Beta-conglycinin alpha subunit [Glycine max (soybean)]/BAE46788


Beta-conglycinin alpha prime subunit [Glycine max (soybean)]/BAB64303


Beta-conglycinin beta subunit [Glycine max (soybean)]/BAB64306


Trypsin inhibitor [Glycine max (soybean)]/AAF87095


Lipoxygenase [Glycine max (soybean)]/CAA39604


Definition [source]/Accession number. Lys-Ala, KA; Ser-Tyr, SY; Val-Lys, VK.


We appear to have isolated novel active peptides with TG-lowering effects from soy protein, including Lys-Ala, Val-Lys, and Ser-Tyr, using in vitro and in vivo screening systems (Figure 8). The physiological properties of these isolates would be, at least in part, attributable to suppressed lipogenesis in the liver. Further studies are necessary to evaluate the effects of these 3 di-peptides on lipid abnormalities in vivo and to determine the lowest effective concentration.
Figure 8
Figure 8

Screening chart of lipid-lowering di-peptides from soy protein isolate.


List of abbreviations


apolipoprotein B100




bovine serum albumin


carnitine palmitoyltransferase


dulbecco's modified eagle medium


fatty acid synthase


glucose 6-phosphate dehydrogenase


gel permeation chromatography


high performance liquid chromatography


3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide


nonalcoholic fatty liver disease




Otsuka Long-Evans Tokushima fatty


phosphatidate phosphohydrolase


soy crude peptide


trifluoroacetic acid




thin-layer chromatography.



This work was supported by a research grant from Fuji Foundation for Protein Research.

Authors’ Affiliations

Department of Applied Biochemistry and Food Science, Saga University, 840-8502 Saga, Japan
Department of Bioscience and Biotechnology, Graduate School of Kyushu University, 812-8581 Fukuoka, Japan
Fuji Oil Co, 598-8540 Osaka, Japan
Graduate School of Agricultural Science, Tohoku University, 981-8555 Miyagi, Japan


