WO2008066070A1 - Dipeptidyl peptidase-iv inhibitor - Google Patents

Dipeptidyl peptidase-iv inhibitor Download PDF

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Publication number
WO2008066070A1
WO2008066070A1 PCT/JP2007/072937 JP2007072937W WO2008066070A1 WO 2008066070 A1 WO2008066070 A1 WO 2008066070A1 JP 2007072937 W JP2007072937 W JP 2007072937W WO 2008066070 A1 WO2008066070 A1 WO 2008066070A1
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WIPO (PCT)
Prior art keywords
gly
amino acid
pro
peptide
dppiv
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PCT/JP2007/072937
Other languages
French (fr)
Japanese (ja)
Inventor
Akiba Hamamoto
Yuji Aoki
Taiji Matsukawa
Takeki Matsui
Yasumasa Yamada
Ichiro Yamada
Original Assignee
Uha Mikakuto Co., Ltd.
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Application filed by Uha Mikakuto Co., Ltd. filed Critical Uha Mikakuto Co., Ltd.
Priority to JP2008547008A priority Critical patent/JP5176964B2/en
Publication of WO2008066070A1 publication Critical patent/WO2008066070A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/06Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/06Antihyperlipidemics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/08Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08Tripeptides
    • C07K5/0802Tripeptides with the first amino acid being neutral
    • C07K5/0804Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Definitions

  • the present invention relates to a collagen or gelatin-derived dipeptidyl peptidase IV inhibitor that has been conventionally used as a food material. Furthermore, the present invention relates to a method for treating diabetes 'a prophylactic agent, preventing diabetes in mammals'.
  • diabetes has increased explosively throughout the world. In Japan, it is said that there are 6 million people with diabetes, and 12 to 15 million reserves. In diabetes, blood vessels are gradually damaged by hyperglycemia, resulting in abnormalities in various organs. Diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy have long been known as three major complications, but in recent years they have been accompanied by metabolic abnormalities such as hyperlipidemia and hyperuricemia, resulting in arteriosclerosis. It is also known that the risk of onset increases. Diabetes mellitus is caused by insulin depletion caused by ⁇ / 3-cell destructive lesions.
  • Type I diabetes and ⁇ / 3-cell dysfunction causes decreased insulin secretion and in target organs such as liver, muscle, and adipose tissue. It is caused by the concomitant decline in insulin sensitivity.
  • Type II diabetes which is increasing rapidly in recent years, is derived from type II and is considered to account for 90 to 95% of diabetes. Type II diabetes is caused by modern social life such as stress, obesity, decreased basic metabolic capacity due to lack of exercise, and intake of high-calorie foods.
  • Incretin which is a gastrointestinal hormone, is attracting attention in the field of research related to diabetes. Incretin is a general term for gastrointestinal hormones that enhance insulin secretion.
  • GIP glucose-d insulin secreted polypeptide, glucose-d endend insulinot ropic polyp tide
  • glucagon-like p-mark tide_l etc. These are known to promote glucose-responsive insulin secretion via a receptor expressed in ⁇ / 3 cells and suppress postprandial blood glucose elevation. In addition to promoting secretion, it has activities such as protecting and proliferating ⁇ / 3 cells, but the strength of incretin is one of the problems of incretin.
  • DPPIV Dipeptidyl peptidase IV
  • Patent Document 1 DPPIV inhibitors, which are being developed as pharmaceuticals, pliability, and pharmaceuticals, are non-naturally occurring chemical compounds and have a safety problem when ingested.
  • Patent Document 2 Collagen has been conventionally used as a food and promotes metabolism by ingesting collagen (Patent Document 2), the hair diameter increases (Non-Patent Document 1), arthropathy It has been reported that it can be used as a therapeutic drug (Patent Document 3).
  • Patents relating to the promotion of skin metabolism by oral intake of collagen protein or its hydrolyzate (Patent Document 2) and patents relating to the promotion of collagen synthesis in vivo (Patent Document 4) are also disclosed. There are many health foods for the market.
  • Diabetic patients often have hyperlipidemia, but there are reports that increase in blood lipid levels is suppressed by DPPIV inhibitors (Non-Patent Documents 7-9). . These DPPIV inhibitors are non-natural chemical compounds and have safety issues when ingested.
  • Diabetes patients often have gout, and often have hyperuricemia! /. Gout / hyperuricemia, like diabetes, is a risk factor for arteriosclerosis. If both blood glucose and uric acid levels are high, sufficient caution is required.
  • Non-patent Document 10 It has also been reported that there is a positive correlation between blood uric acid concentration and neutral fat concentration. However, there is no report that the increase in blood uric acid concentration by collagen or gelatin-derived peptides is suppressed!
  • Patent Document 1 Japanese Patent No. 3681110
  • Patent Document 2 JP-A-7-278012
  • Patent Document 3 JP-A 63-39821
  • Patent Document 4 Japanese Patent No. 3802721
  • Patent Document 5 JP-A-7-82299
  • Patent Document 6 JP-A-6-46875
  • Non-Patent Document 1 Nutrition Reports International, 13, 579, 1976
  • Non-Patent Document 2 Br. J. Pharmacol., 69, 551, 1980
  • Non-Patent Document 3 Arch.Biochem.Biophys., 218, 156, 1982
  • Non-Patent Document 4 Biochem. J., 252, 723, 1988
  • Non-Patent Document 5 Biol. Chem. Hoppe-Seyler., 372, 305, 1991
  • Non-Patent Document 6 J. Antibiot., 37, 422, 1984
  • Non-Patent Document 7 Diabetes, 51, 1461, 2002
  • Non-Patent Document 8 Diabetes, 52, 741, 2003
  • Non-Patent Document 9 Diabetes, 55, 1695, 2006
  • Non-Patent Document 10 J Zhejiang Univ Sci B., 8, 593, 2007
  • An object of the present invention is to provide a highly safe DPPIV inhibitor, a diabetes preventive agent containing the DPPIV inhibitor, a prophylactic agent for diabetes, and a method for preventing diabetes in mammals using the DPPIV inhibitor, as well as diabetes It is an object of the present invention to provide a DPPIV inhibitor having an action for improving hyperlipidemia, gout or hyperuricemia associated therewith. Means for solving the problem
  • this invention is comprised from the following.
  • n is an integer of 0 to 4
  • X is Pro or Leu
  • Y, ⁇ and W are each independently the same or different arbitrary amino acid residues (excluding Gly).
  • a DPPIV inhibitor comprising a peptide comprising the amino acid sequence represented by the above, a peptide comprising the amino acid sequence deleted at the end of the amino acid sequence W amino acid sequence or a salt thereof,
  • the DPPIV inhibitor according to [1] which is a peptide consisting of one or more amino acid sequences selected from the group consisting of
  • a peptide derived from collagen or gelatin comprising a peptide comprising the amino acid sequence represented by the above formula (1), an amino acid residue at the end of the amino acid sequence, and an amino acid sequence deleted from the amino acid sequence
  • a DPPIV inhibitor having an action to improve hyperlipidemia associated with diabetes
  • a peptide derived from collagen or gelatin comprising a peptide having an amino acid sequence represented by the formula (1), an amino acid residue at the end of the amino acid sequence, and a deleted amino acid sequence.
  • a DPPIV inhibitor having an action to improve gout or hyperuricemia associated with diabetes,
  • a peptide derived from collagen or gelatin comprising a peptide comprising the amino acid sequence represented by the formula (1), an amino acid residue at the end of the amino acid sequence, and an amino acid sequence deleted from the amino acid sequence
  • a DPPIV inhibitor comprising a peptide having a molecular weight of 1,500 or less and a peptide having a molecular weight of 50% or more
  • a peptide derived from collagen or gelatin comprising a peptide having the amino acid sequence represented by the formula (1), an amino acid residue at the end of the amino acid sequence, and an amino acid sequence deleted from the amino acid sequence
  • a DPPIV inhibitor characterized by containing 70% or more of a peptide having a molecular weight of 1,500 or less
  • a peptide derived from collagen or gelatin comprising a peptide having the amino acid sequence represented by the formula (1), an amino acid residue at the end of the amino acid sequence, and an amino acid sequence deleted from the amino acid sequence
  • a DPPIV inhibitor characterized by containing 90% or more of a peptide having a molecular weight of 1,500 or less
  • Collagenase-treated collagen or gelatin degradation product is purified by a method using a combination of organic solvent V, precipitation method or resin purification method! /, Shear force, or both.
  • Production of a peptide composition comprising the steps of: obtaining a peptide comprising the amino acid sequence represented by formula (1), and a peptide composition comprising the amino acid sequence deleted from the terminal amino acid residue w force si of the amino acid sequence.
  • the DPPIV inhibitor of the present invention has a high inhibitory activity against DPPIV and has collagen. Highly safe because it is derived from foods such as gelatin and gelatin. Further, the DPPIV inhibitor of the present invention can also be used as a diabetes preventive / treatment agent for mammals. Furthermore, the DPPIV inhibitor of the present invention can also be used as an ameliorating agent for various diseases associated with diabetes (for example, hyperlipidemia, gout or hyperuricemia).
  • FIG. 1 shows the results of analysis of DPPIV inhibition experiments using collagen peptides HACP-01 and HACP-U2, collagen peptide SCP-5000, gelatin, egg white peptide, and soybean peptide.
  • the vertical axis shows the relative activity as an inhibition rate (%), where 100 is the activity when no sample is included, and 100 is the amount of activity when the sample is added.
  • Fig. 2 shows the results of DPPIV inhibition experiments with collagen peptides HACP-01 and HACP-U2 and collagen peptide SCP-5000 adjusted to final concentrations of 2.5, 5.0, and 10 mg / ml. It is the result.
  • the vertical axis shows the relative activity as an inhibition rate (%), where 100 is the activity when no sample is included, and 100 is the activity when the sample is added.
  • FIG. 3 shows the results of examining the DPPIV inhibitory effect of collagen peptides.
  • the plasma DPPIV activity before each test substance administration (-30 minutes) for each individual was defined as the normal activity (0%), and the difference in activity in each plasma was calculated against this activity, and the ratio was calculated. It is expressed as a relative inhibition rate (%).
  • the symbol (*) in the graph indicates that there is a significant difference (P 0.05) compared to the water intake group.
  • FIG. 4 shows the results of examining the effect of increasing the GLP-1 concentration in blood by collagen peptides.
  • the plasma GLP-1 concentration of each individual before administration of the test substance (-30 minutes) was defined as the normal concentration (100%), and the ratio of the concentration in each plasma to this concentration was calculated and expressed relatively. Is. Note that the symbol (*) in the graph is significantly different from the injection water intake group (p 0.05).
  • FIG. 5 shows the results of examining the effect of increasing the insulin concentration in blood by collagen peptides.
  • the insulin concentration in plasma before administration of the test substance (-30 minutes) for each individual is the normal concentration (100%), and the ratio of the concentration in each plasma to this concentration is calculated and expressed relatively. is there.
  • the symbol (*) in the graph indicates that there is a significant difference (p 0.05) compared to the water intake group.
  • Fig. 6 shows the results of examining the inhibitory effect of collagen peptides on the increase in blood glucose concentration.
  • the symbol (*) in the graph indicates that there is a significant difference (p 0.05) compared to the control substance intake group.
  • blood was collected at 0 and 3 weeks on an empty stomach, and at other times during normal times.
  • FIG. 7 shows the results of examining the effect of suppressing the increase in blood triglyceride concentration by collagen peptides.
  • the symbol (*) in the graph indicates that there is a significant difference (p 0.05) compared to the control substance intake group.
  • blood was collected at 0, 3, and 6 on an empty stomach and at other times on a normal basis.
  • Fig. 8 shows the results of investigating the inhibitory effect of collagen peptides on the increase in blood uric acid concentration.
  • the symbol (*) in the graph indicates that there is a significant difference (p 0.05) compared to the control substance intake group.
  • p 0.05 a significant difference
  • Figure 9 shows the results of investigating the effect of collagen peptides to reduce urinary uric acid excretion.
  • FIG. 10 shows the results of examining the effect of suppressing the increase in blood glucose level by collagen peptides.
  • the symbol (*) in the graph indicates a significant difference (p 0.05) compared to the control group.
  • FIG. 11 shows the results of examining the absorbance and DPPIV inhibition rate of each fraction obtained by fractionating the collagen peptide HACP-01.
  • the line graph represents the absorbance at 215 nm.
  • the bar graph shows the results of analyzing the DPPIV inhibition experiment after adjusting the peptide of each fraction and HACP-01 before fractionation to a final concentration of 1.0 mg / ml.
  • FIG. 12 shows the results of examining the molecular weight distribution of MHP-0.
  • the solid line shows the molecular weight marker, and the dotted line shows the molecular weight distribution of MHP-0.
  • the peak at MHP-0 in the arrow indicates that the molecular weight is around 386.3! /.
  • Fig. 13 shows that collagen peptide HACP-01 is used with aqueous ethanol solutions of different concentrations. This is the result of examining the DPPIV inhibition rate of the supernatant fraction and the precipitated fraction fractionated by the precipitation method.
  • the vertical axis represents relative activity as an inhibition rate (%), where 100 is the activity when no sample is included, and 100 is the amount of activity when the sample is added.
  • FIG. 14 shows the DPPIV inhibition rate of a fraction obtained by further fractionating the 85% ethanol concentration fraction obtained by ethanol precipitation with the collagen peptide HACP-01 and further fractionating with the synthetic adsorbent DIAION HP20. It is the result of having investigated.
  • the vertical axis represents relative activity as an inhibition rate (%), where the activity when no sample is included is 100 and the activity when the sample is added is subtracted from 100.
  • FIG. 15 shows the results of examining DPPIV inhibition rates at various concentrations of collagen peptides HACP-01 and E85S_0 (HP20).
  • the vertical axis shows the relative activity as an inhibition rate (%), where the activity when no sample is included is 100, and the activity when the sample is added is subtracted from 100.
  • FIG. 16 shows DPPIV inhibition of the fraction obtained by fractionating the 85% ethanol concentration fraction obtained by fractionating the collagen peptide HACP-01 by ethanol precipitation with the synthetic adsorbent SEPABEADS SP850. It is the result of examining the rate.
  • the vertical axis shows the relative value as the inhibition rate (%), where the activity when no sample is included is 100, and the activity when the sample is added is subtracted from 100.
  • the present invention relates to a peptide having a specific amino acid sequence, which is an inhibitory activity against DPPIV, among peptides obtained by decomposing a polypeptide constituting collagen or gelatin that is widely used as a food. It has been found for the first time that it has excellent anti-diabetic treatment and prevention effects.
  • the DPPIV inhibitor of the present invention has the formula (1):
  • n is an integer of 0 to 4
  • X is Pro or Leu
  • Y, ⁇ and W are each independently the same or different arbitrary amino acid residues (excluding Gly).
  • Gly represents a glycine residue
  • Pro represents a proline residue
  • Leu represents a leucine residue.
  • amino acid residues excluding glycine residues that can be taken by the above ⁇ , ⁇ and W are not particularly limited, and are usually amino acid residues of naturally occurring amino acids (excluding glycine), specifically Specifically, alanine residue, parin residue, leucine residue, isoleucine residue, proline residue, phenylalanine residue, tribubutane residue, methionine residue, serine residue, threonine residue, cysteine residue, It may be any amino acid residue such as gnoretamine residue, asparagine residue, tyrosine residue, lysine residue, arginine residue, histidine residue, aspartate residue, glutamate residue.
  • the amino acid residue may be a modified amino acid residue to which a hydroxyl group or the like is added.
  • an amino acid residue means an L-type amino acid residue.
  • the peptide is composed of one or more amino acid sequences selected from the group consisting of force groups.
  • all amino acid residues are represented according to the known three-letter code, and Hyp is a hydroxyproline residue (3-hydroxyproline residue (3Hyp) or 4-hydroxyproline residue (4Hyp). ).
  • the peptide salt used in the present invention may be an amino acid sequence represented by the formula (1). (Including peptides consisting of amino acid residues at the end of the amino acid sequence of the peptide w amino acid sequence) and alkali metals such as sodium and potassium, alkaline earth metals such as magnesium and calcium , Salts with inorganic bases such as trivalent metals such as aluminum, methylamine, ethylamine, dimethylamine, jetylamine, trimethylamine, triethylamine, monoethanolamine, diethanolamine, triethanolamine, cyclohexylamine , Salts with organic bases such as lysine and ornithine.
  • the peptide represented by the formula (1) or a salt thereof, which is an active ingredient of the present invention may be isolated as a hydrate, various solvates, or a crystalline polymorphic substance. Includes all of these isolates and mixtures.
  • the peptide or salt thereof used in the present invention can be produced by a known method from the above-mentioned raw material containing collagen or gelatin, or a transformant containing DNA encoding the peptide can be cultured. Can also be manufactured. It can also be produced according to known peptide synthesis methods.
  • the DPPIV inhibitor of the present invention only needs to contain the peptide represented by the formula (1) or a salt thereof as an active ingredient.
  • collagen or gelatin decomposed by collagenase treatment It may be a thing.
  • the collagen used as a raw material is not particularly limited, and any of type I to XIII type collagen can be used, and a mixed type collagen, which is a mixture of these, can also be used.
  • collagen is the power that is expected to use mixed collagen obtained from various animals and fish.
  • Animals eg, cattle, pigs, etc.
  • fish eg, cattle, pigs
  • collagen extraction sites can also be bone, skin, tendon, sea buckthorn (fish), etc.
  • Extraction and purification of collagen from these components can be performed by a generally known method. Specifically, for example, after pulverizing collagen-containing tissues such as bone, skin, tendon, and duckweed, washing with water, extraction with a dilute salt solution, extraction with an acid or alkaline solution, enzymes such as pepsin, trypsin and hyaluronidase, etc. It is possible to extract collagen by using a known purification means such as salting-out dialysis or the like and purify the collagen. It can also be obtained as “regenerated collagen” by a generally known method. Also, commercially available collagen It can also be used as a raw material.
  • Gelatin is a water-soluble protein obtained by heating and extracting the aforementioned collagen with water.
  • gelatin produced by a generally known method can be used as a raw material, and a commercially available product can also be used.
  • the peptide used in the present invention can be produced by allowing collagenase to act on collagen or gelatin obtained as described above. Specifically, a degradation product (collagenase degradation product) obtained by subjecting collagen or gelatin to a collagenase treatment is used.
  • Collagenase is not particularly limited, but it is derived from bacteria such as Clostridium histolyticum and Streptomyces parvulus, actinomycetes or fungi, and the like.
  • Gly—A—B) n (where A and B represent amino acid residues other than glycine residues, which may be the same or different from each other, n represents a positive integer): In the following, this amino acid sequence is also referred to as a “specific amino acid sequence”)).
  • collagenase that specifically cleaves the amino group terminal side of the glycine residue, collagenase degradation that contains abundant peptides of this specific amino acid sequence It is possible to obtain a product, which is preferable.
  • the collagenase used here may be a collagenase obtained as a natural product, for example, a modified collagenase having the above-mentioned specificity obtained by modification by a protein engineering technique.
  • the types of amino acid residues excluding glycine residues that can be adopted by A and B are not particularly limited, and are usually amino acid residues of naturally occurring amino acids (excluding glycine), specifically Is alanine residue, valine residue, leucine residue, isoleucine residue, proline residue, phenylalanine residue, tributophan residue, methionine residue, serine residue, threonine residue, cystine residue, glutamine residue It may be any amino acid residue such as a group, an asparagine residue, a tyrosine residue, a lysine residue, an arginine residue, a histidine residue, an aspartate residue, or a glutamate residue.
  • the peptide that can be used in the present invention is free or in accordance with a generally known method, for example, a method described in JP-A-7-82299 or JP-A-9-176196.
  • Collagenase immobilized on an immobilization carrier such as chitobar is mixed with a batch method, a column method, or a combination of these methods, and preferably the reaction temperature is set to 40 to 45 ° C. It can be produced by contacting with collagen or gelatin.
  • the collagenase degradation product may be produced according to the method described above and used as it is, or may be blended in various base materials.
  • the blending amount and the type of base material are not particularly limited, and may be set as appropriate.
  • As the base material for example, an oral administration base material such as a tablet, capsule, candy, gummi or drink is preferable. In addition, it can be produced by any known technique, regardless of whether it is a food or a medicine.
  • a commercially available product such as the collagen name “Tripeptide HACP” (manufactured by Zerais Co., Ltd.) can also be used.
  • a fraction with enhanced DPPIV inhibitory activity can be obtained by further purifying the collagenase degradation product or its commercial product using various resins.
  • the resin to be used include cation exchange resin, anion exchange resin, porous resin, and special resin (chelate resin, synthetic adsorbent, protein separating agent). Since a process is unnecessary, it is preferable to use a synthetic adsorbent.
  • Synthetic adsorbents include, but are not limited to, for example, aromatic (styrene-butylbenzene) -based, aromatic-modified, and talyl (methacrylic) -based.
  • acids, alkalis or various organic solvents such as lower alcohols such as methanol, ethanol, propanol, isopropanol and butanol, esters such as ethyl acetate and butyl acetate, and ketones such as acetone are used.
  • the force S that can be used is not limited to these.
  • These organic solvents may be used alone or in combination of two or more, and may be a mixed solvent of an organic solvent and water or an acid or an alkali. From the viewpoint of economy and safety, it is preferable to elute with ethanol or ethanol aqueous solution.
  • Purification can be performed by a batch method or a column method. The collected fraction can be concentrated by depressurization or ultrafiltration, and if necessary, the solvent can be completely removed and dried or lyophilized.
  • a fraction having enhanced DPPIV inhibitory activity can be obtained by further purifying the produced collagenase degradation product or its commercial product using various organic solvents.
  • organic solvents to be used include methanol, ethanol, The ability to use lower alcohols such as lopanol, isopropanol and butanol, esters such as ethyl acetate and butyl acetate, and ketones such as acetone is not limited thereto.
  • These organic solvents may be used alone or in combination of two or more, and may be a mixed solvent of an organic solvent and water, acid, or alkali.
  • aqueous ethanol solution In view of economy and safety, it is preferable to purify using an aqueous ethanol solution. Purification can be performed by sedimentation. The collected fraction may be concentrated by reduced pressure or ultrafiltration, and if necessary, the solvent may be completely removed and dried or freeze-dried.
  • the peptide used in the present invention can be produced according to a known peptide synthesis method.
  • a peptide synthesis method for example, either a solid phase synthesis method or a liquid phase synthesis method may be used.
  • the peptide used in the present invention can be purified and isolated by combining ordinary purification methods, for example, solvent extraction / distillation 'column chromatography / one-liquid chromatography / recrystallization.
  • ordinary purification methods for example, solvent extraction / distillation 'column chromatography / one-liquid chromatography / recrystallization.
  • the type of peptide salt when the peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, Can be converted to the free form or other salts by a known method or a method analogous thereto.
  • the peptide used in the present invention can also be produced by a genetic engineering technique.
  • the DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned raw materials, cDNA library derived from the above-mentioned raw materials, and synthetic DNA.
  • the polynucleotide can be incorporated into a vector using a known method such as DNA ligase or restriction enzyme, and the vector can then be amplified in a host cell. It is. There are no particular limitations on the vectors, host cells, etc. as long as they are known. A large amount of the peptide used in the present invention can be obtained by separating and purifying the peptide of the present invention from the cultured host cells. The separation and purification method is not particularly limited as long as it is a known method.
  • the peptide represented by the above formula (1) or a salt thereof (hereinafter abbreviated as the peptide of the present invention) obtained as described above can be used as a DPPIV inhibitor.
  • the peptides of the present invention can be used alone or in combination of two or more.
