JP2012116773A - Igf-1 value-elevating agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an IGF-1 (insulin-like growth factor-1) value elevating agent that elevates the IGF-1 concentration in blood by oral administration.SOLUTION: When a collagen peptide extracted by treating a collagen derived from fish scale and/or fish skin with a protease is orally administered, the expression amount of IGF-1 mRNA in the liver is increased and further the expression amounts of IGFBP-2 mRNA and Ci1 mRNA exerting an influence on amounts of IGFBP-2 and Ci1 participating in IGF-1 concentration in blood are increased, so as to function as the IGF-1 value elevating agent.

Description

  The present invention relates to an IGF-1 level-elevating agent mainly comprising a peptide obtained by hydrolyzing fish scale and / or fish skin collagen, and more specifically, IGF-1 in the liver by ingesting the peptide orally. The present invention relates to an agent for increasing IGF-1 level, which can increase the expression level of IGF-1 gene such as mRNA, IGFBP-2 mRNA, Ci1 mRNA, and finally increase the blood concentration of IGF-1.

  Collagen is a major protein that forms the raw skin, tendons, bones, etc. of fish, pigs, cattle, etc., and is universally present in animal tissues. Collagen and collagen peptides hydrolyzed with enzymes, acids, alkalis and the like are widely used as pharmaceutical compositions, food compositions, dietary supplements and the like alone or in combination with other components.

  For example, collagen obtained by heat extraction or pressure heat extraction from fish is enzymatically decomposed with a proteolytic enzyme until the average molecular weight becomes 1000 to 10,000, and this enzyme decomposition product is concentrated and purified, and free amino acids in solid content There is a food and drink containing a collagen peptide derived from fish having a content of 1.0% by mass or less and an arsenic content of 2 ppm or less (Patent Document 1). The present invention provides a method for producing collagen peptides derived from fish that are expected to have various physiological effects of collagen, have no fish-specific taste and smell, and have a low arsenic content, and foods and drinks containing such collagen peptides. The reason why the average molecular weight of the collagen peptide is limited to the above range is that it can be used for food and drink with low viscosity.

  An anti-aging composition comprising a biological collagen synthesis promoter and an abnormal protein removing agent, wherein the degradation product is a degradation product of collagen and / or gelatin having a molecular weight of 200 to 300. There is also an anti-aging composition containing as a collagen synthesis promoter (Patent Document 2). Collagen peptide promotes skin turnover and is said to have an excellent anti-aging effect on skin. The collagen to be used may be extracted from the skin of animals such as cows, pigs and fish, and connective tissues such as bones and tendons, using proteolytic enzymes and having an average molecular weight of around 200 to 300. This is because, if the average molecular weight is in the above range, the tripeptide is highly contained, and the collagen synthesis promoting activity can be dramatically improved.

  Further, it is a degradation product of collagen or gelatin by collagenase, and its amino acid sequence is (Gly-XY) (wherein Gly represents a glycine residue, X and Y represent amino acid residues, and A collagen peptide (Gly-XY) n (n represents a positive integer) composed of a collagen tripeptide represented by X and Y may be the same or different. There is also a hyaluronic acid production promoter (Patent Document 3). The inventors have found that a specific collagen peptide is effective as a hyaluronic acid production promoter. The average molecular weight of the collagen peptide is preferably 280 to 3000, more preferably the content of n = 1 in the collagen peptide (Gly-XY) n is 25 to 100% in the collagen peptide. . Such a collagen peptide can be produced by allowing collagenase to act on collagen or gelatin.

  Furthermore, it is a food composition, 1) collagen having an average molecular weight of 10,000 or less is 10 to 60% by mass with respect to the total amount of the food composition, and 2) guar gum is 0.001 to 0.1 with respect to the total amount of the food composition. And 3) 0.01 to 1% by mass of methylcellulose with respect to the total amount of the food composition, and the guar gum content is 5 to 20% by mass with respect to the cellulose content. There is also a food composition for beautifying skin (Patent Document 4). This skin-beautifying food composition is characterized by being in the form of granules that can be ingested without water. The collagen used is hydrolyzed using fish as a base, and the average molecular weight is 10,000 or less. This is because if it exceeds this, meltability in the mouth is inhibited, making it difficult to swallow.

