WO2007108554A1 - Peptide having anti-hypertensive activity - Google Patents

Peptide having anti-hypertensive activity Download PDF

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Publication number
WO2007108554A1
WO2007108554A1 PCT/JP2007/056143 JP2007056143W WO2007108554A1 WO 2007108554 A1 WO2007108554 A1 WO 2007108554A1 JP 2007056143 W JP2007056143 W JP 2007056143W WO 2007108554 A1 WO2007108554 A1 WO 2007108554A1
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Prior art keywords
collagen
peptide
chicken
action
degradation product
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PCT/JP2007/056143
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French (fr)
Japanese (ja)
Inventor
Ai Saiga
Koji Iwai
Yoshihisa Takahata
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Nippon Meat Packers, Inc.
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Priority to JP2008506354A priority Critical patent/JPWO2007108554A1/en
Publication of WO2007108554A1 publication Critical patent/WO2007108554A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a peptide having a blood pressure increase and inhibitory action. More specifically, it is a product obtained by degrading chicken or pig-derived collagen with a protease, and relates to a peptide having an antihypertensive action, a blood vessel preservation action and the like.
  • Angiotensin Converting Enzyme also referred to as “ACE” in this specification converts angiotensin I (inactive form) into angiotensin II (active form), which has a strong vasoconstrictive action. Decomposes bradykinin, which has vasodilatory effects, into three inactive peptides.
  • ACE inhibitors substances that inhibit the enzyme activity of angiotensin converting enzyme
  • angiotensin converting enzyme such as captopril and enalapril that inhibit ACE activity (the enzyme activity of angiotensin converting enzyme) have been conventionally used as antihypertensive drugs. ing.
  • ACE inhibitors obtained from foods or food ingredients can be expected as low-toxic and highly safe antihypertensives and health-oriented foods, and can be taken in daily eating habits. So far, ACE inhibitors have been reported among many natural products and enzymatic degradation products such as food. In recent years, ingredients derived from foods such as sea bream, bonito and seaweed have been found to suppress the increase in blood pressure, and some of them are marketed as foods suitable for people with high blood pressure (for example, 2-3 1 1 4 9 4 publication, Japanese Patent Laid-Open No. 2 00 0-4 7 9 9 publication, etc.). However, there are very few ACE inhibitors derived from meat and meat products.
  • ACE inhibitors derived from meat 'meat products are not well known, and even after degradation in the digestive tract, substances (peptides) that have a sufficiently high blood pressure rise inhibitory effect (peptides) It is hardly found. Because of these problems, the present inventors have studied ACE inhibitory substances derived from meat and meat products. As a result, protease degradation products of chicken and pork collagen and peptides contained in the degradation products are found. It was also found that it has an excellent antihypertensive effect even in vivo.
  • the decomposed product when collagen extracted from chicken legs or pig skin is decomposed with various proteases and the ACE inhibitory activity of the decomposed product is measured, the decomposed product has an ACE inhibitory activity and further has a molecular weight of 3000.
  • a strong activity was observed in a fraction (Low-Frac.) Having a molecular weight of 3000 or less.
  • protease degradation product of chicken or pig collagen described above has a blood vessel preservation action.
  • the present invention is based on such findings, and has a protease degradation product and peptide of collagen derived from chicken or pig having a blood pressure increase inhibitory action and / or a blood vessel preservation action, and a functional food containing the same, and the protease degradation described above. It provides a method for producing products and peptides. Disclosure of the invention
  • the present invention made in order to solve the above-mentioned problems is a protease degradation product of collagen derived from chicken or pig having a blood pressure increase inhibiting action and / or a blood vessel preservation action.
  • it is a degradation product of collagen derived from chicken or pig collagen and has a molecular weight of 300 or less (ultrafiltration membrane method) in view of the effect of suppressing blood pressure increase and / or blood vessel preservation.
  • a peptide is preferred.
  • the present invention is a peptide having the amino acid sequence represented by SEQ ID NOs: 1 to 4, and the peptide has an antihypertensive action and / or a blood vessel preservation action.
  • the present invention is a functional food having a blood pressure increase-inhibiting action and / or a blood vessel-preserving action, which contains the aforementioned protease degradation product or peptide of chicken or pig-derived collagen.
  • the production method of the present invention is a method for producing a collagen protease degradation product, which comprises enzymatically degrading chicken or pig-derived collagen with a protease to obtain a collagen protease degradation product having an antihypertensive action and a blood vessel preservation action.
  • the collagen protease degradation product is enzymatically degraded with a protease, and if necessary, is subjected to molecular weight fractionation to collect a fraction having a molecular weight of 300 or less (ultrafiltration membrane method).
  • a method for producing a collagen peptide comprising obtaining a collagen peptide having a blood pressure elevation-inhibiting action and a blood vessel preservation action.
  • FIG. 1 is a graph showing the ACE inhibitory activity (final concentration: 0.25 mg / ml) of chicken collagen peptides treated with each enzyme of a chicken collagen protease degradation product.
  • FIG. 2 shows the ACE inhibitory activity (final concentration: 0.25 mg / ml) of chicken collagen peptide (C) and porcine collagen peptide (P). Fractionation with molecular weight 3000 using ultrafiltration membrane, low molecular weight in the figure is molecular weight 3000 or less, and high molecular weight molecular weight 3000 or more.
  • FIG. 3 shows the digestive enzyme resistance of chicken collagen peptide (C) and porcine collagen peptide (P).
  • the ACE inhibitory activity was measured at a final concentration of 0.25 mg / ml.
  • P represents pepsin treatment and T / C represents trypsin / chymotrypsin treatment.
  • fractionation is performed using an ultrafiltration membrane with a molecular weight of 3000.
  • the low molecular fraction in the figure has a molecular weight of 3000 or less, and the high molecular fraction has a molecular weight of 3000 or more.
  • FIG. 4 is a graph showing blood pressure fluctuations of SHR administered with the chicken collagen peptide of the present invention.
  • A indicates a single dose and B indicates a long-term dose.
  • FIG. 5 is a graph showing blood pressure fluctuations of subjects (two persons A and B) who took a single dose of the chicken collagen peptide of the present invention.
  • FIG. 6 is a graph showing blood pressure fluctuations of subjects (14 persons) who took the chicken collagen peptide of the present invention for a long time.
  • FIG. 7 is a graph showing changes in the number of mouthpieces in the EPC colony assay of Example 6.
  • FIG. 8 is a diagram showing changes in morphology in the EPC colony assay of Example 6.
  • Fig. 9 is a chromatogram for purifying peptides from chicken collagen peptides by reversed-phase HPLC. A is the first fraction, B is the second fraction, and C is the third fraction.
  • FIG. 10 is a graph showing the endothelial nitric oxide synthase phosphorylation (eNOS phosphorylation) activity of the chicken collagen peptide of the present invention. The figure in the figure is the final concentration (%).
  • This invention consists of said structure,
  • the protease degradation product and peptide of this invention are derived from a chicken or pig collagen.
  • the type of collagen and the collection site are not particularly limited, and various collagens can be used.
  • type I collagen derived from chicken and pig legs, skin, bones, tendons, intestines and the like is used because of its abundant raw materials.
  • the collagen can be prepared according to a conventional method.
  • the raw material is stirred for 10 hours in an acid (preferably dilute hydrochloric acid) for an appropriate time (for example, about 24 to 30 hours) and decalcified.
  • an acid preferably dilute hydrochloric acid
  • collagen includes gelatin, which is a modified product of collagen, and such gelatin can be prepared from collagen according to a conventional method.
  • the protease is not particularly limited as long as it is a protease capable of degrading collagen, and any of acidic protease, neutral protease, and alkaline protease can be used.
  • an animal-derived protease for example, trypsin, Chymotrypsin, pepsin, etc.
  • plant-derived proteases for example, papain, promeline, physine, etc.
  • microorganism-derived proteases etc.
  • coculase P coculase P (trade name) is preferably used from the viewpoint of enzyme treatment efficiency. Two or more of the above proteases are used in combination You can do it.
  • Protease treatment is carried out by adjusting the pH of the enzyme reaction solution to the optimum pH for each enzyme, reacting at 30 to 80 ° C for about 0.5 to 3 hours, and heating after the completion of the enzyme reaction. This is done by deactivating the enzyme.
  • the amount of enzyme used can be appropriately adjusted according to the desired degree of degradation, but is usually about 0.01 to 2%, preferably about 0.2%, based on collagen.
  • collagen protease degradation product can be obtained by purifying the reaction solution by conventional purification means, for example, ultrafiltration, diatomaceous earth filtration, ion exchange resin, reverse osmosis filtration, activated carbon treatment, etc. It is done. Furthermore, if necessary, the extract can be concentrated to 25-30% and dried by means of a spray dryer or the like to obtain a powder.
  • conventional purification means for example, ultrafiltration, diatomaceous earth filtration, ion exchange resin, reverse osmosis filtration, activated carbon treatment, etc. It is done.
  • the extract can be concentrated to 25-30% and dried by means of a spray dryer or the like to obtain a powder.
  • the protease degradation product thus obtained has an ACE inhibitory action and / or a blood vessel preservation action as shown in Examples below.
  • the protease degradation product obtained above can be further treated with a protease to obtain a collagen peptide having a reduced molecular weight, thereby enhancing the ACE inhibitory action and / or blood vessel preservation action. More specifically, the above protease degradation product is further added to the aforementioned protease (enzyme added to about 1% of the raw material) at the optimum pH of the enzyme and at the optimum temperature for 1 to 24 hours.
  • the collagen peptide is obtained by reacting for preferably 2 to 6 hours, more preferably about 4 hours.
  • the enzyme used for this enzyme reaction is preferably an enzyme different from the enzyme used for the enzyme reaction, that is, an enzyme having a different cleavage site. By using different enzymes, the molecular weight can be reduced efficiently.
  • the collagen peptide obtained above is preferably fractionated with a molecular weight of 3000 using an ultrafiltration membrane to obtain a peptide comprising a fraction with a molecular weight of 3000 or less.
  • the peptide thus obtained and its peptide consisting of a fraction having a molecular weight of 3000 or less can be pulverized by a conventional method such as lyophilization.
  • the peptide has an ACE inhibitory action and / or a blood vessel preservation action, as shown in Examples below.
  • Peptides in the above-mentioned fraction of collagen peptide with a molecular weight of 3000 or less can be purified by conventional purification methods such as gel filtration, ion exchange column chromatography, reversed-phase high-performance liquid. It can be purified and isolated by conventional methods such as chromatography. The structure of the isolated peptide can then be determined by protein sequencer and mass spectrometer.
  • the peptides of SEQ ID NOs: 1 to 4 may be obtained by purification and isolation from collagen peptides by the method described above, but may be synthesized by chemical methods.
  • the chemical synthesis method can be carried out in accordance with a conventional peptide synthesis method, for example, a solid phase synthesis method.
  • the functional food of the present invention comprises the above-mentioned collagen protease degradation product, collagen peptide and Z or the peptides of SEQ ID NOs: 1 to 4 as active ingredients, an antihypertensive action and / or blood vessel preservation It is a functional food having an action.
  • functional food is a concept that includes ordinary food, beverages, confectionery, feed, and the like.
  • the functional food is a functional food useful for the treatment and prevention of active ingredients as they are, added with various nutrients, or contained in foods and drinks for the purpose of suppressing blood pressure rise and / or maintaining blood vessels. It is eaten as (or food material).
  • suitable auxiliaries for example, suitable auxiliaries
  • Suitable forms such as granules, granules, tablets, capsules, pastes, etc. may be used for food, and various foods
  • processed meat products such as ham and sausage, processed fishery products such as kamaboko and chikuwa, confectionery such as snacks, dairy products such as breads, butter and milk powder, and soy products such as tofu and fried chicken
  • used for drinks such as water, fruit juice, milk, and soft drinks You may add and use. Further, it may be in the form of animal feed (including pet food).
  • the intake in the form of such functional foods is appropriately selected and determined according to age, weight, symptoms, degree of disease, form of food, etc.
  • the amount of peptide per day is 1.0 g to 10.0. g, preferably from 2.0 g to 8.0 g. However, even if a large amount is consumed, it does not adversely affect the living body. .
  • the collagen protease degradation product and peptide of the present invention can also be used as a drug having an antihypertensive action and / or a blood vessel preservation action.
  • a preparation comprises the collagen protease degradation product and peptide as active ingredients, If necessary, mix with ingredients necessary for formulation such as appropriate physiologically acceptable additives (for example, carriers, excipients, diluents, etc.) and prepare them in appropriate dosage forms. Examples of such forms include tablets, powders, granules, and capsules.
  • the dose can be appropriately determined according to the patient's symptoms, age, weight, etc., referring to the above intake. Industrial applicability
  • the protease-decomposed product and peptide of chicken or pig-derived collagen of the present invention have strong ACE inhibitory activity, and are used as a functional food or drug having an antihypertensive effect. be able to. In particular, it has the feature that the effect can be maintained even after being absorbed by the living body.