  • the DPPIV inhibitor of the present invention only needs to contain the peptide of the present invention, but from the viewpoint of high DPPIV inhibitory activity with a high content of the peptide of the present invention, the molecular weight is 1,500 or less. It is desirable to contain 50% or more of peptide, preferably 70% or more, more preferably 90% or more.
  • the DPPIV inhibitor of the present invention can be used as an agent for preventing or treating diabetes in mammals.
  • the DPPIV inhibitor When used as a prophylactic / therapeutic agent for diabetes, an effective amount thereof can be administered to a human or non-human mammal according to conventional means.
  • the diabetes treatment 'preventive agent of the present invention comprises an oral or parenteral solid comprising a peptide represented by the formula (1) or a salt thereof and a pharmaceutically acceptable pharmaceutical carrier. It is prepared as a liquid pharmaceutical composition.
  • Examples of the solid composition for oral administration include tablets, pills, capsules, fine granules, granules and the like.
  • one or more active substances are present in at least one inert diluent, such as lactose, mannitol, glucose, hydroxypropylcellulose, crystalline cellulose, various starches, polypyrrole pyrrolidone. And mixed with magnesium aluminate metasilicate.
  • the composition may contain additives other than inert diluents, such as lubricants such as magnesium stearate, disintegrants such as calcium calcium glycolate, stabilizers such as ratatose, glutamic acid or It may contain a solubilizing or solubilizing agent such as aspartic acid.
  • lubricants such as magnesium stearate
  • disintegrants such as calcium calcium glycolate
  • stabilizers such as ratatose
  • glutamic acid glutamic acid
  • a solubilizing or solubilizing agent such as aspartic acid.
  • tablets, pills, granules, and capsules containing granules may be made of sucrose, gelatin, if necessary. Or a film of gastric or enteric material.
  • the preparation can be prepared by performing a known solubilization treatment.
  • Liquid compositions for oral administration include pharmaceutical forms such as pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, etc.
  • Inert diluents that are used specifically, such as purified water, ethanol and the like.
  • This composition contains adjuvants such as wetting agents, suspending agents, sweeteners, flavors, fragrances and preservatives in addition to inert diluents.
  • Injectables for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.
  • Aqueous solutions and suspensions include, for example, distilled water for injection and physiological saline.
  • Non-aqueous solutions and suspensions include, for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, pharmaceutically acceptable alcohols such as ethanol, and polyoxyethylene sorbitan fatty acid esters.
  • a surfactant is included.
  • aqueous and non-aqueous compositions include wetting agents, suspending agents, emulsifying agents, dispersing agents, stabilizing agents (eg, extra leuth), solubilizing agents and solubilizing aids (eg, Auxiliary agents such as glutamic acid aspartic acid) and preservatives may be contained. These are sterilized by, for example, filtration through a bacteria-retaining filter, blending with a bactericide, or irradiation. These can also be produced as a sterile solid composition and dissolved in sterile water or a sterile solvent for injection before use.
  • the peptide of the present invention In order to use the peptide of the present invention for the purpose of preventing or treating the above-mentioned diseases, it is usually administered orally or parenterally.
  • the dose of peptide and the like varies depending on the age, weight, symptom, therapeutic effect, route of administration, etc. of the subject, and is appropriately set in consideration of these. 1 mg to 10 g, preferably 10 mg to 3 g, and 0.1 mg to parenteral administration is preferable. This is given once a day or divided into 2 to several times. Since the dosage varies depending on the purpose of prevention and various other conditions, an amount smaller than the above-mentioned dose range may be sufficient. If the subject is a non-human mammal If you adjust the dose according to the above human.
  • the DPPIV inhibitor of the present invention also has an action of improving various diseases associated with diabetes (eg, hyperlipidemia, gout or hyperuricemia). Therefore, the DPPIV inhibitor of the present invention can also be used as a prophylactic / therapeutic agent for various diseases associated with diabetes (for example, hyperlipidemia, gout or hyperuricemia).
  • the DPPIV inhibitor of the present invention obtained in this way is derived from collagen or gelatin and is safe and low toxic, for example, mammals (for example, humans, rats, mice, guinea pigs, Usagi, bird, hidge, pig, ushi, horse, cat, inu, monkey, chimpanzee, etc.).
  • mammals for example, humans, rats, mice, guinea pigs, Usagi, bird, hidge, pig, ushi, horse, cat, inu, monkey, chimpanzee, etc.
  • Collagen peptide HACP-01 (manufactured by Gelais, processed with collagenase from pork skin) and HACP-U2 (manufactured by Gelais, collagenase treated with fish scale derived gelatin), collagen peptide SCP_5000 (manufactured by Nitta Gelatin) Gelatin, egg white peptide, and soybean peptide were each dissolved in 25 mM Tris-HCl buffer (pH 8.0) to a concentration of 10 mg / ml.
  • the activity inhibition experiment for DPPIV was performed according to the following method. 25 mM Tris-HCl buffer (pH 8.0) 2011 1, buffer or sample solution 5 H 1, diluted DPPIV solution (lng / 1) 5 1 were mixed and incubated at room temperature for 5 minutes. The enzyme reaction was started by adding 20 1 of a substrate solution (0.25 mM glycinoreproline 4-methylcumulyl 7-amide (Gly-Pro-MCA)) in 25 mM Tris-HCl buffer (pH 8.0). went.
  • a substrate solution (0.25 mM glycinoreproline 4-methylcumulyl 7-amide (Gly-Pro-MCA)
  • the amount of 7-amino-4-methylcoumarin (AMC) released by DPPIV was measured with a 96-well plate fluorescence detector (Fluoroscan Ascent: manufactured by Thermo Electron).
  • the excitation wavelength was 390 nm and the measurement wavelength was 460 nm.
  • 25 mM Tris-HCl buffer (pH 8.0) was used in the same manner instead of DPPI V solution.
  • the calculation of the DPPIV inhibition rate includes the sample solution! / ,! The inhibition rate (%) was obtained by subtracting from 100 the activity when the tide solution was added. The inhibition rate was calculated by correcting the pseudo-inhibition effect of the sample itself.
  • the DPPI V inhibition rate of each sample was 15.0% for HACP-01 and 20.5% for HACP-U2 at a sample final concentration of Sl.0 mg / ml, and 0% for SCP_5000, gelatin, egg white peptide, and soybean peptide ( (See Figure 1)
  • Collagen peptides HACP_01 HACP-U2 and SCP-5000 were each dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 25 50 100 mg / ml.
  • Example 2 In the same manner as in Example 1, an activity inhibition experiment on DPPIV was performed.
  • the DPPIV inhibition rate of HACP-01 was 36.5% when the final sample concentration was 2.5 mg / ml, 51.6% when 5.0 mg / ml, and 56.7% when 10 mg / ml. there were.
  • the DPPIV inhibition rate of HACP-U2 was 38.4% at the sample final concentration of 3 ⁇ 4.5 mg / ml, 50.4% at 5.0 mg / ml, and 56.7% at 10 mg / ml.
  • SCP-5000's DPPIV inhibition rate was 0% at the final sample concentration of 3 ⁇ 4.5 mg / ml and 5.0 mg / ml and 23.0% at 10 mg / ml (see Figure 2).
  • Collagen peptide (HACP-01: manufactured by Zelice) was fractionated by high performance liquid chromatography (manufactured by Shimadzu Corporation) connected with a capsule pack C18 UG80 (20 ⁇ 250 mm: manufactured by Shiseido Co.) column and a UV detector. Fractionation was performed under the following conditions.
  • acetonitrile containing 0.1% TFA start with 100% water (including 0.1% trifluoroacetic acid (TFA)) and increase the concentration of acetonitrile (including 0.1% TFA) from 0% to 100% in 100 minutes (hereinafter referred to as acetonitrile containing 0.1% TFA). 0—100% (100 minutes)).
  • Example 1 After concentrating each fraction, each was dissolved in 25 mM Tris-hydrochloric acid buffer (pH 8.0) 200 ⁇ 1 and an activity inhibition experiment on DPPIV was conducted in the same manner as in Example 1 using 51 of them. It was. The DPPIV inhibition rate was determined in the same manner as in Example 1. The DPPIV inhibition rate was 24.1% in the 22nd fraction, 65.0% in the 23rd fraction, 99.9% in the 24th fraction, and 90.6% in the 25th fraction. It was 43.1% in the 30th fraction.
  • the fraction in which DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) connected with an Atlantis dC18 (4.6 ⁇ 250: Waters) column and a UV detector.
  • the purification conditions were elution with 100% water containing 0.1% TFA (flow rate 1 ml / min) at 215 nm, and peak fractionation was performed.
  • a portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (Applied Biosystems). As a result, it was revealed that the amino acid sequence of the active peptide was Gly-Pro-Arg.
  • the peptide having the amino acid sequence was synthesized using a commercially available peptide synthesizer, and the inhibitory activity of DPPIV was examined using Gly-Pro-Arg synthesized as a standard product.
  • Synthetic peptide Gly-Pro-Arg was dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 1.0, 2.5, 5.0 mM, and 51 of these was used to inhibit the activity against DPPIV as in Example 1.
  • An experiment was conducted. When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 31.3% when the final sample concentration was 0.1 mM, 44.7% when 0.25 mM, and 62.6% when 0.5 mM.
  • the concentration at which 50% inhibition of DPPIV activity was determined by drawing an approximate curve was 283.1 ⁇ (see Table 1, “GPR”).
  • the 30th fraction which has a relatively high inhibitory activity, was developed with Develocil C30-UG-5 (20x 250 mm (manufactured by Nomura Chemical Co., Ltd.) Further fractionation was performed by high performance liquid chromatography 1 (manufactured by Shimadzu Corporation) connected with a column and a UV detector. Fractionation conditions were as follows: 0.1% TFA containing acetonitrile 2--20% (100 minutes) linear gradient elution (flow rate 10 ml / min), detection at 215 nm, and fractionation from 0 min to 1 bottle / min. went.
  • the 31st fraction having inhibitory activity was subjected to high performance liquid chromatography (Shimadzu Corporation) connected to a capsule pack Ph UG120 (20x250: manufactured by Shiseido Co., Ltd.) column and a UV detector.
  • the product was further fractionated. Fractionation conditions were linear gradient elution of acetonitrile (0-20% (100 min) containing 0.1% TFA) (flow rate: 10 ml / min) at 215 nm, and fractionation was performed for each peak.
  • the fraction in which DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) connected with a capsule pack C18 MG (4.6x250mm: manufactured by Shiseido) column and a UV detector.
  • the purification conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and peak fractionation.
  • a portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (manufactured by Abride Biosystems). As a result, it was revealed that the amino acid sequence of the active peptide was Gly-Pro-Ser-Gly-Asn-Ala (SEQ ID NO: 1).
  • the inhibitory activity of DPPIV was examined using Gly-Pro-Ser-Gly-Asn-Ala synthesized as a standard. Synthetic peptide Gly-Pro-Ser-Gly-Asn-Ala was dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 1.0, 2.5, and 5.0 mM. Similarly, an activity inhibition experiment for DPPIV was conducted. The DPPIV inhibition rate was determined in the same manner as in Example 1. However, Sampnore final concentration force ⁇ ) .4 ⁇ 9% at lmM, 66 ⁇ 3% at 0.25mM, 79.2 at 0.5mM
  • %Met The concentration at which DPPIV activity was inhibited by 50% by drawing an approximate curve was found to be 135 ⁇ 9 (see Table 1, “GPSGNA”).
  • Example 1 After concentrating each fraction, each was dissolved in 25 mM Tris-HCl buffer (pH 8.0) 2001, and an activity inhibition experiment on DPPIV was conducted in the same manner as in Example 1 using 51 of them.
  • the DPPIV inhibition rate was determined in the same manner as in Example 1.
  • the DPPIV inhibition rate was 13.2% in the 41st fraction, 44.5% in the 2nd fraction, 16.4% in the 43rd fraction, 31.0% in the 44th fraction.
  • the fraction in which DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) coupled with an Atlantis dC18 (4.6x250: Waters) column and a UV detector.
  • the purification conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and peak fractionation.
  • a portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (manufactured by Abride Biosystems). As a result, it was revealed that the amino acid sequence of the active peptide was Gly-Pro-Ala-Gly-Pro-Ala (SEQ ID NO: 2).
  • Example 6 A high-speed liquid in which the 47th and 48th fractions having relatively high inhibitory activity were combined, and a Develocil C30-UG-5 (20x250mm: Nomura Chemical Co.) column and a UV detector were connected. Further fractionation was performed by chromatography (manufactured by Shimadzu Corporation). Fractionation condition is 0.1% TFA Detection was performed at 215 ⁇ m by linear gradient elution (flow rate 10 ml / min) of acetonitrile containing 0-20% (100 min), and fractionation was performed from 0 min to 1 bottle / min.
  • the 54th fraction having a relatively high inhibitory activity was used as a high performance liquid chromatograph to which a capsule pack Phenyl UG120 (4.6x250: manufactured by Shiseido Co., Ltd.) column and a UV detector were connected. Further fractionation was performed by GRAPHI (Waters). Fractionation conditions were as follows: linear gradient elution (acetonitrile 0-20% (100 min) containing 0.1% TFA) (flow rate 1 ml / min), detection at 215 nm, and fractionation for each peak.
  • the fraction in which DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) connected with an Atlantis dC18 (4.6x250: Waters) column and a UV detector.
  • the purification conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and peak fractionation.
  • a portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (manufactured by Abride Biosystems). As a result, it was revealed that the amino acid sequence of the active peptide was Gly-Pro-Va Gly-Ala-Arg (SEQ ID NO: 3).
  • the inhibitory activity of DPPIV was examined using Gly-Pro-Va ⁇ Gly-Ala-Arg synthesized as a standard product. Synthetic peptide Gly-Pro-Va ⁇ Gly-Ala-Arg was dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 1.0, 2.5, 5.0 mM, and 51 of these were used as Example 1 and Similarly, an activity inhibition experiment for DPPIV was conducted. The DPPIV inhibition rate was determined in the same manner as in Example 1. However, the final concentration of Sampnore SO. LmM is 37.7%, 0.25mM is 66.1%, 0.5mM is 76.1%
  • %Met concentration at which DPPIV activity was inhibited by 50% by drawing an approximate curve was 163.0 M (see Table 1, “GPVGAR”).
  • the 55th fraction with relatively high inhibitory activity was added to a capsule pack Phenyl UG120 (4.6x2 50: manufactured by Shiseido) column and a UV detector. Further fractionation was performed by linked high performance liquid chromatography (Wooters). Fractionation conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and fractionation was performed for each peak.
  • the fraction in which DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) connected with an Atlantis dC18 (4.6x250: Waters) column and a UV detector.
  • the purification conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and peak fractionation.
  • a portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (manufactured by Abride Biosystems). As a result, the amino acid sequence of the active peptide was revealed to be Gly-Pro-Ser-Gly-Glu-Arg-Gly-Pro-Hyp (SEQ ID NO: 4).
  • the inhibitory activity of DPPIV was examined using Gly-Pro-Ser-Gly-Glu-Arg-Gly-Pro-Hyp synthesized as a standard product. Tris synthetic peptide Gly-Pro-Ser-Gly- Glu-Arg-Gly-Pro-Hyp of 2 5 m M - 1.0 hydrochloride buffer (pH 8.0), 2.5, were dissolved at a 5.0 mM, 5 1 of which In the same manner as in Example 1, an activity inhibition experiment on DPPIV was conducted using When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 32.5% when the sample final concentration was SO.lmM, 58.5% when 0.25 mM, and 74.4% when 0.5 mM. Also, draw an approximate curve to increase the activity of DPPIV. The concentration at which% inhibition was determined was 190.5 M (see Table 1, “GPSGERG PO”).
  • Capsule pack C18 UG80 (20x250mm: manufactured by Shiseido Co., Ltd.) is a combination of the fractions with relatively high inhibitory activity among the fractions fractionated in the capsule pack C18UG80 column (first time) in Example 3. Further fractionation was performed by high performance liquid chromatography (manufactured by Shimadzu Corporation) connected with a column and a UV detector. The fractionation conditions were linear gradient elution (flow rate 10ml / min) of acetonitrile 0–20% (100 min) containing 0.1% TFA, detection at 215nm, and fractionation from 0 min to 1 bottle / min. .
  • the fraction confirmed to have DPPIV inhibitory activity was subjected to high performance liquid chromatography (water) coupled with an Atlantis dC18 (4.6x250: Waters) column and a UV detector.
  • the product was further purified. Purification conditions were linear gradient elution of acetonitrile (0-20% (100 min) containing 0.1% TFA) (flow rate: 1 ml / min), detection at 215 nm, and peak fractionation.
  • a portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified using a protein sequencer (manufactured by Abride Biosystems). As a result, the amino acid sequence of the active peptide was found to be Gly-Pro-Va Gly-Pro-Ala (SEQ ID NO: 5).
  • the 64th and 65th fractions having inhibitory activity were combined, and the capsule pack Phenyl U G120 (4.6x250: manufactured by Shiseido Co., Ltd.) column and UV detector were combined. Further fractionation was carried out using a high-performance liquid chromatography (manufactured by Waters). The fractionation conditions were linear gradient elution (flow rate 1 ml / min) of acetonitrile 0-20% (100 min) containing 0.1% TFA, detection was performed at 215 nm, and fractionation was performed for each peak.
  • the fraction in which DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) connected with an Atlantis dC18 (4.6x250: Waters) column and a UV detector.
  • the purification conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, peak Sorting was performed.
  • a portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (manufactured by Abride Biosystems). As a result, it was revealed that the amino acid sequence of the active peptide is Gly-Pro-Ile-Gly-Ser-Ala (SEQ ID NO: 6)
  • the inhibitory activity of DPPIV was examined using Gly-Pro-Ile-Gly-Ser-Ala synthesized as a standard product. Synthetic peptide Gly-Pro-Ile-Gly-Ser-Ala was dissolved in 25 mM Tris-hydrochloric acid buffer (pH 8.0) to 0.5, 1.0, 2.5 mM, and 51 of them was used in Example 1. In the same manner as above, an activity inhibition experiment against DPPIV was conducted. When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 32.2% when the final sample concentration was 0.05 mM, 47.3% when O.lmM, and 70.9% when 0.25 mM. The concentration at which 50% inhibition of DPPIV activity was determined by drawing an approximate curve was 107.1 ⁇ M (see Table 1, “GPIGSA”).
  • the 66th and 67th fractions having inhibitory activity were combined, and the capsule pack Phenyl U G120 (4.6x250: manufactured by Shiseido Co.) column and UV detector were combined. Further fractionation was carried out using a high-performance liquid chromatography (manufactured by Waters). The fractionation conditions were linear gradient elution (flow rate 1 ml / min) of acetonitrile 0-20% (100 min) containing 0.1% TFA, detection was performed at 215 nm, and fractionation was performed for each peak.
  • the fifth fraction confirmed to have DPPIV inhibitory activity was further purified by high performance liquid chromatography (Waters) connected with an Atlantis dC 18 (4.6x250: Waters) column and a UV detector. did.
  • the purification conditions were linear gradient elution (acetonitrile 0-20% (100 min) containing 0.1% TFA) (flow rate 1 ml / min), detection at 215 nm, and peak fractionation.
  • a part of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (Applied by Systems). As a result, the active peptide It was revealed that the amino acid sequence of tide was Gly-Leu-Ala-Gly-Pro-Hyp (SEQ ID NO: 7).
  • the inhibitory activity of DPPIV was examined using Gly-Leu-Ala-Gly-Pro-Hyp synthesized as a standard product. Synthetic peptide Gly-Leu-Ala-Gly-Pro-Hyp was dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 1.0, 2.5, and 5.0 mM. Similarly, an activity inhibition experiment for DPPIV was conducted. When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was found to be 31.7% for Sampu Nore final concentration force ⁇ ). LmM, 55.2% for 0.25 mM, and 69.8% for 0.5 mM. The concentration at which 50% inhibition of DPPIV activity was determined by drawing an approximate curve was 211.1 M (see Table 1, “GLAGPO”).
  • Example 11 The 70th, 71st and 72nd fractions having relatively high inhibitory activity were combined, and a Develocil C30-UG-5 (20x250mm: manufactured by Nomura Chemical Co.) column and UV detector were used. Further fractionation was performed by linked high performance liquid chromatography (manufactured by Shimadzu Corporation). The fractionation conditions were linear gradient elution (flow rate 10ml / min) of acetonitrile 0–20% (100 min) containing 0.1% TFA, detection at 215nm, and fractionation from 0 min to 1 bottle / min. .
  • the 76th fraction having inhibitory activity was subjected to high performance liquid chromatography (Waters) connected with a capsule pack Phenyl UG120 (4.6x250: manufactured by Shiseido Co., Ltd.) column and a UV detector. The product was further fractionated. Fractionation conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and fractionation was performed for each peak.
  • the fraction in which the DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) coupled with an Atlantis dC18 (4.6x250: Waters) column and a UV detector.
  • the purification conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and peak fractionation.
  • a portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (manufactured by Abride Biosystems). As a result, it was revealed that the amino acid sequence of the active peptide was Gly-Pro-Arg-Gly-Arg-Thr-Gly-Asp-Ala-Gly-Pro-Val (SEQ ID NO: 8).
  • the 28th fraction having a relatively high inhibitory activity was treated with a capsule pack C18 UG80 (20 x 250 mm: manufactured by Shiseido) column and a UV detector. Further fractionation was performed with a linked high performance liquid chromatography (manufactured by Shimadzu Corporation). Fractionation conditions were as follows: 0.1% TFA containing acetonitrile 2--20% (100 minutes) linear gradient elution (flow rate 10 ml / min), detection at 215 nm, and fractionation from 0 min to 1 bottle / min. went.
  • Fractionation conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and fractionation was performed for each peak. After each fraction was enrichment, respectively 2 5 mM Tris - it was dissolved in hydrochloric acid buffer (pH 8.0) 50 1, was subjected to the activity inhibition experiments against DPPIV in the same manner as in Example 1 using 5 1 of them. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition activity was observed only in the fourth peak portion, and the inhibition rate was 33.1%.
  • the fraction in which DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) connected with an Atlantis dC18 (4.6x250: Waters) column and a UV detector.
  • the purification conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and peak fractionation.
  • a portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (manufactured by Abride Biosystems). As a result, it was revealed that the amino acid sequence of the active peptide was Gly-Pro-Va ⁇ Gly-Pro (SEQ ID NO: 9).
  • Collagen peptide HACP-01 is expected to be included in the 3-residue peptide Gly-Pro-Met, Gly-Pro-Ala, Gly-Pro_Ser, and Gly-Pro-Hyp as standard products. Based on the retention time by, we attempted to isolate these 3-residue peptides from HACP-01. After purification to a single peak for each, the sequencer is targeted by the protein sequencer. Confirmed to match with peptide. In other words, it was revealed that the expected 3-residue peptide was included in HAC P-01. Since these peptides were also expected to have DPPIV inhibitory activity, the DPPIV inhibitory activity of each peptide was examined.
  • a glucose tolerance test was conducted using diabetic model rats. As experimental animals, Zucker Fatty rats were used.
  • the breeding environment was a laboratory animal breeding room with a constant temperature and humidity, 12 hours light / 12 hours dark cycle, and water was freely ingested using a commercially available solid feed (CRF-1, manufactured by Oriental Yeast Co., Ltd.). .
  • the general condition was observed every day for 5 days or more, and it was confirmed that the health condition was good.
  • Body weight was measured at the time of arrival, at the time of ingestion of force and at the time of test substance administration. Surgery for indwelling force for blood collection was performed the day before the glucose tolerance test. Grouping was performed by randomized stratification by weight stratification prior to test substance administration.
  • collagen peptide HACP-01 400 mg / ml, Zerais Co., Ltd.
  • collagen peptide SCP_5000 Nita Gelatin Co., 4 OOmg / ml
  • control Commercially available water for injection manufactured by Otsuka Pharmaceutical Factory
  • 10% glucose was orally administered at 10 ml / kg.