  In addition, there is also a calcium absorption enhancer containing a peptide which is a degradation product of gelatin and / or collagen and has a molecular weight of 1000 or less and an average of 6 mol% or more of glutamic acid units as an active ingredient (patent) Reference 5). It provides a calcium absorption promoter with high absorption efficiency and low odor. Collagen degradation products are water-soluble, so they are excellent in absorption, and by specifying the content and molecular weight of glutamic acid units, It can be used as an active ingredient. Peptides can be produced by degrading collagen with enzymes such as bromelain and flavorzyme M. The molecular weight is preferably 1000 or less, and if it exceeds 1000, the calcium absorption efficiency decreases. The ratio of acidic amino acid units in all amino acid units constituting the peptide is 6 mol% or more on average. It is because calcium absorption efficiency will fall that it is less than 6 mol%.

  On the other hand, one of the peptide hormones is insulin-like growth factor-1 (IGF-1) secreted from the liver by growth hormone (GH) secreted from the pituitary gland. IGF-1 is involved in cell growth and development in addition to insulin-like effects and exerts important endocrine effects such as cortical bone mass, kidney size, prostate size, peripheral vascular resistance, sodium retention, cancer progression, brain function, etc. To do. For this reason, for example, in view of the fact that IGF-1 exhibits a hair-growth effect through activation of hair roots, a technique for directly administering swell thiamarin extracted from plants to hair papilla cells as an IGF-1 production promoter is disclosed. Yes (Patent Document 6).

  On the other hand, there is an IGF-1 elevating agent composed of components obtained by treating the ovary membrane of sputum with a proteolytic enzyme (Patent Document 7). Even if the IGF-1 value decreases due to aging, the IGF-1 value can be increased again.

Japanese Patent No. 4236850 Japanese Patent No. 4286513 Japanese Patent No. 4336486 Japanese Patent No. 4360986 Japanese Patent No. 3881453 JP 2009-263262 A Japanese Patent No. 3946238

  Collagen has different physical properties such as viscosity and solubility depending on the molecular weight, and exhibits different medicinal effects depending on the hydrolysis method and molecular weight. The collagen peptide of Patent Document 1 has various physiological effects, the collagen peptide of Patent Document 2 has an effect of preventing skin aging, the collagen peptide of Patent Document 3 has an effect of promoting hyaluronic acid production, and the collagen peptide of Patent Document 4 has beautiful skin. With respect to the effect and the solubility in the mouth, the collagen peptide of Patent Document 5 exhibits a calcium absorption promoting effect. However, the collagen peptides shown in Patent Documents 1 to 5 are mainly intended for local action on skin and bone.

  On the other hand, IGF-1 is one of peptide hormones. As shown in Patent Document 6, when a specific substance is administered to specific cells such as hair papilla cells, gene expression of IGF-1 is promoted. Thus, cell growth effects such as hair growth effects can be expected through activation of hair roots. In addition, as shown in Patent Document 7, a component obtained by treating and extracting sputum ovarian membrane with proteolytic enzyme increases IGF-1 value even when IGF-1 value decreases after adulthood. It is also known that fatigue, appetite, falling asleep, and sleep are improved.

  On the other hand, collagen and collagen peptides are proteins that are ubiquitous in animal tissues and are highly safe when taken orally. If the blood concentration of IGF-1 is increased by oral intake of collagen or collagen peptide, it becomes an extremely safe IGF-1 level increasing agent.

  In view of the above situation, the relationship with IGF-1 by oral intake of collagen, particularly the involvement in the liver, will be examined, and a new use of the collagen peptide will be provided.

The present inventors verified the effect on the liver by oral intake of collagen peptides derived from fish scales and / or fish skins that are originally discarded, and as shown in the examples described later, ribosomal protein S6 increases, It was found that the amounts of IGF-1 mRNA, IGFBP-2 mRNA, and Ci1 mRNA were significantly increased, and the blood IGF-1 concentration was also increased, and it was found that the collagen peptide can be used as an IGF-1 raising agent. The present invention has been completed. Fish scales are discarded parts, and fish scales and / or fish skin-derived collagen peptides obtained by specific treatment enhance the gene expression level in the liver by ingestion, and insulin-like growth factor-1 (IGF-1) It has never been known to increase blood levels.
That is, the present invention provides an IGF-1 level increasing agent characterized by comprising a collagen peptide extracted by treating fish scale and / or fish skin-derived collagen with a proteolytic enzyme.