  • the chicken or pork-derived collagen protease degradation product and peptide of the present invention are made from foods that can be taken on a daily basis. It is important to prevent and improve high blood pressure, which is a lifestyle-related disease, throughout daily life. By taking such food-derived ingredients well with meals, etc., the QOL (Quality of life) of the subject is impaired. It has the feature that it can lead to normal blood pressure without.
  • the protease degradation product and peptide of collagen derived from chicken or pig of the present invention has an action of promoting the proliferation of EPC (endothelial progenitor cell), which is a cell responsible for repair of vascular endothelium, and damage of vascular endothelium. It is possible to preserve blood vessels such as maintaining functions.
  • EPC endothelial progenitor cell
  • Fresh chicken legs and pig skin were procured from the applicant company's factory.
  • Usagi lung ACE, Hip-HL (Bz-Gly-His-Leu), pepsin, trypsin, chymotrypsin were purchased from Sigma (St. Louis, MO, USA).
  • Cochlase P manufactured by Sankyo Lifetech, Tokyo, Japan
  • Amama Zamano G and Pro Leather (all from Amano Enzym, Aichi, Japan)
  • Sumiteam KP, Sumiteam FP, and Sumiteam LP Nippon Chemical Industry Co., Ltd. (Aichi, Japan) used commercial products.
  • Peptide synthesis reagents were purchased from Shimadzu (Kyoto, Japan). Other reagents were purchased from Wako Pure Chemical (Osaka, Japan).
  • the inhibitory activity against ACE was measured according to the method of Cheung et al. (Cheung et al. J. Biol. Chem. 1980, 255, 401-407). Specifically, add lOOraM boric acid solution (pH 8.3), 5 mM Hip-HL, 500 ⁇ NaCl 20 mU Usagi ACE and the test sample to a final volume of 0.25 ml, and incubate at 37 ° C for 30 minutes. It was. The enzymatic reaction was stopped by adding IN HC1. The degree of hydrolysis of Hip-HL was determined by measuring the amount of hippuric acid released at an absorbance value of 228 nm. The inhibitor concentration at 50% inhibition of ACE is IC 5 . Defined.
  • Example 1 Example 1
  • Raw chicken legs (derived from broiler) were agitated in 10 times the amount of dilute hydrochloric acid for at least 24 hours to decalcify and swell the acid. After thoroughly washing with running water, it was extracted with about twice as much warm water and filtered. The filtrate was subjected to ion exchange resin treatment and further concentrated until the extract content was 10% or more. Coculase P (0.2% based on collagen) was added to this concentrated solution, treated at 50 ° C. for 2 hours, and then heated to inactivate the enzyme. Subsequently, chicken collagen protease degradation product was obtained by purification by diatomaceous earth filtration and activated carbon treatment.
  • the resulting chicken collagen protease degradation product was further enzymatically decomposed using 6 types of enzymes (Coclase ?, Amano N, Aama / G, Pro Leather, Sumiteam KP, Sumiteam FP, and Sumiteam LP). Table 1 shows the properties of the enzymes used.
  • Figure 1 shows the ACE inhibitory activity of chicken collagen protease degradation products and collagen peptides degraded by the above enzymes.
  • Collagen before enzyme degradation did not show inhibitory activity, but chicken collagenase protease digestion product showed about 30% inhibitory activity compared to the case where no inhibitor was added (before treatment in Fig. 1).
  • the collagen peptide obtained by subjecting the chicken collagen protease degradation product to further enzyme treatment showed an increase in activity of almost twice.
  • high activity was observed for those treated with Sumitomo FP (hereinafter simply referred to as FP).
  • the above FP enzyme-treated collagen peptide was fractionated at a molecular weight of 3000 using an ultrafiltration membrane, and the strength of the ACE inhibitory activity of each fraction was compared. The results are shown in Table 2. As shown in Table 2, high activity was observed in the low molecular fraction (molecular weight of 3000 or less). Table 2. Fractionation by P membrane (molecular weight 3000)
  • porcine collagen peptide was prepared in the same manner as in Example 1 (1) using pig skin as a raw material, and its ACE inhibitory activity was measured. The results are shown in Fig. 2.
  • porcine collagen peptide also had ACE inhibitory activity, and the low molecular side had stronger activity than the high molecular side.
  • the FP enzyme-treated product had the strongest activity.
  • a model digestion system was used to investigate whether ACE inhibitory activity was maintained in the body when chicken collagen peptide (FP-treated product) and porcine collagen peptide (FP-treated product) were ingested.
  • FP enzyme-treated chicken collagen peptide and porcine collagen peptide solution were adjusted to 1% of the protein weight, and the pH of the enzyme reaction solution was adjusted to the optimum pH of each enzyme. After that, pepsin (pH 3.0) or trypsin / chymotrypsin (pH 7.0) was added and treated at 37 ° C for 1 hour. After completion of the reaction, fractionation was performed at a molecular weight of 3000 using an ultrafiltration membrane in the same manner as described above, and separated into a low molecular fraction (molecular weight of 3000) and a high molecular fraction (molecular weight> 3000).
  • ACE inhibitory activity was measured for the digested enzyme-treated product and fractions. The results are shown in Fig. 3. As shown in Figure 3, FP enzyme-treated chicken collagen peptide and porcine collagen peptide are also ACE-treated by digestive enzyme treatment (P: pepsin treatment, T / C: trypsin / chymotrypsin treatment). The inhibitory activity was maintained, and in particular, the low molecular fraction of chicken collagen peptide showed strong activity.
  • Example 4 SHR-inhibiting effect on blood pressure increase
  • Chicken collagen peptide (FP-treated product) was administered to SHR, and blood pressure fluctuations were measured over time. Specifically, male SHR (8 weeks old; Charles Liper) was raised in a room adjusted to 23 ° C and 55% humidity with commercial non-purified food (AIN-76; Oriental yeast) and water. In this study, a test subject who was able to confirm that his blood pressure had risen sufficiently was used. At the time of the test, physiological saline (control group) or chicken collagen peptide (FP-treated product) was orally administered (3 or 6 g / kg weight), and blood pressure fluctuation was measured for 24 hours (single administration system).
  • chicken collagen peptide (FP-treated product) was orally administered (6 g / kg weight) daily to male SHR, and blood pressure fluctuations were measured for 4 weeks (long-term administration system).
  • Rat tail artery pressure was measured with a non-invasive blood pressure meter (Softron98A, Softron, Tokyo, Japan). Data analysis was processed by Student's t-test. Figure 4 shows the results.
  • SHR given chicken collagen peptide decreased blood pressure 2 hours after administration and recorded minimum blood pressure at 6 hours compared to SHR in the control group (see Fig. 4A).
  • the action mechanism of the blood pressure elevation inhibiting action of the peptide of the present invention is considered to be the ACE inhibiting action.
  • the resting blood pressure was measured every 2 hours for up to 8 hours.
  • the placebo diet was evaluated on the previous day by comparison with the value at the time of ingestion. The results are shown in Fig. 5. As shown in Figure 5, the median systolic blood pressure tended to decrease 4 hours after ingestion.
  • Fig. 6 As shown in Fig. 6, as a result of the long-term administration test, there was a significant difference between the measured values before intake of the test meal, after 2 weeks, and after 4 weeks. It was confirmed that there is an effect of suppressing movement.
  • the desired daily intake of chicken collagen enzyme degradation product is considered to be 1 to 10 g, more preferably 2 to 8 g as a peptide amount.
  • Example 6 Influence on improvement of vascular repair action: EPC colony assay
  • EPC endothelial progenitor cell
  • the subject blood of the blood pressure test of Example 5 was used. That is, the same amount (10 ml) of the subject blood before and after the collagen peptide ingestion period of the present invention is collected, centrifuged at 1000 g for 30 min, and the precipitated cells are seeded in a petri dish filled with cell culture medium, 37 ° C, 5 % C0 2, after one week of culture, the number of colonies before and after collagen peptide ingestion period of the present invention was examined whether there are differences in whether colony morphology which is force ,, formed was increased.
  • Figure 7 shows the change in the number of cones in the EPC collassey
  • Figure 8 shows the change in the colony morphology.
  • Frac. 4-3 which showed the highest activity, was concentrated to isolate each peptide and subjected to HPLC with milder elution conditions with acetonitrile (0.16% / min) (Fig. See 9C).
  • the amino acid sequence of the obtained peak should be determined using a protein sequencer. .
  • Figure 9 C shows the amino acid sequence of each peak obtained and the results of mass spectrometry. As shown in Table 5, peaks 8, 9, 11 and 12 were peptides having the following amino acid sequences, respectively.
  • the peptides having the amino acid sequences shown in SEQ ID NOs: 1 to 4 were prepared by the Fmoc solid phase method using an automatic peptide synthesizer (PSSM8 Shimadzu). After deprotection, reverse phase HPLC (PEGASIL-300, 20 X 250 mm; Senshu, Tokyo, Japan) and mass spectrometer ESI mass spectrometer LC-Q (Thermo Finnigan, San jose, CA).
  • the peptide consisting of the amino acid sequence represented by SEQ ID NOs: 1 to 4 had ACE inhibitory activity.
  • the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 had strong ACE inhibitory activity.
  • Table 5 ACE P and Harmful Peptide Sequence and Mass Spectrometry
  • a chicken gelatin solution (pH 6.0) was prepared, coculase P (0.2% based on gelatin) was added thereto, treated at 50 ° C. for 3 hours, and then heated to inactivate the enzyme. Subsequently, chicken gelatin protease degradation product was obtained by refine
  • the gelatin peptide obtained above was fractionated at a molecular weight of 3000 using an ultrafiltration membrane, and a low molecular side fraction (molecular weight of 3000 or less) was collected.
  • A2 the collagen peptide before fractionation
  • A2F the low molecular fraction (molecular weight of 3000 or less)
  • vascular endothelial cells In vascular endothelial cells, the action of various cytokines causes the activation of PI3K-protein kinase Akt, followed by the activation of endothelial nitric oxide synthase (eNOS), producing NO. It causes an increase, resulting in improved vascular endothelial function such as angiogenesis, vasodilation and inhibition of monocyte adhesion.
  • eNOS endothelial nitric oxide synthase
  • the activation of eNOS is caused by the phosphorylation of its threonine and serine residues. Therefore, a substance that promotes phosphorylation of eNOS increases NO production and has an effect of improving vascular endothelial function. Become. Therefore, the eNOS phosphorylation effect of the collagen peptide of the present invention was tested.
  • HUVEC Human umbilical vein endothelial cells
  • BAEC ushi aortic vascular endothelial cells
  • ECLplus from Amersham's Pharmacia was used.
  • Piorad's DC protein assay reagent was used as a protein quantification reagent.
  • HUVEC and BAEC are DMEM containing 10% FBS (10% FBS, 100 ⁇ U / ml W
  • Phosphorylated eNOS was detected according to the method of Ju et al. (J. Biol. Chem. 1998, 273, 24025-24029.). That is, after BEAC was treated with 0% FBS-DMEM, the test peptide was added to the medium at 0.1 ° / 0 to 0.001%. In addition, 100 iM bradykinin (BK; Sigma) dissolved in the medium was added as a positive control.
  • BK bradykinin
  • Cells that have been stimulated for a certain period of time (3 minutes) are washed with PBS and washed with Lysis buffer (0.1% triton, 20 mM Tris-HCl (pH 8.0), 20 mM EDTA, ImM PMSF, ImM sodium orthovanadate, lniM leupeptin-pepstatin) Cells were lysed and the cell lysate was collected. The cell lysate that had been subjected to sonication for 15 seconds and heat treatment for 10 minutes was centrifuged (10,000 ⁇ g, lOmin) to obtain a supernatant containing eNOS.
  • Lysis buffer 0.1% triton, 20 mM Tris-HCl (pH 8.0), 20 mM EDTA, ImM PMSF, ImM sodium orthovanadate, lniM leupeptin-pepstatin
  • Samples with the same amount of protein were developed by SDS-PAGE using 7.5% gel and then transferred to a PVDF membrane. After blocking with TBS buffer containing 5% non-fat dry milk at room temperature for 2 hours, ph OS pho-eNOS antibody was used.
  • the chicken collagen peptide of the present invention was found to have a strong phosphorylation signal depending on the addition concentration, and thus was considered to activate eNOS and contribute to the improvement of vascular endothelial function.