  • Blood was collected immediately before glucose administration and 5, 10, 15, 30 minutes after administration. After blood collection, plasma was separated and used for the following experiments.
  • the blood parameters that are considered to undergo changes in concentration and activity when glucose is loaded under conditions that inhibit DPPIV activity include insulin, GLP-1 and DPPIV activity. In this experiment, we attempted to evaluate the effectiveness of collagen peptides by measuring them over time.
  • DPPI V activity in plasma was measured over time according to non-patent literature (J. Med. Chem. 2003 Jun 19; 46 (13): 2774_89). Specifically, the obtained plasma 151 and test buffer (25 mM hepes, 140 mM sodium chloride, 80 mM magnesium chloride, 1% BSA: pH 7.8) 15 to 1 were mixed and left at room temperature for 5 minutes. . Substrate solution diluted to 100 M with test buffer (Gly-Pro-MCA O 1 was added and left at room temperature for 20 minutes. Detection was performed using a 96-well plate fluorescence detector (Fluoroscan Ascent: The amount of AMC released by DP PIV was measured using Thermo Electron Co., Ltd. The excitation wavelength was 390 nm and the measurement wavelength was 460 mm, and the activity was determined from the fluorescence value generated by the reaction for 20 minutes. The fluorescence value shown in the case of containing only was subtracted.
  • GLP-1 concentration in plasma was measured using a GLP-1 detection kit (manufactured by Rinco Research). Each plasma 50 1 was added to the GLP-1 antibody immobilized on a 96-well plate and left at 4 ° C for 20 to 24 hours. By washing anything other than immobilized GLP-1 After removing, an antibody having alkaline phosphatase that binds to immobilized GLP-1 was added, and left at room temperature for 2 hours. After removing non-specifically bound antibody by washing, 200,11 substrate solution (4-methylumbelliferyl phosphate) prepared at 50 g / ml was added to start the enzyme reaction.
  • the reaction was stopped by adding stop solution 50 ⁇ 1 and the fluorescent substance umbelliferone obtained by the reaction was measured with a 96-wall plate fluorescence detector (Fluoroscan Ascent: manufactured by Thermo Electron). .
  • the excitation wavelength was 355 nm and the measurement wavelength was 460 nm.
  • the concentration of GLP-1 in each plasma was determined from a calibration curve obtained by measuring GLP-1 at known concentrations.
  • the effect of collagen peptide on the increase in blood GLP-1 concentration was determined by setting the plasma GLP-1 concentration before administration of the test substance (-30 minutes) to the normal concentration (100%) for each individual. The ratio of the concentration in each plasma relative to was calculated. As a result, the rate of increase in GLP-1 concentration in each experimental group was 120% immediately before glucose administration (0 minutes), 122% 5 minutes after administration, 117% at 10 minutes, and 112% at 15 minutes in HACP-01. It was 116% in 30 minutes. In SCP-5000, 111% immediately before glucose administration (0 minutes), 115% at 5 minutes after administration, 104% at 10 minutes, 103% at 15 minutes, and 98% at 30 minutes.
  • the insulin concentration in plasma was measured using an insulin measurement kit (manufactured by Morinaga Bioscience Institute). Guinea pig anti-insulin serum 50 1 prepared with sample diluent 2 and insulin diluted on 96-well plate, 45 1 sample diluent 2 and 5 ⁇ l of each plasma are added, starting at 4 ° C for 16 hours. Let stand for 20 hours. The non-immobilized insulin was removed by washing, and enzyme-labeled anti-guinea pig IgG antibody that binds to the immobilized insulin was added and allowed to stand at room temperature for 3 hours. After removing non-specifically bound antibody by washing, an enzyme substrate solution 100 ⁇ 1 was added to start the enzyme reaction.
  • a long-term administration test was conducted using diabetic model rats.
  • the experimental animals used were Zucker Fatty rats.
  • the breeding environment is a laboratory animal breeding room with a constant temperature and humidity, 12 hours light / 12 hours dark cycle, and the diet is a high fat diet for diabetes and obesity research (“Quick Fat”) for one week before the grouping (acclimation period).
  • Quick Fat diabetes and obesity research
  • test substance collagen peptide: HACP-01
  • control substance amino acid mixture
  • the amino acid mixture of the control substance was mixed so as to have the same amino acid composition as the test substance collagen peptide.
  • the general condition was observed every day for 5 days or more to confirm that the health condition was good. Grouping was performed by randomized stratification by weight stratification prior to test substance administration.
  • Body weight was measured at the time of arrival, at the time of grouping, and once a week during the test substance administration period (Table 1).
  • Food intake 'Water intake was measured twice a week. Collect blood at the time of grouping. After 3 weeks and 6 weeks, the animals were fasted (fasted for 18 hours or more), and the others were performed once a week at normal times via the caudal vein. After blood collection, plasma was separated and used for the following experiments.
  • Test substance administration period Test substance intake group Control substance intake group
  • the glucose concentration in plasma was determined using a glucose CII test KOKO (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, it was confirmed that the glucose concentration in the test substance intake group was significantly lower at 1 and 3 weeks after the test substance administration compared to the control substance intake group (see Fig. 6). That is, it became clear that ingesting the collagen peptide HACP-01 lowered the blood glucose concentration.
  • the triglyceride E test concentration was obtained using Wako Pure Chemical Industries, Ltd., and the tridallyceride concentration in plasma was determined. As a result, it was confirmed that in the test substance intake group, the tridalylide concentration was significantly lower after 2, 3, 4 and 5 weeks after the test substance administration compared to the control substance intake group. (See Figure 7.) That is, it has been clarified that the intake of collagen peptide HACP-01 decreases the blood tridalylide concentration.
  • the uric acid concentration in plasma was determined using a uric acid C test kit (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, it was confirmed that the uric acid concentration was significantly lower in the test substance intake group 4 weeks and 6 weeks after the test substance administration than in the control substance intake group (see FIG. 8). In other words, it was clarified that the intake of collagen peptide HACP-01 reduces the uric acid concentration in the blood.
  • Urine was collected 3 weeks and 6 weeks after administration of the test substance.
  • a 24-hour urine collection was collected, and the uric acid concentration in the urine was measured using a uric acid C test kit (manufactured by Wako Pure Chemical Industries, Ltd.) to determine the amount of uric acid excretion per day.
  • the test substance intake group compared to the control substance intake group.
  • uric acid excretion decreased by 15.2% after 3 weeks of test substance administration and by 22.7% after 6 weeks of test substance administration (see Fig. 9). That is, it became clear that the amount of uric acid excretion decreased by ingesting the collagen peptide HACP-01.
  • the intake of the collagen peptide HACP-01 also reduces the uric acid concentration in the blood, which is thought to suppress uric acid synthesis! /.
  • a blood glucose measurement test with 10 in-house volunteers was conducted at the crossover. In accordance with the spirit of the Declaration of Helsinki, the subjects were fully explained to the subject of the study, and the study was conducted with written consent. The subject was fasted for more than 9 hours from the previous day, and after measuring the blood glucose level with a blood glucose self-monitoring device (One Touch Ultra: Johnson 'End' Johnson), collagen peptide (HACP-Ol) lOg and water, egg white peptide 10g and water Took either water only. Thirty minutes later, the blood glucose level was measured again, and an oral glucose tolerance test sugar solution (Traillan G75: Ajinomoto Co., Inc.) was ingested.
  • each subject measured blood glucose level at 10, 20, 30, 45, 60, 90, and 120 minutes after ingesting the sugar solution.
  • blood glucose in each intake group was 87.5 mg / dl before collagen peptide intake (-30 minutes) and 92. lmg / dl immediately before sugar solution intake (0 minutes) in the collagen peptide intake group.
  • 115.6 mg / dl after 10 minutes 130.1 mg / dl at 20 minutes, 130.0 mg / dl at 30 minutes, 113.6 mg / dl at 45 minutes, 101.6 mg / dl at 60 minutes, 117.4 mg / dl at 90 minutes, It was 107. lmg / dl in 120 minutes.
  • Collagen peptide HACP-01 (manufactured by Zelice) was fractionated using a synthetic adsorbent DIAION HP20 (manufactured by Mitsubishi Chemical Corporation). 100 g of the synthetic adsorbent and 10 g of collagen peptide were mixed in water, stirred for 1 hour, allowed to stand, and the supernatant was filtered and collected. Thereafter, 100 ml of a 10% aqueous ethanol solution was added, stirred for 10 minutes, and allowed to stand, and the supernatant was filtered and collected. Repeat the same operation for 20%, 30%, 40%, 50%, 75% aqueous ethanol and 100% ethanol. The filtrate was recovered even after the addition of the sol solution.
  • the absorbance of the collected 8 fraction filtrates was measured with a spectrophotometer (manufactured by JASCO Corporation). The measurement at 215 nm was performed by diluting the filtrate 1000 times. The measurement at 280 nm was performed using the filtrate as it was. As a result, the absorbance at 215 nm was 0.205 for the ethanol concentration fraction at 0%, 0.087 for the 10% elution fraction, 0.163 for the 20% elution fraction, 0.243 for the 30% elution fraction, 0.219 for the 40% elution fraction, It was 0.179 for the 50% elution fraction, 0.117 for the 75% elution fraction, and 0.027 for the 100% elution fraction (see Figure 11).
  • the absorbance at 280 nm is 0.288 for the ethanol concentration 0% elution fraction, 0.187 for the 10% elution fraction, 0.351 for the 20% elution fraction, 0.552 for the 30% elution fraction, and 1.075 for the 40% elution fraction. It was 1.573 for the 50% elution fraction, 1.592 for the 75% elution fraction, and 1.053 for the 100% elution fraction. Absorption at 280nm is probably due to contaminants in HACP-01. When sensory tests were performed on each of these fractions, the unpleasant odor (collagen odor) was significantly reduced in the elution fractions with ethanol concentration of 10%, 20%, and 30% or more. It was.
  • the collected 8 fraction filtrates were concentrated with a rotary evaporator under reduced pressure to remove ethanol, and then lyophilized to obtain peptide powder.
  • the peptides obtained in this way are MHP-0, 10, 20, 30, 40, 50, 75, 100 (ethanol concentration%, 10%, 20%, 30%, 40%, 50%, 75% and 100% fractions).
  • Each of these peptide powders was dissolved in 25 mM Tris-HCl buffer (pH 8.0) so as to be 10 mg / ml.
  • an activity inhibition experiment on DPPIV was conducted in the same manner as in Example 1.
  • MHP-0 was 36.8%
  • MHP-10 was 4.7%
  • MHP-20 was 22.1%
  • MHP-30 was 11.9%
  • MHP- 40 was 3 ⁇ 8%
  • MHP-50 was 0%
  • MHP-75 was 10 ⁇ 1%
  • MHP-100 was 0% (see Figure 11).
  • MHP-0 contained many low molecular weight peptides having a molecular weight of 500 or less (see FIG. 12).
  • collagen adsorbent H was added to synthetic adsorbent DIAION HP20 using water as a solvent.
  • ACP-01 adsorbing ACP-01 and collecting the non-adsorbed fraction, it is possible to obtain a fraction MHP-0 that further enhances the DPPIV inhibitory activity and contains a large amount of low-molecular-weight peptides.
  • the peptide obtained by this method is a small molecule that is likely to be absorbed in the digestive tract and has a high DPPIV inhibitory activity, so it requires less intake compared to HACP-01 before purification. Conceivable.
  • the unpleasant odor (collagen odor) is reduced, it is considered easy to take.
  • Collagen peptide HACP-01 (manufactured by Zerais) was fractionated by precipitation using ethanol. 1.5 g of collagen peptide and 15 ml of 70%, 75%, 80%, 85%, 90% and 95% ethanol aqueous solution were mixed well and left at -20 ° C. The supernatant fraction was collected and concentrated with a rotary evaporator under reduced pressure to remove ethanol, and then freeze-dried to obtain peptide powder. The precipitate fraction was dissolved in water and then freeze-dried to obtain peptide powder.
  • the supernatant fraction is 27.0%, the precipitation fraction is 4.4%, the supernatant fraction with ethanol concentration of 80% is 36.5%, the precipitation fraction is 5.6%, the supernatant fraction with ethanol concentration of 85% is 41.7%, the precipitation fraction The fraction was 13.5%, the 90% supernatant fraction was 38.0%, the precipitate fraction was 15.8%, the 95% supernatant fraction was 42.0%, and the precipitate fraction was 17.5%.
  • the DPPIV inhibition rate of HACP-01 before fractionation was 18.9% (see Figure 13).
  • the supernatant fraction having an ethanol concentration of 85% obtained in Example 18 was further fractionated using a synthetic adsorbent DIAIO N HP20 (manufactured by Mitsubishi Chemical Corporation).
  • the synthetic adsorbent and the supernatant fraction with an ethanol concentration of 85% were mixed in water, stirred for 1 hour, allowed to stand, and the supernatant was filtered and collected. Thereafter, a 100% ethanol solution was added and stirred for 10 minutes, and then allowed to stand, and the supernatant was filtered and collected. Concentrate each fraction on a rotary evaporator under reduced pressure and freeze. The peptide powder was obtained by drying.
  • the peptides thus obtained were E85S_0 (HP20) and E85S_100 (HP20) (the ethanol fraction 85% fraction was fractionated using HP20 and the ethanol concentrations were 0% and 100%, respectively. Minutes).
  • Each of these peptide powders was dissolved in 25 mM Tris-HCl buffer (pH 8.0) so as to be 20 mg / ml.
  • an activity inhibition experiment against DPPIV was conducted in the same manner as in Example 1.
  • the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 58.1% for E85S_0 (HP20) and 41.2% for E85S_100 (HP20) (see FIG. 14).
  • Collagen peptides HACP-01 and E85S_0 were dissolved in 25 mM Tris-HCl buffer (pH 8.0) to a concentration of 20, 50, 100 mg / ml and 10, 20, 50 mg / ml, respectively.
  • the concentration at which DPPIV activity was inhibited by 50% by drawing an approximated curve was determined to be 8.4 mg / ml for HACP-01 and 1.4 mg / ml for E85S_0 (HP20).
  • the ability to increase the DPPIV inhibitory activity 6-fold by purifying the collagen peptide by combining the ethanol precipitation method and the purification method using a synthetic adsorbent was achieved.
  • the supernatant fraction having an ethanol concentration of 85% obtained in the same manner as in Example 18 was further fractionated in the same manner as in Example 16 using a synthetic adsorbent SEPABEADS SP850 (manufactured by Mitsubishi Chemical Corporation).
  • the collected filtrate of each fraction was concentrated with a rotary evaporator under reduced pressure to remove ethanol, and then freeze-dried to obtain peptide powder.
  • the peptide thus obtained was E85 S-0, 10, 20, 30, 40, 50, 100 (SP850) (ethanol concentration 85% supernatant fraction fractionated using SP850, ethanol concentration 0%, 10%, 20%, 30%, 40%, 50%, 100% fraction) I will do it.
  • the DPPIV inhibitor of the present invention can be used as an agent for preventing or treating diabetes in mammals.
  • the DPPIV inhibitor of the present invention can also be used as an ameliorating agent for various diseases associated with diabetes (for example, hyperlipidemia, gout or hyperuricemia).
  • the peptides and the like of the present invention are excellent in safety, it is possible to combine an effective amount with food, confectionery and the like. It is also possible to develop foods and confectionery that can prevent the onset of diabetes by continuously ingesting these foods and confectionery. Furthermore, various diseases associated with diabetes (for example, hyperlipidemia, gout or hyperuricemia) can be prevented, or foods and confectionery for patients suffering from these diseases can be developed.
  • various diseases associated with diabetes for example, hyperlipidemia, gout or hyperuricemia

Abstract

Disclosed is a dipeptidyl peptidase-IV inhibitor comprising a peptide derived from collagen or gelatin and comprising an amino acid sequence represented by the formula (1): Gly-X-Y-(Gly-Z-W)n [wherein n represents an integer of 0 to 4; X represents Pro or Leu; and Y, Z and W independently represent an amino acid residue (excluding Gly), a peptide comprising an amino acid sequence having the deletion of an amino acid residue W at the terminus of the above-mentioned amino acid sequence, or a salt thereof. Also disclosed is a therapeutic/prophylactic agent for diabetes, which comprises the dipeptidyl peptidase-IV inhibitor as an active ingredient. Further disclosed is a method for the prevention/treatment of diabetes, which comprises administering an effective amount of the dipeptidyl peptidase-IV inhibitor to a mammal. The dipeptidyl peptidase-IV inhibitor can be used as an ameliorating agent for diabetes or a disease associated with diabetes (e.g., hyperlipemia, gout, hyperuricemia).

Description

明 細 書  Specification
ジぺプチジ ぺプチダーゼ IV阻害剤  Dipeptidyl peptidase IV inhibitor
技術分野  Technical field
[0001] 本発明は、従来から食材として用いられてきたコラーゲンあるいはゼラチン由来の ジぺプチジルぺプチダーゼ IV阻害剤に関する。さらに本発明は、糖尿病治療 '予防 剤、哺乳動物の糖尿病の予防'治療法に関する。  [0001] The present invention relates to a collagen or gelatin-derived dipeptidyl peptidase IV inhibitor that has been conventionally used as a food material. Furthermore, the present invention relates to a method for treating diabetes 'a prophylactic agent, preventing diabetes in mammals'.
背景技術  Background art
[0002] 近年、全世界において糖尿病が爆発的に増加している。 日本では糖尿病患者 600 万人、その予備軍は 1200万人〜 1500万人といわれている。糖尿病では高血糖が続く ことによって血管が徐々に障害を受け、さまざまな臓器に異常が生じる。糖尿病性腎 症、糖尿病性網膜症、糖尿病性神経症が三大合併症として以前から知られているが 、近年では高脂血症や高尿酸血症などの代謝異常を併発し、動脈硬化症発症のリス クが高くなることも知られている。糖尿病には「瞵 /3細胞の破壊的病変でインスリンの 欠乏が生じて起こる」 I型糖尿病と「瞵 /3細胞の機能異常によるインスリン分泌能低下 と肝、筋、脂肪組織等の標的臓器におけるインスリン感受性低下が併発することによ つて発症する」 II型糖尿病がある。昨今激増する糖尿病は II型に由来するものであり 、糖尿病の 90〜95%を占めていると考えられている。 II型糖尿病は「生活習慣病」とい われているように、ストレス、肥満、運動不足による基礎代謝能低下と、それに加えて の高カロリー食摂取等、現代型社会生活によって引き起こされている。  In recent years, diabetes has increased explosively throughout the world. In Japan, it is said that there are 6 million people with diabetes, and 12 to 15 million reserves. In diabetes, blood vessels are gradually damaged by hyperglycemia, resulting in abnormalities in various organs. Diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy have long been known as three major complications, but in recent years they have been accompanied by metabolic abnormalities such as hyperlipidemia and hyperuricemia, resulting in arteriosclerosis. It is also known that the risk of onset increases. Diabetes mellitus is caused by insulin depletion caused by 瞵 / 3-cell destructive lesions. Type I diabetes and 瞵 / 3-cell dysfunction causes decreased insulin secretion and in target organs such as liver, muscle, and adipose tissue. It is caused by the concomitant decline in insulin sensitivity. ”Type II diabetes. Diabetes, which is increasing rapidly in recent years, is derived from type II and is considered to account for 90 to 95% of diabetes. Type II diabetes is caused by modern social life such as stress, obesity, decreased basic metabolic capacity due to lack of exercise, and intake of high-calorie foods.
[0003] このような糖尿病に関する研究分野において、消化管ホルモンであるインクレチン が注目されつつある。インクレチンはインスリン分泌を増強する消化管ホルモンの総 称で、 GIP (グルコース依存性インスリン分泌ポリペプチド (glucose-d印 endent insulinot ropic polyp印 tide》や GLP- 1(グノレ力ゴン様ペプチド- l(glucagon-like p印 tide_l》等 が知られている。これらは、瞵 /3細胞に発現する受容体を介したグルコース応答性ィ ンスリン分泌を促進し、食後の血糖上昇を抑制する。また、インスリン分泌促進以外 に、瞵 /3細胞の保護および増殖作用といった活性を持っている。し力もながら、インク レチンの問題点として、安定性が挙げられる。すなわち、インクレチンは体内に普遍 的に存在するジぺプチジルぺプチダーゼ IV (DPPIV)によって速やかに不活性なも のへと分解され、数分間で半減してしまう。そこで、 DPPIV阻害剤の開発が進められ ている(たとえば特許文献 1参照)。し力、しな力 、医薬品として開発されている DPPIV 阻害剤は非天然物の化学合成物であり、摂取する際の安全性に問題がある。 [0003] Incretin, which is a gastrointestinal hormone, is attracting attention in the field of research related to diabetes. Incretin is a general term for gastrointestinal hormones that enhance insulin secretion. GIP (glucose-d insulin secreted polypeptide, glucose-d endend insulinot ropic polyp tide) glucagon-like p-mark tide_l ”etc. These are known to promote glucose-responsive insulin secretion via a receptor expressed in 瞵 / 3 cells and suppress postprandial blood glucose elevation. In addition to promoting secretion, it has activities such as protecting and proliferating 瞵 / 3 cells, but the strength of incretin is one of the problems of incretin. Dipeptidyl peptidase IV (DPPIV) is rapidly degraded to inactive and halved in a few minutes. Therefore, development of DPPIV inhibitors is underway (see, for example, Patent Document 1). DPPIV inhibitors, which are being developed as pharmaceuticals, pliability, and pharmaceuticals, are non-naturally occurring chemical compounds and have a safety problem when ingested.
[0004] コラーゲンは従来力 食品として利用されており、コラーゲンを摂取することにより新 陳代謝が促進されること (特許文献 2)や頭髪の直径が太くなること (非特許文献 1)、 関節症治療用薬剤として利用可能なこと (特許文献 3)等が報告されている。また、コ ラーゲンタンパクもしくはその加水分解物の経口摂取による皮膚の新陳代謝促進に 関する特許 (特許文献 2)や生体内でのコラーゲン合成の促進に関する特許 (特許文 献 4)も開示され、主に美容向けの健康食品が多数販売されている。  [0004] Collagen has been conventionally used as a food and promotes metabolism by ingesting collagen (Patent Document 2), the hair diameter increases (Non-Patent Document 1), arthropathy It has been reported that it can be used as a therapeutic drug (Patent Document 3). Patents relating to the promotion of skin metabolism by oral intake of collagen protein or its hydrolyzate (Patent Document 2) and patents relating to the promotion of collagen synthesis in vivo (Patent Document 4) are also disclosed. There are many health foods for the market.
[0005] コラーゲンおよびその熱変性体であるゼラチンは粘性が高く凝固し易い性質を持 つため、加工適性を向上させるため、タンパク質分解酵素を用いて処理したもの、あ るいは酸-塩基分解により部分加水分解処理したものを使用することが多い。また、タ ンパク質であるため抗原性を有し、アレルギー体質のヒトの摂取には問題がある。そ のため、コラーゲンをコラゲナーゼによって、低分子化することにより抗原性をなくしァ レルギ一患者向けのタンパク質源あるいは輸液製剤成分としての利用が開示されて いる(特許文献 5)。また、コラゲナーゼによるコラーゲンの分解物の生理活性につい ては、フイブリン凝集阻害活性 (特許文献 6)、麻酔作用(非特許文献 2)が知られてい る。し力、しながら、コラーゲンあるいはその分解物に DPPIVの阻害活性があるといった 報告はない。  [0005] Collagen and its heat-denatured gelatin, which is highly viscous and easily coagulated, have been processed with proteolytic enzymes to improve processability, or by acid-base decomposition. In many cases, a partially hydrolyzed product is used. Moreover, since it is a protein, it has antigenicity, and there is a problem in the intake of humans with allergies. For this reason, the use of collagen as a protein source or an infusion preparation component for allergy patients by reducing the molecular weight of collagen with collagenase has been disclosed (Patent Document 5). As for the physiological activity of collagen degradation products by collagenase, fibrin aggregation inhibitory activity (Patent Document 6) and anesthetic action (Non-Patent Document 2) are known. However, there has been no report that collagen or its degradation products have DPPIV inhibitory activity.
