JP5177778B2 - peptide - Google Patents

Description

The present invention relates to a peptide having an antioxidant action derived from milk protein. The present invention also relates to an antioxidant containing the peptide as an active ingredient and an antioxidant food and drink or feed containing the peptide.
Furthermore, the present invention relates to a peptide having an adiponectin production promoting action derived from milk protein. Furthermore, the present invention relates to an adiponectin production promoter containing the peptide as an active ingredient and a food or drink or feed for promoting adiponectin production containing the peptide.

Peroxides and free radicals generated by the oxidation of unsaturated fatty acids not only impair the flavor and nutritional value of food and cause quality deterioration, but also in the living body due to its strong oxidizing power, intracellular proteins and genetic DNA It is said to attack cell lipids and make lipid peroxides such as highly toxic hydroperoxide to cause cell damage and tissue damage. The accumulation of harmful effects on living bodies due to such active oxygen and free radicals may be related to one of the causes of so-called lifestyle-related diseases such as aging, cancer, arteriosclerosis, and heart disease. It has become clear. In particular, lifestyle-related diseases are closely related to dietary habits, and thus the importance of disease prevention by improving dietary content has attracted attention. Therefore, from the viewpoint of preventing or suppressing oxidative stress due to food components, research on the relationship between the antioxidant properties of foods and food components and the search for components having antioxidant properties are being conducted.

Plant-derived vitamins and polyphenols have been known for antioxidant components. There are many reports on vitamins, especially vitamin C, vitamin E and β
-Antioxidant properties are recognized in carotenes. As for polyphenols, catechins and flavonoids have been shown to have strong antioxidant properties. Furthermore, a wide variety of antioxidant peptides have been separated and identified from protein protease hydrolysates, and there are reports that three types of antioxidant peptides have been separated from the enzyme degradation products of ovalbumin (for example, non-patent literature). 1). There is also a report that six kinds of antioxidant peptides were separated and identified from protease hydrolyzate of β-conglycinin of soybean protein (
For example, see Non-Patent Document 2. ).

On the other hand, many physiological actions such as opioid activity, calcium absorption promoting action, cell proliferation action, antibacterial action, angiotensin I converting enzyme inhibitory action, etc. have been clarified for enzymatic degradation products of milk proteins. A method of suppressing fish oil odor by emulsifying eicosapentaenoic acid-containing fats and oils such as fish oil with a water-soluble protein solution (for example, see Patent Document 1), or emulsifying highly unsaturated fatty acid-containing fats and oils with a partial hydrolyzate of milk. A method for obtaining highly unsaturated fatty acid-containing fats and oils having high oxidation stability (for example, see Patent Document 2) is disclosed. In addition, highly unsaturated fatty acid-containing fats and oils, cheese and water are emulsified to prepare antioxidant emulsions to prevent oxidation of highly unsaturated fatty acid-containing fats and oils. A method (for example, refer to Patent Document 3) of masking the off-flavor of odor is disclosed. However, all of these are highly unsaturated fatty acid-containing fats and oils, each of which is a water-soluble protein solution, a partially hydrolyzed milk product, or an emulsion of highly unsaturated fatty acid-containing fats and oils emulsified with cheese. This prevents the fishy odor of the highly unsaturated fatty acid-containing oil itself and the off-flavor during storage. However, there are few reports that a milk protein-derived peptide alone has antioxidant properties without being mixed with a highly unsaturated fatty acid-containing oil and emulsified. The present inventors have found that the water-soluble peptide fraction of cheese has an antioxidant action, and filed a patent application (see Patent Document 4).
). Moreover, it discovered that the peptide which has a specific amino acid sequence has an antioxidant activity (
See Patent Document 5. ). However, these peptides are particularly abundant in mold-ripened cheeses. Looking at cheese production worldwide, cheese ripened only with lactic acid bacteria is produced and consumed much more than mold-ripened cheese. Cheese ripened only with lactic acid bacteria is often used as a raw material for processed cheese. Therefore, it is desired to isolate a peptide component exhibiting a high antioxidant effect in cheese ripened only with lactic acid bacteria.