  Further, the present invention is characterized in that the proteolytic enzyme is GF produced from any one of Aspergillus oryzae, Bacillus subtilis or Bacillus licheniformis, GF, -1 value raising agent is provided.

  Moreover, this invention provides the said IGF-1 value raiser whose weight average molecular weights of the said collagen peptide are 1000-10000.

  IGF-1 is generally a hormone that is secreted from the liver by growth hormone and is involved in cell growth and development in addition to insulin-like effects. According to the present invention, it is possible to increase the expression level of IGF-1 mRNA in the liver and increase the blood concentration of IGF-1 simply by ingesting the fish scale and / or fish skin collagen peptide that has been specifically treated. be able to.

The results of Experiment 1 are shown, and a group (10C group) orally fed a diet containing 10% casein, a group (6C + 4CP group) orally fed a diet containing 6% casein and 4% collagen peptide, It is a figure which shows the difference in the gene expression level of the ribosome protein small 6 in the liver. **: p <0.01 The results of Experiment 1 are shown, and a group (10C group) orally fed a diet containing 10% casein, a group (6C + 4CP group) orally fed a diet containing 6% casein and 4% collagen peptide, It is a figure which shows the difference of the amount of ribosomal protein small 6 in the liver. The results of Experiment 2 are shown, and a group (10C group) orally fed a diet containing 10% casein, a group (6C + 4CP group) orally fed a diet containing 6% casein and 4% collagen peptide, and It is a figure which shows the difference in the expression level of IGF-1 mRNA in the liver in. *: P <0.05 The results of Experiment 2 are shown, and a group (10C group) orally fed a diet containing 10% casein, a group (6C + 4CP group) orally fed a diet containing 6% casein and 4% collagen peptide, and It is a figure which shows the difference of the expression level of IGFBP-2 mRNA in 10C group and 6C + 4CP group in the liver. **: p <0.01 The results of Experiment 2 are shown, and a group (10C group) orally fed a diet containing 10% casein, a group (6C + 4CP group) orally fed a diet containing 6% casein and 4% collagen peptide, and It is a figure which shows the time-dependent change of the body weight in. *: P <0.05, **: p <0.01 The results of Experiment 2 are shown, and a group (10C group) orally fed a diet containing 10% casein, a group (6C + 4CP group) orally fed a diet containing 6% casein and 4% collagen peptide, and It is a figure which shows the time-dependent change of the food intake of the 10C group in 6C + 4CP group. The results of Experiment 2 are shown, and a group (10C group) orally fed a diet containing 10% casein, a group (6C + 4CP group) orally fed a diet containing 6% casein and 4% collagen peptide, and It is a figure which shows the bone density in each site | part of the femur of 10C group and 6C + 4CP group. *: P <0.05 The results of Experiment 3 are shown, and a group (10C group) orally fed a diet containing 10% casein and a group (6C + 4CP group) orally fed a diet containing 6% casein and 4% collagen peptide. It is a figure which shows the difference of the expression level of Ci1 mRNA in 10C group and 6C + 4CP group in the liver. *: P <0.05

The present invention is an IGF-1 level increasing agent characterized by comprising a collagen peptide extracted by treating fish scale and / or fish skin-derived collagen with a proteolytic enzyme. Preferably, the proteolytic enzyme is a protease produced from any one of Aspergillus oryzae, Bacillus subtilis, or Bacillus licheniformis, and the average molecular weight of the obtained molecular peptide is 1000 -10000. Hereinafter, the present invention will be described in detail.
(1) Method for producing IGF-1 level increasing agent The IGF-1 level increasing agent of the present invention comprises a collagen peptide extracted by treating fish scale and / or fish skin-derived collagen with a proteolytic enzyme. Although there is no limitation in the manufacturing method of this collagen peptide, what pre-processed fish scale and / or a fish skin, then extracted collagen, and hydrolyzed the obtained collagen with the proteolytic enzyme can be used preferably. The hydrolyzate may be purified by filtration or the like and adjusted to pH 5 to 7 by ion exchange.