  1. Kissebah AH, Krakower GR: Regional adiposity and morbidity. Physiol Rev. 1994, 74: 761-811.PubMedGoogle Scholar
  2. Formiguera X, Canton A: Obesity: epidemiology and clinical aspects. Best Pract Res Clin Gastroenterol. 2004, 18: 1125-1146.View ArticlePubMedGoogle Scholar
  3. Nagao K, Yanagita T: Bioactive lipids in metabolic syndrome. Prog Lipid Res. 2008, 47: 127-146. 10.1016/j.plipres.2007.12.002View ArticlePubMedGoogle Scholar
  4. Tovar AR, Torres N: The role of dietary protein on lipotoxicity. Biochim Biophys Acta. 2010, 1801: 367-371.View ArticlePubMedGoogle Scholar
  5. Appel LJ: The effects of protein intake on blood pressure and cardiovascular disease. Curr Opin Lipidol. 2003, 14: 55-59. 10.1097/00041433-200302000-00010View ArticlePubMedGoogle Scholar
  6. Sirtori CR, Galli C, Anderson JW, Arnoldi A: Nutritional and nutraceutical approaches to dyslipidemia and atherosclerosis prevention: Focus on dietary proteins. Atherosclerosis. 2009, 203: 8-17. 10.1016/j.atherosclerosis.2008.06.019View ArticlePubMedGoogle Scholar
  7. Elliott P, Stamler J, Dyer AR, Appel L, Dennis B, Kesteloot H, Ueshima H, Okayama A, Chan Q, Garside DB, Zhou B: Association between protein intake and blood pressure: the INTERMAP Study. Arch Intern Med. 2006, 166: 79-87. 10.1001/archinte.166.1.79View ArticlePubMedGoogle Scholar
  8. Sakono M, Yoshida K, Yahiro M: Combined effects of dietary protein and fat on lipid metabolism in rats. J Nutr Sci Vitaminol (Tokyo). 1993, 39: 335-343.View ArticleGoogle Scholar
  9. Koba K, Liu JW, Bobik E, Mills DE, Sugano M, Huang YS: Effect of phytate in soy protein on the serum and liver cholesterol levels and liver fatty acid profile in rats. Biosci Biotechnol Biochem. 2003, 67: 15-22. 10.1271/bbb.67.15View ArticlePubMedGoogle Scholar
  10. Nagata Y, Ishiwaki N, Sugano M: Studies on the mechanism of antihypercholesterolemic action of soy protein and soy protein-type amino acid mixtures in relation to the casein counterparts in rats. J Nutr. 1982, 112: 1614-1625.PubMedGoogle Scholar
  11. Erdmann K, Cheung BW, Schröder H: The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease. J Nutr Biochem. 2008, 19: 643-654. 10.1016/j.jnutbio.2007.11.010View ArticlePubMedGoogle Scholar
  12. Shahidi F, Zhong Y: Bioactive peptides. J AOAC Int. 2008, 91: 914-931.PubMedGoogle Scholar
  13. Motoi H, Kodama T: Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides from wheat gliadin hydrolysate. Nahrung. 2003, 47: 354-358. 10.1002/food.200390081View ArticlePubMedGoogle Scholar
  14. Matsufuji H, Matsui T, Ohshige S, Kawasaki T, Osajima K, Osajima Y: Antihypertensive effects of angiotensin fragments in SHR. Biosci Biotechnol Biochem. 1995, 59: 1398-3401. 10.1271/bbb.59.1398View ArticlePubMedGoogle Scholar
  15. Kawasaki T, Seki E, Osajima K, Yoshida M, Asada K, Matsui T, Osajima Y: Antihypertensive effect of valyl-tyrosine, a short chain peptide derived from sardine muscle hydrolyzate, on mild hypertensive subjects. J Hum Hypertens. 2000, 14: 519-523. 10.1038/sj.jhh.1001065View ArticlePubMedGoogle Scholar
  16. Nagaoka S, Futamura Y, Miwa K, Awano T, Yamauchi K, Kanamaru Y, Tadashi K, Kuwata T: Identification of novel hypocholesterolemic peptides derived from bovine milk beta-lactoglobulin. Biochem Biophys Res Commun. 2001, 281: 11-17. 10.1006/bbrc.2001.4298View ArticlePubMedGoogle Scholar
  17. Morikawa K, Kondo I, Kanamaru Y, Nagaoka S: A novel regulatory pathway for cholesterol degradation via lactostatin. Biochem Biophys Res Commun. 2007, 352: 697-702. 10.1016/j.bbrc.2006.11.090View ArticlePubMedGoogle Scholar