  • CCOP a synthetic peptide, eNOS phosphate was also observed in an additive concentration-dependent manner, so CCOP is one of the components that contributes to the improvement of vascular endothelial function in chicken-derived collagen protease degradation products. I thought it was. Production example 1
  • Natural fruit juice (concentrated juice reduction) is mixed with 50 O mg of chicken collagen peptide (FP-treated product) per 20 ml of the juice, then sterilized according to conventional methods and packaged in a boutique to obtain a juice product. It was. Production example 2

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Abstract

The object is to provide an enzymatic collagen hydrolysate or a peptide which has an ACE (angiotensin converting enzyme)-inhibiting activity (i.e., an anti-hypertensive activity)/a blood vessel protection activity. Disclosed is a protease hydrolysate of a chicken or porcine collagen, which has an anti-hypertensive activity and/or a blood vessel protection activity, particularly a peptide which is a protease hydrolysate of a chicken or porcine collagen and has a molecular weight of 3000 or less. The protease hydrolysate of a chicken or porcine collagen or the peptide has an ACE-inhibiting activity and/or a blood vessel protection activity, and therefore is useful as a functional food having an anti-hypertensive activity and/or a blood vessel protection activity or the like.

Description

明細書 血圧上昇抑制作用を有するぺプチド 技術分野  Peptide having blood pressure elevation inhibiting effect Technical Field
本発明は血圧上昇,抑制作用を有するペプチドに関する。 より詳細には、 鶏又は豚 由来コラーゲンをプロテアーゼで分解した産物であり、 血圧上昇抑制作用、 血管保 全作用などを有するぺプチドに関する。 背景技術  The present invention relates to a peptide having a blood pressure increase and inhibitory action. More specifically, it is a product obtained by degrading chicken or pig-derived collagen with a protease, and relates to a peptide having an antihypertensive action, a blood vessel preservation action and the like. Background art
高血圧患者は、 アメリカ ' ョ一口ッパ · 日本で約 2億人いると言われ、 危険域の ヒ トを含めると 10億人に達すると推測されている。高血圧発症のメカニズムは種々 存在するが、 レニン ·アンジォテンシン系とカリクレイン 'キニン系が大きく関係 するとレヽわれて ヽる。アンジォテンシン (Angiotensin Converting Enzyme, 本明細書では、 「ACE」 とも言う。） は、 アンジォテンシン I (inactive form)を強い 血管収縮作用をもつアンジォテンシン II (active form)に変換すると共に、 血管拡 張作用のあるブラジキニンを不活性な 3種のペプチドに分解する。そこで、 ACE活性 (アンジォテンシン変換酵素の酵素活性） を阻害するカプトプリルやェナラプリル などの ACE阻害物質 （アンジォテンシン変換酵素の酵素活性を阻害する物質） が高 血圧治療薬として、 従来から利用されている。  There are approximately 200 million people with hypertension in the United States, Japan, and it is estimated that 1 billion people will be included in the risk zone. There are various mechanisms for the development of hypertension, but it is believed that the renin-angiotensin system and the kallikrein'kinin system are closely related. Angiotensin Converting Enzyme (also referred to as “ACE” in this specification) converts angiotensin I (inactive form) into angiotensin II (active form), which has a strong vasoconstrictive action. Decomposes bradykinin, which has vasodilatory effects, into three inactive peptides. Therefore, ACE inhibitors (substances that inhibit the enzyme activity of angiotensin converting enzyme) such as captopril and enalapril that inhibit ACE activity (the enzyme activity of angiotensin converting enzyme) have been conventionally used as antihypertensive drugs. ing.
食品又は食品原料から得られる ACE阻害物質は低毒性で安全性の高い降圧剤、 健 康志向食品として期待でき、 日常の食生活において摂取可能となる。 これまで多く の天然物や食品等の酵素分解物の中から ACE阻害物質が報告されている。 近年、 ィ ヮシ、 カツォ、 ワカメなど食品由来成分に血圧の上昇を抑制する作用が見出されて おり、 一部は血圧が高めの人に適した食品として市販されている （例えば、 特開平 2 - 3 1 1 4 9 4公報、 特開 2 0 0 0— 4 7 9 9公報など)。 し力 し、 食肉 ·食肉製 品に由来する ACE阻害物質は非常に少ない。  ACE inhibitors obtained from foods or food ingredients can be expected as low-toxic and highly safe antihypertensives and health-oriented foods, and can be taken in daily eating habits. So far, ACE inhibitors have been reported among many natural products and enzymatic degradation products such as food. In recent years, ingredients derived from foods such as sea bream, bonito and seaweed have been found to suppress the increase in blood pressure, and some of them are marketed as foods suitable for people with high blood pressure (for example, 2-3 1 1 4 9 4 publication, Japanese Patent Laid-Open No. 2 00 0-4 7 9 9 publication, etc.). However, there are very few ACE inhibitors derived from meat and meat products.
また、 食品由来の ACE阻害物質の中には、 消化器官で分解されたりして活性が低 下するものもあり、 その有効性が疑問視されるものもある。 そのため、 通常の食事 における摂取、 つまり消化器官で分解などがされた後でも、 十分に高い血圧上昇抑 制作用を有する物質 （ペプチド） が望まれるが、 係る物質はほとんど見出されてい なかった。 In addition, some food-derived ACE inhibitors are degraded in the digestive tract and their activity decreases, and some of them are questionable for their effectiveness. For this reason, even after ingestion in a normal diet, that is, after decomposition in the digestive tract, a sufficiently high suppression of blood pressure increase is achieved. Substances (peptides) for production purposes are desired, but few such substances have been found.
上述のように、食肉'食肉製品に由来する ACE阻害物質はあまり知られておらず、 更に消化器官で分解などがされた後でも、 十分に高い血圧上昇抑制作用を有する物 質 （ペプチド） はほとんど見出されていない。 このような問題点から、 本願発明者 らは、 食肉 ·食肉製品に由来する ACE阻害物質を研究したところ、 鶏及び豚由来コ ラーゲンのプロテアーゼ分解物及びこの分解物に含有されているぺプチドが、 in vivoにおいても優れた血圧上昇抑制作用を有することを見出した。  As mentioned above, ACE inhibitors derived from meat 'meat products are not well known, and even after degradation in the digestive tract, substances (peptides) that have a sufficiently high blood pressure rise inhibitory effect (peptides) It is hardly found. Because of these problems, the present inventors have studied ACE inhibitory substances derived from meat and meat products. As a result, protease degradation products of chicken and pork collagen and peptides contained in the degradation products are found. It was also found that it has an excellent antihypertensive effect even in vivo.
より具体的には、 鶏足又は豚皮等から抽出したコラーゲンを各種プロテアーゼで 分解し、 当該分解物の ACE阻害活性を測定したところ、 当該分解物は ACE阻害活性 を有し、 更に分子量 3000の限外濾過膜で分画したところ、 分子量 3000以下の画分 (Low-Frac. )に強い活性が認められた。  More specifically, when collagen extracted from chicken legs or pig skin is decomposed with various proteases and the ACE inhibitory activity of the decomposed product is measured, the decomposed product has an ACE inhibitory activity and further has a molecular weight of 3000. When fractionated with an ultrafiltration membrane, a strong activity was observed in a fraction (Low-Frac.) Having a molecular weight of 3000 or less.
また、 Low-Frac.を咼'血圧自然発 ¾£ラッ卜 (Spontaneously Hypertensive Rat, SHR) に投与したところ、 2時間後から血圧の低下が認められ、 4週間の長期投与試験にお いても、 有意に血圧の上昇を抑制することが明らかとなった。 次いで、 この画分を さらに HPLCで分画し、 高い ACE阻害活性を有する画分のぺプチドを得、 それにつ'い てプロテインシークェンサ一でアミノ酸配列を決定した。  In addition, when Low-Frac. Was administered to spontaneously hypertensive rats (SHR), blood pressure decreased after 2 hours. In a 4-week long-term administration study, It was revealed that the increase in blood pressure was significantly suppressed. Subsequently, this fraction was further fractionated by HPLC to obtain a peptide having a high ACE inhibitory activity, and the amino acid sequence was determined with a protein sequencer.
更に、 上述の鶏又は豚由来コラーゲンのプロテァーゼ分解物は血管保全作用を有 することも判明した。  Furthermore, it was also found that the protease degradation product of chicken or pig collagen described above has a blood vessel preservation action.
本発明は係る知見に基づくもので、血圧上昇抑制作用及び/又は血管保全作用を有 する、 鶏又は豚由来コラーゲンのプロテアーゼ分解物及びぺプチド並びにそれを含 有する機能性食品、 更に上記のプロテアーゼ分解物及びぺプチドを製造する方法を 提供するものである。 . 発明の開示  The present invention is based on such findings, and has a protease degradation product and peptide of collagen derived from chicken or pig having a blood pressure increase inhibitory action and / or a blood vessel preservation action, and a functional food containing the same, and the protease degradation described above. It provides a method for producing products and peptides. Disclosure of the invention
上記の課題を解決するためになされた本発明は、血圧上昇抑制作用及び/又は血管 保全作用を有する鶏又は豚由来コラーゲンのプロテアーゼ分解物である。 特に、 血 圧上昇抑制作用及び/又血管保全作用の効果の面から、鶏又は豚由来コラーゲンのプ 口テアーゼ分解物であって、 分子量が 3 0 0 0以下 （限外濾過膜法） であるべプチ ドが好ましい。 また、 本発明は、 配列番号 1〜4で示されるアミノ酸配列からなるペプチドで、 係るペプチドは血圧上昇抑制作用及び/又は血管保全作用を有する。 The present invention made in order to solve the above-mentioned problems is a protease degradation product of collagen derived from chicken or pig having a blood pressure increase inhibiting action and / or a blood vessel preservation action. In particular, it is a degradation product of collagen derived from chicken or pig collagen and has a molecular weight of 300 or less (ultrafiltration membrane method) in view of the effect of suppressing blood pressure increase and / or blood vessel preservation. A peptide is preferred. In addition, the present invention is a peptide having the amino acid sequence represented by SEQ ID NOs: 1 to 4, and the peptide has an antihypertensive action and / or a blood vessel preservation action.
更に、 本発明は、 上述した鶏又は豚由来コラーゲンのプロテアーゼ分解物又はぺ プチドを含有する、血圧上昇抑制作用及び/又は血管保全作用を有する機能性食品で ある。  Furthermore, the present invention is a functional food having a blood pressure increase-inhibiting action and / or a blood vessel-preserving action, which contains the aforementioned protease degradation product or peptide of chicken or pig-derived collagen.
また、 本発明の製造方法は、 鶏又は豚由来コラーゲンをプロテアーゼで酵素分解 して、 血圧上昇抑制作用及び血管保全作用を有するコラーゲンプロテアーゼ分解物 を得ることからなるコラーゲンプロテアーゼ分解物の製造方法であり、 また当該コ ラーゲンプロテアーゼ分解物をプロテアーゼで酵素分解して、 また必要に応じて分 子量分画に付しして分子量 3 0 0 0以下 （限外濾過膜法） の画分を採取し、 血圧上 昇抑制作用及び血管保全作用を有するコラーゲンべプチドを得ることからなるコラ 一ゲンべプチドの製造方法である。 図面の簡単な説明  The production method of the present invention is a method for producing a collagen protease degradation product, which comprises enzymatically degrading chicken or pig-derived collagen with a protease to obtain a collagen protease degradation product having an antihypertensive action and a blood vessel preservation action. In addition, the collagen protease degradation product is enzymatically degraded with a protease, and if necessary, is subjected to molecular weight fractionation to collect a fraction having a molecular weight of 300 or less (ultrafiltration membrane method). A method for producing a collagen peptide comprising obtaining a collagen peptide having a blood pressure elevation-inhibiting action and a blood vessel preservation action. Brief Description of Drawings
図 1は、 鶏コラーゲンプロテアーゼ分解物を、 各酵素で処理した鶏コラーゲンぺ プチドの ACE阻害活性 (終濃度： 0. 25mg/ml)を示す図である。  FIG. 1 is a graph showing the ACE inhibitory activity (final concentration: 0.25 mg / ml) of chicken collagen peptides treated with each enzyme of a chicken collagen protease degradation product.
図 2は、鶏コラーゲンぺプチド（C)と豚コラーゲンぺプチド (P)の ACE阻害活性 (終 濃度： 0. 25mg/ml)を示す図である。 限外濾過膜を用いて分子量 3000で分画し、 図中 の低分子は分子量 3000以下、 高分子は分子量 3000以上である。  FIG. 2 shows the ACE inhibitory activity (final concentration: 0.25 mg / ml) of chicken collagen peptide (C) and porcine collagen peptide (P). Fractionation with molecular weight 3000 using ultrafiltration membrane, low molecular weight in the figure is molecular weight 3000 or less, and high molecular weight molecular weight 3000 or more.