As described above, it has been clarified that accumulation of harmful effects on living bodies due to active oxygen and free radicals is related as one of the causes of so-called lifestyle-related diseases, but high blood pressure, hyperlipidemia, It is important to prevent risk factors for the development and development of lifestyle-related diseases such as diabetes. Currently, deaths due to heart disease and cerebrovascular disease tend to increase year by year in Japan. Up to now, adipose tissue has been considered only as a reservoir of energy, but in recent years, the importance of adipose tissue in considering the pathology of metabolic disorders centering on lifestyle-related diseases has attracted attention. It has become. That is, adipose tissue secretes endocrine factors such as plasminogen activator inhibitor, tumor necrosis factor (TNF-α), leptin, and adiponectin and contributes to the maintenance of homeostasis in the body. It has become clear that the breakdown of balance, such as excess or deficiency, is deeply related to the onset and progression of abnormal sugar / lipid metabolism, hypertension, and arteriosclerosis.
Adiponectin, one of endocrine factors derived from adipose tissue, is 2
It is a 30 kDa hormone consisting of 44 amino acids.
It is said to have an effect of promoting fat burning in the liver and muscles. Adiponectin has been shown to have a function of promoting the uptake of glucose and fatty acids in blood into cells. If fat accumulates in muscles or liver, sugar intake becomes worse, leading to diabetes. However, adiponectin usually seems to maintain nutritional balance in the body by breaking down excess fat and sugar, and as obesity progresses, the function of fat cells that secrete adiponectin becomes weaker, It is said that the nutritional balance will be lost.
Recently, research on food ingredients having a function of suppressing the progression of disease states as much as possible through eating habits has attracted attention rather than treatment with synthetic pharmaceuticals.
As for adiponectin, adiponectin suppresses liver fibrosis, normal hepatocyte growth promoting effect and anti-inflammatory effect (see, for example, Patent Document 6), and as an adiponectin production promoting substance derived from food and drink, fermented tea extract A composition containing as the active ingredient (see, for example, Patent Document 7), and a composition for enhancing secretory protein in blood adipose tissue, consisting of a sub-tree extract of deciduous leaves belonging to the genus Komikansoh, called Amler (for example, see Patent Document 8). Is disclosed.

JP-A-60-102168 JP-A-2-305898 Japanese Patent Laid-Open No. 7-274823 JP 2004-352958 A Japanese Patent Application No. 2005-294358 JP 2000-256208 A JP 2002-363094 A JP 2006-56836 A

Nobuaki Tsuge et al., Japanese Journal of Agricultural Chemistry, 65, p. 1635, 1991 H.M. et al., J. Agric. Food Chem., 43, p.574, 1995

The present invention not only impairs the flavor and nutritional value of food and causes deterioration of quality, but also suppresses oxidative damage of the living body due to active oxygen and free radicals that adversely affect diseases and aging in the living body. It is an object of the present invention to provide a peptide having an effective antioxidative effect. Another object of the present invention is to provide a peptide that suppresses arteriosclerosis and promotes fat burning in the liver and muscles by promoting adiponectin production in the living body.

As a result of intensive studies to solve the above problems, the present inventors have found that peptides comprising any one of the amino acid sequences represented by the following formulas (1) to (3) have an antioxidant effect and are low It has been found that there is an effect at a dose, and the present invention has been completed.
His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln Formula (1)
His-Pro-Ile-Lys formula (2)
His-Gln-Gly-Leu-Pro-Gln formula (3)
That is, the present invention relates to a peptide having an antioxidative action consisting of an amino acid sequence represented by any one of the above formulas (1) to (3).
The present invention also relates to a peptide having an antioxidative action comprising an amino acid sequence represented by any one of formulas (1) to (3) derived from milk protein.
Furthermore, this invention relates to the antioxidant which uses the 1 or more peptide represented by either of Formula (1)-(3) as an active ingredient.
Furthermore, this invention relates to the food-drinks or feed for antioxidants which mix | blended 1 or more peptides represented by either of Formula (1)-(3).
In addition, the present inventors have found that the peptide consisting of the amino acid sequence represented by the above formula (1) has an effect of promoting adiponectin production, and has an effect at a low dose, thereby completing the present invention. It came to do.
That is, the present invention relates to a peptide having an adiponectin production promoting action comprising the amino acid sequence represented by the above formula (1).
The present invention also relates to a peptide having an adiponectin production promoting action consisting of an amino acid sequence represented by formula (1) derived from milk protein.
Furthermore, this invention relates to the adiponectin production promoter which uses the peptide represented by Formula (1) as an active ingredient.
Furthermore, this invention relates to the food-drinks or feed for adiponectin production promotion which mix | blended the peptide represented by Formula (1).