  18. Yoshikawa M, Fujita H, Matoba N, Takenaka Y, Yamamoto T, Yamauchi R, Tsuruki H, Takahata K: Bioactive peptides derived from food proteins preventing lifestyle-related diseases. Biofactors. 2000, 12: 143-146. 10.1002/biof.5520120122View ArticlePubMedGoogle Scholar
  19. Kodera T, Nio N: Identification of an Angiotensin I-converting Enzyme Inhibitory Peptides from Protein Hydrolysates by a Soybean Protease and the Antihypertensive Effects of Hydrolysates in 4 Spontaneously Hypertensive Model Rats. J Food Sci. 2006, 71: C164-C173. 10.1111/j.1365-2621.2006.tb15612.x. 10.1111/j.1365-2621.2006.tb15612.xView ArticleGoogle Scholar
  20. Chen HM, Muramoto K, Yamauchi F, Fujimoto K, Nokihara K: Antioxidative Properties of Histidine-Containing Peptides Designed from Peptide Fragments Found in the Digests of a Soybean Protein. J Agric Food Chem. 1998, 46: 49-53. 10.1021/jf970649wView ArticlePubMedGoogle Scholar
  21. Nishi T, Hara H, Asano K, Tomita F: The soybean beta-conglycinin beta 51-63 fragment suppresses appetite by stimulating cholecystokinin release in rats. J Nutr. 2003, 133: 2537-2542.PubMedGoogle Scholar
  22. Kagawa K, Matsutaka H, Fukuhama C, Watanabe Y, Fujino H: Globin digest, acidic protease hydrolysate, inhibits dietary hypertriglyceridemia and Val-Val-Tyr-Pro, one of its constituents, possesses most superior effect. Life Sci. 1996, 58: 1745-1755. 10.1016/0024-3205(96)00156-7View ArticlePubMedGoogle Scholar
  23. Dixon JL, Ginsberg HN: Regulation of hepatic secretion of apolipoprotein B-containing lipoproteins: information obtained from cultured liver cells. J Lipid Res. 1993, 34: 167-179.PubMedGoogle Scholar
  24. Yagi K, Kim S, Wanibuchi H, Yamashita T, Yamamura Y, Iwao H: Characteristics of diabetes, blood pressure, and cardiac and renal complications in Otsuka Long-Evans Tokushima Fatty rats. Hypertension. 1997, 29: 728-735.View ArticlePubMedGoogle Scholar
  25. Takiguchi S, Taketa Y, Funakoshi A, Miyasaka K, Kataoka K, Fujimura Y, Goto T, Kono A: Disrupted cholecystokinin type-A receptor (CCKAR) gene in OLETF rats. Gene. 1997, 197: 169-175. 10.1016/S0378-1119(97)00259-XView ArticlePubMedGoogle Scholar
  26. Hida K, Wada J, Zhang H, Hiragushi K, Tsuchiyama Y, Shikata K, Makino H: Identification of genes specifically expressed in the accumulated visceral adipose tissue of OLETF rats. J Lipid Res. 2000, 41: 1615-1622.PubMedGoogle Scholar
  27. Tamaru S, Kurayama T, Sakono M, Fukuda N, Nakamori T, Furuta H, Tanaka K, Sugano M: Effects of dietary soybean peptides on hepatic production of ketone bodies and secretion of triglyceride by perfused rat liver. Biosci Biotechnol Biochem. 2007, 71: 2451-2457. 10.1271/bbb.70221View ArticlePubMedGoogle Scholar
  28. Van Harken DR, Dixon CW, Heimberg M: Hepatic lipid metabolism in experimental diabetes V. The effect of concentration of oleate on metabolism of triglycerides and on ketogenesis. J Biol Chem. 1969, 244: 2278-2285.PubMedGoogle Scholar
  29. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem. 1951, 193: 265-275.PubMedGoogle Scholar
  30. Sorensen K, Brodbeck U: Assessment of coating-efficiency in ELISA plates by direct protein determination. J Immunol Methods. 1986, 95: 291-293. 10.1016/0022-1759(86)90419-9View ArticlePubMedGoogle Scholar
  31. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959, 37: 911-917. 10.1139/o59-099View ArticlePubMedGoogle Scholar
  32. , : Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J Nutr. 1977, 107: 1340-1348.Google Scholar
  33. Folch J, Lees M, Sloane-Stanley GH: A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957, 226: 497-509.PubMedGoogle Scholar
  34. Fletcher MJ: A colorimetric method for estimating serum triglycerides. Clin Chim Acta. 1968, 22: 393-397. 10.1016/0009-8981(68)90041-7View ArticlePubMedGoogle Scholar