[0006] 一方で、ペプチド性の DPPIV阻害剤に関する報告力 Sいくつかある(非特許文献 3〜  [0006] On the other hand, there are several reporting capabilities S related to peptidic DPPIV inhibitors (Non-patent Documents 3 to
6)。し力もながら、これらのペプチド性の DPPIV阻害剤は、食品由来ではないため、 摂取する際の安全性の点で充分とはいえない。また、コラーゲンあるいはゼラチン由 来のペプチドによる DPPIV阻害に関する報告はない。  6). However, these peptidic DPPIV inhibitors are not derived from food and are not sufficient in terms of safety when ingested. There is no report on DPPIV inhibition by collagen or gelatin derived peptides.
[0007] 糖尿病患者は高脂血症を合併していることが多いが、 DPPIV阻害剤によって血中 の脂質濃度の上昇が抑制されるとレ、う報告もある(非特許文献 7〜9)。し力、しな力 Sら、 これらの DPPIV阻害剤は非天然物の化学合成物であり、摂取する際の安全性に問 題がある。 [0008] 糖尿病患者は痛風あるレ、は高尿酸血症を併発してレ、ることも多!/、。痛風 ·高尿酸血 症は糖尿病同様、動脈硬化症の危険因子であり、血糖値 ·尿酸値ともに高い場合は 十分な注意が必要となる。 [0007] Diabetic patients often have hyperlipidemia, but there are reports that increase in blood lipid levels is suppressed by DPPIV inhibitors (Non-Patent Documents 7-9). . These DPPIV inhibitors are non-natural chemical compounds and have safety issues when ingested. [0008] Diabetes patients often have gout, and often have hyperuricemia! /. Gout / hyperuricemia, like diabetes, is a risk factor for arteriosclerosis. If both blood glucose and uric acid levels are high, sufficient caution is required.
[0009] また、血中の尿酸濃度と中性脂肪濃度に正の相関があることも報告されている(非 特許文献 10)。し力もながら、コラーゲンあるいはゼラチン由来のペプチドによる血中 尿酸濃度の上昇が抑制されるとレ、つた報告はな!/、。 [0009] It has also been reported that there is a positive correlation between blood uric acid concentration and neutral fat concentration (Non-patent Document 10). However, there is no report that the increase in blood uric acid concentration by collagen or gelatin-derived peptides is suppressed!
特許文献 1:特許第 3681110号号公報  Patent Document 1: Japanese Patent No. 3681110
特許文献 2 :特開平 7— 278012号公報  Patent Document 2: JP-A-7-278012
特許文献 3 :特開昭 63— 39821号公報  Patent Document 3: JP-A 63-39821
特許文献 4 :特許第 3802721号号公報  Patent Document 4: Japanese Patent No. 3802721
特許文献 5:特開平 7— 82299号公報  Patent Document 5: JP-A-7-82299
特許文献 6 :特開平 6— 46875号公報  Patent Document 6: JP-A-6-46875
非特許文献 1 : Nutrition Reports International, 13巻, 579頁, 1976年  Non-Patent Document 1: Nutrition Reports International, 13, 579, 1976
非特許文献 2 : Br.J.Pharmacol.,69巻, 551頁, 1980年  Non-Patent Document 2: Br. J. Pharmacol., 69, 551, 1980
非特許文献 3 : Arch.Biochem.Biophys., 218巻, 156頁, 1982年  Non-Patent Document 3: Arch.Biochem.Biophys., 218, 156, 1982
非特許文献 4 : Biochem.J., 252巻, 723頁, 1988年  Non-Patent Document 4: Biochem. J., 252, 723, 1988
非特許文献 5 : Biol.Chem.Hoppe-Seyler., 372巻, 305頁, 1991年  Non-Patent Document 5: Biol. Chem. Hoppe-Seyler., 372, 305, 1991
非特許文献 6 : J.Antibiot., 37巻, 422頁, 1984年  Non-Patent Document 6: J. Antibiot., 37, 422, 1984
非特許文献 7 : Diabetes, 51巻, 1461頁, 2002年  Non-Patent Document 7: Diabetes, 51, 1461, 2002
非特許文献 8 : Diabetes, 52巻, 741頁, 2003年  Non-Patent Document 8: Diabetes, 52, 741, 2003
非特許文献 9 : Diabetes, 55巻, 1695頁, 2006年  Non-Patent Document 9: Diabetes, 55, 1695, 2006
非特許文献 10 : J Zhejiang Univ Sci B., 8巻, 593頁, 2007年  Non-Patent Document 10: J Zhejiang Univ Sci B., 8, 593, 2007
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0010] 本発明の課題は、安全性の高い DPPIV阻害剤および該 DPPIV阻害剤を含有する 糖尿病治療'予防剤、ならびに前記 DPPIV阻害剤を用いる哺乳動物の糖尿病の予 防 '治療法、さらに糖尿病に随伴する高脂血症、痛風あるいは高尿酸血症の改善作 用を有する DPPIV阻害剤を提供することにある。 課題を解決するための手段 [0010] An object of the present invention is to provide a highly safe DPPIV inhibitor, a diabetes preventive agent containing the DPPIV inhibitor, a prophylactic agent for diabetes, and a method for preventing diabetes in mammals using the DPPIV inhibitor, as well as diabetes It is an object of the present invention to provide a DPPIV inhibitor having an action for improving hyperlipidemia, gout or hyperuricemia associated therewith. Means for solving the problem
本発明者らは、安全性の観点から、食材として用いられているコラーゲンあるいは ゼラチン由来のペプチドに着目し、研究を行った結果、 DPPIV阻害活性を有するぺ プチドを見出し、本発明の完成に至った。すなわち、本発明は、以下から構成される From the viewpoint of safety, the present inventors have focused on collagen or gelatin-derived peptides used as foodstuffs, and as a result of their research, have found peptides having DPPIV inhibitory activity and have completed the present invention. It was. That is, this invention is comprised from the following.
Yes
〔1〕コラーゲンまたはゼラチン由来のペプチドであって、式(1):  [1] A peptide derived from collagen or gelatin having the formula (1):
Gly-X-Y- (Gly-Z-W) n (1) Gly-X-Y- (Gly-Z-W) n (1)
(式中、 nは 0〜4の整数、 Xは Proまたは Leu、 Y、 Ζおよび Wはそれぞれ独立して同 一または異なる任意のアミノ酸残基 (ただし、 Glyを除く)を示す。 )  (In the formula, n is an integer of 0 to 4, X is Pro or Leu, Y, Ζ and W are each independently the same or different arbitrary amino acid residues (excluding Gly).)
で表されるアミノ酸配列からなるペプチド、前記のアミノ酸配列の末端のアミノ酸残基 W力 個欠失されたアミノ酸配列からなるペプチドあるいはその塩を含有する DPPIV 阻害剤、 A DPPIV inhibitor comprising a peptide comprising the amino acid sequence represented by the above, a peptide comprising the amino acid sequence deleted at the end of the amino acid sequence W amino acid sequence or a salt thereof,
〔2〕前記ペプチドが  [2] The peptide is
Giy_Pro_Argゝ Giy_Pro_Arg ゝ
Giy-Pro-Ser-Giy-Asn-Aia  Giy-Pro-Ser-Giy-Asn-Aia
Gly-Pro-Ala-Gly-Pro-Ala,  Gly-Pro-Ala-Gly-Pro-Ala,
Gly-Pro-Val-Gly-Ala-Arg,  Gly-Pro-Val-Gly-Ala-Arg,
Gly-Pro-Val-Gly-Pro-Ala,  Gly-Pro-Val-Gly-Pro-Ala,
Giy-Pro-Ile-Gly-¾er-Ala  Giy-Pro-Ile-Gly-¾er-Ala
Giy_Pro_Ser_Giy_Lrlu_Arg_Lrly_Pro_Hypゝ  Giy_Pro_Ser_Giy_Lrlu_Arg_Lrly_Pro_Hyp ゝ
Giy-Pro-Arg- ly-Arg- hr_Gly_Asp_Ala_Giy_Pro_Val、  Giy-Pro-Arg- ly-Arg- hr_Gly_Asp_Ala_Giy_Pro_Val,
Gly-Pro-Val-Gly-Pro,  Gly-Pro-Val-Gly-Pro,
Gly_Leu_Ala_Gly_Pro_Hyp、  Gly_Leu_Ala_Gly_Pro_Hyp,
力、らなる群より選ばれる 1種類以上のアミノ酸配列からなるペプチドである前記〔1〕記 載の DPPIV阻害剤、 The DPPIV inhibitor according to [1], which is a peptide consisting of one or more amino acid sequences selected from the group consisting of
〔3〕前記〔1〕または〔2〕に記載の DPPIV阻害剤を有効成分として含有する糖尿病治 療-予防剤、  [3] Diabetes treatment-prevention agent containing the DPPIV inhibitor according to [1] or [2] as an active ingredient,
〔4〕哺乳動物に対して、前記〔1〕または〔2〕に記載の DPPIV阻害剤の有効量を投与 することを特徴とする糖尿病の予防'治療法。 [4] An effective amount of the DPPIV inhibitor according to [1] or [2] is administered to a mammal Diabetes prevention and treatment characterized by.
〔5〕コラーゲンまたはゼラチン由来のペプチドであって、前記式(1)で表されるァミノ 酸配列からなるペプチド、前記のアミノ酸配列の末端のアミノ酸残基 W力 個欠失さ れたアミノ酸配列からなるペプチドあるいはその塩を含有し、糖尿病に随伴する高脂 血症の改善作用を有する DPPIV阻害剤、  [5] A peptide derived from collagen or gelatin, comprising a peptide comprising the amino acid sequence represented by the above formula (1), an amino acid residue at the end of the amino acid sequence, and an amino acid sequence deleted from the amino acid sequence A DPPIV inhibitor having an action to improve hyperlipidemia associated with diabetes,
〔6〕コラーゲンまたはゼラチン由来のペプチドであって、前記式(1)で表されるァミノ 酸配列からなるペプチド、前記のアミノ酸配列の末端のアミノ酸残基 W力 個欠失さ れたアミノ酸配列からなるペプチドあるいはその塩を含有し、糖尿病に随伴する痛風 あるいは高尿酸血症の改善作用を有する DPPIV阻害剤、  [6] A peptide derived from collagen or gelatin, comprising a peptide having an amino acid sequence represented by the formula (1), an amino acid residue at the end of the amino acid sequence, and a deleted amino acid sequence. A DPPIV inhibitor having an action to improve gout or hyperuricemia associated with diabetes,
〔7〕コラーゲンまたはゼラチン由来のペプチドであって、前記式(1)で表されるァミノ 酸配列からなるペプチド、前記のアミノ酸配列の末端のアミノ酸残基 W力 個欠失さ れたアミノ酸配列からなるペプチドあるいはその塩を含有し、分子量が 1 , 500以下の ペプチドを 50%以上含有することを特徴とする DPPIV阻害剤、  [7] A peptide derived from collagen or gelatin, comprising a peptide comprising the amino acid sequence represented by the formula (1), an amino acid residue at the end of the amino acid sequence, and an amino acid sequence deleted from the amino acid sequence A DPPIV inhibitor comprising a peptide having a molecular weight of 1,500 or less and a peptide having a molecular weight of 50% or more,
〔8〕コラーゲンまたはゼラチン由来のペプチドであって、前記式(1)で表されるァミノ 酸配列からなるペプチド、前記のアミノ酸配列の末端のアミノ酸残基 W力 個欠失さ れたアミノ酸配列からなるペプチドあるいはその塩を含有し、分子量が 1 , 500以下の ペプチドを 70%以上含有することを特徴とする DPPIV阻害剤、 [8] A peptide derived from collagen or gelatin, comprising a peptide having the amino acid sequence represented by the formula (1), an amino acid residue at the end of the amino acid sequence, and an amino acid sequence deleted from the amino acid sequence A DPPIV inhibitor characterized by containing 70% or more of a peptide having a molecular weight of 1,500 or less,
〔9〕コラーゲンまたはゼラチン由来のペプチドであって、前記式(1)で表されるァミノ 酸配列からなるペプチド、前記のアミノ酸配列の末端のアミノ酸残基 W力 個欠失さ れたアミノ酸配列からなるペプチドあるいはその塩を含有し、分子量が 1 , 500以下の ペプチドを 90%以上含有することを特徴とする DPPIV阻害剤、 [9] A peptide derived from collagen or gelatin, comprising a peptide having the amino acid sequence represented by the formula (1), an amino acid residue at the end of the amino acid sequence, and an amino acid sequence deleted from the amino acid sequence A DPPIV inhibitor characterized by containing 90% or more of a peptide having a molecular weight of 1,500 or less,
〔10〕コラゲナーゼ処理されたコラーゲンまたはゼラチンの分解物を、有機溶媒を用 V、た沈殿法あるいは樹脂を用いた精製法の!/、ずれ力、もしくは両方を組み合わせた方 法により精製し、前記式(1)で表されるアミノ酸配列からなるペプチド、前記のアミノ酸 配列の末端のアミノ酸残基 w力 si個欠失されたアミノ酸配列からなるペプチド組成物 を得る工程を含む、ペプチド組成物の製造方法。 [10] Collagenase-treated collagen or gelatin degradation product is purified by a method using a combination of organic solvent V, precipitation method or resin purification method! /, Shear force, or both. Production of a peptide composition comprising the steps of: obtaining a peptide comprising the amino acid sequence represented by formula (1), and a peptide composition comprising the amino acid sequence deleted from the terminal amino acid residue w force si of the amino acid sequence. Method.
発明の効果 The invention's effect
本発明の DPPIV阻害剤は、 DPPIVに対して高い阻害活性を有し、かつ、コラーゲ ン、ゼラチン等の食品由来のため安全性が高い。また、本発明の DPPIV阻害剤は、 哺乳動物の糖尿病予防 ·治療剤としても使用することができる。さらには、本発明の D PPIV阻害剤は、糖尿病に随伴する各種の疾患(例えば、高脂血、症痛風あるいは高 尿酸血症)の改善剤としても使用することができる。 The DPPIV inhibitor of the present invention has a high inhibitory activity against DPPIV and has collagen. Highly safe because it is derived from foods such as gelatin and gelatin. Further, the DPPIV inhibitor of the present invention can also be used as a diabetes preventive / treatment agent for mammals. Furthermore, the DPPIV inhibitor of the present invention can also be used as an ameliorating agent for various diseases associated with diabetes (for example, hyperlipidemia, gout or hyperuricemia).
図面の簡単な説明 Brief Description of Drawings
[図 1]図 1は、コラーゲンペプチド HACP-01および HACP-U2、コラーゲンペプチド SCP -5000、ゼラチン、卵白ペプチド、大豆ペプチドを用いて DPPIV阻害実験を行って、 分析した結果である。縦軸は、サンプルを含んでいない場合の活性を 100とし、サン プルを添加した場合の活性を 100から差し引いた分を阻害率(%)として相対的に表し たものである。 [FIG. 1] FIG. 1 shows the results of analysis of DPPIV inhibition experiments using collagen peptides HACP-01 and HACP-U2, collagen peptide SCP-5000, gelatin, egg white peptide, and soybean peptide. The vertical axis shows the relative activity as an inhibition rate (%), where 100 is the activity when no sample is included, and 100 is the amount of activity when the sample is added.
[図 2]図 2は、コラーゲンペプチド HACP-01および HACP-U2とコラーゲンペプチド SC P-5000を終濃度 2.5, 5.0, 10mg/mlとなるように調整し、 DPPIV阻害実験を行って、分 析した結果である。縦軸は、サンプルを含んでいない場合の活性を 100とし、サンプ ルを添加した場合の活性を 100から差し引いた分を阻害率(%)として相対的に表した ものである。  [Fig. 2] Fig. 2 shows the results of DPPIV inhibition experiments with collagen peptides HACP-01 and HACP-U2 and collagen peptide SCP-5000 adjusted to final concentrations of 2.5, 5.0, and 10 mg / ml. It is the result. The vertical axis shows the relative activity as an inhibition rate (%), where 100 is the activity when no sample is included, and 100 is the activity when the sample is added.
[図 3]図 3は、コラーゲンペプチドによる DPPIV阻害効果を調べた結果である。各個体 の被験物質投与前 (-30分)の血漿中 DPPIV活性を通常時の活性 (0%)とし、この活性 に対して各血漿での活性の差を算出し、その比を取ることによって阻害率(%)として 相対的に表したものである。なお、グラフ中の記号(* )は注射用水摂取群と比較し て有意差 (Pく 0.05)があることを示すものである。  FIG. 3 shows the results of examining the DPPIV inhibitory effect of collagen peptides. The plasma DPPIV activity before each test substance administration (-30 minutes) for each individual was defined as the normal activity (0%), and the difference in activity in each plasma was calculated against this activity, and the ratio was calculated. It is expressed as a relative inhibition rate (%). The symbol (*) in the graph indicates that there is a significant difference (P 0.05) compared to the water intake group.
[図 4]図 4は、コラーゲンペプチドによる血中 GLP-1濃度上昇効果を調べた結果であ る。各個体の被験物質投与前 (-30分)の血漿中 GLP-1濃度を通常時の濃度 (100%)と し、この濃度に対する各血漿での濃度の割合を算出して相対的に表したものである。 なお、グラフ中の記号( * )は注射用水摂取群と比較して有意差 (pく 0.05)があること  [FIG. 4] FIG. 4 shows the results of examining the effect of increasing the GLP-1 concentration in blood by collagen peptides. The plasma GLP-1 concentration of each individual before administration of the test substance (-30 minutes) was defined as the normal concentration (100%), and the ratio of the concentration in each plasma to this concentration was calculated and expressed relatively. Is. Note that the symbol (*) in the graph is significantly different from the injection water intake group (p 0.05).
[図 5]図 5は、コラーゲンペプチドによる血中インスリン濃度上昇効果を調べた結果で ある。各個体の被験物質投与前 (-30分)の血漿中インスリン濃度を通常時の濃度 (100 %)とし、この濃度に対する各血漿での濃度の割合を算出して相対的に表したもので ある。なお、グラフ中の記号(* )は注射用水摂取群と比較して有意差 (pく 0.05)があ ることを示すものである。 FIG. 5 shows the results of examining the effect of increasing the insulin concentration in blood by collagen peptides. The insulin concentration in plasma before administration of the test substance (-30 minutes) for each individual is the normal concentration (100%), and the ratio of the concentration in each plasma to this concentration is calculated and expressed relatively. is there. In addition, the symbol (*) in the graph indicates that there is a significant difference (p 0.05) compared to the water intake group.
園 6]図 6は、コラーゲンペプチドによる血中グルコース濃度上昇抑制効果を調べた 結果である。なお、グラフ中の記号(* )は対照物質摂取群と比較して有意差 (pく 0.05 )があることを示すものである。また、 0、 3週目は空腹時、その他は通常時に採血を 行った。 6] Fig. 6 shows the results of examining the inhibitory effect of collagen peptides on the increase in blood glucose concentration. The symbol (*) in the graph indicates that there is a significant difference (p 0.05) compared to the control substance intake group. In addition, blood was collected at 0 and 3 weeks on an empty stomach, and at other times during normal times.
[図 7]図 7は、コラーゲンペプチドによる血中トリグリセライド濃度上昇抑制効果を調べ た結果である。なお、グラフ中の記号(* )は対照物質摂取群と比較して有意差 (pく 0. 05)があることを示すものである。また、 0、 3、 6週目は空腹時、その他は通常時に採 血を行った。  [FIG. 7] FIG. 7 shows the results of examining the effect of suppressing the increase in blood triglyceride concentration by collagen peptides. The symbol (*) in the graph indicates that there is a significant difference (p 0.05) compared to the control substance intake group. In addition, blood was collected at 0, 3, and 6 on an empty stomach and at other times on a normal basis.
園 8]図 8は、コラーゲンペプチドによる血中尿酸濃度上昇抑制効果を調べた結果で ある。なお、グラフ中の記号(* )は対照物質摂取群と比較して有意差 (pく 0.05)があ ることを示すものである。また、 0、 3、 6週目は空腹時、その他は通常時に採血を行つ た。 Fig. 8 shows the results of investigating the inhibitory effect of collagen peptides on the increase in blood uric acid concentration. The symbol (*) in the graph indicates that there is a significant difference (p 0.05) compared to the control substance intake group. On week 0, 3, and 6, blood was collected on an empty stomach and in other cases on a normal basis.
園 9]図 9は、コラーゲンペプチドによる尿中尿酸排泄量減少効果を調べた結果であ 9] Figure 9 shows the results of investigating the effect of collagen peptides to reduce urinary uric acid excretion.
[図 10]図 10は、コラーゲンペプチドによる血中グルコース濃度上昇抑制効果を調べ た結果である。なお、グラフ中の記号(* )はコントロール群と比較して有意差 (pく 0.05 )があることを示すものである。 [FIG. 10] FIG. 10 shows the results of examining the effect of suppressing the increase in blood glucose level by collagen peptides. The symbol (*) in the graph indicates a significant difference (p 0.05) compared to the control group.
[図 11]図 11は、コラーゲンペプチド HACP-01を分画したそれぞれの画分の吸光度と DPPIV阻害率を調べた結果である。折れ線グラフは 215nmの吸光度を表したもので ある。棒グラフはそれぞれの画分のペプチドおよび分画前の HACP-01を終濃度 1.0m g/mlとなるように調整し、 DPPIV阻害実験を行って、分析した結果を表したものである FIG. 11 shows the results of examining the absorbance and DPPIV inhibition rate of each fraction obtained by fractionating the collagen peptide HACP-01. The line graph represents the absorbance at 215 nm. The bar graph shows the results of analyzing the DPPIV inhibition experiment after adjusting the peptide of each fraction and HACP-01 before fractionation to a final concentration of 1.0 mg / ml.
Yes
[図 12]図 12は、 MHP-0の分子量分布を調べた結果である。実線は分子量マーカー、 点線は MHP-0の分子量分布を示している。矢印の MHP-0における最大のピークは 分子量 386.3付近にあることを示して!/、る。  FIG. 12 shows the results of examining the molecular weight distribution of MHP-0. The solid line shows the molecular weight marker, and the dotted line shows the molecular weight distribution of MHP-0. The peak at MHP-0 in the arrow indicates that the molecular weight is around 386.3! /.
[図 13]図 13は、コラーゲンペプチド HACP-01を異なる濃度のエタノール水溶液を用 いた沈殿法により分画した上清画分および沈殿画分の DPPIV阻害率を調べた結果 である。縦軸は、サンプルを含んでいない場合の活性を 100とし、サンプルを添加した 場合の活性を 100から差し引いた分を阻害率(%)として相対的に表したものである。 [Fig. 13] Fig. 13 shows that collagen peptide HACP-01 is used with aqueous ethanol solutions of different concentrations. This is the result of examining the DPPIV inhibition rate of the supernatant fraction and the precipitated fraction fractionated by the precipitation method. The vertical axis represents relative activity as an inhibition rate (%), where 100 is the activity when no sample is included, and 100 is the amount of activity when the sample is added.