(I) Fish scales and / or fish skins Fish scales and / or fish skins used in the IGF-1 level increasing agent of the present invention may be any fish species such as saltwater fish and freshwater fish. For example, fresh water fish such as tilapia, salmon and crucian fish, and saltwater fish such as salmon, salmon and salmon can be preferably used. The fish scale and / or fish skin collagen peptide used in the present invention is obtained by hydrolyzing the collagen contained in the fish scale and / or fish skin with a proteolytic enzyme and having a weight average molecular weight of 1000 to 10,000.

(ii) Pretreatment It is preferable to use the fish scales and / or fish skins after washing them with water to remove deposits. You may perform a decalcification process after washing with water. This is because fish scales contain inorganic substances such as calcium phosphate, so that collagen extraction is facilitated by performing decalcification treatment.

  The deashing treatment is not particularly limited, and for example, fish scales are stirred for 2 to 48 hours in a treatment solution such as hydrochloric acid, ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid disodium, ethylenediaminetetraacetic acid tetrasodium. The amount of the treatment liquid used may be set as appropriate and is not particularly limited. The deashing treatment may be performed a plurality of times as necessary, and may be washed with water after deashing as necessary. The fish scales and / or fish skins may be crushed.

(Iii) Collagen extraction In the present invention, collagen is extracted from pretreated fish scales and / or fish skin. As a collagen extraction method, for example, a method of heating extraction or pressure heating extraction by adding water is preferably used. According to this method, insoluble protein such as elastin which is hardly soluble in water does not elute, and collagen can be extracted efficiently. Collagen extraction may be performed by adding 100 to 500 parts by mass of water to 100 parts by mass of fish scales and / or fish skin, and heating and extracting at 60 to 100 ° C. for 0.5 to 10 hours. In addition, what is necessary is just to extract for 0.5 to 3 hours at 110-120 degreeC, when extracting by pressurization heating.

(Iv) Hydrolysis Next, the collagen obtained in the extraction step is hydrolyzed by treatment with a protein hydrolase.

  The proteolytic enzyme to be used is not particularly limited, and neutral protease, alkaline protease, acidic protease, plant-derived or microorganism-derived protease can be used.

  Plant origins include papaya papain, chymopapain, pineapple bromelain, fig ficin, kiwifruit actinidine, and the use of collagenase derived from papaya, ginger, kiwifruit, mango, pineapple, etc. it can.

  In addition, the microbial origins include rennet derived from Rhizomucor miehei and Rhizomucor pusillus, sp. ) And Rhizopus niveus, protease derived from Aspergillus oryzae, neutral or alkaline protease derived from Bacillus subtilis, Bacillus bis There are alkaline proteases derived from Licheniformis, actinase derived from Streptomyces grieseus, Streptomyces parvulus, Clostridium histolium and the like. In the present invention, the enzyme having endo-type protease activity is desirable, and a mixture of an enzyme having endo-type protease activity and an enzyme having exo-type protease activity (aminopeptidase, carboxypeptidase) may be used.

  As conditions for producing a collagen peptide from fish scale and / or collagen in fish skin, the optimum temperature, pH, time and the like for the proteolytic enzyme used can be appropriately selected. Typical temperatures are in the range of 30-80 ° C. The time is 1 to 36 hours, preferably 1 to 20 hours.

  Before allowing the proteolytic enzyme to act on fish scales and / or fish skin, the fish scales and / or fish skin is 30 ° C. or higher and lower than 100 ° C., preferably 50 ° C. or higher and lower than 100 ° C., particularly preferably 80 ° C. or higher and lower than 100 ° C. It is preferable to immerse and hold in temperature water for 10 hours or less, preferably 2 hours or less. Collagen is gelatinized by being immersed in water at a temperature of 30 ° C. or more and less than 100 ° C. and pretreated, and a part thereof is eluted in water, so that hydrolysis by a proteolytic enzyme is performed smoothly.

  In the present invention, a collagen peptide may be directly extracted without extracting collagen from fish scales and / or fish skin. For example, in fish scales and / or fish skin, among bacterial alkaline proteases from the genus Bacillus, papain, bromelain, thermolysin, trypsin, pronase, chymotrypsin, subtilisin, staphylococcus protease, pepsin, proctase A, proctase B Collagen peptides can be obtained by the action of neutral or alkaline proteolytic enzymes.