  35. Walton PA, Possmayer F: Mg2+-dependent phosphatidate phosphohydrolase of rat lung: development of an assay employing a defined chemical substrate which reflects the phosphohydrolase activity measured using membrane-bound substrate. Anal Biochem. 1985, 151: 479-486. 10.1016/0003-2697(85)90208-8View ArticlePubMedGoogle Scholar
  36. Kelley DS, Nelson GJ, Hunt JE: Effect of prior nutritional status on the activity of lipogenic enzymes in primary monolayer cultures of rat hepatocytes. Biochem J. 1986, 235: 87-90.PubMed CentralView ArticlePubMedGoogle Scholar
  37. Ochoa S: Malic enzyme. Methods in Enzymology. Edited by: Colowick SP, Kaplan NO. 1955, 1: 739-753. Academic Press: New York, NYGoogle Scholar
  38. Kelley DS, Kletzien RF: Ethanol modulation of the hormonal and nutritional regulation of glucose 6-phosphate dehydrogenase activity in primary cultures of rat hepatocytes. Biochem J. 1984, 217: 543-549.PubMed CentralView ArticlePubMedGoogle Scholar
  39. Markwell MA, McGroarty EJ, Bieber LL, Tolbert NE: The subcellular distribution of carnitine acyltransferases in mammalian liver and kidney. J Biol Chem. 1973, 248: 3426-3432.PubMedGoogle Scholar
  40. Fan JG, Li F, Cai XB, Peng YD, Gao Y: Effects of nonalcoholic fatty iver disease on the development of metabolic disorders. J Gastroenterol Hepatol. 2007, 22: 1086-1091. 10.1111/j.1440-1746.2006.04781.xView ArticlePubMedGoogle Scholar
  41. Harrison SA, Diehl AM: Fat and the liver--a molecular overview. Semin Gastrointest Dis. 2002, 13: 3-16.PubMedGoogle Scholar
  42. Youssef W, McCullough AJ: Diabetes mellitus, obesity, and hepatic steatosis. Semin Gastrointest Dis. 2002, 13: 17-30.PubMedGoogle Scholar
  43. Yotsumoto H, Hara E, Naka S, Adlof RO, Emken EA, Yanagita T: 10trans, 12cis-Linoleic acid reduces apolipoprotein B secretion in HepG2 cells. Food Res Int. 1998, 31: 403-409. 10.1016/S0963-9969(98)00103-3. 10.1016/S0963-9969(98)00103-3View ArticleGoogle Scholar
  44. Wang YM, Nagao K, Inoue N, Ujino Y, Shimada Y, Nagao T, Iwata T, Kamegai T, Yamauchi-Sato Y, Yanagita T: Isomer-specific anti-obese and hypolipidemic properties of conjugated linoleic acid in obese OLETF rats. Biosci Biotechnol Biochem. 2006, 70: 355-362. 10.1271/bbb.70.355View ArticlePubMedGoogle Scholar
  45. Arao K, Yotsumoto H, Han SY, Nagao K, Yanagita T: The 9cis, 11trans, 13cis isomer of conjugated linolenic acid reduces apolipoprotein B100 secretion and triacylglycerol synthesis in HepG2 cells. Biosci Biotechnol Biochem. 2004, 68: 2643-2645. 10.1271/bbb.68.2643View ArticlePubMedGoogle Scholar
  46. Arao K, Wang YM, Inoue N, Hirata J, Cha JY, Nagao K, Yanagita T: Dietary effect of pomegranate seed oil rich in 9cis, 11trans, 13cis conjugated linolenic acid on lipid metabolism in obese, hyperlipidemic OLETF Rats. Lipids Health Dis. 2004, 3: 24- 10.1186/1476-511X-3-24PubMed CentralView ArticlePubMedGoogle Scholar
  47. Nagao K, Yamano N, Shirouchi B, Inoue N, Murakami S, Sasaki T, Yanagita T: Effects of citrus auraptene (7-geranyloxycoumarin) on hepatic lipid metabolism in vitro and in vivo. J Agric Food Chem. 2010, 58: 9028-9032.View ArticlePubMedGoogle Scholar
  48. Pena-Ramos EA, Xiong YL, Arteaga GE: Fractionation and characterisation for antioxidant activity of hydrolysed whey protein. J Sci Food Agric. 2004, 84: 1908-1918. 10.1002/jsfa.1886. 10.1002/jsfa.1886View ArticleGoogle Scholar
  49. Iwami K, Sakakibara H, Ibuki F: Involvement of post-digestion 'hydrophobic' peptides in plasma cholesterol-low ering effect of dietary plant proteins. Agric Biol Chem. 1986, 50: 1217-1222.View ArticleGoogle Scholar