図 3は、 鶏コラーゲンペプチド （C) と豚コラーゲンペプチド （P) の消化酵素耐 性を示す図であり、ACE阻害活性は終濃度 0. 25mg/mlで測定した。 Pはペプシン処理、 T/Cはトリプシン/キモトリプシン処理を示す。 また限外濾過膜を用いて分子量 3000 で分画し、 図中の低分子画分は分子量 3000以下、 高分子画分は分子量 3000以上で ある。  FIG. 3 shows the digestive enzyme resistance of chicken collagen peptide (C) and porcine collagen peptide (P). The ACE inhibitory activity was measured at a final concentration of 0.25 mg / ml. P represents pepsin treatment and T / C represents trypsin / chymotrypsin treatment. In addition, fractionation is performed using an ultrafiltration membrane with a molecular weight of 3000. The low molecular fraction in the figure has a molecular weight of 3000 or less, and the high molecular fraction has a molecular weight of 3000 or more.
図 4は、 本発明の鶏コラーゲンペプチドを投与した SHRの血圧変動を示す図であ る。 Aは単回投与、 Bは長期投与を示す。  FIG. 4 is a graph showing blood pressure fluctuations of SHR administered with the chicken collagen peptide of the present invention. A indicates a single dose and B indicates a long-term dose.
図 5は、 本発明の鶏コラーゲンペプチドを単回摂取した被験者 （A及び Bの 2名） の血圧変動を示す図である。  FIG. 5 is a graph showing blood pressure fluctuations of subjects (two persons A and B) who took a single dose of the chicken collagen peptide of the present invention.
図 6は、 本発明の鶏コラーゲンペプチドを長期摂取した被験者 （1 4名） の血圧 変動を示す図である。 図 7は、 実施例 6の E P Cコロニーアッセィにおけるコ口ニー数の変化を示す図 である。 FIG. 6 is a graph showing blood pressure fluctuations of subjects (14 persons) who took the chicken collagen peptide of the present invention for a long time. FIG. 7 is a graph showing changes in the number of mouthpieces in the EPC colony assay of Example 6.
図 8は、実施例 6の E P Cコロニーアッセィにおける形態の変化を示す図である。 図 9は、鶏コラーゲンぺプチドから逆相 HPLCでぺプチドを精製するクロマト図で あり、 Aは第 1フラクション、 Bは第 2フラクション、 Cは第 3フラクションであ る。  FIG. 8 is a diagram showing changes in morphology in the EPC colony assay of Example 6. Fig. 9 is a chromatogram for purifying peptides from chicken collagen peptides by reversed-phase HPLC. A is the first fraction, B is the second fraction, and C is the third fraction.
図 1 0は、 本発明の鶏コラーゲンぺプチドの内皮型一酸化窒素合成酵素リン酸化 (eNOSリン酸化)活性を示す図である。 図中の数値は終濃度 （％) である。 発明を実施するための最良の形態  FIG. 10 is a graph showing the endothelial nitric oxide synthase phosphorylation (eNOS phosphorylation) activity of the chicken collagen peptide of the present invention. The figure in the figure is the final concentration (%). BEST MODE FOR CARRYING OUT THE INVENTION
本発明は上記の構成からなり、 本発明のプロテアーゼ分解物及びペプチドは、 鶏 又は豚コラーゲンに由来する。  This invention consists of said structure, The protease degradation product and peptide of this invention are derived from a chicken or pig collagen.
使用されるコラーゲンに関し、 コラーゲンのタイプ及び採取部位などは特に限定 されず、 種々のコラーゲンを使用することができる。 好適には、 原料が豊富である ことから、 鶏及び豚の足、 皮、 骨、 腱、 腸などに由来する I型コラーゲンが使用さ れる。  Regarding the collagen to be used, the type of collagen and the collection site are not particularly limited, and various collagens can be used. Preferably, type I collagen derived from chicken and pig legs, skin, bones, tendons, intestines and the like is used because of its abundant raw materials.
上記コラーゲンは常法に準じて調製することができ、例えば、原料を 10倍量程度 の酸 （好ましくは希塩酸） 中で、 適当な時間 （例えば 24時間〜 30時間程度） 撹拌 処理し、 脱灰と酸膨潤を行い、 十分に流水洗浄した後、 適当量 （例えば、 約 2倍量） の温水で抽出 '濾過し、 濾液を必要に応じてイオン交換担体などで処理し、 適当な 濃度 （例えば、 1 0 %程度） に濃縮することにより得ることができる。  The collagen can be prepared according to a conventional method. For example, the raw material is stirred for 10 hours in an acid (preferably dilute hydrochloric acid) for an appropriate time (for example, about 24 to 30 hours) and decalcified. After swelling with acid and thoroughly washing with running water, extract and filter with an appropriate amount (for example, about 2 times the amount of warm water), treat the filtrate with an ion exchange carrier, etc., if necessary. , About 10%).
なお、 本発明において、 コラーゲンにはコラーゲンの変性物であるゼラチンも包 含され、 係るゼラチンは常法に準じてコラーゲンより調製することができる。  In the present invention, collagen includes gelatin, which is a modified product of collagen, and such gelatin can be prepared from collagen according to a conventional method.
次いで、 上記で調製されたコラーゲン溶液をプロテアーゼで処理する。 プロテア ーゼとしては、 コラーゲンを酵素分解できるプロテアーゼであれば特に限定はされ ず、 酸性プロテアーゼ、 中性プロテアーゼ、 アルカリ性プロテアーゼのいずれも使 用することができ、 例えば、 動物由来プロテアーゼ （例えば、 トリプシン、 キモト リプシン、 ペプシン等）、 植物由来プロテアーゼ （例えば、 パパイン、 プロメリン、 フイシン等）、 微生物由来のプロテアーゼなどが挙げられ、 酵素処理効率の点からコ クラーゼ P (商品名） が好適に使用される。 上記のプロテアーゼは 2種以上を併用 してもよレ、。 Next, the collagen solution prepared above is treated with protease. The protease is not particularly limited as long as it is a protease capable of degrading collagen, and any of acidic protease, neutral protease, and alkaline protease can be used. For example, an animal-derived protease (for example, trypsin, Chymotrypsin, pepsin, etc.), plant-derived proteases (for example, papain, promeline, physine, etc.), microorganism-derived proteases, etc., and coculase P (trade name) is preferably used from the viewpoint of enzyme treatment efficiency. Two or more of the above proteases are used in combination You can do it.
プロテアーゼ処理は、 酵素反応液の p Hを各酵素の至適 p Hに調整した後、 3 0 〜8 0 °C、 0 . 5〜3時間程度反応させ、 酵素反応終了後加熱などの方法により酵 素を失活させることにより行われる。 酵素の使用量は所望する分解度に応じて適宜 調整することができるが、コラーゲンに対して通常 0. 01〜 2 %程度、好ましくは 0. 2% 程度で使用される。  Protease treatment is carried out by adjusting the pH of the enzyme reaction solution to the optimum pH for each enzyme, reacting at 30 to 80 ° C for about 0.5 to 3 hours, and heating after the completion of the enzyme reaction. This is done by deactivating the enzyme. The amount of enzyme used can be appropriately adjusted according to the desired degree of degradation, but is usually about 0.01 to 2%, preferably about 0.2%, based on collagen.
反応終了後、 必要に応じて、 反応液を慣用の精製手段、 例えば、 限外濾過、 珪藻 土濾過、 イオン交換樹脂、 逆浸透濾過、 活性炭処理などにより精製することにより コラーゲンのプロテアーゼ分解物が得られる。更に必要に応じて、エキス分 25-30% になるまで濃縮し、 スプレードライヤーなどの手段で乾燥して粉末状とすることが できる。  After completion of the reaction, collagen protease degradation product can be obtained by purifying the reaction solution by conventional purification means, for example, ultrafiltration, diatomaceous earth filtration, ion exchange resin, reverse osmosis filtration, activated carbon treatment, etc. It is done. Furthermore, if necessary, the extract can be concentrated to 25-30% and dried by means of a spray dryer or the like to obtain a powder.
かくして得られたプロテアーゼ分解物は、 後記実施例に示されるように ACE阻害 作用及び/又は血管保全作用を有する。  The protease degradation product thus obtained has an ACE inhibitory action and / or a blood vessel preservation action as shown in Examples below.
上記で得られたプロテアーゼ分解物は、 更にプロテアーゼで処理して分子量を低 減ィ匕したコラーゲンぺプチドとすることにより、 ACE 阻害作用及び/又は血管保全 作用を強めることができる。 より具体的には、 上記のプロテアーゼ分解物を、 更に 前述のプロテアーゼ（原料に対し、 1%程度酵素添加）を用いて、 酵素の至適 p H、 至 適温度で、 1〜 2 4時間、 好ましくは 2〜 6時間、 更に好ましくは 4時間程度反応 させ、 コラーゲンペプチドが得られる。  The protease degradation product obtained above can be further treated with a protease to obtain a collagen peptide having a reduced molecular weight, thereby enhancing the ACE inhibitory action and / or blood vessel preservation action. More specifically, the above protease degradation product is further added to the aforementioned protease (enzyme added to about 1% of the raw material) at the optimum pH of the enzyme and at the optimum temperature for 1 to 24 hours. The collagen peptide is obtained by reacting for preferably 2 to 6 hours, more preferably about 4 hours.
この酵素反応に使用される酵素は、 前記の酵素反応に使用された酵素とは異なる 酵素、 即ち切断部位が異なる酵素を使用するのが好ましい。 異なる酵素を使用する ことにより、 低分子化を効率よく行うことができる。  The enzyme used for this enzyme reaction is preferably an enzyme different from the enzyme used for the enzyme reaction, that is, an enzyme having a different cleavage site. By using different enzymes, the molecular weight can be reduced efficiently.
上記で得られたコラーゲンぺプチドは、限外濾過膜を用いて分子量 3000で分画を 行い、 分子量 3000以下の画分からなるぺプチドとするのが好ましい。  The collagen peptide obtained above is preferably fractionated with a molecular weight of 3000 using an ultrafiltration membrane to obtain a peptide comprising a fraction with a molecular weight of 3000 or less.
かくして得られるコラーゲンぺプチド及びそれの分子量 3000以下の画分からなる ペプチドは、 凍結乾燥などの慣用の方法にて粉末化することができる。 また、 当該 ぺプチドは後記実施例に示されるように ACE阻害作用及び/又は血管保全作用を有す る。  The peptide thus obtained and its peptide consisting of a fraction having a molecular weight of 3000 or less can be pulverized by a conventional method such as lyophilization. In addition, the peptide has an ACE inhibitory action and / or a blood vessel preservation action, as shown in Examples below.
上記のコラーゲンぺプチドの分子量 3000以下の画分の中のぺプチドは、慣用の精 製手段、 例えば、 ゲル濾過、 イオン交換カラムクロマトグラフィー、 逆相高速液体 クロマトグラフィーなどの慣用の方法で精製し、 単離することができる。 次いで、 単離したペプチドの構造をプロテインシーケンサーと質量分析計により決定するこ とができる。 Peptides in the above-mentioned fraction of collagen peptide with a molecular weight of 3000 or less can be purified by conventional purification methods such as gel filtration, ion exchange column chromatography, reversed-phase high-performance liquid. It can be purified and isolated by conventional methods such as chromatography. The structure of the isolated peptide can then be determined by protein sequencer and mass spectrometer.
その結果、 ACE阻害作用 ·血管保全作用を有するぺプチドとして、 下記のぺプチド が得られた。  As a result, the following peptides were obtained as peptides having an ACE inhibitory action / vascular preservation action.
Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro (配列番号 1 )  Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro (SEQ ID NO: 1)
Gly-Ala-Hyp-Gly-Pro-Ala-Gly-Pro-Gly-Gly-Ile-Hyp-Gly-Glu-Arg-Gly (配列番号 2 )  Gly-Ala-Hyp-Gly-Pro-Ala-Gly-Pro-Gly-Gly-Ile-Hyp-Gly-Glu-Arg-Gly (SEQ ID NO: 2)
Gly-Leu-Hyp-Gly-Ser-Arg-Gly-Glu-Arg-Gly-Leu-Hyp-Gly (配列番号 3 )  Gly-Leu-Hyp-Gly-Ser-Arg-Gly-Glu-Arg-Gly-Leu-Hyp-Gly (SEQ ID NO: 3)
Gly-Ile-Hyp-Gly-Glu-Arg-Gly-Glu-Hyp-Gly-Pro-Val-Gly-Pro-Ser-Gly (配列番号 4 ) Gly-Ile-Hyp-Gly-Glu-Arg-Gly-Glu-Hyp-Gly-Pro-Val-Gly-Pro-Ser-Gly (SEQ ID NO: 4)
上記の配列番号 1〜4のペプチドは、 前述の方法により、 コラーゲンペプチドよ り精製 ·単離して取得してもよいが、 化学的方法で合成してもよい。 化学的合成方 法は、 慣用のぺプチド合成法に準じて行うことができ、 例えば固相合成法などが例 示できる。  The peptides of SEQ ID NOs: 1 to 4 may be obtained by purification and isolation from collagen peptides by the method described above, but may be synthesized by chemical methods. The chemical synthesis method can be carried out in accordance with a conventional peptide synthesis method, for example, a solid phase synthesis method.