The peptide having an antioxidative action consisting of the amino acid sequence represented by any one of the formulas (1) to (3) of the present invention not only deteriorates the flavor and nutritional value of food, but also causes deterioration in quality. Is effective in suppressing oxidative damage of living organisms due to active oxygen and free radicals, which adversely affect diseases and aging, etc. As an antioxidant containing this peptide as an active ingredient,
Moreover, it is useful as an antioxidant food or drink containing this peptide.
In addition, a peptide having an adiponectin production promoting action comprising the amino acid sequence represented by the formula (1) of the present invention is effective for promoting the production of adiponectin, and is used as an adiponectin production promoter containing this peptide as an active ingredient. Moreover, it is useful as a food or drink for promoting adiponectin production or feed containing this peptide.

The separation chromatogram of the water-soluble peptide fraction which uses gouda cheese as a raw material is shown (Example 1). The separation chromatogram of a synthetic peptide fraction is shown (Example 2). The antioxidant activity evaluation of a synthetic peptide fraction is shown (Example 2, Test Example 2). The separation chromatogram of the peptide fraction after trypsin digestion is shown (Example 3). The antioxidant activity evaluation of the peptide fraction after trypsin digestion is shown (Example 3, Test Example 3). The adiponectin production amount (per unit DNA amount) according to the difference in peptide concentration is shown (Example 1, Test Example 4). The amount of accumulated fat according to the difference in peptide concentration is shown (Example 1, Test Example 4).

His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln, His-Pro-Ile that can be used in the present invention
-Lys, or a peptide consisting of an amino acid sequence represented by His-Gln-Gly-Leu-Pro-Gln,
For example, it can be obtained by suspending cheese in a solvent and removing insoluble substances by degreasing and centrifugation. Furthermore, you may remove protein from the obtained fraction. In the present invention, suspending cheese in a solvent means adding a solvent to the cheese to homogenize it or crushing it in the solvent to make it easy to obtain a water-soluble peptide fraction. As the solvent, an aqueous solvent such as water or a phosphate buffer can be used. Thereafter, desalting may be performed with a dialysis membrane, an ion exchange resin, or the like, or powder may be formed by drying by freeze drying or spray drying.

Moreover, as a cheese raw material for obtaining the peptide which consists of an amino acid sequence represented by Formula (1)-(3), Parmesan cheese, Gruyère cheese, Maribo cheese, Gouda cheese, cheddar cheese, Emmental cheese, Edam cheese, Camembert cheese, Brie Natural cheeses such as cheese, manstail cheese, Pon reveck cheese, stilton cheese, Dana blue cheese, blue cheese, and process cheese made from these natural cheeses can be used, but the degree of ripening has advanced It is desirable to use lactic acid bacteria matured natural cheese.

Furthermore, after suspending cheese in a solvent, a fraction containing a peptide consisting of the amino acid sequence represented by the formulas (1) to (3) obtained by degreasing, removal of insoluble substances and removal of protein is obtained using a C18 column. Further purification is possible by the reverse phase chromatography used. When the fraction containing this peptide is passed through a C18 column under acidic conditions such as trifluoroacetic acid (TFA) or neutral conditions such as distilled water, the fraction having antioxidant activity is not adsorbed on the column. And the fraction adsorbed on the column and eluted with 80% acetonitrile.
Furthermore, after the fraction containing this peptide was dissolved in distilled water, YMC-Pack ODS-A
The peptide is fractionated by reversed-phase HPLC using a column (4.6 mm × 150 mm; YMC).
Chromatography should be performed under the conditions of solvent (A solution: 50 mM ammonium acetate aqueous solution; B solution: 80% acetonitrile), concentration gradient (12% B → 60% B, 60 min), flow rate 0.8 ml / min, detection wavelength 215 nm. Is desirable.