  50. Making S, Nakashima H, Minami K, Moriyama R, Takao S: Bile acid-binding protein from soybean seed: isolation, partial characterization and insulin-stimulating activity. Agric Biol Chem. 1988, 52: 803-809.View ArticleGoogle Scholar
  51. Webb KE: Intestinal absorption of protein hydrolysis products. J Anim Sci. 1990, 68: 3011-3022.PubMedGoogle Scholar
  52. Fei YJ, Kanai Y, Nussberger S, Ganapathy V, Leibach FH, Romero MF, Singh SK, Boron WF, Hediger MA: Expression cloning of a mammalian proton-coupled oligopeptide transporter. Nature. 1994, 368: 563-566. 10.1038/368563a0View ArticlePubMedGoogle Scholar
  53. Liu W, Liang R, Ramamoorthy S, Fei YJ, Ganapathy ME, Hediger MA, Ganapathy V, Leibach FH: Molecular cloning of PEPT 2, a new member of the H+/peptide cotransporter family, from human kidney. Biochim Biophys Acta. 1995, 1235: 461-466. 10.1016/0005-2736(95)80036-FView ArticlePubMedGoogle Scholar
  54. Daniel H, Spanier B, Kottra G, Weitz D: From bacteria to man: archaic proton-dependent peptide transporters at work. Physiology (Bethesda). 2006, 21: 93-102.View ArticleGoogle Scholar
  55. Matsui T, Tamaya K, Seki E, Osajima K, Matsumoto K, Kawasaki T: Val-Tyr as a natural antihypertensive dipeptide can be absorbed into the human circulatory blood system. Clin Exp Pharmacol Physiol. 2002, 29: 204-208. 10.1046/j.1440-1681.2002.03628.xView ArticlePubMedGoogle Scholar
  56. Matsui T, Tamaya K, Seki E, Osajima K, Matsumo K, Kawasaki T: Absorption of Val-Tyr with in vitro angiotensin I-converting enzyme inhibitory activity into the circulating blood system of mild hypertensive subjects. Biol Pharm Bull. 2002, 25: 1228-1230. 10.1248/bpb.25.1228View ArticlePubMedGoogle Scholar
  57. Matsui T, Imamura M, Oka H, Osajima K, Kimoto K, Kawasaki T, Matsumoto K: Tissue distribution of antihypertensive dipeptide, Val-Tyr, after its single oral administration to spontaneously hypertensive rats. J Pept Sci. 2004, 10: 535-545. 10.1002/psc.568View ArticlePubMedGoogle Scholar
  58. Fong DG, Nehra V, Lindor KD, Buchman AL: Metabolic and nutritional considerations in nonalcoholic fatty liver. Hepatology. 2000, 32: 3-10.View ArticlePubMedGoogle Scholar
  59. Tsai J, Zhang R, Qiu W, Su Q, Naples M, Adeli K: Inflammatory NG-kappaB activation promotes hepatic apolipoprotein B100 secretion: evidence for a link between hepatic inflammation and lipoprotein production. Am J Physiol Gstrointest Liver Physiol. 2009, 296: G1287-1298. 10.1152/ajpgi.90540.2008. 10.1152/ajpgi.90540.2008View ArticleGoogle Scholar
  60. Cave M, Deaciuc I, Mendez C, Song Z, Joshi-Barve S, Barve S, McClain C: Nonalcoholic fatty liver disease: predisposing factors and the role of nutrition. J Nutr Biochem. 2007, 18: 184-195. 10.1016/j.jnutbio.2006.12.006View ArticlePubMedGoogle Scholar
  61. Yanagita T, Han SY, Hu Y, Nagao K, Kitajima H, Murakami S: Taurine reduces the secretion of apolipoprotein B100 and lipids in HepG2 cells. Lipids Health Dis. 2008, 7: 38- 10.1186/1476-511X-7-38PubMed CentralView ArticlePubMedGoogle Scholar
  62. Inoue N, Yamano N, Sakata K, Nagao K, Hama Y, Yanagita T: The sulfated polysaccharide porphyran reduces apolipoprotein B100 secretion and lipid synthesis in HepG2 cells. Biosci Biotechnol Biochem. 2009, 73: 447-449. 10.1271/bbb.80688View ArticlePubMedGoogle Scholar
  63. Inoue N, Yamano N, Sakata K, Arao K, Kobayashi T, Nagao T, Shimada Y, Nagao K, Yanagita T: Linoleic acid-menthyl ester reduces the secretion of apolipoprotein B100 in HepG2 cells. J Oleo Sci. 2009, 58: 171-175. 10.5650/jos.58.171View ArticlePubMedGoogle Scholar


© Inoue et al; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.