本発明の機能性食品は、 前記のコラーゲンプロテアーゼ分解物、 コラーゲンぺプ チド及ぴ Z又は配列番号 1〜 4のぺプチドを有効成分として含有することからなる、 血圧上昇抑制作用及び/又は血管保全作用を有する機能性食品である。 なお、 本明細 書において、 機能性食品とは、 通常の食品、 飲料、 菓子類、 飼料などを含む概念で ある。  The functional food of the present invention comprises the above-mentioned collagen protease degradation product, collagen peptide and Z or the peptides of SEQ ID NOs: 1 to 4 as active ingredients, an antihypertensive action and / or blood vessel preservation It is a functional food having an action. In this specification, functional food is a concept that includes ordinary food, beverages, confectionery, feed, and the like.
当該機能性食品は、 有効成分そのまま、 又は種々の栄養分を加えて、 若しくは飲 食品中に含有せしめて、血圧上昇抑制及び/又は血管保全を目的として、 その治療及 び予防に有用な機能性食品 （又は食品素材） として食される。 例えば、 適当な助剤 The functional food is a functional food useful for the treatment and prevention of active ingredients as they are, added with various nutrients, or contained in foods and drinks for the purpose of suppressing blood pressure rise and / or maintaining blood vessels. It is eaten as (or food material). For example, suitable auxiliaries
(例えば、 グルコース、 乳糖、 ショ糖、 澱粉、 マンニトール、 デキストリン、 ポリ エチレングリコ一ノレ、 ヒ ドロキシェチノレデンプン、 エチレングリコーノレ、 アミノ酸 等） を添加した後、 慣用の手段を用いて、 食用に適した形態、 例えば、 顆粒状、 粒 状、 錠剤、 カプセル、 ペース ト等に成形して食用に供してもよく、 また種々の食品(E.g. glucose, lactose, sucrose, starch, mannitol, dextrin, polyethylene glycol mononole, hydroxychuccinole starch, ethylene glyconole, amino acid, etc.) Suitable forms such as granules, granules, tablets, capsules, pastes, etc. may be used for food, and various foods
(例えば、 ハム、 ソーセージ等の食肉加工食品、 かまぼこ、 ちくわ等の水産加工食 品、 スナック菓子等の菓子類、 パン類、 バター、 粉乳等の乳製品、 豆腐、 油揚げ等 の大豆製品など） に添加して使用されたり、 水、 果汁、 牛乳、 清涼飲料等の飲物に 添加して使用してもよい。 また、 動物用の飼料 （ペットフードなどを含む） の形態 であってもよい。 (For example, processed meat products such as ham and sausage, processed fishery products such as kamaboko and chikuwa, confectionery such as snacks, dairy products such as breads, butter and milk powder, and soy products such as tofu and fried chicken) Used for drinks such as water, fruit juice, milk, and soft drinks You may add and use. Further, it may be in the form of animal feed (including pet food).
係る機能性食品の形態における摂取量は、 年齢、 体重、 症状、 疾患の程度、 食品 の形態等により、 適宜選択 ·決定され、 例えば、 1日当りペプチドの量として、 1. 0 g〜10. 0 g程度、 好ましくは 2. 0 g〜8. 0 gとされるが、 多量に摂取しても生体に悪 影響を与えなレ、利点を有することから、 それ以上の量を摂取してもよい。  The intake in the form of such functional foods is appropriately selected and determined according to age, weight, symptoms, degree of disease, form of food, etc. For example, the amount of peptide per day is 1.0 g to 10.0. g, preferably from 2.0 g to 8.0 g. However, even if a large amount is consumed, it does not adversely affect the living body. .
また、 本発明のコラーゲンプロテアーゼ分解物及びペプチドは、 血圧上昇抑制作 用及び/又は血管保全作用を有する薬剤としても利用することができ、 係る製剤は、 コラーゲンプロテアーゼ分解物及びペプチドを有効成分とし、 必要に応じて、 適宜 の生理的に許容される添加剤 （例えば、 担体、 賦形剤、 希釈剤等） などの製剤上必 要な成分と混合し、 適宜な剤形の形態に調製することにより得られ、 係る形態とし ては錠剤状、 粉末状、 顆粒状、 カプセル剤状などが例示できる。 投与量は、 患者の 症状、 年齢、 体重などに応じて、 上記の摂取量を参考にして適宜決定することがで きる。 産業上の利用可能性  The collagen protease degradation product and peptide of the present invention can also be used as a drug having an antihypertensive action and / or a blood vessel preservation action. Such a preparation comprises the collagen protease degradation product and peptide as active ingredients, If necessary, mix with ingredients necessary for formulation such as appropriate physiologically acceptable additives (for example, carriers, excipients, diluents, etc.) and prepare them in appropriate dosage forms. Examples of such forms include tablets, powders, granules, and capsules. The dose can be appropriately determined according to the patient's symptoms, age, weight, etc., referring to the above intake. Industrial applicability
後記実施例に示されるよう、 本発明の鶏又は豚由来コラーゲンのプロテアーゼ分 解物及びべプチドは強い ACE阻害活性を有しており、 血圧上昇抑制作用を有する機 能性食品又は薬剤として利用することができる。 特に生体に吸収された後において も効果を持続することができるという特長を有している。 本発明の鶏又は豚由来コ ラーゲンのプロテアーゼ分解物及びぺプチドは、 日常的に摂取可能な食品を原料と している。 生活習慣病である高血圧は、 日常の生活を通して、 予防 ·改善すること が重要であり、 このような食品由来成分を食事などによりうまく摂取することで、 対象者の QOL (Quality of life) を損なうことなく、 正常血圧へ導くことができる という特長を有する。  As shown in the examples described later, the protease-decomposed product and peptide of chicken or pig-derived collagen of the present invention have strong ACE inhibitory activity, and are used as a functional food or drug having an antihypertensive effect. be able to. In particular, it has the feature that the effect can be maintained even after being absorbed by the living body. The chicken or pork-derived collagen protease degradation product and peptide of the present invention are made from foods that can be taken on a daily basis. It is important to prevent and improve high blood pressure, which is a lifestyle-related disease, throughout daily life. By taking such food-derived ingredients well with meals, etc., the QOL (Quality of life) of the subject is impaired. It has the feature that it can lead to normal blood pressure without.
また、 本発明の鶏又は豚由来コラーゲンのプロテアーゼ分解物及びぺプチドは、 血管内皮の修復を担う細胞である EPC (endothelial progenitor cell)の増殖を促進 させる作用を有しており、 血管内皮の障害、 機能保持などの血管保全を図ることが できる。  In addition, the protease degradation product and peptide of collagen derived from chicken or pig of the present invention has an action of promoting the proliferation of EPC (endothelial progenitor cell), which is a cell responsible for repair of vascular endothelium, and damage of vascular endothelium. It is possible to preserve blood vessels such as maintaining functions.
更に、 本発明の製造方法によれば、 上記のプロテアーゼ分解物及ぴペプチドを簡 便且つ確実に製造することができる 実施例 Furthermore, according to the production method of the present invention, the above protease degradation product and peptide are simplified. Examples that can be manufactured with convenience and reliability
以下、 実施例に基づいて本発明をより詳細に説明するが、 本発明はこれらの例に 限定されるものではない。 なお、 使用した材料及び ACE阻害活性測定法は以下のと おりである。  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these examples. The materials used and the ACE inhibitory activity measurement method are as follows.
(1)材料  (1) Material
新鮮な鶏足と豚皮は出願人会社の工場から調達した。 ゥサギ肺 ACE、 Hip-HL (Bz-Gly- His- Leu)、ペプシン、 トリプシン、キモトリプシンはシグマ (St. Louis, MO, USA) から購入した。 コクラーゼ P (三共ライフテック社製、東京、 日本）、 アマ Ν、 Αァマノ G及びプロレザー（いずれも天野ェンザィム社製、愛知、 日本）、 スミチーム KP、 スミチーム FP及びスミチーム LP (レ、ずれも新日本化学工業社製、 愛知、 日本） は市販品を使用した。 ペプチド合成試薬は島津 （京都、 日本） より購入した。 その 他の試薬は和光純薬 （大阪、 日本） より購入した。  Fresh chicken legs and pig skin were procured from the applicant company's factory. Usagi lung ACE, Hip-HL (Bz-Gly-His-Leu), pepsin, trypsin, chymotrypsin were purchased from Sigma (St. Louis, MO, USA). Cochlase P (manufactured by Sankyo Lifetech, Tokyo, Japan), Amama, Zamano G and Pro Leather (all from Amano Enzym, Aichi, Japan), Sumiteam KP, Sumiteam FP, and Sumiteam LP Nippon Chemical Industry Co., Ltd. (Aichi, Japan) used commercial products. Peptide synthesis reagents were purchased from Shimadzu (Kyoto, Japan). Other reagents were purchased from Wako Pure Chemical (Osaka, Japan).
(2) ACE阻害活性測定  (2) ACE inhibitory activity measurement
ACEに対する阻害活性の測定は Cheungらの方法 (Cheung et al. J. Biol. Chem. 1980, 255, 401-407) に従って行った。 具体的には、 最終容量が 0. 25mlとなるよう に lOOraMホウ酸溶液 （pH8. 3)、 5mM Hip-HL, 500πιΜ NaCl 20mUゥサギ ACE及び試験 試料を加え、 37°Cで 30分間インキュベートをおこなった。 酵素反応は IN HC1 の添 加で停止させた。 Hip- HLの加水分解の程度は放出された馬尿酸 （Hippuric acid)量 を吸光値 228nmで測定した。 ACEを 50%阻害するときの阻害剤濃度を IC5。と定義した。 実施例 1 The inhibitory activity against ACE was measured according to the method of Cheung et al. (Cheung et al. J. Biol. Chem. 1980, 255, 401-407). Specifically, add lOOraM boric acid solution (pH 8.3), 5 mM Hip-HL, 500πιΜ NaCl 20 mU Usagi ACE and the test sample to a final volume of 0.25 ml, and incubate at 37 ° C for 30 minutes. It was. The enzymatic reaction was stopped by adding IN HC1. The degree of hydrolysis of Hip-HL was determined by measuring the amount of hippuric acid released at an absorbance value of 228 nm. The inhibitor concentration at 50% inhibition of ACE is IC 5 . Defined. Example 1
(1)コラーゲンプロテアーゼ分解物及びコラーゲンぺプチドの調製  (1) Preparation of collagen protease degradation product and collagen peptide
原料の鶏足（ブロイラー由来）を 10倍量の希塩酸中で、 24時間以上撹拌処理し、 脱灰と酸膨潤を行った。そして、十分に流水洗浄した後、約 2倍量の温水で抽出し、 濾過した。 濾液をイオン交換榭脂処理し、 更に、 エキス分 10%以上になるまで濃縮 した。 この濃縮液にコクラーゼ P (コラーゲンに対して 0.2%) を加え、 50°C, 2時 間処理した後、 加熱して酵素を失活させた。 次いで、 珪藻土濾過、 活性炭処理によ り精製することにより鶏コラーゲンプロテアーゼ分解物を得た。 得られた鶏コラーゲンプロテアーゼ分解物は、 更に 6種類の酵素 （コクラ一ゼ？、 ァマノ N、 Aアマ / G、 プロレザー、 スミチーム KP、 スミチーム FP、 スミチーム LP) を用いて酵素分解した。 使用した酵素の性状を表 1に示す。 Raw chicken legs (derived from broiler) were agitated in 10 times the amount of dilute hydrochloric acid for at least 24 hours to decalcify and swell the acid. After thoroughly washing with running water, it was extracted with about twice as much warm water and filtered. The filtrate was subjected to ion exchange resin treatment and further concentrated until the extract content was 10% or more. Coculase P (0.2% based on collagen) was added to this concentrated solution, treated at 50 ° C. for 2 hours, and then heated to inactivate the enzyme. Subsequently, chicken collagen protease degradation product was obtained by purification by diatomaceous earth filtration and activated carbon treatment. The resulting chicken collagen protease degradation product was further enzymatically decomposed using 6 types of enzymes (Coclase ?, Amano N, Aama / G, Pro Leather, Sumiteam KP, Sumiteam FP, and Sumiteam LP). Table 1 shows the properties of the enzymes used.
この酵素分解反応は、 50° (：、 プロレザーについては pH10. 0、 その他の酵素につい ては pH6. 0で 4時間から 24時間酵素処理し（原料に対し、 1%酵素添加）、 各酵素処 理は 20分間の加熱により酵素を失活させ、遠心処理を行って上清を採取することに よりコラーゲンぺプチドを得た。 表 1 . 使用酵素一覧 The enzymatic degradation reaction, 5 0 ° (:.. , PH10 For Proleather 0, with respect to other about the enzyme pH6 for 24 hours the enzyme treatment from 0 for 4 h (starting material, 1% enzyme added), each Enzyme treatment was performed by heating for 20 minutes to inactivate the enzyme, centrifuging and collecting the supernatant to obtain collagen peptides Table 1. List of enzymes used
Figure imgf000010_0001
Figure imgf000010_0001
鶏コラーゲンプロテアーゼ分解物及び上記の各酵素で分解したコラーゲンぺプチ ドの ACE阻害活性を図 1に示す。 Figure 1 shows the ACE inhibitory activity of chicken collagen protease degradation products and collagen peptides degraded by the above enzymes.