The peptide consisting of the amino acid sequence represented by the formulas (1) to (3) of the present invention and the fraction containing the peptide can be blended in food and drink and used for preventing quality deterioration of the food and drink. When blended in food or drink, after the cheese is ground in water, insoluble substances are removed by degreasing and centrifugation, and then the water-soluble peptide of cheese obtained by removing protein can be blended as it is. In addition, those desalted with a dialysis membrane, an ion exchange resin or the like, and further powdered by drying by freeze drying or spray drying can be blended.
Moreover, it can utilize also about the peptide obtained by synthesize | combining.

The peptide having an antioxidative action consisting of the amino acid sequence represented by the formulas (1) to (3) of the present invention and the fraction containing the peptide are orally or parenterally administered to the body to react with active oxygen or free By eliminating radicals and the like, it is possible to prevent progression of illness and aging.
In addition, the peptide having the adiponectin production promoting action comprising the amino acid sequence represented by the formula (1) of the present invention and the fraction containing the peptide are administered orally or parenterally to promote adiponectin production in the living body. Thus, arteriosclerosis can be suppressed and the burning of fat in the liver and muscles can be promoted. When administered orally or parenterally, the dosage form of the peptide of the present invention having the antioxidant action or adiponectin production promoting action includes tablets, capsules, fine granules, powders, pills, troches, sublinguals or liquids. And the like, and preparations for parenteral administration such as injections and suppositories.

A peptide having an antioxidant action consisting of an amino acid sequence represented by formulas (1) to (3) of the present invention, or a peptide having an adiponectin production promoting action consisting of an amino acid sequence represented by formula (1) of the present invention The oral dose may be appropriately determined in consideration of the purpose of treatment and prevention, symptoms, weight, age, sex, etc., but is usually represented by formulas (1) to (3) per day for an adult. 10 μg to 500 μg as a peptide having an antioxidant action consisting of an amino acid sequence or a peptide having an adiponectin production promoting action consisting of an amino acid sequence represented by the formula (1) of the present invention
When mg is administered, an effect of suppressing oxidative damage of the living body due to active oxygen or free radicals that adversely affect diseases, aging, etc., or promoting adiponectin production can be obtained. Thus, the present invention is effective at low doses.

Moreover, the peptide consisting of the amino acid sequence represented by the formulas (1) to (3) of the present invention has an antioxidant action or adiponectin production promoting action in vivo by ingesting foods and drinks or feeds containing them. In addition to exerting, it also prevents deterioration of the food or drink blended in the food or drink or feed itself due to oxidation. Examples of the food or drink for antioxidant or adiponectin production promotion according to the present invention include cheese, butter, milk beverage, soft drink, juice, yogurt, jelly, bread, ice cream, noodle, sausage, infant formula and baby food be able to.

Furthermore, the peptide having an antioxidant action consisting of the amino acid sequences represented by the formulas (1) to (3) of the present invention can be used as an antioxidant with only one type of each peptide as an active ingredient, or only one type can be formulated. Antioxidant food or drink or feed can also be used, but one or more of each peptide can be used as an active ingredient to make an antioxidant, or one or more can be blended to make an antioxidant food or drink or feed.

EXAMPLES The present invention will be described in more detail below with reference to examples and test examples, but these are merely illustrative and the present invention is not limited by these.

(Preparation of Gouda cheese peptide)
Add 160 ml of distilled water to 40 g of Gouda cheese and add 15 to Waring Blender (Nippon Seiki Seisakusho)
After grinding for 30 minutes, it was further crushed for 30 seconds with a Polytron homogenizer (Kinematica).
The milk fat generated during crushing is removed, and the resulting cheese slurry is centrifuged (6,000 × g, 20
Min, 4 ° C) was used to remove insoluble matter, and the supernatant was filtered through filter paper (No. 113; Whatman). Ethanol was added to the obtained filtrate to a concentration of 70%, and the mixture was allowed to stand overnight at 4 ° C. Then, insoluble matters were removed by centrifugation (9,000 xg, 20 min, 4 ° C), and ethanol was removed using an evaporator. After removal, it was freeze-dried to obtain a water-soluble peptide fraction of Gouda cheese.
After dissolving this water-soluble peptide fraction with distilled water, a YMC-Pack ODS-A column (
YMC (4.6 mm × 150 mm) was used for reverse phase HPLC and fractionated into a water-soluble peptide purified fraction. Chromatography consists of solvent (A solution: 50 mM ammonium acetate aqueous solution; B solution: 80% acetonitrile), concentration gradient (12% B → 60% B, 60 min), flow rate 0.8 ml / min, detection wavelength 215 nm.
It went on condition of. The results are shown in FIG. When the antioxidant activity of the fractionated fraction was measured by the method of Test Example 1 shown below, a strong antioxidant activity was confirmed in the fraction indicated by the arrow in the chromatogram.
The molecular weight of the obtained Gouda cheese peptide was estimated using a liquid chromatograph mass spectrometer (Thermo Electron), and the amino acid sequence was analyzed with a peptide sequencer (Applied Biosystems). Number 6 shown is His-Pro-I
It was confirmed that the sequence was le-Lys-His-Gln-Gly-Leu-Pro-Gln.
The peptide thus obtained can be used as it is as the antioxidant or the adiponectin production promoter of the present invention.