[図 14]図 14は、コラーゲンペプチド HACP-01をエタノール沈殿法により分画したエタ ノール濃度 85%の上清画分をさらに合成吸着剤 DIAION HP20を用いて分画した画 分の DPPIV阻害率を調べた結果である。縦軸は、サンプルを含んでいない場合の活 性を 100とし、サンプルを添加した場合の活性を 100から差し引いた分を阻害率(%)と して相対的に表したものである。  [FIG. 14] FIG. 14 shows the DPPIV inhibition rate of a fraction obtained by further fractionating the 85% ethanol concentration fraction obtained by ethanol precipitation with the collagen peptide HACP-01 and further fractionating with the synthetic adsorbent DIAION HP20. It is the result of having investigated. The vertical axis represents relative activity as an inhibition rate (%), where the activity when no sample is included is 100 and the activity when the sample is added is subtracted from 100.
[図 15]図 15は、コラーゲンペプチド HACP-01および E85S_0(HP20)の各濃度における DPPIV阻害率を調べた結果である。縦軸は、サンプルを含んでいない場合の活性を 100とし、サンプルを添加した場合の活性を 100から差し引いた分を阻害率(%)として 相対的に表したものである。  FIG. 15 shows the results of examining DPPIV inhibition rates at various concentrations of collagen peptides HACP-01 and E85S_0 (HP20). The vertical axis shows the relative activity as an inhibition rate (%), where the activity when no sample is included is 100, and the activity when the sample is added is subtracted from 100.
[図 16]図 16は、コラーゲンペプチド HACP-01をエタノール沈殿法により分画したエタ ノール濃度 85%の上清画分をさらに合成吸着剤 SEPABEADS SP850を用いて分画し た画分の DPPIV阻害率を調べた結果である。縦軸は、サンプルを含んでいない場合 の活性を 100とし、サンプルを添加した場合の活性を 100から差し引いた分を阻害率( %)として相対的に表したものである。  [FIG. 16] FIG. 16 shows DPPIV inhibition of the fraction obtained by fractionating the 85% ethanol concentration fraction obtained by fractionating the collagen peptide HACP-01 by ethanol precipitation with the synthetic adsorbent SEPABEADS SP850. It is the result of examining the rate. The vertical axis shows the relative value as the inhibition rate (%), where the activity when no sample is included is 100, and the activity when the sample is added is subtracted from 100.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0014] 本発明は、食品として広く一般的に利用されているコラーゲンやゼラチンを構成し ているポリペプチドを分解したペプチドの中でも、特定のアミノ酸配列からなるぺプチ ドが DPPIVに対して阻害活性を有し、しかも糖尿病治療、予防効果にも優れることを 初めて見出し、完成させたものである。  [0014] The present invention relates to a peptide having a specific amino acid sequence, which is an inhibitory activity against DPPIV, among peptides obtained by decomposing a polypeptide constituting collagen or gelatin that is widely used as a food. It has been found for the first time that it has excellent anti-diabetic treatment and prevention effects.
[0015] 即ち、本発明の DPPIV阻害剤は、式(1):  That is, the DPPIV inhibitor of the present invention has the formula (1):
Gly-X-Y- (Gly-Z-W) n (1)  Gly-X-Y- (Gly-Z-W) n (1)
(式中、 nは 0〜4の整数、 Xは Proまたは Leu、 Y、 Ζおよび Wはそれぞれ独立して同 一または異なる任意のアミノ酸残基 (ただし、 Glyを除く)を示す。 )  (In the formula, n is an integer of 0 to 4, X is Pro or Leu, Y, Ζ and W are each independently the same or different arbitrary amino acid residues (excluding Gly).)
で表されるアミノ酸配列からなるペプチド、前記のアミノ酸配列の末端のアミノ酸残基 w力 個欠失されたアミノ酸配列からなるペプチドあるいはその塩を含有する。 [0016] 前記式中、 Glyはグリシン残基、 Proはプロリン残基、 Leuはロイシン残基を示す。ま た、上記の Υ、 Ζおよび Wが採り得る、グリシン残基を除くアミノ酸残基の種類は、特に 限定されず、通常は、天然に存在するアミノ酸 (グリシンを除く)のアミノ酸残基、具体 的には、ァラニン残基、パリン残基、ロイシン残基、イソロイシン残基、プロリン残基、 フエ二ルァラニン残基、トリブトファン残基、メチォニン残基、セリン残基、トレオニン残 基、システィン残基、グノレタミン残基、ァスパラギン残基、チロシン残基、リシン残基、 アルギニン残基、ヒスチジン残基、ァスパラギン酸残基、グルタミン酸残基のいずれの アミノ酸残基であってもよい。また、前記アミノ酸残基は、ヒドロキシル基等が付与され た修飾アミノ酸残基であってもよい。本明細書においては、特に断わらない限り、アミ ノ酸残基は、 L型アミノ酸残基を意味する。 A peptide consisting of the amino acid sequence represented by the above, a peptide consisting of the amino acid sequence deleted at the terminal amino acid residue w of the amino acid sequence, or a salt thereof. In the above formula, Gly represents a glycine residue, Pro represents a proline residue, and Leu represents a leucine residue. In addition, the types of amino acid residues excluding glycine residues that can be taken by the above Υ, Ζ and W are not particularly limited, and are usually amino acid residues of naturally occurring amino acids (excluding glycine), specifically Specifically, alanine residue, parin residue, leucine residue, isoleucine residue, proline residue, phenylalanine residue, tribubutane residue, methionine residue, serine residue, threonine residue, cysteine residue, It may be any amino acid residue such as gnoretamine residue, asparagine residue, tyrosine residue, lysine residue, arginine residue, histidine residue, aspartate residue, glutamate residue. The amino acid residue may be a modified amino acid residue to which a hydroxyl group or the like is added. In this specification, unless otherwise specified, an amino acid residue means an L-type amino acid residue.
[0017] また、前記式中、 ηが;!〜 4の整数の場合、前記アミノ酸配列の末端アミノ酸残基 W 力 a個欠失されたアミノ酸配歹 IJも本発明で用いられるペプチドに含まれる。  [0017] In the above formula, when η is an integer of !! to 4, an amino acid sequence IJ from which a terminal amino acid residue W force a has been deleted from the amino acid sequence is also included in the peptide used in the present invention. .
[0018] 中でも、本発明では、前記ペプチドが DPPIV阻害活性に優れる観点から、  [0018] Among them, in the present invention, from the viewpoint that the peptide is excellent in DPPIV inhibitory activity,
Giy_Pro_Argゝ  Giy_Pro_Arg ゝ
Gly-Pro-Ser-Gly-Asn-Ala (配列番号 1)、  Gly-Pro-Ser-Gly-Asn-Ala (SEQ ID NO: 1),
Gly-Pro-Ala-Gly-Pro-Ala (配列番号 2)、  Gly-Pro-Ala-Gly-Pro-Ala (SEQ ID NO: 2),
Gly-Pro-Val-Gly-Ala-Arg (配列番号 3)、  Gly-Pro-Val-Gly-Ala-Arg (SEQ ID NO: 3),
Gly-Pro-Val-Gly-Pro-Ala (配列番号 5)、  Gly-Pro-Val-Gly-Pro-Ala (SEQ ID NO: 5),
Gly-Pro-Ile-Gly-Ser-Ala (配列番号 6)、  Gly-Pro-Ile-Gly-Ser-Ala (SEQ ID NO: 6),
Gly-Pro-Ser-Gly-Glu-Arg-Gly-Pro-Hyp (配列番号 4)、  Gly-Pro-Ser-Gly-Glu-Arg-Gly-Pro-Hyp (SEQ ID NO: 4),
Gly-Pro-Arg-Gly-Arg-Thr-Gly-Asp-Ala-Gly-Pro-Val (配列番号 8)、  Gly-Pro-Arg-Gly-Arg-Thr-Gly-Asp-Ala-Gly-Pro-Val (SEQ ID NO: 8),
Gly-Pro-Val-Gly-Pro (配列番号 9)および  Gly-Pro-Val-Gly-Pro (SEQ ID NO: 9) and
Gly-Leu-Ala-Gly-Pro-Hyp (配列番号 7)  Gly-Leu-Ala-Gly-Pro-Hyp (SEQ ID NO: 7)
力、らなる群より選ばれる 1種類以上のアミノ酸配列からなるペプチドであることが好ま しい。なお、上記式中において、アミノ酸残基はいずれも公知の 3文字表記に準じて 表示され、 Hypはヒドロキシプロリン残基(3—ヒドロキシプロリン残基(3Hyp)または 4 ーヒドロキシプロリン残基(4Hyp) )を示す。  It is preferable that the peptide is composed of one or more amino acid sequences selected from the group consisting of force groups. In the above formula, all amino acid residues are represented according to the known three-letter code, and Hyp is a hydroxyproline residue (3-hydroxyproline residue (3Hyp) or 4-hydroxyproline residue (4Hyp). ).
[0019] また、本発明で用いるペプチドの塩としては、前記式(1)で表されるアミノ酸配列か らなるペプチド(該ペプチドのアミノ酸配列の末端のアミノ酸残基 w力 si個欠失された アミノ酸配列からなるペプチドを含む)とナトリウム、カリウム等のアルカリ金属、マグネ シゥム、カルシウム等のアルカリ土類金属、アルミニウム等の三価金属等の無機塩基 との塩、メチルァミン、ェチルァミン、ジメチルァミン、ジェチルァミン、トリメチルァミン 、トリエチノレアミン、モノエタノールァミン、ジエタノールァミン、トリエタノーノレアミン、シ クロへキシルァミン、リジン、オル二チン等の有機塩基との塩が挙げられる。本発明の 有効成分である式(1)で表されるペプチドまたはその塩は、水和物、各種溶媒和物と して、または結晶多形の物質として単離される場合もあり、本発明にはこれらの単離 されたものおよび混合物の全てが包含される。 [0019] The peptide salt used in the present invention may be an amino acid sequence represented by the formula (1). (Including peptides consisting of amino acid residues at the end of the amino acid sequence of the peptide w amino acid sequence) and alkali metals such as sodium and potassium, alkaline earth metals such as magnesium and calcium , Salts with inorganic bases such as trivalent metals such as aluminum, methylamine, ethylamine, dimethylamine, jetylamine, trimethylamine, triethylamine, monoethanolamine, diethanolamine, triethanolamine, cyclohexylamine , Salts with organic bases such as lysine and ornithine. The peptide represented by the formula (1) or a salt thereof, which is an active ingredient of the present invention, may be isolated as a hydrate, various solvates, or a crystalline polymorphic substance. Includes all of these isolates and mixtures.
[0020] 本発明で用いるペプチドまたはその塩は、前述したコラーゲンまたはゼラチンを含 む原料から公知の方法によって製造することもできるし、ペプチドをコードする DNA を含有する形質転換体を培養することによつても製造することができる。また、公知の ペプチド合成法に準じて製造することもできる。  [0020] The peptide or salt thereof used in the present invention can be produced by a known method from the above-mentioned raw material containing collagen or gelatin, or a transformant containing DNA encoding the peptide can be cultured. Can also be manufactured. It can also be produced according to known peptide synthesis methods.
[0021] 例えば、本発明の DPPIV阻害剤は、前記式(1)で表されるペプチドまたはその塩を 有効成分として含有しているものであればよぐ例えば、コラーゲンまたはゼラチンを コラゲナーゼ処理した分解物であってもよい。原料となるコラーゲンは、特に限定され ず、 I型から XIII型のコラーゲンのいずれをも用いることが可能であり、これらの混合物 である混合型のコラーゲンを用いることもできる。現実的には、コラーゲンは、各種の 動物や魚類から得られる、混合型のコラーゲンを用いることが想定される力 このコラ 一ゲンの出所となる動物(例えば、牛、豚等)や魚類 (例えば、ヒラメ、サケ、イワシ、マ グロ等)の種類や、コラーゲンの抽出部位も、骨、皮、腱、ゥキブクロ(魚類)等が可能 である。  [0021] For example, the DPPIV inhibitor of the present invention only needs to contain the peptide represented by the formula (1) or a salt thereof as an active ingredient. For example, collagen or gelatin decomposed by collagenase treatment It may be a thing. The collagen used as a raw material is not particularly limited, and any of type I to XIII type collagen can be used, and a mixed type collagen, which is a mixture of these, can also be used. Realistically, collagen is the power that is expected to use mixed collagen obtained from various animals and fish. Animals (eg, cattle, pigs, etc.) and fish (eg, cattle, pigs) , Flounder, salmon, sardines, tuna, etc.) and collagen extraction sites can also be bone, skin, tendon, sea buckthorn (fish), etc.
[0022] これらの成分からのコラーゲンの抽出 ·精製は、通常公知の方法を用いて行うことが できる。具体的には、例えば、骨、皮、腱、ゥキブクロ等のコラーゲンを含有する組織 を粉砕した後、水洗、希塩溶液による抽出、酸あるいはアルカリ溶液による抽出、ぺ プシン,トリプシンゃヒアルロニダーゼ等の酵素による抽出を行い、塩析ゃ透析等の 公知の精製手段を施して、コラーゲンを精製して得ること力 Sできる。また、通常公知の 方法により、「再生コラーゲン」として得ることも可能である。また、市販のコラーゲンを 、原料として用いることも可能である。 [0022] Extraction and purification of collagen from these components can be performed by a generally known method. Specifically, for example, after pulverizing collagen-containing tissues such as bone, skin, tendon, and duckweed, washing with water, extraction with a dilute salt solution, extraction with an acid or alkaline solution, enzymes such as pepsin, trypsin and hyaluronidase, etc. It is possible to extract collagen by using a known purification means such as salting-out dialysis or the like and purify the collagen. It can also be obtained as “regenerated collagen” by a generally known method. Also, commercially available collagen It can also be used as a raw material.
[0023] そして、ゼラチンは、上述のコラーゲンを、水で加熱抽出して得られる水溶性タンパ ク質である。本発明においては、通常公知の方法により製造したゼラチンを原料とし て用いることも可能であり、市販品を用いることも可能である。  [0023] Gelatin is a water-soluble protein obtained by heating and extracting the aforementioned collagen with water. In the present invention, gelatin produced by a generally known method can be used as a raw material, and a commercially available product can also be used.
[0024] 本発明に用いるペプチドは、上述のようにして得られるコラーゲンまたはゼラチンに 、コラゲナーゼを作用させた製造することができる。具体的には、コラーゲンまたはゼ ラチンにコラゲナーゼ処理を施した分解物(コラゲナーゼ分解物)を使用する。  [0024] The peptide used in the present invention can be produced by allowing collagenase to act on collagen or gelatin obtained as described above. Specifically, a degradation product (collagenase degradation product) obtained by subjecting collagen or gelatin to a collagenase treatment is used.
[0025] コラゲナーゼとしては、特に限定されないが、クロストリジゥム 'ヒストリティカム(Clostr idium histolyticum)、ストレプトミセス'パノレブラス(Streptomyces parvulus)等の細菌、 放線菌または真菌等由来で、コラーゲン特有のアミノ酸配列〔(Gly— A— B) n (式中 、 A, Bは、グリシン残基を除くアミノ酸残基を表し、互いに同一であっても、異なって もよぐ、 nは、正の整数を表す):以下、このアミノ酸配列を、「特有アミノ酸配列」ともい う)〕のグリシン残基のアミノ基末端側を、特異的に切断するコラゲナーゼを用いること で、この特有アミノ酸配列のペプチドを豊富に含むコラゲナーゼ分解物を得ることが 可能であり、好ましい。また、ここで用いるコラゲナーゼは、天然物として得られるコラ ゲナーゼは勿論のこと、例えば、タンパク工学的な手法で改変して得られる、上記の 特異性を有する改変コラゲナーゼであってもよい。  [0025] Collagenase is not particularly limited, but it is derived from bacteria such as Clostridium histolyticum and Streptomyces parvulus, actinomycetes or fungi, and the like. Gly—A—B) n (where A and B represent amino acid residues other than glycine residues, which may be the same or different from each other, n represents a positive integer): In the following, this amino acid sequence is also referred to as a “specific amino acid sequence”)). By using collagenase that specifically cleaves the amino group terminal side of the glycine residue, collagenase degradation that contains abundant peptides of this specific amino acid sequence It is possible to obtain a product, which is preferable. Further, the collagenase used here may be a collagenase obtained as a natural product, for example, a modified collagenase having the above-mentioned specificity obtained by modification by a protein engineering technique.
[0026] 上記の A、 Bが採り得る、グリシン残基を除くアミノ酸残基の種類は、特に限定され ず、通常は、天然に存在するアミノ酸 (グリシンを除く)のアミノ酸残基、具体的には、 ァラニン残基、バリン残基、ロイシン残基、イソロイシン残基、プロリン残基、フエニル ァラニン残基、トリブトファン残基、メチォニン残基、セリン残基、トレオニン残基、シス ティン残基、グルタミン残基、ァスパラギン残基、チロシン残基、リシン残基、アルギニ ン残基、ヒスチジン残基、ァスパラギン酸残基、グルタミン酸残基のいずれのアミノ酸 残基であってもよい。  [0026] The types of amino acid residues excluding glycine residues that can be adopted by A and B are not particularly limited, and are usually amino acid residues of naturally occurring amino acids (excluding glycine), specifically Is alanine residue, valine residue, leucine residue, isoleucine residue, proline residue, phenylalanine residue, tributophan residue, methionine residue, serine residue, threonine residue, cystine residue, glutamine residue It may be any amino acid residue such as a group, an asparagine residue, a tyrosine residue, a lysine residue, an arginine residue, a histidine residue, an aspartate residue, or a glutamate residue.
[0027] また、本発明に用いられ得るペプチドは、通常公知の方法、例えば、特開平 7— 82 299号公報ゃ特開平 9— 176196号公報に記載されている方法に準じて、遊離また はキトバール等の固定化担体に固定化されたコラゲナーゼを、バッチ法、カラム法ま たはこれらの方法を組み合わせ、好ましくは、反応温度を 40〜45°Cに設定して、前 記コラーゲンまたはゼラチンと接触させることで製造することができる。 In addition, the peptide that can be used in the present invention is free or in accordance with a generally known method, for example, a method described in JP-A-7-82299 or JP-A-9-176196. Collagenase immobilized on an immobilization carrier such as chitobar is mixed with a batch method, a column method, or a combination of these methods, and preferably the reaction temperature is set to 40 to 45 ° C. It can be produced by contacting with collagen or gelatin.
[0028] コラゲナーゼ分解物は、上述の方法に従い、これを製造してそのまま用いてもよい 、各種基材に配合してもよい。配合量や基材の種類は特に限定されるものではなく 、適時設定すればよい。基材としてはたとえば、錠剤、カプセル、飴、グミあるいは飲 料等の経口投与基材が好ましい。また、食品'医薬品を問わず、公知の技術を用い て、いずれにおいても製造することができる。なお、コラゲナーゼ分解物として市販品 、例えば、商品名コラーゲン 'トリペプチド HACP (ゼライス社製)等を用いることも可能 である。 [0028] The collagenase degradation product may be produced according to the method described above and used as it is, or may be blended in various base materials. The blending amount and the type of base material are not particularly limited, and may be set as appropriate. As the base material, for example, an oral administration base material such as a tablet, capsule, candy, gummi or drink is preferable. In addition, it can be produced by any known technique, regardless of whether it is a food or a medicine. As the collagenase degradation product, a commercially available product such as the collagen name “Tripeptide HACP” (manufactured by Zerais Co., Ltd.) can also be used.
これらのコラゲナーゼ分解物は、各種の精製方法に供することが好まし!/、。  These collagenase degradation products are preferably used for various purification methods!
[0029] 例えば、前記コラゲナーゼ分解物あるいはその市販品を、さらに種々の樹脂を用い て精製することによって、 DPPIVの阻害活性を高めた画分を得ることもできる。使用す る樹脂としてはたとえば、陽イオン交換樹脂、陰イオン交換樹脂、多孔性樹脂、特殊 樹脂 (キレート樹脂、合成吸着剤、蛋白分離剤)等が挙げられるが、回収した画分の 脱塩処理工程が不要であることから、合成吸着剤を用いるのが好ましい。合成吸着 剤としてはたとえば、芳香族 (スチレン-ビュルベンゼン)系、芳香族系修飾型、アタリ ル (メタクリル)系等が挙げられる力 これらに限定されるものではない。吸着した有機 物の溶離には、酸、アルカリまたは種々の有機溶媒、たとえばメタノール、エタノール 、プロパノール、イソプロパノール、ブタノール等の低級アルコールや酢酸ェチル、酢 酸ブチル等のエステル類、アセトン等のケトン類を用いることできる力 S、これらに限定 されるものではない。また、これらの有機溶媒は単独または 2種類以上を混合して用 いてもよく、有機溶媒と水あるいは酸、アルカリとの混合溶媒としてもよい。なお、経済 性と安全性の点からは、エタノールまたはエタノール水溶液を用いて溶離するのが好 ましい。精製は、バッチ法あるいはカラム法にて行うことができる。回収した画分は減 圧または限外ろ過により濃縮し、さらに必要に応じて溶媒を完全に除去して乾固する か凍結乾燥を行ってもょレ、。 [0029] For example, a fraction with enhanced DPPIV inhibitory activity can be obtained by further purifying the collagenase degradation product or its commercial product using various resins. Examples of the resin to be used include cation exchange resin, anion exchange resin, porous resin, and special resin (chelate resin, synthetic adsorbent, protein separating agent). Since a process is unnecessary, it is preferable to use a synthetic adsorbent. Synthetic adsorbents include, but are not limited to, for example, aromatic (styrene-butylbenzene) -based, aromatic-modified, and talyl (methacrylic) -based. For elution of adsorbed organic substances, acids, alkalis or various organic solvents such as lower alcohols such as methanol, ethanol, propanol, isopropanol and butanol, esters such as ethyl acetate and butyl acetate, and ketones such as acetone are used. The force S that can be used is not limited to these. These organic solvents may be used alone or in combination of two or more, and may be a mixed solvent of an organic solvent and water or an acid or an alkali. From the viewpoint of economy and safety, it is preferable to elute with ethanol or ethanol aqueous solution. Purification can be performed by a batch method or a column method. The collected fraction can be concentrated by depressurization or ultrafiltration, and if necessary, the solvent can be completely removed and dried or lyophilized.
[0030] また、上述の方法に従!/、製造したコラゲナーゼ分解物あるいはその市販品を、さら に種々の有機溶媒を用いて精製することによって、 DPPIVの阻害活性を高めた画分 を得ることもできる。使用する有機溶媒としてはたとえば、メタノール、エタノール、プ ロパノール、イソプロパノール、ブタノール等の低級アルコールや酢酸ェチル、酢酸 ブチル等のエステル類、アセトン等のケトン類を用いることできる力 これらに限定さ れるものではない。また、これらの有機溶媒は単独または 2種類以上を混合して用い てもよく、有機溶媒と水あるいは酸、アルカリとの混合溶媒としてもよい。なお、経済性 と安全性の点からは、エタノール水溶液を用いて精製するのが好ましい。精製は、沈 殿法にて行うことができる。回収した画分は減圧または限外ろ過により濃縮し、さらに 必要に応じて溶媒を完全に除去して乾固するか凍結乾燥を行ってもよい。 [0030] Further, according to the above-mentioned method !, a fraction having enhanced DPPIV inhibitory activity can be obtained by further purifying the produced collagenase degradation product or its commercial product using various organic solvents. You can also. Examples of organic solvents to be used include methanol, ethanol, The ability to use lower alcohols such as lopanol, isopropanol and butanol, esters such as ethyl acetate and butyl acetate, and ketones such as acetone is not limited thereto. These organic solvents may be used alone or in combination of two or more, and may be a mixed solvent of an organic solvent and water, acid, or alkali. In view of economy and safety, it is preferable to purify using an aqueous ethanol solution. Purification can be performed by sedimentation. The collected fraction may be concentrated by reduced pressure or ultrafiltration, and if necessary, the solvent may be completely removed and dried or freeze-dried.