  The fish scale and / or fish skin-derived collagen peptide used in the present invention preferably has a weight average molecular weight in the range of 1000 to 10,000, more preferably 2000 to 10,000, particularly preferably 3000 to 7000. Such a collagen peptide having a weight average molecular weight can be carried out, for example, by adjusting the amount of enzyme or adjusting the treatment time in the hydrolysis step.

(V) Purification In the present invention, the solution containing the fish scale and / or fish skin-derived collagen peptide obtained as described above is deactivated after enzymatic hydrolysis, and hardly soluble in water by filtration, salting out or aciding out. It is preferable to remove insoluble components. Furthermore, pH and concentration may be adjusted after desalting and purification by ion exchange resin treatment, dialysis treatment with a semipermeable membrane, electrodialysis treatment, and the like.

  For example, the obtained collagen peptide solution is treated with an evaporator, a reverse osmosis membrane or the like, and the concentrated solution is recovered. Concentrated and purified using reverse osmosis membranes, fish scale and / or fish skin-specific taste and odor components such as amino acids, oligopeptides, nucleic acids, organic acids, minerals, volatile sulfur compounds, fatty acids, nitrogen compounds , Carbonyl compounds and the like can be removed. The collected concentrated liquid can be used as it is or after being appropriately dried and powdered.

  Examples of the reverse osmosis membrane used here include trade name “NTR-7410”, trade name “NTR-7430”, trade name “NTR-7450” (all manufactured by Nitto Denko).

If the reverse osmosis membrane salt rejection is outside the above range, impurities such as taste, odor, and arsenic may not be sufficiently removed, and the loss of collagen peptide will be unfavorable. The conditions for the reverse osmosis membrane treatment can be set as appropriate. Usually, the treatment liquid is adjusted to a solid content concentration of 15% by mass or less, and the solid content concentration is circulated at a pH of 4-7 and a liquid temperature of 60 ° C. It is preferable to concentrate until it becomes 25 mass% or more. At this time, it is preferable to add 1 to 10 times, preferably 3 to 5 times the amount of water of the stock solution while appropriately adding water to allow the solution to permeate. Impurities can be efficiently removed by repeating the hydration operation.
(2) IGF-1 Value Raising Agent The collagen peptide obtained above is in the form of an aqueous solution, but it may be dried by freeze drying or spray drying to prepare a powder. The IGF-1 level-increasing agent of the present invention may be ingested as an aqueous solution or powder of the above-mentioned collagen peptide as it is, but is added with excipients such as lactose and corn starch, and further, other amino acids, vitamins, fats and oils Or tablets may be added to prepare tablets, pills, liquids and other preparations.

  The agent for increasing IGF-1 level of the present invention is characterized in that blood IGF-1 concentration can be increased by oral administration. Although the details of the mechanism of action are unknown, as shown in the examples described later, the expression level of IGFBP-2 mRNA that regulates the blood concentration of IGF-1 increases with the increase in the amount of IGF-1 mRNA. However, the expression level of mRNA of Ci1, which is one of proton-coupled oligopeptide transporters, is also increasing. By ingesting collagen peptides having specific sequences, digestive tract absorption products of tripeptides and oligopeptides, which are digested products, are transported to the liver, and these peptides express the expression levels of IGF-1 mRNA, IGFBP-2 mRNA, and Ci1 mRNA. It is presumed that the blood concentration of IGF-1 was finally improved by increasing the protein synthesis function by increasing the ribosomal protein.

  The fish scale and / or fish skin-derived collagen peptide used in the present invention is limited to those hydrolyzed with a proteolytic enzyme. Specific amino acid sequences are conserved in collagen peptides due to the specificity of proteolytic enzymes, and as a result of transporting such peptides to the liver, expression of IGF-1 mRNA, IGFBP-2 mRNA, and Ci1 mRNA as described above. It is thought to increase the amount.

The collagen peptide used in the present invention preferably has a weight average molecular weight in the range of 1000 to 10,000. It is because it is excellent in solubility.
The IGF-1 level-increasing agent of the present invention may be taken in an amount of 3 to 30 g, more preferably 5 to 10 g per day, and can be added to foods and used as health foods.