酵素分解前のコラーゲンでは阻害活性が認めらないなかったが、 鶏コラーゲンプ 口テアーゼ分解物は阻害剤無添加の場合に対し、 約 30%の阻害活性を示した （図 1 の処理前)。  Collagen before enzyme degradation did not show inhibitory activity, but chicken collagenase protease digestion product showed about 30% inhibitory activity compared to the case where no inhibitor was added (before treatment in Fig. 1).
鶏コラーゲンプロテア一ゼ分解物を、 更に酵素処理を行って得られたコラーゲン ペプチドは、 2倍近い活性の上昇が認められた。 各酵素処理の中でもスミチ一ム FP (以下、 単に FPという） 酵素処理したものに、 高い活性が認められた。  The collagen peptide obtained by subjecting the chicken collagen protease degradation product to further enzyme treatment showed an increase in activity of almost twice. Among the enzyme treatments, high activity was observed for those treated with Sumitomo FP (hereinafter simply referred to as FP).
(2)分子量分画した画分の ACE阻害作用 (2) ACE inhibitory action of molecular weight fractions
上記 FP酵素処理したコラーゲンべプチドを、 限外濾過膜を用いて分子量 3000で 分画を行い、 各々の画分の ACE阻害活性の強さを比較した。 その結果を表 2に示し た。 表 2に示されるように、 低分子側画分 (分子量 3000以下）に高い活性が認められ た。 表 2 . P艮外濾過膜による分画 (分子量 3000)
Figure imgf000011_0001
The above FP enzyme-treated collagen peptide was fractionated at a molecular weight of 3000 using an ultrafiltration membrane, and the strength of the ACE inhibitory activity of each fraction was compared. The results are shown in Table 2. As shown in Table 2, high activity was observed in the low molecular fraction (molecular weight of 3000 or less). Table 2. Fractionation by P membrane (molecular weight 3000)
Figure imgf000011_0001
実施例 2 Example 2
畜種によるコラーゲンペプチドの ACE阻害活性を比較するため、 豚皮を原料とし て、 実施例 1 (1)と同様の方法で豚コラーゲンぺプチドを調製し、 その ACE阻害活性 を測定した。 その結果を図 2に示す。  In order to compare the ACE inhibitory activity of collagen peptides by livestock, porcine collagen peptide was prepared in the same manner as in Example 1 (1) using pig skin as a raw material, and its ACE inhibitory activity was measured. The results are shown in Fig. 2.
図 2に示されるように、 豚コラーゲンペプチドも ACE阻害活性を有し、 高分子側 に比べて低分子側の方が強い活性を有していた。鶏と同様に FP酵素処理したものの 活性が最も強かった。  As shown in Fig. 2, porcine collagen peptide also had ACE inhibitory activity, and the low molecular side had stronger activity than the high molecular side. As with chickens, the FP enzyme-treated product had the strongest activity.
し力 し、 その ACE阻害活性は鶏コラーゲンペプチドの方が強かった。 この理由と しては、 （a)豚コラーゲンの方がプロテアーゼによる分解を受けやすく、 ACE阻害活 性を示せないほどに低分子化 （単体のアミノ酸） しゃすいことと、 （b)鶏コラーゲン の方が、 内因的に ACE阻害活性を発現しやすいアミノ酸配列を有していることが推 察された。 実施例 3 (消化酵素処理による ACE阻害活性への影響）  However, chicken collagen peptide had stronger ACE inhibitory activity. The reasons for this are: (a) porcine collagen is more susceptible to protease degradation and is so low in molecular weight that it does not show ACE inhibitory activity (a single amino acid); (b) chicken collagen Therefore, it was speculated that it had an amino acid sequence that tends to express ACE inhibitory activity endogenously. Example 3 (Effect of digestive enzyme treatment on ACE inhibitory activity)
鶏コラーゲンぺプチド（FP処理物）及ぴ豚コラーゲンぺプチド (FP処理物）を摂取し た際に、 体内でも ACE阻害活性が維持されるのか、 モデル消化系を用いて検討を行 つた。  A model digestion system was used to investigate whether ACE inhibitory activity was maintained in the body when chicken collagen peptide (FP-treated product) and porcine collagen peptide (FP-treated product) were ingested.
即ち、 FP酵素処理鶏コラーゲンべプチド及ぴ豚コラーゲンぺプチド溶液 (pH3. 0) に、 タンパク質重量に対し 1%となるようにし、 酵素反応液の p Hを各酵素の至適 p Hに調整した後、ペプシン（pH3. 0)又はトリプシン/キモトリプシン（pH7. 0)を添加し、 37°Cで 1時間処理を行った。 反応終了後、 前述と同様に限外濾過膜を用いて分子量 3000で分画を行い、 低分子画分 (分子量く 3000) と高分子画分 (分子量 > 3000) に分 離した。  In other words, FP enzyme-treated chicken collagen peptide and porcine collagen peptide solution (pH 3.0) were adjusted to 1% of the protein weight, and the pH of the enzyme reaction solution was adjusted to the optimum pH of each enzyme. After that, pepsin (pH 3.0) or trypsin / chymotrypsin (pH 7.0) was added and treated at 37 ° C for 1 hour. After completion of the reaction, fractionation was performed at a molecular weight of 3000 using an ultrafiltration membrane in the same manner as described above, and separated into a low molecular fraction (molecular weight of 3000) and a high molecular fraction (molecular weight> 3000).
上記の消化酵素処理物及ぴ分画画分について、 ACE阻害活性の測定を行った。その 結果を図 3に示す。 図 3に示されるように、 FP酵素処理鶏コラーゲンぺプチド及ぴ豚コラーゲンぺプ チドは、 消化酵素処理 (P：ペプシン処理、 T/C： トリプシン/キモトリプシン処理)す ることによつても ACE阻害活性を維持しており、 特に鶏コラーゲンぺプチドの低分 子画分は強レ、活性を示した。 実施例 4 (SHRにおける血圧上昇抑制作用） ' ACE inhibitory activity was measured for the digested enzyme-treated product and fractions. The results are shown in Fig. 3. As shown in Figure 3, FP enzyme-treated chicken collagen peptide and porcine collagen peptide are also ACE-treated by digestive enzyme treatment (P: pepsin treatment, T / C: trypsin / chymotrypsin treatment). The inhibitory activity was maintained, and in particular, the low molecular fraction of chicken collagen peptide showed strong activity. Example 4 (SHR-inhibiting effect on blood pressure increase) '
鶏コラーゲンぺプチド (FP処理物）を SHRに投与し、血圧変動を経時的に測定した。 具体的には、 雄 SHR (8週齢； チャールズリパー） は市販の非精製餌 （AIN- 76；ォ リエンタル酵母） と水で 23°C、湿度 55%に調整された部屋で飼育した。本試験には、 血圧が十分に上昇したことが確認できた週齢のものを使用した。 試験時に、 生理的 食塩水 （コントロール群） 又は鶏コラーゲンペプチド (FP処理物）を経口的に投与 (3 又は 6g/kg weight) し、 血圧変動を 2 4時間測定した （単回投与系）。  Chicken collagen peptide (FP-treated product) was administered to SHR, and blood pressure fluctuations were measured over time. Specifically, male SHR (8 weeks old; Charles Liper) was raised in a room adjusted to 23 ° C and 55% humidity with commercial non-purified food (AIN-76; Oriental yeast) and water. In this study, a test subject who was able to confirm that his blood pressure had risen sufficiently was used. At the time of the test, physiological saline (control group) or chicken collagen peptide (FP-treated product) was orally administered (3 or 6 g / kg weight), and blood pressure fluctuation was measured for 24 hours (single administration system).
また、 同様に雄 SHR に、 鶏コラーゲンペプチド (FP処理物）を毎日経口的に投与 (6g/kg weight) し、 血圧変動を 4週間測定した （長期投与系）。  Similarly, chicken collagen peptide (FP-treated product) was orally administered (6 g / kg weight) daily to male SHR, and blood pressure fluctuations were measured for 4 weeks (long-term administration system).
なお、 ラット尾動脈圧は非観血式血圧測定器（Softron98A、 Softron、東京、 日本） で測定を行なった。 データの解析は Student' s t-testにより処理した。 その結果 を図 4に示す。  Rat tail artery pressure was measured with a non-invasive blood pressure meter (Softron98A, Softron, Tokyo, Japan). Data analysis was processed by Student's t-test. Figure 4 shows the results.
単回投与系においては、 コントロール群 SHRに対し、 鶏コラーゲンペプチドを投 与した SHRは投与後 2時間後から血圧が低下し、 6時間で最低血圧を記録した（図 4A 参照）。  In the single-dose system, SHR given chicken collagen peptide decreased blood pressure 2 hours after administration and recorded minimum blood pressure at 6 hours compared to SHR in the control group (see Fig. 4A).
また、長期投与を行った場合は、鶏コラーゲンペプチドの投与で、 1週目より血圧 の低下が認められ、 3週目には有意な血圧低下が認められた (p〈0. 05) (図 4B参照）。 ナトリウム、 カリウム、 カルシウムなどの血清ミネラル成分は血圧に影響を及ぼ すことが知られているが、 4週間にわたる長期投与の後でもコント口ール群と鶏コラ 一ゲンべプチド投与群に違いは認められなかった（表 3参照）。 血圧が上昇するメカ 二ズムには種々の理由が存在する。 アドレナリン、 ノルアドレナリンなどのカテコ ールァミンは神経性の調圧因子としてよく知られているし、 血清ミネラル成分のナ トリウム、 カリウム、 カルシウムも血圧に影響する。 今回鶏コラーゲンペプチドを 投与した SHRにおいても血清ミネラル成分が多く検出されると予想されたが、 4週 間にわたる長期投与の後でも血圧に影響すると考えられるこれらの成分に大きな違 いは認められなかった。 従って、 本発明のペプチドの血圧上昇抑制作用の作用機序 は ACEの阻害作用であると考えられる。 血液成分分析 In addition, in the case of long-term administration, a decrease in blood pressure was observed from the 1st week after administration of chicken collagen peptide, and a significant decrease in blood pressure was observed in the 3rd week (p 〈0. 05) (Fig. (See 4B). Serum mineral components such as sodium, potassium, and calcium are known to affect blood pressure, but even after a long-term administration over 4 weeks, there is no difference between the control mouth group and the chicken collagen gene treatment group. Not recognized (see Table 3). There are various reasons for the mechanism by which blood pressure rises. Catecholamines such as adrenaline and noradrenaline are well known as neural regulators, and the serum minerals sodium, potassium, and calcium also affect blood pressure. Although it was expected that serum mineral components were often detected even in SHR administered chicken collagen peptide this time, there was a big difference in these components that are thought to affect blood pressure even after long-term administration over 4 weeks. Was not recognized. Therefore, the action mechanism of the blood pressure elevation inhibiting action of the peptide of the present invention is considered to be the ACE inhibiting action. Blood component analysis
Figure imgf000013_0001
Figure imgf000013_0001
実施例 5 (ヒトでの臨床試験） Example 5 (clinical trials in humans)
鶏コラーゲンペプチド (FP処理物）のヒトでの効果を調べるために、 被験者の事前 の了解を得て、 以下の試験条件で単回摂取試験と長期摂取試験を実施した。  In order to examine the human effect of chicken collagen peptide (FP-treated product), a single intake test and a long-term intake test were performed under the following test conditions with the prior consent of the subjects.
(1)単回摂取試験（1日間、 被験者： A及び Bの 2名）  (1) Single intake test (1 day, subjects: A and B)
被験者に、 試験食 (ぺプチド量として 7. 8g)を摂取後、 安静時血圧を 2時間毎に 8 時間まで測定した。  After ingesting the test meal (7.8 g peptide amount) to the subjects, the resting blood pressure was measured every 2 hours for up to 8 hours.
前日にプラセボ食を摂取時の値との前後比較で評価した。 その結果を図 5に示す。 図 5に示されるように、 摂取 4時間後に最高血圧の中央値が低下する傾向を示し た。  The placebo diet was evaluated on the previous day by comparison with the value at the time of ingestion. The results are shown in Fig. 5. As shown in Figure 5, the median systolic blood pressure tended to decrease 4 hours after ingestion.