[Test Example 1]
(Measurement of antioxidant activity of peptides)
Antioxidant activity of the peptide fractionated in Example 1 was measured by a method utilizing the action of the linoleic acid oxide to fade β-carotene. That is, 0.5 ml of β-carotene solution (1 mg / ml chloroform), 0.2 ml of linoleic acid solution (100 mg / ml chloroform), Tween 40 solution (
(200 mg / ml chloroform) 1.0 ml was placed in a 300 ml Erlenmeyer flask, chloroform was completely removed with nitrogen gas, and then 100 ml distilled water was added to dissolve. Furthermore, 0.2M phosphate buffer (pH 7.
0) 9.0 ml was added to prepare a linoleic acid / β-carotene solution. Next, 0.18 ml of the above linoleic acid / β-carotene solution was added to a 98-well microwell plate into which 0.02 ml of the peptide fraction had been dispensed in advance, and the absorbance (S ₀ ) at 490 nm was immediately measured. Linoleic acid
In preparing the β-carotene solution, the amount of β-carotene solution added was appropriately increased or decreased so that S ₀ was about 1.2 (1.1 to 1.3). After S ₀ measured, immediately placed microwell plates in a thermostat at 50 ° C., and incubated for 30 min. Immediately after the incubation, the absorbance (S ₃₀ ) was measured, and the decrease in absorbance at 490 nm over 30 minutes, ΔS = S ₀ -S ₃₀ was calculated. The blank was subjected to the same operation using 70% ethanol instead of the sample. That is, linoleic acid / β-
Absorbance immediately after adding the carotene solution (B ₀ ) and absorbance after holding at 50 ° C. for 30 minutes (B ₃₀
) And the decrease in absorbance at 490 nm over 30 minutes, ΔB = B ₀ -B ₃₀ was determined. Antioxidant activity was substituted into the following formula and expressed as an antioxidant rate (%).

As positive control, carnosine (β-Ala-L-His; Peptide Institute), a muscle-derived antioxidant activity peptide, prepared to 0.01 μg / ml, 0.1 μg / ml, and 1 μg / ml was used.

The results of measuring the antioxidant activity of purified peptide and carnosine as a positive control are shown in Table 1.

As can be seen in Table 1, the addition of peptides showed concentration-dependent antioxidant activity.
From the results of this test example, the cheese-derived peptide consisting of the amino acid sequence represented by His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln has antioxidant activity (radical scavenger activity). It was found that it is useful for the prevention and improvement of oxidative cell damage caused by active oxygen and lipid peroxide.

(Peptide synthesis)
The peptide consisting of the amino acid sequence represented by the above formula (1) was synthesized with a peptide synthesizer. Peptide synthesizer 431A (Applied Biosystems) using p-hydroxymethylphenoxymethyl polystyrene (HMP) resin and using 9-fluorenylmethyloxycarbonyl (Fmoc) group as amino-terminal protecting group from the C-terminal side to the peptide chain A linearly protected peptide was synthesized on a 0.25 mmol scale by sequentially extending. The obtained HMP resin-bound protected peptide was converted to HMP of the peptide with trifluoroacetic acid (TFA) in the presence of phenol, 1,2-ethanedithiol and thioanisole.
Cleavage from the resin and removal of the protecting group were performed simultaneously. After removing TFA by concentration under reduced pressure, the crude peptide was crystallized with ethyl ether, dissolved in 5% acetic acid, and lyophilized.
The obtained linear crude peptide was converted into a YMC-Pack ODS-A column (YMC; 4.6 mm × 15
0 mm). Chromatography is performed using a solvent (solution A: 50 mmol ammonium acetate aqueous solution; solution B: 80% acetonitrile), concentration gradient (12% B → 60% B, 60 min).
, Under conditions of a flow rate of 0.8 ml / min and a detection wavelength of 215 nm. The purity of the obtained linear purified peptide is
The result of analysis by HPLC was 98%. The results are shown in FIG.
The peptide thus obtained can be used as it is as the antioxidant or the adiponectin production promoter of the present invention.