EXAMPLES Next, although an Example is given and this invention is demonstrated concretely, these Examples do not restrict | limit this invention at all.
(Production Example 1)
5 kg of fresh tilapia scales were washed with water and once drained, 24 L of water and 1.6 L of 35% strength hydrochloric acid were added. After stirring for 2 hours, hydrochloric acid in which calcium phosphate was dissolved was removed, and washing was performed until the pH of the washing water reached 6 or more. After draining water, 12 L of hot water was added and extracted at 73 ° C. for 5 hours.

0.1 parts by mass of protease derived from Aspergillus oryzae, a proteolytic enzyme, is added to 100 parts by mass of the solid content of the extracted collagen solution, the pH is adjusted to 6.0, and the temperature is 50 ° C. for 2 hours. Processed. The enzyme was inactivated by heating at a temperature of 85 ° C. for 30 minutes, and then subjected to auxiliary filtration and ion exchange treatment with diatomaceous earth. This solution was concentrated to a concentration of 30% by mass and dried by spray drying. It was 5400 when the weight average molecular weight of the obtained collagen was measured by the following method.
(Measuring method)
The weight average molecular weight was measured by gel filtration using a high performance liquid chromatograph.

The measurement conditions were as follows: Column: Showa Denko KK, Shodex OHpak SB802.5HQ and SB803HQ, one each mounted in series, at a column temperature of 40 ° C., eluent (0.1 mol / potassium dihydrogen phosphate solution and 0.1 mol) / Dipotassium hydrogen phosphate solution mixed to pH 6.90 and filtered through a 0.45 μm filter) is flowed at a flow rate of 0.8 ml / min, and 20 μl of sample is injected to detect the differential refractive index. Measured with a vessel. About the above-mentioned HPLC for 10 kinds of polyethylene glycol solutions (peak molecular weights 106, 194, 400, 620, 1080, 1900, 4020, 6450, 11840, 22450) of molecular weight measurement standard for gel filtration (manufactured by Polymer Laboratories) with known peak molecular weights The weight average molecular weight of the sample was obtained based on a calibration curve prepared by measuring similarly under the conditions.
(Experiment 1)
(1) Twenty-four mice at 6 weeks of age divided into two groups of 12 mice per group, and a feed containing 10% casein and 4% corn starch in the product name “AIN-93M” manufactured by Oriental Yeast Co., Ltd. ( 10C group), manufactured by Oriental Yeast Co., Ltd., trade name “AIN-93M” was fed with 6% casein, 4% collagen peptide prepared in Production Example 1 and 4% corn starch (6C + 4CP group).

  (2) For each group, 10 weeks after feeding, the whole liver was removed, and the first lobe (about 5 mm × 5 mm × 3 mm) was added at −80 ° C. together with an RNA stabilization solution (trade name “RNAlater”, manufactured by Ambion). Saved with.

(3) RNA was extracted from the preserved liver tissue using a nucleic acid extraction reagent (Nippon Gene Co., Ltd., trade name “ISOGEN”).
(4) Using a cDNA reverse transcription kit (manufactured by Applied Biosystems, trade name “High Capacity cDNA Reverse Transcription Kit”), cDNA was synthesized from the RNA obtained in (3) above.

  Primer of ribosomal protein small 6 (Applied Biosystems, “TaqMan gene expression analysis; ID Mm02342456_g1”), Primer primer of ribosomal protein small 10 (Applied Biosystems, “TaqMan gene expression analysis; ID Mm02391992_g1”), Ribosomal protein small 17 primers (Applied Biosystems, “TaqMan Gene Expression Analysis; ID Mm0131492_g1”), Ribosomal Protein Large 13a Primer (Applied Biosystems, “TaqMan Gene Expression Analysis; ID Mm01612986_gH”) and GAPDH Primer as an Endogenous Control (Applied Biosystems made , “TaqMan gene expression analysis; ID Mm0330249_gl”), and real-time PCR (Applied Biosystems; 7300 real-time PCR system) was performed. Specifically, 20 μl of a reaction solution consisting of 10 μl of TaqMan universal PCR master mix, 1 μl of primer and 9 μl of water was administered to each well, and PCR was performed using two wells for each sample. As amplification conditions, Ct values were obtained by performing 40 cycles of 50 minutes at 50 ° C., 10 minutes at 95 ° C., then 15 seconds at 95 ° C. and 1 minute at 60 ° C.