[0031] 中でも、上述の方法に従い製造したコラゲナーゼ分解物あるいはその市販品を、さ らに種々の有機溶媒を用いた沈殿法および前記のような種々の樹脂を用いた精製 法を組み合わせることによって、前記式(1)で表されるアミノ酸配列からなるペプチド を含む画分 (ペプチド組成物)を効率的に得て、 DPPIVの阻害活性を高めたぺプチ ド組成物を製造することができるので好ましレ、。  [0031] Above all, by combining a collagenase degradation product produced according to the above-mentioned method or a commercial product thereof with a precipitation method using various organic solvents and a purification method using various resins as described above, Since a peptide composition having a peptide comprising the amino acid sequence represented by the formula (1) (peptide composition) can be efficiently obtained and a peptide composition having enhanced DPPIV inhibitory activity can be produced, it is preferable. Masle.
[0032] また、本発明で用いられるペプチドは、公知のペプチドの合成法に従って製造する ことができる。ペプチドの合成法としては、例えば、固相合成法、液相合成法のいず れによっても良い。  [0032] The peptide used in the present invention can be produced according to a known peptide synthesis method. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used.
[0033] また、合成反応後は通常の精製法、例えば、溶媒抽出 ·蒸留'カラムクロマトグラフィ 一-液体クロマトグラフィー.再結晶等を組み合わせて本発明で用いられるペプチドを 精製単離すること力できる。また、ペプチドの塩の種類については、上記方法で得ら れるペプチドが遊離体である場合は、公知の方法あるいはそれに準じる方法によつ て適当な塩に変換することができるし、逆に塩で得られた場合は、公知の方法あるい はそれに準じる方法によって遊離体または他の塩に変換することができる。  [0033] Further, after the synthesis reaction, the peptide used in the present invention can be purified and isolated by combining ordinary purification methods, for example, solvent extraction / distillation 'column chromatography / one-liquid chromatography / recrystallization. As for the type of peptide salt, when the peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, Can be converted to the free form or other salts by a known method or a method analogous thereto.
[0034] また、本発明で用いられるペプチドを遺伝工学的な手法で製造することもできる。  [0034] The peptide used in the present invention can also be produced by a genetic engineering technique.
例えば、前記ペプチドをコードする塩基配列を含有するポリヌクレオチド、好ましくは DNAを作製して行うことができる。 DNAとしては、ゲノム DNA、ゲノム DNAライブラ リー、前記した原料由来の cDNA、前記した原料由来の cDNAライブラリー、合成 D NAのいずれでもよい。  For example, it can be carried out by preparing a polynucleotide containing the base sequence encoding the peptide, preferably DNA. The DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned raw materials, cDNA library derived from the above-mentioned raw materials, and synthetic DNA.
[0035] 前記のポリヌクレオチドは、例えば、 DNAリガーゼ、制限酵素等の公知の方法を用 いてベクターに組み込み、次いでそのベクターを宿主細胞中で増幅させることも可能 である。ベクター、宿主細胞等については公知のものであれば特に限定はない。培 養した宿主細胞から本発明のペプチドを分離精製することで、本発明に用いられる ペプチドを大量に得ることができる。分離精製方法としては、公知の方法であれば特 に限定はない。 [0035] The polynucleotide can be incorporated into a vector using a known method such as DNA ligase or restriction enzyme, and the vector can then be amplified in a host cell. It is. There are no particular limitations on the vectors, host cells, etc. as long as they are known. A large amount of the peptide used in the present invention can be obtained by separating and purifying the peptide of the present invention from the cultured host cells. The separation and purification method is not particularly limited as long as it is a known method.
[0036] 以上のようにして得られる前記式(1)で表されるペプチドまたはその塩(以下、本発 明のペプチド等と略す)は、 DPPIV阻害剤として使用することができる。  [0036] The peptide represented by the above formula (1) or a salt thereof (hereinafter abbreviated as the peptide of the present invention) obtained as described above can be used as a DPPIV inhibitor.
[0037] また、本発明のペプチド等は、それぞれ単独でまたは 2種以上を混合して用いるこ と力 Sできる。  [0037] The peptides of the present invention can be used alone or in combination of two or more.
本発明の DPPIV阻害剤としては、本発明のペプチド等を含有していればよいが、本 発明のペプチド等の含有率が高ぐ DPPIV阻害活性が高いという観点から、分子量 が 1 , 500以下のペプチドを 50%以上、好ましくは 70%以上、より好ましくは 90%以上 含有することが望ましい。  The DPPIV inhibitor of the present invention only needs to contain the peptide of the present invention, but from the viewpoint of high DPPIV inhibitory activity with a high content of the peptide of the present invention, the molecular weight is 1,500 or less. It is desirable to contain 50% or more of peptide, preferably 70% or more, more preferably 90% or more.
[0038] また、本発明の DPPIV阻害剤は、哺乳動物の糖尿病の予防'治療剤として使用す ること力 Sでさる。 [0038] In addition, the DPPIV inhibitor of the present invention can be used as an agent for preventing or treating diabetes in mammals.
前記 DPPIV阻害剤を糖尿病の予防 ·治療剤として使用する場合は、常套手段に従 つて、その有効量をヒトまたは非ヒトの哺乳動物に対して投与することができる。  When the DPPIV inhibitor is used as a prophylactic / therapeutic agent for diabetes, an effective amount thereof can be administered to a human or non-human mammal according to conventional means.
[0039] 本発明の糖尿病治療'予防剤は、前記式(1)で表されるペプチドまたはその塩と、 薬学的に許容される製薬用の担体とを配合して、経口、非経口の固体、液体等の医 薬組成物として調製される。  [0039] The diabetes treatment 'preventive agent of the present invention comprises an oral or parenteral solid comprising a peptide represented by the formula (1) or a salt thereof and a pharmaceutically acceptable pharmaceutical carrier. It is prepared as a liquid pharmaceutical composition.
[0040] 前記の経口投与のための固体組成物としては、錠剤、丸剤、カプセル剤、細粒剤、 顆粒剤等が挙げられる。このような固体組成物においては、一つまたはそれ以上の 活性物質が、少なくとも一つの不活性な希釈剤、例えば乳糖、マンニトール、ぶどう 糖、ヒドロキシプロピルセルロース、結晶セルロース、各種でんぷん、ポリビュルピロリ ドン、メタケイ酸アルミン酸マグネシウム等と混合される。組成物は、常法に従って、不 活性な希釈剤以外の添加剤、例えばステアリン酸マグネシウムのような滑沢剤、繊維 素グリコール酸カルシウムのような崩壊剤、ラタトースのような安定化剤、グルタミン酸 またはァスパラギン酸のような溶解乃至溶解補助剤を含有していてもよい。また、錠 剤、丸剤、顆粒剤、顆粒を含有するカプセル剤の顆粒は、必要により、しょ糖、ゼラチ のような糖衣、または胃溶性もしくは腸溶性物質のフィルムで被覆してもよい。また、 製剤の溶解性を向上させるために、公知の可溶化処理を施して製剤化を行うこともで きる。 [0040] Examples of the solid composition for oral administration include tablets, pills, capsules, fine granules, granules and the like. In such solid compositions, one or more active substances are present in at least one inert diluent, such as lactose, mannitol, glucose, hydroxypropylcellulose, crystalline cellulose, various starches, polypyrrole pyrrolidone. And mixed with magnesium aluminate metasilicate. In accordance with conventional methods, the composition may contain additives other than inert diluents, such as lubricants such as magnesium stearate, disintegrants such as calcium calcium glycolate, stabilizers such as ratatose, glutamic acid or It may contain a solubilizing or solubilizing agent such as aspartic acid. In addition, tablets, pills, granules, and capsules containing granules may be made of sucrose, gelatin, if necessary. Or a film of gastric or enteric material. Further, in order to improve the solubility of the preparation, the preparation can be prepared by performing a known solubilization treatment.
[0041] 経口投与のための液体組成物としては、製薬学的に許容される乳濁剤、溶液剤、 懸濁剤、シロップ剤、エリキシル剤等の製剤形態を含み、組成物の成分として一般的 に用いられる不活性な希釈剤、例えば精製水、エタノール等を含む。この組成物は 不活性な希釈剤以外に湿潤剤、懸濁化剤などの補助剤、甘味剤、風味剤、芳香剤、 防腐剤を含有してレ、てもよレ、。  [0041] Liquid compositions for oral administration include pharmaceutical forms such as pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, etc. Inert diluents that are used specifically, such as purified water, ethanol and the like. This composition contains adjuvants such as wetting agents, suspending agents, sweeteners, flavors, fragrances and preservatives in addition to inert diluents.
[0042] また、非経口投与のための注射剤としては、無菌の水性または非水性の、溶液剤、 懸濁剤および乳濁剤を包含する。水性の溶液剤や懸濁剤には、例えば注射剤用蒸 留水および生理食塩水が含まれる。非水性の溶液剤、懸濁剤には、例えばプロピレ ングリコール、ポリエチレングリコール、ォリーブ油のような植物油、エタノールのような 製薬学的に許容されるアルコール類、ポリオキシエチレンソルビタン脂肪酸エステル のような界面活性剤が含まれる。このような水性、非水性の組成物はこれらの添加剤 以外に湿潤剤、懸濁化剤、乳化剤、分散剤、安定化剤 (例えばラ外ース)、溶解乃 至溶解補助剤(例えば、グルタミン酸ゃァスパラギン酸など)などの補助剤や防腐剤 を含有していてもよい。これらは例えばバクテリア保留フィルターを通す濾過、殺菌剤 の配合または照射によって無菌化される。これらはまた無菌の固体組成物として製造 し、使用前に無菌水または無菌の注射用溶媒に溶解して使用する形態とすることも できる。  [0042] Injectables for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Aqueous solutions and suspensions include, for example, distilled water for injection and physiological saline. Non-aqueous solutions and suspensions include, for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, pharmaceutically acceptable alcohols such as ethanol, and polyoxyethylene sorbitan fatty acid esters. A surfactant is included. In addition to these additives, such aqueous and non-aqueous compositions include wetting agents, suspending agents, emulsifying agents, dispersing agents, stabilizing agents (eg, extra leuth), solubilizing agents and solubilizing aids (eg, Auxiliary agents such as glutamic acid aspartic acid) and preservatives may be contained. These are sterilized by, for example, filtration through a bacteria-retaining filter, blending with a bactericide, or irradiation. These can also be produced as a sterile solid composition and dissolved in sterile water or a sterile solvent for injection before use.
[0043] 本発明のペプチド等を上記の疾患の予防'治療目的で用いるには、通常経口また は非経口で投与される。ペプチド等の投与量は被検体の年齢、体重、症状、治療効 果、投与ルート等により異なり、これらを考慮して適宜設定されるが、例えば、ヒトであ れば通常成人一日当たり、経口投与で lmg〜; 10g、好ましくは 10mg〜3g、また、非 経口投与で 0. lmg〜; lgが好ましぐこれを 1日 1回であるいは 2〜数回に分けて投 与される。投与量は予防目的やその他の種々の条件によって変動するので、上記投 与量範囲より少ない量で十分な場合もある。被検体がヒト以外の哺乳動物であれば 上記のヒトに準じて投与量を調整すればょレ、。 [0043] In order to use the peptide of the present invention for the purpose of preventing or treating the above-mentioned diseases, it is usually administered orally or parenterally. The dose of peptide and the like varies depending on the age, weight, symptom, therapeutic effect, route of administration, etc. of the subject, and is appropriately set in consideration of these. 1 mg to 10 g, preferably 10 mg to 3 g, and 0.1 mg to parenteral administration is preferable. This is given once a day or divided into 2 to several times. Since the dosage varies depending on the purpose of prevention and various other conditions, an amount smaller than the above-mentioned dose range may be sufficient. If the subject is a non-human mammal If you adjust the dose according to the above human.
[0044] また、本発明の DPPIV阻害剤は、糖尿病に随伴する各種の疾患(例えば、高脂血、 症痛風あるいは高尿酸血症)の改善作用も有する。したがって、本発明の DPPIV阻 害剤は、糖尿病に随伴する各種の疾患(例えば、高脂血、症痛風あるいは高尿酸血 症)の予防 ·治療剤としても使用できる。 [0044] The DPPIV inhibitor of the present invention also has an action of improving various diseases associated with diabetes (eg, hyperlipidemia, gout or hyperuricemia). Therefore, the DPPIV inhibitor of the present invention can also be used as a prophylactic / therapeutic agent for various diseases associated with diabetes (for example, hyperlipidemia, gout or hyperuricemia).
[0045] このようにして得られる本発明の DPPIV阻害剤は、コラーゲンまたはゼラチン由来で あることから、安全で低毒性であるので、例えば、哺乳動物(例えば、ヒト、ラット、マウ ス、モルモット、ゥサギ、トリ、ヒッジ、ブタ、ゥシ、ゥマ、ネコ、ィヌ、サル、チンパンジー 等)に対して投与することができる。 [0045] Since the DPPIV inhibitor of the present invention obtained in this way is derived from collagen or gelatin and is safe and low toxic, for example, mammals (for example, humans, rats, mice, guinea pigs, Usagi, bird, hidge, pig, ushi, horse, cat, inu, monkey, chimpanzee, etc.).
実施例  Example
[0046] 以下、本発明を実施例により具体的に説明するが、本発明はこれらによって制限さ れるものではない。  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.
[0047] (実施例 1) [Example 1]
コラーゲンペプチド HACP-01 (ゼライス社製、豚皮由来のゼラチンのコラゲナーゼ 処理品)および HACP-U2 (ゼライス社製、魚鱗由来のゼラチンのコラゲナーゼ処理品 )、コラーゲンペプチド SCP_5000 (新田ゼラチン社製)、ゼラチン、卵白ペプチド、大 豆ペプチドをそれぞれ 25mMのトリス-塩酸バッファー(pH 8.0)に 10mg/mlとなるように 溶解した。  Collagen peptide HACP-01 (manufactured by Gelais, processed with collagenase from pork skin) and HACP-U2 (manufactured by Gelais, collagenase treated with fish scale derived gelatin), collagen peptide SCP_5000 (manufactured by Nitta Gelatin) Gelatin, egg white peptide, and soybean peptide were each dissolved in 25 mM Tris-HCl buffer (pH 8.0) to a concentration of 10 mg / ml.
[0048] DPPIVに対する活性阻害実験は以下の方法に従った。 25mMのトリス-塩酸バッファ 一 (pH 8.0) 2011 1、バッファーもしくはサンプル溶液 5 H 1、希釈した DPPIV溶液(lng/ 1) 5 1を混合し、室温で 5分間インキュベートした。酵素反応開始は 25mMのトリス- 塩酸バッファー(pH 8.0)に溶力もた基質溶液(0.25mMのグリシノレプロリン一 4—メチ ルクマリルー7—アミド(Gly-Pro-MCA) )を 20 1添加することによって行った。室温で 10分間反応後、 96ゥエルプレート対応蛍光検出器 (フルォロスキャンアセント:サーモ エレクトロン社製)で、 DPPIVによって遊離される 7—アミノー 4—メチルクマリン (AMC) 量を測定した。なお、励起波長は 390nm、測定波長 460nmで行った。対照区は DPPI V溶液の代わりに 25mMのトリス-塩酸バッファー(pH 8.0)を用いて同様に行った。  [0048] The activity inhibition experiment for DPPIV was performed according to the following method. 25 mM Tris-HCl buffer (pH 8.0) 2011 1, buffer or sample solution 5 H 1, diluted DPPIV solution (lng / 1) 5 1 were mixed and incubated at room temperature for 5 minutes. The enzyme reaction was started by adding 20 1 of a substrate solution (0.25 mM glycinoreproline 4-methylcumulyl 7-amide (Gly-Pro-MCA)) in 25 mM Tris-HCl buffer (pH 8.0). went. After reacting at room temperature for 10 minutes, the amount of 7-amino-4-methylcoumarin (AMC) released by DPPIV was measured with a 96-well plate fluorescence detector (Fluoroscan Ascent: manufactured by Thermo Electron). The excitation wavelength was 390 nm and the measurement wavelength was 460 nm. In the control group, 25 mM Tris-HCl buffer (pH 8.0) was used in the same manner instead of DPPI V solution.
[0049] DPPIV阻害率の計算は、サンプル溶液を含んで!/、な!/、場合の活性を 100とし、ぺプ チド溶液を添加した場合の活性を 100から差し引いた分を阻害率(%)とした。サンプ ル自体の擬似阻害効果を補正して阻害率を求めた。その結果、各サンプルの DPPI V阻害率はサンプル終濃度力 Sl.0mg/mlのとき HACP-01で 15.0% HACP-U2で 20.5% SCP_5000、ゼラチン、卵白ペプチドおよび大豆ペプチドでは 0%であった(図 1を参 昭) [0049] The calculation of the DPPIV inhibition rate includes the sample solution! / ,! The inhibition rate (%) was obtained by subtracting from 100 the activity when the tide solution was added. The inhibition rate was calculated by correcting the pseudo-inhibition effect of the sample itself. As a result, the DPPI V inhibition rate of each sample was 15.0% for HACP-01 and 20.5% for HACP-U2 at a sample final concentration of Sl.0 mg / ml, and 0% for SCP_5000, gelatin, egg white peptide, and soybean peptide ( (See Figure 1)
[0050] (実施例 2)  [0050] (Example 2)
コラーゲンペプチド HACP_01 HACP-U2および SCP-5000をそれぞれ 25mMのトリ ス -塩酸バッファー(pH 8.0)に 25 50 100mg/mlとなるように溶解した。  Collagen peptides HACP_01 HACP-U2 and SCP-5000 were each dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 25 50 100 mg / ml.
[0051] 実施例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV 阻害率を求めたところ、 HACP-01の DPPIV阻害率はサンプル終濃度が 2.5mg/mlの とき 36.5% 5.0mg/mlのとき 51.6% 10mg/mlのとき 56.7%であった。 HACP—U2の DPPIV 阻害率はサンプル終濃度力 ¾.5mg/mlのとき 38.4% 5.0mg/mlのとき 50.4% 10mg/mlの とき 56.7%であった。 SCP-5000の DPPIV阻害率はサンプル終濃度力 ¾.5mg/mlおよび 5.0mg/mlのとき 0% 10mg/mlのとき 23.0%であった(図 2を参照)。  [0051] In the same manner as in Example 1, an activity inhibition experiment on DPPIV was performed. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition rate of HACP-01 was 36.5% when the final sample concentration was 2.5 mg / ml, 51.6% when 5.0 mg / ml, and 56.7% when 10 mg / ml. there were. The DPPIV inhibition rate of HACP-U2 was 38.4% at the sample final concentration of ¾.5 mg / ml, 50.4% at 5.0 mg / ml, and 56.7% at 10 mg / ml. SCP-5000's DPPIV inhibition rate was 0% at the final sample concentration of ¾.5 mg / ml and 5.0 mg / ml and 23.0% at 10 mg / ml (see Figure 2).
[0052] (実施例 3)  [0052] (Example 3)
コラーゲンペプチド(HACP-01:ゼライス社製)をカプセルパック C18 UG80 (20x250 mm :資生堂社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィー(島 津製作所社製)によって分画した。分画は、以下の条件で行った。  Collagen peptide (HACP-01: manufactured by Zelice) was fractionated by high performance liquid chromatography (manufactured by Shimadzu Corporation) connected with a capsule pack C18 UG80 (20 × 250 mm: manufactured by Shiseido Co.) column and a UV detector. Fractionation was performed under the following conditions.
試料濃度: 10mg/ml  Sample concentration: 10mg / ml
負荷量: 2ml  Load amount: 2ml
流速: 10ml/分  Flow rate: 10ml / min
溶出条件:リニアグラジェント溶出  Elution condition: Linear gradient elution
水(0.1%トリフルォロ酢酸 (TFA)を含む) 100%から開始し、ァセトニトリル(0.1%TFA を含む)濃度を 100分間で 0%から 100%まで上昇させる(以下、 0.1%TFAを含むァセ トニトリル 0— 100% (100分)と表記)。  Start with 100% water (including 0.1% trifluoroacetic acid (TFA)) and increase the concentration of acetonitrile (including 0.1% TFA) from 0% to 100% in 100 minutes (hereinafter referred to as acetonitrile containing 0.1% TFA). 0—100% (100 minutes)).
検出条件: 215nm  Detection condition: 215nm
分取条件: 0分から開始、 1本/分  Preparative conditions: Start from 0 minutes, 1 bottle / minute
この分画操作を 5回繰り返し、同じフラクションを集めて濃縮した後に、それぞれを 25 mMのトリス-塩酸バッファー(pH 8.0) lmlに溶解し、そのうちの 5 1を用いて実施例 1と 同様に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求 めたところ、 DPPIV阻害率は 14番目のフラクションで 35.8%、 15番目のフラクションで 4 2.4%、 16番目のフラクションで 15.1%、 17番目のフラクションで 22.5%、 19番目のフラ クシヨンで 32.5%、 20番目のフラクションで 24.9%、 21番目のフラクションで 26.3%、 22 番目のフラクションで 25.0%、 23番目のフラクションで 35.4%、 24番目のフラクションで 3 1.8%、 25番目のフラクションで 12.1%、 26番目のフラクションで 19.1%、 27番目のフラ クションで 16.1 %、 28番目のフラクションで 31.8%であつた。 Repeat this fractionation 5 times, collect and concentrate the same fractions, An activity inhibition experiment on DPPIV was carried out in the same manner as in Example 1 using 51 of them dissolved in 1 ml of mM Tris-HCl buffer (pH 8.0). When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition rate was 35.8% in the 14th fraction, 4 2.4% in the 15th fraction, 15.1% in the 16th fraction, and 22.5 in the 17th fraction. %, 32.5% in the 19th fraction, 24.9% in the 20th fraction, 26.3% in the 21st fraction, 25.0% in the 22nd fraction, 35.4% in the 23rd fraction, 3 1.8 in the 24th fraction %, 12.1% in the 25th fraction, 19.1% in the 26th fraction, 16.1% in the 27th fraction, and 31.8% in the 28th fraction.
[0053] 前記のように分画したフラクションのうち比較的阻害活性の高力、つた 14番目と 15番 目のフラクションを合わせて、カプセルパック C 18 UG80 (20x250mm :資生堂社製)力 ラムおよび UV検出器を連結した高速液体クロマトグラフィー(島津製作所社製)によ つてさらに分画した。分画条件は、 0.1%丁?八を含むァセトニトリル0— 20% (100分)の リニアグラジェント溶出(流速 10ml/分)で、 215nmで検出を行い、 0分から 1本/分で 分取を行った。各フラクションを濃縮した後に、それぞれを 25mMのトリス-塩酸バッフ ァー(pH 8.0) 200 ^ 1に溶解し、そのうちの 5 1を用いて実施例 1と同様に DPPIVに対 する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めたところ、 DPPIV 阻害率は 22番目のフラクションで 24.1%、 23番目のフラクションで 65.0%、 24番目のフ ラタシヨンで 99.9%、 25番目のフラクションで 90.6%、 30番目のフラクションで 43.1%で あった。 [0053] Of the fractions fractionated as described above, the comparatively high inhibitory activity, the 14th and 15th fractions, capsule pack C 18 UG80 (20x250mm: manufactured by Shiseido Co., Ltd.) Ram and UV Further fractionation was performed by high-performance liquid chromatography (manufactured by Shimadzu Corporation) connected to a detector. Is the fractionation condition 0.1%? Detection was performed at 215 nm by linear gradient elution (flow rate 10 ml / min) of 0 to 20% (100 min) of acetonitrile containing 8 and fractionation was performed from 0 min to 1 bottle / min. After concentrating each fraction, each was dissolved in 25 mM Tris-hydrochloric acid buffer (pH 8.0) 200 ^ 1 and an activity inhibition experiment on DPPIV was conducted in the same manner as in Example 1 using 51 of them. It was. The DPPIV inhibition rate was determined in the same manner as in Example 1. The DPPIV inhibition rate was 24.1% in the 22nd fraction, 65.0% in the 23rd fraction, 99.9% in the 24th fraction, and 90.6% in the 25th fraction. It was 43.1% in the 30th fraction.