[Test Example 2]
(Measurement of antioxidant activity of synthetic peptides)
For the peptide obtained in Example 2, the antioxidant activity was measured in the same manner as in Test Example 1.
As a positive control, carnosine (β-Ala) is a muscle-derived antioxidant peptide.
-L-His (Peptide Institute) and catechin (Wako Pure Chemical Industries) prepared to 1 μm, 10 μm, and 100 μm were used. The results of evaluating the antioxidant activity of the synthetic peptides are shown in Table 2 and FIG. As shown in Table 2 and FIG. 3, strong antioxidant activity was confirmed in the obtained synthetic peptide.

(Preparation of trypsin digest)
A tryptic digest of the peptide obtained in Example 2 was prepared.
The peptide obtained in Example 2 and trypsin (Sigma Aldrich Japan) were
Each was dissolved in an enzyme reaction solution (50 mM Tris-HCl, 20 mM CaCl ₂ , pH 8.0) to a concentration of 1 mg / ml. 5 μl of a trypsin solution was added to 500 μl of a peptide solution and reacted at 37 ° C. for 2 hours. Then, 5 μl of a trypsin solution was further added and reacted at 37 ° C. for 18 hours to obtain a trypsin digest.
After this trypsin digest was dissolved in distilled water, a YMC-Pack ODS-A column (Y
MC, 4.6 mm × 150 mm) was used for HPLC. The HPLC analysis was performed under the same conditions as the peptide before trypsin digestion in Example 2. The results are shown in FIG. As shown in the figure, it was divided into two peaks.
Two fragments digested with trypsin (His-Pro-Ile-Lys, peptide 1) and (His
Two peaks corresponding to -Gln-Gly-Leu-Pro-Gln and peptide 2) were collected 10 times each and dried using a centrifugal evaporator to obtain two peptides which are trypsin digests. The antioxidant activity of the obtained two peptides and the tryptic digest before fractionation was measured by the method of Test Example 3 shown below. As shown in FIG. 5, the synthetic peptide confirmed strong antioxidant activity. It was done.
The peptide having the antioxidant action thus obtained can be used as it is as the antioxidant of the present invention.

[Test Example 3]
(Measurement of antioxidant activity of peptides)
Antioxidant activity was measured using the carotene fading method in the same manner as in Test Example 2 for the two peptides that were trypsin digests obtained in Example 3 and the trypsin digest before fractionation. The peptide concentration was measured by dissolving each peptide in 500 μl of pure water and using Micro BCA Protein assay kit (Pierce). As positive controls, carnosine (β-Ala-L-His; Peptide Institute) and catechin (Wako Pure Chemical Industries, Ltd.) were used as in Test Example 2. Dilution series of 1 ng / ml, 10 ng / ml, and 100 ng / ml were prepared for each fragment, and antioxidant activity was evaluated using β-carotene bleaching method. The results of evaluating the antioxidant activity of trypsin digested peptides are shown in FIG. As shown in FIG. 5, strong antioxidative activity was confirmed in the obtained trypsin digested peptide.