(2)長期摂取試験 (28日間、 被験者： 1 4名）  (2) Long-term intake test (28 days, subjects: 1 to 4)
被験者に、 試験食（1日当たりのペプチド摂取量として 5. 2g)を毎日摂取させ、 2 週間毎に 4週間後まで血圧変動を測定した。 各人について、 摂取前の値との前後比 較で評価した。 その結果を図 6に示す。 図 6に示されるように、 長期投与試験の結果、 本試験食の摂取前と 2週後と 4週 後の測定値の各々に有意差が認められ、 本発明の鶏コラーゲンペプチドは、 血圧変 動を抑制する効果があることが認められた。 Subjects were allowed to ingest a test meal (5.2 g of peptide intake per day) every day, and blood pressure fluctuations were measured every 2 weeks until 4 weeks later. Each person was evaluated by comparison before and after intake. The result is shown in Fig. 6. As shown in Fig. 6, as a result of the long-term administration test, there was a significant difference between the measured values before intake of the test meal, after 2 weeks, and after 4 weeks. It was confirmed that there is an effect of suppressing movement.
本試験の結果より、 鶏コラーゲン酵素分解物の 1日当たりの望ましい摂取量は、 ペプチド量として 1〜1 0 g、 より好ましくは 2〜8 gと考えられる。 実施例 6 (血管修復作用向上への影響： E P Cコロニーアッセィ）  From the results of this test, the desired daily intake of chicken collagen enzyme degradation product is considered to be 1 to 10 g, more preferably 2 to 8 g as a peptide amount. Example 6 (Influence on improvement of vascular repair action: EPC colony assay)
本発明のコラーゲンペプチドの摂取が、 血圧を下げるだけでなく、 血管機能の改 善効果があることを EPC (endothelial progenitor cell)コロニーァッセィで確認し た。 この EPCは骨髄で産生される血管内皮細胞の前駆体で、 末梢血中の EPCは炎症 ゃ虚血、 酸ィ匕ストレスなどの組織障害を感知し、 細胞の修復を担う。 高血圧症や高 脂血症、 糖尿病の患者では EPCの機能が低下しており、 血管障害の程度と高い相関 を示すことが知られている。  It was confirmed by EPC (endothelial progenitor cell) colony assay that the intake of the collagen peptide of the present invention not only lowered blood pressure but also improved blood vessel function. This EPC is a precursor of vascular endothelial cells produced in the bone marrow, and EPC in peripheral blood senses tissue damage such as inflammation, ischemia and acid stress, and is responsible for cell repair. In patients with hypertension, hyperlipidemia, and diabetes, EPC function is reduced, and it is known to correlate with the degree of vascular disorder.
より具体的には、 上記実施例 5の血圧試験の被験者血液を使用した。 つまり、 本 発明のコラーゲンぺプチド摂取期間前後の被験者血液を同量（10ml)採取し、 1000g、 30min で遠心し、 沈殿した細胞を細胞培養液を満たしたシャーレに播種し、 37°C, 5%C02, 1週間培養後、 本発明のコラーゲンペプチド摂取期間前後でコロニー数が増 加したかどう力、、 形成されるコロニー形態に違いがないかを調べた。 E P Cコロェ ーァッセィにおけるコ口ニー数の変化を図 7に、 コロニーの形態の変化を図 8に示 す。 More specifically, the subject blood of the blood pressure test of Example 5 was used. That is, the same amount (10 ml) of the subject blood before and after the collagen peptide ingestion period of the present invention is collected, centrifuged at 1000 g for 30 min, and the precipitated cells are seeded in a petri dish filled with cell culture medium, 37 ° C, 5 % C0 2, after one week of culture, the number of colonies before and after collagen peptide ingestion period of the present invention was examined whether there are differences in whether colony morphology which is force ,, formed was increased. Figure 7 shows the change in the number of cones in the EPC collassey, and Figure 8 shows the change in the colony morphology.
本試験の結果として、 EPCコロニーアツセィにより、本発明のコラーゲンぺプチド の摂取後において、 形成されるコロニー数が増加し、 コロ—ニーの形態が改善される ことが判明し、 本発明のコラーゲンペプチドは、 血圧を下げるだけでなく、 血管の 状態を改善する作用 （血管保全作用） を有することが確認された。 実施例 7 (ACE阻害ぺプチドの単離 .精製）  As a result of this test, it was found that EPC colony assembly increased the number of colonies formed after ingestion of the collagen peptide of the present invention and improved colony morphology. It was confirmed that the peptide not only lowers blood pressure but also has an action to improve the state of blood vessels (blood vessel preservation action). Example 7 (Isolation of ACE inhibitory peptide. Purification)
酵素処理によって得られた鶏コラーゲンペプチドから ACE阻害ペプチドを単離す るため、 逆相 HPLC (CH3CN; 8-40% in 0. 1% CF3C00H) によって分画を行った。 溶出ピ ークは 220nmで検出を行つた。 - まず、 図 9Aに示されるように、溶出時間に従い、すべてのクロマトを 6つに分画 (Frac. 1-Frac. 6) し、 ACE阻害活性の比較を行った （表 4A参照）。 In order to isolate the ACE inhibitory peptide from chicken collagen peptide obtained by enzyme treatment, fractionation was performed by reverse phase HPLC (CH 3 CN; 8-40% in 0.1% CF 3 C00H). The elution peak was detected at 220 nm. -First, fractionate all chromatographs into 6 according to elution time as shown in Figure 9A (Frac. 1-Frac. 6) and compared ACE inhibitory activity (see Table 4A).
その結果、 Frac. 4に強い活性が認められたので、 画分を濃縮し、 ァセトニトリル での溶出条件を穏やかにして（0. 4%/min)再度 HPLCに供した（図 9B参照）。 得られた ピークを溶出時間毎に分画し（Frac. 4-l〜Frac. 4-4)、 ACE阻害活性を比較した（表 4 B 参照)。  As a result, since strong activity was observed in Frac. 4, the fraction was concentrated and subjected to HPLC again with mild elution conditions with acetonitrile (0.4% / min) (see FIG. 9B). The obtained peaks were fractionated for each elution time (Frac. 4-l to Frac. 4-4), and ACE inhibitory activities were compared (see Table 4B).
最も高い活性が認められた Frac. 4-3は、各ペプチドを単離するため、濃縮を行い、 ァセトニトリルでの溶出条件を更に穏やかにして（0. 16%/min)HPLC に供した（図 9C 参照）。得られたピークはプロテインシークェンサ一によりアミノ酸配列の決定を行 つすこ。 .  Frac. 4-3, which showed the highest activity, was concentrated to isolate each peptide and subjected to HPLC with milder elution conditions with acetonitrile (0.16% / min) (Fig. See 9C). The amino acid sequence of the obtained peak should be determined using a protein sequencer. .
各画分の ACE阻害活性 (A; 1回目の HPLC分画精製 B; 2回目の HPLC分画精製)
Figure imgf000015_0001
ACE inhibitory activity of each fraction (A; 1st HPLC fraction purification B; 2nd HPLC fraction purification)
Figure imgf000015_0001
B
Figure imgf000015_0002
ァミノ酸配列は常法に準じて、プロティンシークェンサ一 G1005A (Hewlett PackardB
Figure imgf000015_0002
The amino acid sequence was determined according to the conventional method, using the protein sequencer G1005A (Hewlett Packard
Co. Wilmington, DE) で決定した。 Co. Wilmington, DE).
図 9 C得られた各ピークのァミノ酸配列及び質量分析の結果を表 5に示した。 表 5に示されるように、 ピーク 8、 9、 1 1及び 1 2は、 それぞれ下記のァミノ 酸配列を有するぺプチドであった。  Figure 9 C shows the amino acid sequence of each peak obtained and the results of mass spectrometry. As shown in Table 5, peaks 8, 9, 11 and 12 were peptides having the following amino acid sequences, respectively.
Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro (配列番号 1 )  Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro (SEQ ID NO: 1)
Gly-Ala-Hyp-Gly-Pro-Ala-Gly-Pro-Gly-Gly-Ile-Hyp-Gly-Glu-Arg-Gly (配列番号 2 )  Gly-Ala-Hyp-Gly-Pro-Ala-Gly-Pro-Gly-Gly-Ile-Hyp-Gly-Glu-Arg-Gly (SEQ ID NO: 2)
Gly-Leu-Hyp-Gly-Ser-Arg-Gly-Glu-Arg-Gly-Leu-Hyp-Gly (配列番号 3 )  Gly-Leu-Hyp-Gly-Ser-Arg-Gly-Glu-Arg-Gly-Leu-Hyp-Gly (SEQ ID NO: 3)
Gly-Ile-Hyp-Gly-Glu-Arg-Gly-Glu-Hyp-Gly-Pro-Val-Gly-Pro-Ser-Gly (配列番号 4 ) Gly-Ile-Hyp-Gly-Glu-Arg-Gly-Glu-Hyp-Gly-Pro-Val-Gly-Pro-Ser-Gly (SEQ ID NO: 4)
上記配列番号 1〜4に示されるァミノ酸配列を有するぺプチドを、 Fmoc固相法に より自動ペプチド合成機 (PSSM8 島津） を用いて作製した。 脱保護の後、 逆相 HPLC (PEGASIL- 300， 20 X 250 謹； Senshu, Tokyo, Japan) 及び質量分析計 ESI mass spectrometer LC-Q (Thermo Finnigan, San jose, CA) で確 を行つ 7こ。 The peptides having the amino acid sequences shown in SEQ ID NOs: 1 to 4 were prepared by the Fmoc solid phase method using an automatic peptide synthesizer (PSSM8 Shimadzu). After deprotection, reverse phase HPLC (PEGASIL-300, 20 X 250 mm; Senshu, Tokyo, Japan) and mass spectrometer ESI mass spectrometer LC-Q (Thermo Finnigan, San jose, CA).
合成されたペプチドの ACE阻害活性を測定し、 その活性 (IC5。）を表 5に示した。表The ACE inhibitory activity of the synthesized peptides was measured and the activity (IC 5 ) is shown in Table 5. table
5に示されるように、 配列番号 1〜4で示されるァミノ酸配列からなるペプチドは ACE阻害活性を有していた。特に配列番号 1で示されるアミノ酸配列からなるぺプチ ドは強い ACE阻害活性を有していた。 表 5 . ACE P且害ぺプチドの配列および質量分析 As shown in 5, the peptide consisting of the amino acid sequence represented by SEQ ID NOs: 1 to 4 had ACE inhibitory activity. In particular, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 had strong ACE inhibitory activity. Table 5. ACE P and Harmful Peptide Sequence and Mass Spectrometry
Figure imgf000016_0001
Figure imgf000016_0001
実施例 8 (鶏コラーゲンべプチドの調製） Example 8 (Preparation of chicken collagen peptide)
鶏ゼラチン溶液 (pH6.0)を調製し、 これにコクラーゼ P (ゼラチンに対して 0.2%) を加え、 50°C， 3時間処理した後、 加熱して酵素を失活させた。 次いで、 珪藻土濾 過、 活性炭処理により精製することにより鶏ゼラチンプロテアーゼ分解物を得た。 得られた鶏ゼラチンプロテアーゼ分解物の溶液 (pH6.0)に、スミチーム FP (原料に 対し、 0. 5%酵素添加）を添加し、 50°Cで 4時間酵素処理し、 20分間の加熱により酵 素を失活させ、 遠心処理を行って上清を採取することによりゼラチンペプチドを得A chicken gelatin solution (pH 6.0) was prepared, coculase P (0.2% based on gelatin) was added thereto, treated at 50 ° C. for 3 hours, and then heated to inactivate the enzyme. Subsequently, chicken gelatin protease degradation product was obtained by refine | purifying by diatomaceous earth filtration and activated carbon treatment. The resulting chicken gelatin protease degradation product solution (pH 6.0) is added to Sumiteam FP (raw material). In contrast, 0.5% enzyme was added), the enzyme treatment was performed at 50 ° C for 4 hours, the enzyme was inactivated by heating for 20 minutes, and the supernatant was collected by centrifugation and the gelatin peptide was collected. Gain
7 o 7 o
上記で得られたゼラチンぺプチドを、限外濾過膜を用いて分子量 3000で分画を行 い、 低分子側画分 (分子量 3000以下）を採取した。 以下、 分画前のコラーゲンぺプチ ドを A 2と称し、 低分子側画分 (分子量 3000以下）を A 2 Fと称する。  The gelatin peptide obtained above was fractionated at a molecular weight of 3000 using an ultrafiltration membrane, and a low molecular side fraction (molecular weight of 3000 or less) was collected. Hereinafter, the collagen peptide before fractionation is referred to as A2, and the low molecular fraction (molecular weight of 3000 or less) is referred to as A2F.