[0054] 前記のように分画したフラクションのうち比較的阻害活性の高力、つた 23番目と 24番 目と 25番目のフラクションを合わせて、デベロシル C30-UG-5 (20x250mm:野村化学 社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィー(島津製作所社 製)によってさらに分画した。分画条件は、 0.1%TFAを含むァセトニトリル 0— 20% (1 00分)のリニアグラジェント溶出(流速 10ml/分)で、 215nmで検出を行い、 0分から 1本 /分で分取を行った。各フラクションを濃縮した後に、それぞれを 25mMのトリス-塩酸 バッファー(pH 8.0) 200 1に溶解し、そのうちの 5 1を用いて実施例 1と同様に DPPI Vに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めたところ、 DPPIV阻害率はそれぞれ、 27番目のフラクションで 33.6%、 28番目のフラクションで 9 2.9 %、 41番目のフラクションで 11.7 %であった。 [0054] Among the fractions fractionated as described above, relatively high inhibitory activity, ie, combining the 23rd, 24th and 25th fractions, Develocil C30-UG-5 (20x250mm: manufactured by Nomura Chemical Co., Ltd.) ) Further fractionation was performed by high performance liquid chromatography (manufactured by Shimadzu Corporation) connected with a column and a UV detector. Fractionation conditions were: linear gradient elution of 0 to 20% (100 min) of acetonitrile containing 0.1% TFA (flow rate 10 ml / min), detection at 215 nm, and fractionation from 0 min to 1 bottle / min. It was. After concentrating each fraction, each was dissolved in 25 mM Tris-hydrochloric acid buffer (pH 8.0) 2001, and an activity inhibition experiment against DPPI V was conducted in the same manner as in Example 1 using 51 of them. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition rate was 33.6% in the 27th fraction and 9% in the 28th fraction, respectively. It was 2.9% and 11.7% in the 41st fraction.
[0055] 前記のように分画したフラクションのうち比較的阻害活性の高力、つた 28番目のフラク シヨンを、アトランティス dC18 (4.6x250 :ウォーターズ社製)カラムおよび UV検出器を 連結した高速液体クロマトグラフィー(ウォーターズ社製)によってさらに分画した。分 画条件は、 0.1%TFAを含む水 100%のァイソクラティック溶出(流速 lml/分)で、 215η mで検出を行い、ピークごとに分取を行った。各フラクションを濃縮した後に、それぞ れを 25mMのトリス-塩酸バッファー(pH 8.0) 100〃 1に溶解し、そのうちの 5〃 1を用いて 実施例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV 阻害率を求めたところ、 DPPIV阻害活性は 1番目のピーク部分にのみに認められ、阻 害率は 16.9%であった。 [0055] Among the fractions fractionated as described above, relatively high inhibitory activity, that is, the 28th fraction, was subjected to high-performance liquid chromatography connected to an Atlantis dC18 (4.6x250: Waters) column and a UV detector. Further fractionation was performed by GRAPHI (manufactured by Waters). Fractionation conditions were as follows: isocratic elution of 100% water containing 0.1% TFA (flow rate 1 ml / min), detection at 215 ηm, and fractionation for each peak. After each fraction was concentrated, each was dissolved in 100 mM 1 of 25 mM Tris-HCl buffer (pH 8.0), and an activity inhibition experiment against DPPIV was conducted in the same manner as in Example 1 using 5 to 1 of them. . When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition activity was observed only in the first peak portion, and the inhibition rate was 16.9%.
[0056] DPPIV阻害活性を確認したフラクションを、アトランティス dC18 (4.6x250 :ウォータ ーズ社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィー(ウォーター ズ社製)によってさらに精製した。精製条件は、 0.1%TFAを含む水 100%のァイソクラ ティック溶出(流速 lml/分)で、 215nmで検出を行い、ピーク分取を行った。分取した 一部を濃縮し、超純水に溶解し、プロテインシーケンサ(アプライドバイオシステムズ 社製)によりアミノ酸配列を同定した。その結果、活性ペプチドのアミノ酸配列は Gly- Pro-Argであることが明らかになった。  The fraction in which DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) connected with an Atlantis dC18 (4.6 × 250: Waters) column and a UV detector. The purification conditions were elution with 100% water containing 0.1% TFA (flow rate 1 ml / min) at 215 nm, and peak fractionation was performed. A portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (Applied Biosystems). As a result, it was revealed that the amino acid sequence of the active peptide was Gly-Pro-Arg.
[0057] そこで、市販のペプチド合成装置を用いて前記アミノ酸配列のペプチドを合成し、 標準品として合成された Gly-Pro-Argを用いて DPPIVの阻害活性を調べた。合成ぺ プチド Gly- Pro-Argを 25mMのトリス-塩酸バッファー(pH 8.0)に 1.0, 2.5, 5.0mMとなる ように溶解し、そのうちの 5 1を用いて実施例 1と同様に DPPIVに対する活性阻害実 験を行った。実施例 1と同様に DPPIV阻害率を求めたところ、サンプル終濃度が 0.1m Mのとき 31·3%、 0.25mMのとき 44·7%、 0.5mMのとき 62.6%であった。また、近似曲泉を 引いて DPPIVの活性を 50%阻害するときの濃度を求めたところ、 283.1 \でぁった( 表 1を参照。「GPR」)。  [0057] Therefore, the peptide having the amino acid sequence was synthesized using a commercially available peptide synthesizer, and the inhibitory activity of DPPIV was examined using Gly-Pro-Arg synthesized as a standard product. Synthetic peptide Gly-Pro-Arg was dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 1.0, 2.5, 5.0 mM, and 51 of these was used to inhibit the activity against DPPIV as in Example 1. An experiment was conducted. When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 31.3% when the final sample concentration was 0.1 mM, 44.7% when 0.25 mM, and 62.6% when 0.5 mM. The concentration at which 50% inhibition of DPPIV activity was determined by drawing an approximate curve was 283.1 \ (see Table 1, “GPR”).
[0058] (実施例 4)  [Example 4]
実施例 3においてカプセルパック C18UG80カラム (2回目)で分画したフラクションの うち比較的阻害活性の高力、つた 30番目のフラクションを、デベロシル C30-UG-5 (20x 250mm:野村化学社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィ 一(島津製作所社製)によってさらに分画した。分画条件は、 0.1%TFAを含むァセト 二トリル 0— 20% (100分)のリニアグラジェント溶出(流速 10ml/分)で、 215nmで検出を 行い、 0分から 1本/分で分取を行った。各フラクションを濃縮した後に、それぞれを 2 5mMのトリス-塩酸バッファー(pH 8.0) 200 μ 1に溶解し、そのうちの 5 μ 1を用いて実施 例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害 率を求めたところ、 DPPIV阻害率は、 31番目のフラクションで 29.4%であった。 Among the fractions fractionated in the capsule pack C18UG80 column in Example 3 (second time), the 30th fraction, which has a relatively high inhibitory activity, was developed with Develocil C30-UG-5 (20x 250 mm (manufactured by Nomura Chemical Co., Ltd.) Further fractionation was performed by high performance liquid chromatography 1 (manufactured by Shimadzu Corporation) connected with a column and a UV detector. Fractionation conditions were as follows: 0.1% TFA containing acetonitrile 2--20% (100 minutes) linear gradient elution (flow rate 10 ml / min), detection at 215 nm, and fractionation from 0 min to 1 bottle / min. went. After concentrating each fraction, each was dissolved in 25 mM Tris-hydrochloric acid buffer (pH 8.0) 200 μl, and using 5 μl of the same, an activity inhibition experiment on DPPIV was conducted in the same manner as in Example 1. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition rate was 29.4% in the 31st fraction.
[0059] 前記のように分画したフラクションのうち阻害活性のあった 31番目のフラクションを、 カプセルパック Ph UG120 (20x250:資生堂社製)カラムおよび UV検出器を連結した 高速液体クロマトグラフィー(島津製作所社製)によってさらに分画した。分画条件は 、 0.1%TFAを含むァセトニトリル 0— 20% (100分)のリニアグラジェント溶出(流速 10ml /分)で、 215nmで検出を行い、ピークごとに分取を行った。各フラクションを濃縮した 後に、それぞれを 25mMのトリス-塩酸バッファー(ρΗδ.θ θΟ ^ Ιに溶解し、そのうちの 5 1を用いて実施例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1と同 様に DPPIV阻害率を求めたところ、 DPPIV阻害活性は 2番目のピーク部分にのみに 認められ、阻害率は 33.2%であった。  [0059] Among the fractions fractionated as described above, the 31st fraction having inhibitory activity was subjected to high performance liquid chromatography (Shimadzu Corporation) connected to a capsule pack Ph UG120 (20x250: manufactured by Shiseido Co., Ltd.) column and a UV detector. The product was further fractionated. Fractionation conditions were linear gradient elution of acetonitrile (0-20% (100 min) containing 0.1% TFA) (flow rate: 10 ml / min) at 215 nm, and fractionation was performed for each peak. After concentrating each fraction, each was dissolved in 25 mM Tris-hydrochloric acid buffer (ρΗδ.θ θ 、 ^ 5, and 51 of them were used to conduct an activity inhibition experiment on DPPIV in the same manner as in Example 1. When the DPPIV inhibition rate was determined in the same manner as in 1, DPPIV inhibition activity was observed only in the second peak, and the inhibition rate was 33.2%.
[0060] DPPIV阻害活性を確認したフラクションを、カプセルパック C18 MG (4.6x250mm :資 生堂社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィー(ウォーター ズ社製)によってさらに精製した。精製条件は、 0.1%TFAを含むァセトニトリル 0— 20 % (100分)のリニアグラジェント溶出(流速 lml/分)で、 215nmで検出を行い、ピーク 分取を行った。分取した一部を濃縮し、超純水に溶解し、プロテインシーケンサ (アブ ライドバイオシステムズ社製)によりアミノ酸配列を同定した。その結果、活性ペプチド のアミノ酸配列は Gly-Pro-Ser-Gly-Asn-Ala (配列番号 1)であることが明らかになつ た。  [0060] The fraction in which DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) connected with a capsule pack C18 MG (4.6x250mm: manufactured by Shiseido) column and a UV detector. The purification conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and peak fractionation. A portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (manufactured by Abride Biosystems). As a result, it was revealed that the amino acid sequence of the active peptide was Gly-Pro-Ser-Gly-Asn-Ala (SEQ ID NO: 1).
[0061] 標準品として合成された Gly-Pro-Ser-Gly-Asn-Alaを用いて DPPIVの阻害活性を 調べた。合成ペプチド Gly-Pro-Ser-Gly-Asn-Alaを 25mMのトリス-塩酸バッファー(p H 8.0)に 1.0, 2.5, 5.0mMとなるように溶解し、そのうちの 5 1を用いて実施例 1と同様 に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めた ところ、サンプノレ終濃度力^) . lmMのとき 41·9%、 0.25mMのとき 66·3%、 0.5mMのとき 79.2[0061] The inhibitory activity of DPPIV was examined using Gly-Pro-Ser-Gly-Asn-Ala synthesized as a standard. Synthetic peptide Gly-Pro-Ser-Gly-Asn-Ala was dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 1.0, 2.5, and 5.0 mM. Similarly, an activity inhibition experiment for DPPIV was conducted. The DPPIV inhibition rate was determined in the same manner as in Example 1. However, Sampnore final concentration force ^) .4 · 9% at lmM, 66 · 3% at 0.25mM, 79.2 at 0.5mM
%であった。また、近似曲線を引いて DPPIVの活性を 50%阻害するときの濃度を求め たところ、 135·9(Μであった(表 1を参照。「GPSGNA」)。 %Met. The concentration at which DPPIV activity was inhibited by 50% by drawing an approximate curve was found to be 135 · 9 (see Table 1, “GPSGNA”).
[0062] (実施例 5) [Example 5]
実施例 3においてカプセルパック C18UG80カラム (1回目)で分画したフラクションの うち比較的阻害活性の高かった 19番目と 20番目のフラクションを合わせて、カプセノレ ノ /ク C18 UG80 (20x250mm :資生堂社製)カラムおよび UV検出器を連結した高速液 体クロマトグラフィー(島津製作所社製)によってさらに分画した。分画条件は、 0.1% TFAを含むァセトニトリル 0— 20% (100分)のリニアグラジェント溶出(流速 10ml/分)で 、 215nmで検出を行い、 0分から 1本/分で分取を行った。各フラクションを濃縮した 後に、それぞれを 25mMのトリス-塩酸バッファー(pH 8.0) 200 1に溶解し、そのうちの 5 1を用いて実施例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1と同 様に DPPIV阻害率を求めたところ、 DPPIV阻害率は 41番目のフラクションで 13.2%、 4 2番目のフラクションで 34.5%、 43番目のフラクションで 16.4%、 44番目のフラクション で 31.0%、 45番目のフラクションで 23.5%、 46番目のフラクションで 20.8%、 47番目のフ ラタシヨンで 49.2%、 48番目のフラクションで 47.2%、 49番目のフラクションで 16.7%、 5 0番目のフラクションで 19.8%、 51番目のフラクションで 22.0%、 52番目のフラクション で 11.1 %、 53番目のフラクションで 12.6 %であった。  Among the fractions fractionated in the capsule pack C18UG80 column (first time) in Example 3, the 19th and 20th fractions, which had relatively high inhibitory activity, were combined, and Capseno Leno / C18 UG80 (20x250mm: manufactured by Shiseido Co., Ltd.) Further fractionation was performed by high-performance liquid chromatography (manufactured by Shimadzu Corporation) connected with a column and a UV detector. The fractionation conditions were linear gradient elution (flow rate 10 ml / min) of acetonitrile 0–20% (100 min) containing 0.1% TFA, detection was performed at 215 nm, and fractionation was performed from 0 min to 1 tube / min. . After concentrating each fraction, each was dissolved in 25 mM Tris-HCl buffer (pH 8.0) 2001, and an activity inhibition experiment on DPPIV was conducted in the same manner as in Example 1 using 51 of them. The DPPIV inhibition rate was determined in the same manner as in Example 1. The DPPIV inhibition rate was 13.2% in the 41st fraction, 44.5% in the 2nd fraction, 16.4% in the 43rd fraction, 31.0% in the 44th fraction. %, 23.5% in the 45th fraction, 20.8% in the 46th fraction, 49.2% in the 47th fraction, 47.2% in the 48th fraction, 16.7% in the 49th fraction, 19.8 in the 50th fraction %, 22.0% in the 51st fraction, 11.1% in the 52nd fraction, and 12.6% in the 53rd fraction.
[0063] 前記のように分画したフラクションのうち比較的阻害活性の高かった 42番目と 43番 目と 44番目のフラクションを合わせて、デベロシル C30-UG-5 (20x250mm:野村化学 社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィー(島津製作所社 製)によってさらに分画した。分画条件は、 0.1%TFAを含むァセトニトリル 0— 20% (1 00分)のリニアグラジェント溶出(流速 10ml/分)で、 215nmで検出を行い、 0分から 1本 /分で分取を行った。各フラクションを濃縮した後に、それぞれを 25mMのトリス-塩酸 バッファー(pH 8.0) 200 1に溶解し、そのうちの 5 1を用いて実施例 1と同様に DPPI Vに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めたところ、 DPPIV阻害活性は 49番目のフラクションにのみ認められ、阻害率は 11.7%であった。  [0063] The 42th, 43rd, and 44th fractions having relatively high inhibitory activity among the fractions fractionated as described above were combined, and the Develocil C30-UG-5 (20x250mm: manufactured by Nomura Chemical Co.) column Further fractionation was performed by high performance liquid chromatography (manufactured by Shimadzu Corporation) connected with a UV detector. Fractionation conditions were: linear gradient elution of 0 to 20% (100 min) of acetonitrile containing 0.1% TFA (flow rate 10 ml / min), detection at 215 nm, and fractionation from 0 min to 1 bottle / min. It was. After concentrating each fraction, each was dissolved in 25 mM Tris-hydrochloric acid buffer (pH 8.0) 2001, and an activity inhibition experiment against DPPI V was conducted in the same manner as in Example 1 using 51 of them. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition activity was found only in the 49th fraction, and the inhibition rate was 11.7%.
[0064] 前記のように分画したフラクションのうち阻害活性のあった 49番目のフラクションを、 カプセルパック Phenyl UG120 (4.6x250:資生堂社製)カラムおよび UV検出器を連結 した高速液体クロマトグラフィー(ウォーターズ社製)によってさらに分画した。分画条 件は、 0.1%TFAを含むァセトニトリル 0— 20% (100分)のリニアグラジェント溶出(流 速 lml/分)で、 215nmで検出を行い、ピークごとに分取を行った。各フラクションを濃 縮した後に、それぞれを 25mMのトリス-塩酸バッファー(pH 8.0) 50 1に溶解し、その うちの 5 1を用いて実施例 1と同様に DPPIVに対する活性阻害実験を行った。実施 例 1と同様に DPPIV阻害率を求めたところ、 DPPIV阻害活性は 4番目のピーク部分に のみに認められ、阻害率は 48.8%であった。 [0064] The 49th fraction having inhibitory activity among the fractions fractionated as described above, Further fractionation was performed by high performance liquid chromatography (Waters) connected to a capsule pack Phenyl UG120 (4.6x250: Shiseido) column and a UV detector. Fractionation conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and fractionation was performed for each peak. After each fraction was enrichment, respectively 2 5 mM Tris - it was dissolved in hydrochloric acid buffer (pH 8.0) 50 1, was subjected to the activity inhibition experiments against DPPIV in the same manner as in Example 1 using 5 1 of them. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition activity was observed only in the fourth peak portion, and the inhibition rate was 48.8%.
[0065] DPPIV阻害活性を確認したフラクションを、アトランティス dC18 (4.6x250 :ウォータ ーズ社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィー(ウォーター ズ社製)によってさらに精製した。精製条件は、 0.1%TFAを含むァセトニトリル 0— 20 % (100分)のリニアグラジェント溶出(流速 lml/分)で、 215nmで検出を行い、ピーク 分取を行った。分取した一部を濃縮し、超純水に溶解し、プロテインシーケンサ (アブ ライドバイオシステムズ社製)によりアミノ酸配列を同定した。その結果、活性ペプチド のアミノ酸配列は Gly-Pro-Ala-Gly-Pro-Ala (配列番号 2)であることが明らかになつ た。 [0065] The fraction in which DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) coupled with an Atlantis dC18 (4.6x250: Waters) column and a UV detector. The purification conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and peak fractionation. A portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (manufactured by Abride Biosystems). As a result, it was revealed that the amino acid sequence of the active peptide was Gly-Pro-Ala-Gly-Pro-Ala (SEQ ID NO: 2).
[0066] 標準品として合成された Gly-Pro-Ala-Gly-Pro-Alaを用いて DPPIVの阻害活性を 調べた。合成ペプチド Gly_Pro-Ala-Gly_Pro-Alaを 25mMのトリス-塩酸バッファー(p H 8.0)に 0.5, 1.0, 2.5mMとなるように溶解し、そのうちの 5 1を用いて実施例 1と同様 に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めた ところ、サンプル終濃度が 0.05mMのとき 27.9%、 O. lmMのとき 48.3%、 0.25mMのとき 71. 1%であった。また、近似曲線を引いて DPPIVの活性を 50%阻害するときの濃度を求 めたところ、 111.3〃 Mであった(表 1を参照。「GPAGPA」)。  [0066] The inhibitory activity of DPPIV was examined using Gly-Pro-Ala-Gly-Pro-Ala synthesized as a standard product. Synthetic peptide Gly_Pro-Ala-Gly_Pro-Ala was dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 0.5, 1.0, 2.5 mM, and 51 of them was used to treat DPPIV as in Example 1. Activity inhibition experiments were performed. When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 27.9% when the final sample concentration was 0.05 mM, 48.3% when O.lmM, and 71.1% when 0.25 mM. The concentration at which DPPIV activity was inhibited by 50% by drawing an approximate curve was 111.3 M (see Table 1, “GPAGPA”).
[0067] (実施例 6) 較的阻害活性の高かった 47番目と 48番目のフラクションを合わせて、デベロシル C30 -UG-5 (20x250mm:野村化学社製)カラムおよび UV検出器を連結した高速液体クロ マトグラフィー(島津製作所社製)によってさらに分画した。分画条件は、 0.1%TFAを 含むァセトニトリル 0— 20% (100分)のリニアグラジェント溶出(流速 10ml/分)で、 215η mで検出を行い、 0分から 1本/分で分取を行った。各フラクションを濃縮した後に、そ れぞれを 25mMのトリス-塩酸バッファー(pH 8.0) 200 μ 1に溶解し、そのうちの 5 μ 1を用 いて実施例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPP IV阻害率を求めたところ、 DPPIV阻害率は、 53番目のフラクションで 17.1%、 54番目 のフラクションで 36.5%、 55番目のフラクションで 24.2%、 56番目のフラクションで 17.4 %であった。 [0067] (Example 6) A high-speed liquid in which the 47th and 48th fractions having relatively high inhibitory activity were combined, and a Develocil C30-UG-5 (20x250mm: Nomura Chemical Co.) column and a UV detector were connected. Further fractionation was performed by chromatography (manufactured by Shimadzu Corporation). Fractionation condition is 0.1% TFA Detection was performed at 215 ηm by linear gradient elution (flow rate 10 ml / min) of acetonitrile containing 0-20% (100 min), and fractionation was performed from 0 min to 1 bottle / min. After concentrating each fraction, each was dissolved in 200 μ1 of 25 mM Tris-HCl buffer (pH 8.0), and 5 μ1 of this was used to conduct an activity inhibition experiment against DPPIV in the same manner as in Example 1. It was. When the DPP IV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition rate was 17.1% in the 53rd fraction, 36.5% in the 54th fraction, 24.2% in the 55th fraction, and 17.4 in the 56th fraction. %Met.
[0068] 前記のように分画したフラクションのうち比較的阻害活性の高かった 54番目のフラク シヨンを、カプセルパック Phenyl UG120 (4.6x250:資生堂社製)カラムおよび UV検出 器を連結した高速液体クロマトグラフィー(ウォーターズ社製)によってさらに分画した 。分画条件は、 0.1%TFAを含むァセトニトリル 0— 20% (100分)のリニアグラジェント 溶出(流速 lml/分)で、 215nmで検出を行い、ピークごとに分取を行った。各フラクショ ンを濃縮した後に、それぞれを 25mMのトリス-塩酸バッファー(pH 8.0) 50 1に溶解し 、そのうちの 5 1を用いて実施例 1と同様に DPPIVに対する活性阻害実験を行った。 実施例 1と同様に DPPIV阻害率を求めたところ、 DPPIV阻害活性は 1番目のピーク部 分にのみに認められ、阻害率は 13.5%であった。  [0068] Among the fractions fractionated as described above, the 54th fraction having a relatively high inhibitory activity was used as a high performance liquid chromatograph to which a capsule pack Phenyl UG120 (4.6x250: manufactured by Shiseido Co., Ltd.) column and a UV detector were connected. Further fractionation was performed by GRAPHI (Waters). Fractionation conditions were as follows: linear gradient elution (acetonitrile 0-20% (100 min) containing 0.1% TFA) (flow rate 1 ml / min), detection at 215 nm, and fractionation for each peak. After concentrating each fraction, each was dissolved in 25 mM Tris-HCl buffer (pH 8.0) 501, and an activity inhibition experiment on DPPIV was conducted in the same manner as in Example 1 using 51 of them. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition activity was observed only in the first peak portion, and the inhibition rate was 13.5%.