[Test Example 4]
(Peptide adipocyte administration experiment)
About the peptide isolate | separated from the cheese obtained in Example 1, the administration experiment to the primary culture | cultivation built-in fat cell was conducted.
Experiments were performed using rat primary cultured visceral adipocytes (VAC01, Cell Garage) and built-in adipocyte differentiation induction medium (Cell Garage). Thaw the cryopreserved cells according to the protocol of Cell Garage and the day of seeding in a 24-well plate is defined as day 0, and the medium (peptide concentration: 100 μM, 50 μM, 10 μM, 0 μM) in which the synthetic peptide is dissolved on day 2, day 5, and day 7 is used. In addition, the cells were cultured for 8 days. The culture supernatant was collected on day 5, day 7 and day 8, and the adiponectin concentration was measured. Also da
Cells were collected at y8, and intracellular glycerol 3-phosphate dehydrogenase (GPDH) enzyme activity, DNA amount in each well, and intracellular accumulated fat amount were measured. GPDH is an enzyme that works when synthesizing triglyceride from glucose, and is expressed in mature adipocytes. In this culture system, the effect of the preadipocyte differentiation was confirmed by administering the peptide of the present invention.
The amount of DNA was quantified using a DNA quantification kit manufactured by Cell Garage.
Cellular glycerol 3-phosphate dehydrogenase (GPDH) enzyme activity is measured by Cell Garage GPD
Measurement was performed according to the Cell Garage protocol using an H activity measurement kit. The measurement results were standardized by the amount of DNA extracted from each well.
Adiponectin concentration was measured using an adiponectin ELISA kit from Otsuka Pharmaceutical Co., Ltd.
The adiponectin concentration in the culture supernatant was measured. The measurement results were standardized by the amount of DNA extracted from each well.
The amount of accumulated fat in the cells was determined by fat staining with oil red 0 and quantification of accumulated fat by absorbance measurement using a fat staining kit manufactured by Cell Garage.

(Results of peptide adipocyte administration experiment)
As a result of quantifying the cell-derived DNA in each well, in Student`s t-test, no significant difference was detected in the amount of cellular DNA between the group to which the peptide of the present invention was not added and the group to which the peptide was added.
As a result of measuring the GPDH activity (per unit DNA amount), the GPDH activity standardized by the amount of cellular DNA in each well showed significantly higher activity in the group containing 10 μM or 50 μM of the peptide of the present invention than in the group containing no peptide. No significant difference was detected between the peptide 100 μM added group and the non-added group.
According to the measurement result of the change in the concentration of adiponectin depending on the number of days of culture, it was found that the amount of adiponectin production reached the maximum on the 7th day of culture, but rapidly decreased on the 8th day. FIG. 6 shows the difference in adiponectin production amount (per unit DNA amount) due to the difference in the administered peptide concentration on day 7 when the adiponectin concentration in the culture supernatant was the highest.
Cells with added peptide produced significantly more adiponectin than cells cultured without added peptide. This tendency was particularly noticeable when the peptide concentration was 10 μM or 50 μM.
FIG. 7 shows the result of quantification of the amount of accumulated fat by fat staining with oil red 0 and absorbance measurement on the 8th day of culture.
Since fixation is necessary for staining of adipocytes, the number of cells in the well where fat was quantified (cell DNA
Quantity) cannot be quantified. However, from the results of DNA quantification of cells cultured under the same conditions, it was estimated that there was almost no number of cells in each well since no effect on the number of cells due to the addition of peptide was observed. In Student`s t-test, there was no significant difference in the amount of accumulated fat between those cultured without the peptide and those cultured with the peptide added. As shown, there was a tendency for fat accumulation to decrease slightly.

(Summary of peptide fat cell administration experiment)
When 10-50 μM of the peptide of the present invention is added to the preadipocytes and cultured, (1) GPDH activity increases,
(2) Adiponectin production increases,
(3) There was no change in the amount of accumulated fat.
In this case, it is considered that the addition of the peptide promotes differentiation from preadipocytes into adipocytes, or promotes adipogenic activity in differentiated mature adipocytes. However, since there was no difference between the amount of accumulated fat and cells without added peptide, the synthesized fat may be degraded by some other molecular mechanism. Adiponectin is a good cytokine secreted from mature adipocytes, and this adiponectin production promoting effect was confirmed by the addition of the peptide of the present invention. From the above, it is recognized that the peptide of the present invention has an effect of promoting functional differentiation from preadipocytes to adipocytes and increasing the production of adiponectin, and suppresses fat accumulation in cells. It was suggested that there is a possibility of effect.
Adiponectin is said to have an effect of promoting fat burning in the liver and muscles in addition to the action of inhibiting arteriosclerosis. It is considered that the peptide of the present invention has a function of producing adipocytes that are likely to produce adiponectin and preventing metabolic syndrome.
On the other hand, when 100 μM of the peptide of the present invention was added to the preadipocytes and cultured, (1) GPDH activity did not change
(2) Adiponectin production increased,
(3) There is no change in the amount of accumulated fat (although there is no significant difference, there seems to be a decreasing trend)
.
From these results, the effect of the peptide of the present invention on adipocytes varies depending on the concentration, suggesting the possibility that an optimal concentration exists. In any case, the effect of promoting the production of adiponectin was confirmed by the addition of the peptide of the present invention. Moreover, there is no doubt that there is an effect of suppressing fat accumulation, and the possibility that the peptide of the present invention has a preventive effect on metabolic syndrome was shown.
It should be noted that His-Pro-Ile-Lys and His-Gln-Gly are trypsin degradation products of peptides consisting of the amino acid sequence represented by this His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln. It was confirmed that the peptide having the amino acid sequence represented by -Leu-Pro-Gln also promoted the same adiponectin production amount.