  Ct values obtained by amplification of each target were compared between samples after normalization using GAPDH expression levels. The results are shown in Table 1. As a result, in the 6C + 4CP group, a significant increase by the t-test was observed in the gene expression of ribosomal protein small 6 mRNA and ribosomal protein large 13a mRNA. The result of mRNA of ribosomal protein small 6 is shown in FIG.

(4) The liver obtained in (2) above is homogenized and subjected to Western blotting by electrophoresis, transcription, and primary / secondary antibody reaction according to a conventional method, using GAPDH as an internal control, ribosomal protein small 6, ribosomal protein small 14 Ribosomal protein large 7a and ribosomal protein large 10 were measured. The protein amount of ribosomal protein small 6 was higher in the 6C + 4CP group than in the 10C group. The results are shown in FIG.
(Experiment 2)
(1) Twenty-four mice at 6 weeks of age divided into two groups of 12 mice per group, and a feed containing 10% casein and 4% corn starch in the product name “AIN-93M” manufactured by Oriental Yeast Co., Ltd. ( 10C group), manufactured by Oriental Yeast Co., Ltd., trade name “AIN-93M” was fed with 6% casein, 4% collagen peptide prepared in Production Example 1 and 4% corn starch (6C + 4CP group).

(2) About each group, the time-dependent change of the body weight and the food intake was measured.
(3) For each group, blood was collected from the heart 10 weeks after feeding to obtain plasma. In addition, the whole liver was removed, and the first lobe (about 5 mm × 5 mm × 3 mm) was stored at −80 ° C. together with an RNA stabilization solution (trade name “RNAlater” manufactured by Ambion Co., Ltd.). In addition, femurs were harvested and stored in 70% ethanol. The bone density at each position obtained by dividing the femur into 20 equal parts was measured with a dual energy X-ray absorption measurement apparatus (trade name “CS-600R” manufactured by Aroka Co., Ltd.).

  (4) cDNA synthesis was performed from the total RNA of each sample using a cDNA reverse transcription kit (product name “High-capacity cDNA reverse transcription kit” manufactured by Applied Biosystems).

  Mouse IGF-1 primer (Applied Biosystems, “TaqMan gene expression analysis; ID Mm01233960_ml”), IGFBP-1 primer (Applied Biosystems, “TaqMan gene expression analysis; ID Mm00833447_ml”), IGFBP-2 primer (Applied Biosystems, “TaqMan gene expression analysis; ID Mm00492632_ml”), IGFBP-3 primer (Applied Biosystems, “TaqMan gene expression analysis; ID Mm00515156_ml”), IGFBP-4 Primer Applied Biosystems, “ TaqMan gene expression analysis; ID Mm01296351_ml "), IGFBP-5 primer (App Id biosystems, "TaqMan gene expression analysis; ID Mm00516037_ml"), IGFBP-6 primer (Applied Biosystems, "TaqMan gene expression analysis; ID Mm00599696_ml") and GAPDH primer (product of Applied Biosystems) as an endogenous control , “TaqMan gene expression analysis; ID Mm0330249_gl”), and real-time PCR (Applied Biosystems; 7300 real-time PCR system) was performed. 20 μl of a reaction solution consisting of 10 μl of TaqMan universal PCR master mix, 1 μl of primer and 9 μl of water was administered to each well, and PCR was performed using two wells for each sample. As amplification conditions, Ct values were obtained by performing 40 cycles of 50 minutes at 50 ° C., 10 minutes at 95 ° C., then 15 seconds at 95 ° C. and 1 minute at 60 ° C.

  Ct values obtained by amplification of each target were compared between samples after normalization using GAPDH expression levels. As a result, the expression levels of IGF-1 mRNA and IGFBP-2 mRNA showed significantly high values by t-test. The results are shown in FIGS.

(5) Changes over time in body weight and food intake are shown in FIGS.
As shown in FIG. 5, the average value of the body weight of the 6C + 4CP group was significantly lower than that of the 10C group by t-test. Moreover, as shown in FIG. 6, there was little difference in the amount of food intake in each group.

(6) Regarding the femur collected in (3) above, the bone density at each site divided into 20 equal parts from the waist to the knee was measured. The results are shown in FIG.
(Experiment 3)
Using the liver tissue obtained in Experiment 2, TaqMan gene expression analysis for proton-coupled oligopeptide transporters PEPT1, PEPT2, PHT1 and Ci1 which is a mouse homologous gene of PHT2 in humans; ID: Mm00453524_m1, Mm00451610_m1 , Mm00505709_m1 and Mm00491666_m1 were used for real-time PCR. The results are shown in Table 2.