上記の A 2及び A 2 Fについて、 ACE阻害活性を測定したところ、 それぞれの IC50 は 0. 586mg/ml及び 0. 326mg/mlであった。 実施例 9 (コラーゲンべプチドによる血管内皮機能改善作用） When the ACE inhibitory activity of the above A 2 and A 2 F was measured, the respective IC 50 values were 0.586 mg / ml and 0.326 mg / ml. Example 9 (Improvement of vascular endothelial function by collagen peptide)
血管内皮細胞においては、 種々のサイトカインの作用により、 PI3K—プロテイン キナーゼ Akt の活性化が起り、 それに続いて内皮型一酸化窒素合成酵素 (Endothelial Nitric-oxide synthase, eNOS)が活性化され、 NO産生増加を引き起こ し、 血管新生、血管拡張、 単球接着阻害などの血管内皮機能改善をもたらす。 eNOS の活性化は、そのスレオニン残基とセリン残基のリン酸ィ匕により生じるので、 eNOS のリン酸化を促進する物質は N O産生を増加させ、 血管内皮機能の改善効果を有す ることになる。 そこで、 本発明のコラーゲンペプチドの eNOS リン酸化作用を試験 した。  In vascular endothelial cells, the action of various cytokines causes the activation of PI3K-protein kinase Akt, followed by the activation of endothelial nitric oxide synthase (eNOS), producing NO. It causes an increase, resulting in improved vascular endothelial function such as angiogenesis, vasodilation and inhibition of monocyte adhesion. The activation of eNOS is caused by the phosphorylation of its threonine and serine residues. Therefore, a substance that promotes phosphorylation of eNOS increases NO production and has an effect of improving vascular endothelial function. Become. Therefore, the eNOS phosphorylation effect of the collagen peptide of the present invention was tested.
(1)供試材料  (1) Test material
ヒト臍帯静脈内皮細胞 （HUVEC) はタカラバイオから、 ゥシ大動脈血管内皮細胞 (BAEC) は大日本住友製薬から購入した。 実験に供した HUVEC及ぴ BAECは継 代数 4から 6代目までのものを用いた。 細胞培養に用いた試薬類 （DMEM, FBS, Trypsin-EDTA) はィンビトロジェンから購入した。 phospho-eNOS(Serll77)抗体 は Cell Signaling社から購入した。 また抗体の検出には、 アマシャム ' フアルマシ ァの ECLplusを用いた。 タンパク定量試薬にはパイオラッドの DC protein assay reagentを使用した。  Human umbilical vein endothelial cells (HUVEC) were purchased from Takara Bio, and ushi aortic vascular endothelial cells (BAEC) were purchased from Sumitomo Dainippon Pharma. The HUVEC and BAEC used in the experiments were from passage 4 to 6th generation. Reagents (DMEM, FBS, Trypsin-EDTA) used for cell culture were purchased from Invitrogen. The phospho-eNOS (Serll77) antibody was purchased from Cell Signaling. For detection of antibodies, ECLplus from Amersham's Pharmacia was used. Piorad's DC protein assay reagent was used as a protein quantification reagent.
試験べプチドであるコラーゲンぺプチドとしては、 配列番号 1に示されるォクタ ペプチド（合成品、以下 C C O Pという）並びに上記の A 2及び A 2 Fを試験した。 As collagen peptides as test peptides, the Octa peptide shown in SEQ ID NO: 1 (synthetic product, hereinafter referred to as C COP) and the above A 2 and A 2 F were tested.
(2)細胞培養 (2) Cell culture
HUVEC 及び BAEC は 10%FBS を含む DMEM ( 10%FBS, 100 μ U/ml W HUVEC and BAEC are DMEM containing 10% FBS (10% FBS, 100 μU / ml W
17 streptomycin-penicillin, 50pg/mlへパリン）にて目的の継代数に至るまで培養を行 つた。 リン酸化試験に供する際は 0% FBS-DMEMで 12時間培養し、 FBSに含まれ る成長促進因子などがリン酸ィヒに及ぼす影響を除去した。  17 Streptomycin-penicillin, 50 pg / ml heparin), and cultured until the desired passage number was reached. When subjected to the phosphorylation test, the cells were cultured in 0% FBS-DMEM for 12 hours to eliminate the effects of growth-promoting factors contained in FBS on phosphoric acid.
(3)リン酸ィ匕 eNOSの検出  (3) Phosphate eNOS detection
リン酸化 eNOSの検出は Juら （J. Biol. chem. 1998， 273， 24025-24029.) の方法 に準じて行った。 即ち、 BEACを 0% FBS-DMEMで処理した後、 培地に試験ぺプ チドを 0.1°/0〜0.001%で添加した。 また、 ポジティブコントローノレとして培地に、溶 解させた 100 i Mブラジキニン （BK;シグマ） を添カ卩した。 一定時間 （3分間） 刺激 を与えた細胞を PBSで洗浄し、 Lysis buffer (0.1% triton, 20mM Tris-HCl (pH8.0), 20mM EDTA, ImM PMSF, ImM sodium orthovanadate, lniM leupeptin-pepstatin) で細胞を溶解させて、 細胞溶解液を回収した。 15秒間のソニ ケーシヨン、 および 10分間の加熱処理を行った細胞溶解液を遠心処理 （10,000xg, lOmin) し、 eNOSを含む上清を得た。 タンパク質量を揃えたサンプルは 7.5%ゲル を用いた SDS-PAGEで展開させた後、 PVDF膜に転写した。 5%脱脂粉乳を含む TBS バッファーを用いて室温にて 2時間ブロッキングした後、 phOSpho-eNOS 抗体でPhosphorylated eNOS was detected according to the method of Ju et al. (J. Biol. Chem. 1998, 273, 24025-24029.). That is, after BEAC was treated with 0% FBS-DMEM, the test peptide was added to the medium at 0.1 ° / 0 to 0.001%. In addition, 100 iM bradykinin (BK; Sigma) dissolved in the medium was added as a positive control. Cells that have been stimulated for a certain period of time (3 minutes) are washed with PBS and washed with Lysis buffer (0.1% triton, 20 mM Tris-HCl (pH 8.0), 20 mM EDTA, ImM PMSF, ImM sodium orthovanadate, lniM leupeptin-pepstatin) Cells were lysed and the cell lysate was collected. The cell lysate that had been subjected to sonication for 15 seconds and heat treatment for 10 minutes was centrifuged (10,000 × g, lOmin) to obtain a supernatant containing eNOS. Samples with the same amount of protein were developed by SDS-PAGE using 7.5% gel and then transferred to a PVDF membrane. After blocking with TBS buffer containing 5% non-fat dry milk at room temperature for 2 hours, ph OS pho-eNOS antibody was used.
4 °C、 一晩処理した。 1次抗体を洗レ、流した後、 HRPを付けた 2次抗体で 1時間反 応させた。 1次抗体、 2次抗体をマゥントさせた PVDF膜は TBS-Tで洗つた後、 化学発光試薬により phospho-eNOSを検出し X線フィルムに焼き付けた。 Treated overnight at 4 ° C. The primary antibody was washed and washed, and then reacted with a secondary antibody with HRP for 1 hour. PVDF membranes mounted with primary and secondary antibodies were washed with TBS-T, then phospho-eNOS was detected with a chemiluminescent reagent and baked on X-ray film.
(4)結果  (4) Results
上記の結果を図 1 0に示す。 本発明の鶏コラーゲンペプチドは、 添加濃度依存的 にリン酸化シグナルが強くみとめられたことから、 eNOS を活性化させ、 血管内皮 機能の改善に寄与すると考えられた。 合成ペプチドである C C O Pにおいても、 添 加濃度依存的に eNOSのリン酸ィ匕が認められたことから、 C C O Pが鶏由来コラー ゲンプロテアーゼ分解物中の血管内皮機能改善に寄与する成分の一つであると考え られた。 製造例 1  The above results are shown in FIG. The chicken collagen peptide of the present invention was found to have a strong phosphorylation signal depending on the addition concentration, and thus was considered to activate eNOS and contribute to the improvement of vascular endothelial function. In CCOP, a synthetic peptide, eNOS phosphate was also observed in an additive concentration-dependent manner, so CCOP is one of the components that contributes to the improvement of vascular endothelial function in chicken-derived collagen protease degradation products. I thought it was. Production example 1
天然果汁 （濃縮果汁還元） に、 当該果汁 2 0 0 m l当り鶏コラーゲンペプチド (FP 処理物） 5 0 O m gを混合した後、 常法に準じて殺菌し、 ァセブティック包装して、 果汁製品を得た。 製造例 2 Natural fruit juice (concentrated juice reduction) is mixed with 50 O mg of chicken collagen peptide (FP-treated product) per 20 ml of the juice, then sterilized according to conventional methods and packaged in a boutique to obtain a juice product. It was. Production example 2
ウインナソーセージ用練り肉に、 当該練り肉 1 5 g当り鶏コラーゲンペプチド (FP 処理物） 5 0 0 m gを混合した後、 常法に準じてソーセージケーシングに充填し、 燻煙し、 殺菌し、 冷却後に包装し、 ウインナソーセージを得た。  Mix the chicken collagen peptide (FP-treated product) 500 mg per 15 g of the kneaded meat for wiener sausage, fill it into the sausage casing according to the conventional method, smoke, sterilize, cool Later, packaging was performed to obtain wiener sausage.

Claims

請求の範囲 The scope of the claims
1 . 血圧上昇抑制作用を有する、鶏又は豚由来コラーゲンのプロテアーゼ分解物。1. A protease degradation product of collagen derived from chickens or pigs, which has a blood pressure increase inhibitory effect.
2 . 血管保全作用を有する、 鶏又は豚由来コラーゲンのプロテアーゼ分解物。2. A protease degradation product of collagen derived from chicken or pig having a blood vessel preservation action.
3 . 鶏又は豚由来コラーゲンのプロテアーゼ分解物であって、 分子量が 3 0 0 0 以下 （限外濾過膜法） である、 血圧上昇抑制作用を有するペプチド。 3. A protease degradation product of collagen derived from chicken or pig, which has a molecular weight of 300 or less (ultrafiltration membrane method) and has a blood pressure increase inhibitory effect.
4 . 鶏又は豚由来コラーゲンのプロテアーゼ分解物であって、 分子量が 3 0 0 0 以下 （限外濾過膜法） である、 血管保全作用を有するペプチド。  4. A protease degradation product of collagen derived from chicken or pig, which has a molecular weight of 300 or less (ultrafiltration membrane method) and has a blood vessel preservation action.
5 . 配列番号 1〜4で示されるァミノ酸配列からなるぺプチド。  5. A peptide comprising the amino acid sequence represented by SEQ ID NOs: 1 to 4.
6 . 請求項 1〜 5の何れかに記載されるプロテアーゼ分解物又はべプチドを含有 する、 血圧上昇抑制作用及び/又は血管保全作用を有する機能性食品。  6. A functional food having an antihypertensive action and / or a blood vessel preservation action, comprising the protease degradation product or peptide according to any one of claims 1 to 5.
7 . 食品の形態が、 ハム、 ソーセージ等の食肉加工食品、 かまぼこ、 ちくわ等の 水産加工食品、 スナック菓子等の菓子類、パン類、バター、粉乳等の乳製品、豆腐、 油揚げ等の大豆製品、 水、 果汁、 牛乳、 清涼飲料等の飲料である請求項 6記載の機 能性食品。  7. The food form is processed meat products such as ham and sausage, processed fish foods such as kamaboko and chikuwa, confectionery products such as snacks, dairy products such as breads, butter and powdered milk, soy products such as tofu and fried chicken, The functional food according to claim 6, which is a beverage such as water, fruit juice, milk, or soft drink.
8 . 鶏又は豚由来コラーゲンをプロテアーゼで酵素分解して、 血圧上昇抑制作用 及び血管保全作用を有するコラーゲンプロテアーゼ分解物を得ることを特徴とする コラーゲンプロテアーゼ分解物の製造方法。  8. A method for producing a collagen protease degradation product, characterized in that collagen derived from chicken or pig is enzymatically degraded with a protease to obtain a collagen protease degradation product having an antihypertensive action and a blood vessel preservation action.
9 . 請求項 8記載のコラーゲンプロテアーゼ分解物を、 更にプロテアーゼで酵素 分解して、 血圧上昇抑制作用及び血管保全作用を有するコラーゲンペプチドを得る ことを特徵とするコラーゲンぺプチドの製造方法。  9. A method for producing a collagen peptide, characterized in that the collagen protease degradation product according to claim 8 is further enzymatically degraded with a protease to obtain a collagen peptide having an antihypertensive action and a blood vessel preservation action.
10. 請求項 9記載のコラーゲンぺプチドを、 分子量分画に付し、 分子量 3 0 0 0 以下 （限外濾過膜法） の画分を採取し、 血圧上昇抑制作用及び血管保全作用を有す るコラーゲンぺプチドを得ることを特徴とするコラーゲンぺプチドの製造方法。  10. The collagen peptide according to claim 9 is attached to a molecular weight fraction, and a fraction having a molecular weight of 300 or less (ultrafiltration membrane method) is collected to have a blood pressure increase inhibiting action and a blood vessel preservation action. A method for producing a collagen peptide, comprising obtaining a collagen peptide.
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