(Manufacture of tablets)
Antioxidant peptide obtained in Example 1 (His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln) 20
% By weight, lactose (DMV) 46% by weight, crystalline cellulose (Wako Pure Chemical Industries) 31% by weight, water 3
After fully mixing the parts by weight, the mixture was tableted with a tableting machine (Fuji Pharmaceutical Co., Ltd.) to produce the antioxidant tablet of the present invention.

(Manufacture of fruit juice drinks)
Each component was mixed with the composition shown in Table 3, filled into a container, then heat sterilized, and the synthetic antioxidant peptide (His-Pro-Ile-Lys-His-Gln) obtained in Example 2 of the present invention. An antioxidant fruit juice beverage according to the present invention containing -Gly-Leu-Pro-Gln) was produced.

(Manufacture of soft drinks)
Each component was mixed with the composition shown in Table 4, filled into a container, and then heat sterilized, and the trypsin digested peptide (His-Gln-Gly-Leu-Pro-Gln) obtained in Example 3 of the present invention was used. The antioxidant soft drink of this invention which mix | blended this was manufactured.

(Manufacture of soft drinks)
Each component was mixed with the composition shown in Table 5, filled into a container, and then heat-sterilized, and the trypsin-digested peptide obtained in Example 3 of the present invention (His-Gln-Gly-Leu-Pro-Gln and (His-Pro-Ile-Lys)
The antioxidant soft drink of this invention which mix | blended this was manufactured.

(Manufacture of biscuits)
A dough having the composition shown in Table 6 was prepared, molded, and then baked to obtain the peptide (His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln) obtained in Example 1 of the present invention. The antioxidant biscuits of the present invention were prepared.

(Manufacture of feed for dog breeding)
Each component was mixed in the composition shown in Table 7, and the antioxidant peptide (His−) obtained in Example 1 of the present invention was mixed.
Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln) blended feed for dog breeding of the present invention was produced.

(Manufacture of tablets)
Antioxidant peptide obtained in Example 1 (His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln) 20
% By weight, lactose (DMV) 46% by weight, crystalline cellulose (Wako Pure Chemical Industries) 31% by weight, water 3
After fully mixing the weight parts, the mixture was tableted with a tableting machine (Fuji Pharmaceutical Co., Ltd.) to produce the tablet for promoting adiponectin production of the present invention.

(Manufacture of fruit juice drinks)
Each component was mixed with the composition shown in Table 8, filled into a container, then sterilized by heating, and the synthetic peptide (His-Pro-Ile-Lys-His-Gln-Gly) obtained in Example 2 of the present invention was used. -A juice drink for promoting adiponectin production according to the present invention containing Leu-Pro-Gln) was produced.

[FIG. 1] The arrow indicates the amino acid sequence of His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln of the present invention.
[FIG. 4] In the figure, 1 is the amino acid sequence of His-Pro-Ile-Lys of the present invention, and 2 is His-G of the present invention.
The amino acid sequence of ln-Gly-Leu-Pro-Gln is shown.
[FIG. 5] Peptide digest 1 shows the amino acid sequence of His-Pro-Ile-Lys of the present invention, and peptide digest 2 shows the amino acid sequence of His-Gln-Gly-Leu-Pro-Gln of the present invention.

Claims (1)

The adiponectin production promoter which uses as an active ingredient one or more peptides of the peptide which consists of an amino acid sequence represented by either of following formula (1) -(3) .
His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln Formula (1)
His-Pro-Ile-Lys formula (2)
His-Gln-Gly-Leu-Pro-Gln formula (3)