As a result, in Ci1 mRNA, the 6C + 4CP group showed a significantly higher value than the 10C group. The results are shown in FIG.
(Experiment 4)
Using the plasma obtained in Experiment 2, GOT, GPT, and IGF-1 concentrations were measured. The results are shown in Table 3.

(結果)
（１）表３に示すように、本発明のＩＧＦ−１値上昇剤を１０週間に亘って経口投与した６Ｃ＋４ＣＰ群は、ＩＧＦ−１値上昇剤を投与しない１０Ｃ群と比較して血中のＩＧＦ−１濃度が上昇した。なお、ＧＯＴ値やＧＰＴ値は正常値であるから、ＩＧＦ−１値上昇剤の安全は極めて高い。

(result)
(1) As shown in Table 3, the 6C + 4CP group in which the IGF-1 level-increasing agent of the present invention was orally administered for 10 weeks was compared with the 10C group in which the IGF-1 level-increasing agent was not administered. IGF-1 concentration increased. In addition, since a GOT value and a GPT value are normal values, the safety | security of an IGF-1 value raising agent is very high.

  (2) There are approximately 80 types of ribosomal proteins in the liver. When the agent for increasing IGF-1 level of the present invention was orally administered over 10 weeks, the expression level of ribosomal protein mRNA increased by about 80% (not shown). When a more detailed increase in expression level was examined, as shown in FIG. 1, the expression level of ribosomal protein small 6 mRNA was significantly increased in the 6C + 4CP group as compared to the 10C group. As shown in FIG. 2, the amount of ribosomal protein small 6 was also high in the 6C + 4CP group. It was found that oral administration of the IGF-1 level increasing agent of the present invention increases the amount of ribosomal protein in the liver and activates protein synthesis.

  (3) When the IGF-1 level increasing agent of the present invention was orally administered, as shown in FIG. 5, a decrease in body weight was observed in the 6C + 4CP group, but as shown in FIG. There were few. It is considered that the IGF-1 level-increasing agent taken orally in this way is digested and absorbed from the digestive tract. By ingesting the IGF-1 level increasing agent of the present invention, the expression level of Ci1 mRNA in the liver is significantly increased as shown in FIG. For this reason, the increase in the blood concentration of IGF-1 may increase the amount of peptide transporter in the liver by the IGF-1 level-increasing agent of the present invention, and thereby the action of the peptide in the liver may proceed smoothly. There is sex.

  (4) When the IGF-1 level elevating agent of the present invention is orally administered, the expression levels of IGF-1 mRNA and IGFBP-2 mRNA are increased as shown in FIGS. In fact, as shown in Table 3, the blood concentration of IGF-1 also increased. From this, the expression level of Ci1 mRNA is increased by oral ingestion of the IGF-1 level increasing agent, whereby the action of the peptide derived from the IGF-1 level increasing agent of the present invention in the liver smoothly proceeds in the liver, It is thought that the expression level of IGF-1 mRNA and IGFBP-2 mRNA and protein synthesis of IGF-1 and IGFBP-2 were increased. In particular, since IGFBP-2 has an action of binding to IGF-1 and transporting it from the blood circulation to the periphery, the produced IGF-1 quickly migrates to the periphery after moving into the blood and into the tissue. It is considered that a specific action of IGF-1 is exhibited.

  According to the present invention, a collagen peptide obtained by treating and extracting a collagen peptide obtained from originally discarded fish scales and / or fish skin with a proteolytic enzyme can be used as an agent for increasing IGF-1. This agent for raising IGF-1 level is useful because it is highly safe and can increase blood IGF-1 concentration by ingestion.

Claims (3)

  An agent for increasing IGF-1 value, comprising a collagen peptide extracted by treating fish scale and / or fish skin-derived collagen with a proteolytic enzyme.   The GF according to claim 1, wherein the proteolytic enzyme is a protease produced from one of Aspergillus oryzae, Bacillus subtilis or Bacillus licheniformis. Value raising agent.   The IGF-1 value increasing agent according to claim 1 or 2, wherein the collagen peptide has a weight average molecular weight of 1,000 to 10,000.

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