[0069] DPPIV阻害活性を確認したフラクションを、アトランティス dC18 (4.6x250 :ウォータ ーズ社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィー(ウォーター ズ社製)によってさらに精製した。精製条件は、 0.1%TFAを含むァセトニトリル 0— 20 % (100分)のリニアグラジェント溶出(流速 lml/分)で、 215nmで検出を行い、ピーク 分取を行った。分取した一部を濃縮し、超純水に溶解し、プロテインシーケンサ (アブ ライドバイオシステムズ社製)によりアミノ酸配列を同定した。その結果、活性ペプチド のアミノ酸配列は Gly-Pro-Va卜 Gly-Ala-Arg (配列番号 3)であることが明らかになつ た。  [0069] The fraction in which DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) connected with an Atlantis dC18 (4.6x250: Waters) column and a UV detector. The purification conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and peak fractionation. A portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (manufactured by Abride Biosystems). As a result, it was revealed that the amino acid sequence of the active peptide was Gly-Pro-Va Gly-Ala-Arg (SEQ ID NO: 3).
[0070] 標準品として合成された Gly-Pro-Va卜 Gly-Ala-Argを用いて DPPIVの阻害活性を 調べた。合成ペプチド Gly-Pro-Va卜 Gly-Ala-Argを 25mMのトリス-塩酸バッファー(p H 8.0)に 1.0, 2.5, 5.0mMとなるように溶解し、そのうちの 5 1を用いて実施例 1と同様 に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めた ところ、サンプノレ終濃度力 SO. lmMのとき 37·7%、 0.25mMのとき 61·6%、 0.5mMのとき 76.1[0070] The inhibitory activity of DPPIV was examined using Gly-Pro-Va 卜 Gly-Ala-Arg synthesized as a standard product. Synthetic peptide Gly-Pro-Va 卜 Gly-Ala-Arg was dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 1.0, 2.5, 5.0 mM, and 51 of these were used as Example 1 and Similarly, an activity inhibition experiment for DPPIV was conducted. The DPPIV inhibition rate was determined in the same manner as in Example 1. However, the final concentration of Sampnore SO. LmM is 37.7%, 0.25mM is 66.1%, 0.5mM is 76.1%
%であった。また、近似曲線を引いて DPPIVの活性を 50%阻害するときの濃度を求め たところ、 163.0 Mであった(表 1を参照。「GPVGAR」)。 %Met. The concentration at which DPPIV activity was inhibited by 50% by drawing an approximate curve was 163.0 M (see Table 1, “GPVGAR”).
[0071] (実施例 7) [Example 7]
実施例 6においてデベロシル C30-UG-5カラムで分画したフラクションのうち比較 的阻害活性の高かった 55番目のフラクションを、カプセルパック Phenyl UG120 (4.6x2 50:資生堂社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィー(ゥォ 一ターズ社製)によってさらに分画した。分画条件は、 0.1%TFAを含むァセトニトリル 0— 20% (100分)のリニアグラジェント溶出(流速 lml/分)で、 215nmで検出を行い、ピ ークごとに分取を行った。各フラクションを濃縮した後に、それぞれを 25mMのトリス- 塩酸バッファー(pH 8.0) 10011 1に溶解し、そのうちの 5 H 1を用いて実施例 1と同様に D PPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めたとこ ろ、 DPPIV阻害活性は 4番目のピーク部分にのみ認められ、阻害率は 21.8%であった Among the fractions fractionated with the Develocil C30-UG-5 column in Example 6, the 55th fraction with relatively high inhibitory activity was added to a capsule pack Phenyl UG120 (4.6x2 50: manufactured by Shiseido) column and a UV detector. Further fractionation was performed by linked high performance liquid chromatography (Wooters). Fractionation conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and fractionation was performed for each peak. After concentrating each fraction, each was dissolved in 25 mM Tris-hydrochloric acid buffer (pH 8.0) 100111, and activity inhibition experiment on DPPIV was conducted in the same manner as in Example 1 using 5 H 1 thereof. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition activity was observed only in the fourth peak portion, and the inhibition rate was 21.8%.
Yes
[0072] DPPIV阻害活性を確認したフラクションを、アトランティス dC18 (4.6x250 :ウォータ ーズ社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィー(ウォーター ズ社製)によってさらに精製した。精製条件は、 0.1%TFAを含むァセトニトリル 0— 20 % (100分)のリニアグラジェント溶出(流速 lml/分)で、 215nmで検出を行い、ピーク 分取を行った。分取した一部を濃縮し、超純水に溶解し、プロテインシーケンサ (アブ ライドバイオシステムズ社製)によりアミノ酸配列を同定した。その結果、活性ペプチド のアミノ酸配列は Gly-Pro-Ser-Gly-Glu-Arg-Gly-Pro-Hyp (配列番号 4)であることが 明らかになった。  [0072] The fraction in which DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) connected with an Atlantis dC18 (4.6x250: Waters) column and a UV detector. The purification conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and peak fractionation. A portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (manufactured by Abride Biosystems). As a result, the amino acid sequence of the active peptide was revealed to be Gly-Pro-Ser-Gly-Glu-Arg-Gly-Pro-Hyp (SEQ ID NO: 4).
[0073] 標準品として合成された Gly-Pro-Ser-Gly-Glu-Arg-Gly-Pro-Hypを用いて DPPIV の阻害活性を調べた。合成ペプチド Gly-Pro-Ser-Gly-Glu-Arg-Gly-Pro-Hypを 25m Mのトリス-塩酸バッファー(pH 8.0)に 1.0, 2.5, 5.0mMとなるように溶解し、そのうちの 5 1を用いて実施例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1と同 様に DPPIV阻害率を求めたところ、サンプル終濃度力 SO. lmMのとき 32.5%、 0.25mMの とき 58.5%、 0.5mMのとき 74.4%であった。また、近似曲線を引いて DPPIVの活性を 50 %阻害するときの濃度を求めたところ、 190.5 Mであった(表 1を参照。「GPSGERG PO」)。 [0073] The inhibitory activity of DPPIV was examined using Gly-Pro-Ser-Gly-Glu-Arg-Gly-Pro-Hyp synthesized as a standard product. Tris synthetic peptide Gly-Pro-Ser-Gly- Glu-Arg-Gly-Pro-Hyp of 2 5 m M - 1.0 hydrochloride buffer (pH 8.0), 2.5, were dissolved at a 5.0 mM, 5 1 of which In the same manner as in Example 1, an activity inhibition experiment on DPPIV was conducted using When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 32.5% when the sample final concentration was SO.lmM, 58.5% when 0.25 mM, and 74.4% when 0.5 mM. Also, draw an approximate curve to increase the activity of DPPIV. The concentration at which% inhibition was determined was 190.5 M (see Table 1, “GPSGERG PO”).
[0074] (実施例 8) [Example 8]
実施例 3においてカプセルパック C18UG80カラム(1回目)で分画したフラクション のうち比較的阻害活性の高力、つた 23番目と 24番目のフラクションを合わせて、カプセ ルパック C18 UG80 (20x250mm :資生堂社製)カラムおよび UV検出器を連結した高速 液体クロマトグラフィー(島津製作所社製)によってさらに分画した。分画条件は、 0.1 %TFAを含むァセトニトリル 0— 20% (100分)のリニアグラジェント溶出(流速 10ml/分 )で、 215nmで検出を行い、 0分から 1本/分で分取を行った。各フラクションを濃縮し た後に、それぞれを 25mMのトリス-塩酸バッファー(pH 8.0) 200 1に溶解し、そのうち の 5 1を用いて実施例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1と 同様に DPPIV阻害率を求めたところ、 DPPIV阻害率は 59番目のフラクションで 42.4% 、 60番目のフラクションで 13.0%、 61番目のフラクションで 43.6%、 62番目のフラクショ ンで 56.4%、 64番目のフラクションで 21.9%、 65番目のフラクションで 26.3%、 66番目の フラクションで 33.7%、 67番目のフラクションで 17.7%、 70番目のフラクションで 27.9% 、 71番目のフラクションで 34.5%、 72番目のフラクションで 25.9%、 75番目のフラクショ ンで 10.8%であった。  Capsule pack C18 UG80 (20x250mm: manufactured by Shiseido Co., Ltd.) is a combination of the fractions with relatively high inhibitory activity among the fractions fractionated in the capsule pack C18UG80 column (first time) in Example 3. Further fractionation was performed by high performance liquid chromatography (manufactured by Shimadzu Corporation) connected with a column and a UV detector. The fractionation conditions were linear gradient elution (flow rate 10ml / min) of acetonitrile 0–20% (100 min) containing 0.1% TFA, detection at 215nm, and fractionation from 0 min to 1 bottle / min. . After concentrating each fraction, each was dissolved in 25 mM Tris-HCl buffer (pH 8.0) 2001, and an activity inhibition experiment on DPPIV was conducted in the same manner as in Example 1 using 51 of them. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition rate was 42.4% in the 59th fraction, 13.0% in the 60th fraction, 43.6% in the 61st fraction, and 56.4% in the 62nd fraction. 21.9% in the 64th fraction, 26.3% in the 65th fraction, 33.7% in the 66th fraction, 17.7% in the 67th fraction, 27.9% in the 70th fraction, 34.5% in the 71st fraction, 72 It was 25.9% in the 1st fraction and 10.8% in the 75th fraction.
[0075] 前記のように分画したフラクションのうち比較的阻害活性の高力、つた 61番目と 62番 ラムおよび UV検出器を連結した高速液体クロマトグラフィー(ウォーターズ社製)によ つてさらに分画した。分画条件は、 0.1%丁?八を含むァセトニトリル0— 20% (100分)の リニアグラジェント溶出(流速 lml/分)で、 215nmで検出を行い、ピークごとに分取を 行った。各フラクションを濃縮した後に、それぞれを 25mMのトリス-塩酸バッファー(p H 8.0) 50 1に溶解し、そのうちの 5 1を用いて実施例 1と同様に DPPIVに対する活 性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めたところ、 DPPIV阻害 活性は 5番目のピーク部分にのみ認められ、阻害率は 39.3%であった。  [0075] Among the fractions fractionated as described above, the fractions were further fractionated by high-performance liquid chromatography (Waters Co., Ltd.) connected to the 61st and 62nd rams and UV detector. did. Is the fractionation condition 0.1%? Detection was carried out at 215 nm using linear gradient elution (flow rate 1 ml / min) of 0 to 20% (100 min) of acetonitrile containing eight, and fractionation was performed for each peak. After concentrating each fraction, each was dissolved in 25 mM Tris-HCl buffer (pH 8.0) 501, and an activity inhibition experiment on DPPIV was conducted in the same manner as in Example 1 using 51 of them. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition activity was observed only in the fifth peak portion, and the inhibition rate was 39.3%.
[0076] DPPIV阻害活性を確認したフラクションを、アトランティス dC18 (4.6x250 :ウォータ ーズ社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィー(ウォーター ズ社製)によってさらに精製した。精製条件は、 0.1%TFAを含むァセトニトリル 0— 20 % (100分)のリニアグラジェント溶出(流速 lml/分)で、 215nmで検出を行い、ピーク 分取を行った。分取した一部を濃縮し、超純水に溶解し、プロテインシーケンサ (アブ ライドバイオシステムズ社製)によりアミノ酸配列を同定した。その結果、活性ペプチド のアミノ酸配列は Gly-Pro-Va卜 Gly-Pro-Ala (配列番号 5)であることが明らかになつ た。 [0076] The fraction confirmed to have DPPIV inhibitory activity was subjected to high performance liquid chromatography (water) coupled with an Atlantis dC18 (4.6x250: Waters) column and a UV detector. The product was further purified. Purification conditions were linear gradient elution of acetonitrile (0-20% (100 min) containing 0.1% TFA) (flow rate: 1 ml / min), detection at 215 nm, and peak fractionation. A portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified using a protein sequencer (manufactured by Abride Biosystems). As a result, the amino acid sequence of the active peptide was found to be Gly-Pro-Va Gly-Pro-Ala (SEQ ID NO: 5).
[0077] 標準品として合成された Gly-Pro-Va卜 Gly-Pro-Alaを用いて DPPIVの阻害活性を 調べた。合成ペプチド Gly_Pro-Val_Gly_Pro-Alaを 25mMのトリス-塩酸バッファー(p H 8.0)に 0.5, 1.0, 2.5mMとなるように溶解し、そのうちの 5 1を用いて実施例 1と同様 に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めた ところ、サンプル終濃度が 0.05mMのとき 45.3%、 O. lmMのとき 61.7%、 0.25mMのとき 81. 6%であった。また、近似曲線を引いて DPPIVの活性を 50%阻害するときの濃度を求 めたところ、 60.8〃Mであった(表 1を参照。「GPVGPA」)。  [0077] The inhibitory activity of DPPIV was examined using Gly-Pro-Va Gly-Pro-Ala synthesized as a standard product. Synthetic peptide Gly_Pro-Val_Gly_Pro-Ala was dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 0.5, 1.0, 2.5 mM, and 51 of these was used to inhibit the activity against DPPIV as in Example 1. The experiment was conducted. When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 45.3% when the final sample concentration was 0.05 mM, 61.7% when O.lmM, and 81.6% when 0.25 mM. The concentration at which DPPIV activity was inhibited by 50% by drawing an approximate curve was 60.8 〃M (see Table 1, “GPVGPA”).
[0078] (実施例 9)  [0078] (Example 9)
実施例 8においてカプセルパック C18UG80カラムで分画したフラクションのうち阻 害活性のあった 64番目と 65番目のフラクションを合わせて、カプセルパック Phenyl U G120 (4.6x250:資生堂社製)カラムおよび UV検出器を連結した高速液体クロマトダラ フィー(ウォーターズ社製)によってさらに分画した。分画条件は、 0.1%TFAを含むァ セトニトリル 0— 20% (100分)のリニアグラジェント溶出(流速 lml/分)で、 215nmで検 出を行い、ピークごとに分取を行った。各フラクションを濃縮した後に、それぞれを 25 mMのトリス-塩酸バッファー(pH 8.0) 50 1に溶解し、そのうちの 5 1を用いて実施例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率 を求めたところ、 DPPIV阻害活性は 2番目のピーク部分にのみ認められ、阻害率は 38 • 2%であった。  Among the fractions fractionated by the capsule pack C18UG80 column in Example 8, the 64th and 65th fractions having inhibitory activity were combined, and the capsule pack Phenyl U G120 (4.6x250: manufactured by Shiseido Co., Ltd.) column and UV detector were combined. Further fractionation was carried out using a high-performance liquid chromatography (manufactured by Waters). The fractionation conditions were linear gradient elution (flow rate 1 ml / min) of acetonitrile 0-20% (100 min) containing 0.1% TFA, detection was performed at 215 nm, and fractionation was performed for each peak. After concentrating each fraction, each was dissolved in 25 mM Tris-hydrochloric acid buffer (pH 8.0) 501, and an activity inhibition experiment on DPPIV was conducted in the same manner as in Example 1 using 51 of them. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition activity was observed only in the second peak portion, and the inhibition rate was 38 • 2%.
[0079] DPPIV阻害活性を確認したフラクションを、アトランティス dC18 (4.6x250 :ウォータ ーズ社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィー(ウォーター ズ社製)によってさらに精製した。精製条件は、 0.1%TFAを含むァセトニトリル 0— 20 % (100分)のリニアグラジェント溶出(流速 lml/分)で、 215nmで検出を行い、ピーク 分取を行った。分取した一部を濃縮し、超純水に溶解し、プロテインシーケンサ(アブ ライドバイオシステムズ社製)によりアミノ酸配列を同定した。その結果、活性ペプチド のアミノ酸配列は Gly-Pro-Ile-Gly-Ser-Ala (配列番号 6)であることが明らかになった[0079] The fraction in which DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) connected with an Atlantis dC18 (4.6x250: Waters) column and a UV detector. The purification conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, peak Sorting was performed. A portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (manufactured by Abride Biosystems). As a result, it was revealed that the amino acid sequence of the active peptide is Gly-Pro-Ile-Gly-Ser-Ala (SEQ ID NO: 6)
Yes
[0080] 標準品として合成された Gly-Pro-Ile-Gly-Ser-Alaを用いて DPPIVの阻害活性を調 ベた。合成ペプチド Gly-Pro-Ile-Gly-Ser-Alaを 25mMのトリス-塩酸バッファー(pH 8. 0)に 0.5, 1.0, 2.5mMとなるように溶解し、そのうちの 5 1を用いて実施例 1と同様に D PPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めたとこ ろ、サンプル終濃度が 0.05mMのとき 32.2%、 O. lmMのとき 47.3%、 0.25mMのとき 70.9% であった。また、近似曲線を引いて DPPIVの活性を 50%阻害するときの濃度を求め たところ、 107.1〃Mであった(表 1を参照。「GPIGSA」)。  [0080] The inhibitory activity of DPPIV was examined using Gly-Pro-Ile-Gly-Ser-Ala synthesized as a standard product. Synthetic peptide Gly-Pro-Ile-Gly-Ser-Ala was dissolved in 25 mM Tris-hydrochloric acid buffer (pH 8.0) to 0.5, 1.0, 2.5 mM, and 51 of them was used in Example 1. In the same manner as above, an activity inhibition experiment against DPPIV was conducted. When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 32.2% when the final sample concentration was 0.05 mM, 47.3% when O.lmM, and 70.9% when 0.25 mM. The concentration at which 50% inhibition of DPPIV activity was determined by drawing an approximate curve was 107.1 μM (see Table 1, “GPIGSA”).
[0081] (実施例 10)  [Example 10]
実施例 8においてカプセルパック C18UG80カラムで分画したフラクションのうち阻 害活性のあった 66番目と 67番目のフラクションを合わせて、カプセルパック Phenyl U G120 (4.6x250:資生堂社製)カラムおよび UV検出器を連結した高速液体クロマトダラ フィー(ウォーターズ社製)によってさらに分画した。分画条件は、 0.1%TFAを含むァ セトニトリル 0— 20% (100分)のリニアグラジェント溶出(流速 lml/分)で、 215nmで検 出を行い、ピークごとに分取を行った。各フラクションを濃縮した後に、それぞれを 25 mMのトリス-塩酸バッファー(pH 8.0) 50 1に溶解し、そのうちの 5 1を用いて実施例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率 を求めたところ、 DPPIV阻害活性は 5番目と 6番目のピーク部分に認められ、阻害率 は 5番目のピークで 37.4%、 6番目のピークで 11.0%であった。  Among the fractions fractionated by the capsule pack C18UG80 column in Example 8, the 66th and 67th fractions having inhibitory activity were combined, and the capsule pack Phenyl U G120 (4.6x250: manufactured by Shiseido Co.) column and UV detector were combined. Further fractionation was carried out using a high-performance liquid chromatography (manufactured by Waters). The fractionation conditions were linear gradient elution (flow rate 1 ml / min) of acetonitrile 0-20% (100 min) containing 0.1% TFA, detection was performed at 215 nm, and fractionation was performed for each peak. After concentrating each fraction, each was dissolved in 25 mM Tris-hydrochloric acid buffer (pH 8.0) 501, and an activity inhibition experiment on DPPIV was conducted in the same manner as in Example 1 using 51 of them. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition activity was observed at the fifth and sixth peaks, and the inhibition rate was 37.4% at the fifth peak and 11.0% at the sixth peak. It was.
[0082] DPPIV阻害活性を確認した 5番目のフラクションを、アトランティス dC 18 (4.6x250:ゥ オーターズ社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィー(ゥォ 一ターズ社製)によってさらに精製した。精製条件は、 0.1%TFAを含むァセトニトリル 0— 20% (100分)のリニアグラジェント溶出(流速 lml/分)で、 215nmで検出を行い、ピ 一ク分取を行った。分取した一部を濃縮し、超純水に溶解し、プロテインシーケンサ( アプライドバイォシステムズ社製)によりアミノ酸配列を同定した。その結果、活性ぺプ チドのアミノ酸配列は Gly-Leu-Ala-Gly-Pro-Hyp (配列番号 7)であることが明らかに なった。 [0082] The fifth fraction confirmed to have DPPIV inhibitory activity was further purified by high performance liquid chromatography (Waters) connected with an Atlantis dC 18 (4.6x250: Waters) column and a UV detector. did. The purification conditions were linear gradient elution (acetonitrile 0-20% (100 min) containing 0.1% TFA) (flow rate 1 ml / min), detection at 215 nm, and peak fractionation. A part of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (Applied by Systems). As a result, the active peptide It was revealed that the amino acid sequence of tide was Gly-Leu-Ala-Gly-Pro-Hyp (SEQ ID NO: 7).
[0083] 標準品として合成された Gly-Leu-Ala-Gly-Pro-Hypを用いて DPPIVの阻害活性を 調べた。合成ペプチド Gly-Leu-Ala-Gly-Pro-Hypを 25mMのトリス-塩酸バッファー(p H 8.0)に 1.0, 2.5, 5.0mMとなるように溶解し、そのうちの 5 1を用いて実施例 1と同様 に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めた ところ、サンプノレ終濃度力^) . lmMのとき 31.7%、 0.25mMのとき 55.2%、 0.5mMのとき 69.8 %であった。また、近似曲線を引いて DPPIVの活性を 50%阻害するときの濃度を求め たところ、 211.1 Mであった(表 1を参照。「GLAGPO」)。  [0083] The inhibitory activity of DPPIV was examined using Gly-Leu-Ala-Gly-Pro-Hyp synthesized as a standard product. Synthetic peptide Gly-Leu-Ala-Gly-Pro-Hyp was dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 1.0, 2.5, and 5.0 mM. Similarly, an activity inhibition experiment for DPPIV was conducted. When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was found to be 31.7% for Sampu Nore final concentration force ^). LmM, 55.2% for 0.25 mM, and 69.8% for 0.5 mM. The concentration at which 50% inhibition of DPPIV activity was determined by drawing an approximate curve was 211.1 M (see Table 1, “GLAGPO”).
[0084] (実施例 11) 較的阻害活性の高かった 70番目と 71番目と 72番目のフラクションを合わせて、デベロ シル C30-UG-5 (20x250mm:野村化学社製)カラムおよび UV検出器を連結した高速 液体クロマトグラフィー(島津製作所社製)によってさらに分画した。分画条件は、 0.1 %TFAを含むァセトニトリル 0— 20% (100分)のリニアグラジェント溶出(流速 10ml/分 )で、 215nmで検出を行い、 0分から 1本/分で分取を行った。各フラクションを濃縮し た後に、それぞれを 25mMのトリス-塩酸バッファー(pH 8.0) 200 1に溶解し、そのうち の 5 1を用いて実施例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1と 同様に DPPIV阻害率を求めたところ、 DPPIV阻害活性は 76番目のフラクションにの み認められ、阻害率は 5.9%であった。  [0084] (Example 11) The 70th, 71st and 72nd fractions having relatively high inhibitory activity were combined, and a Develocil C30-UG-5 (20x250mm: manufactured by Nomura Chemical Co.) column and UV detector were used. Further fractionation was performed by linked high performance liquid chromatography (manufactured by Shimadzu Corporation). The fractionation conditions were linear gradient elution (flow rate 10ml / min) of acetonitrile 0–20% (100 min) containing 0.1% TFA, detection at 215nm, and fractionation from 0 min to 1 bottle / min. . After concentrating each fraction, each was dissolved in 25 mM Tris-HCl buffer (pH 8.0) 2001, and an activity inhibition experiment on DPPIV was conducted in the same manner as in Example 1 using 51 of them. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition activity was observed only in the 76th fraction, and the inhibition rate was 5.9%.
[0085] 前記のように分画したフラクションのうち阻害活性のあった 76番目のフラクションを、 カプセルパック Phenyl UG120 (4.6x250:資生堂社製)カラムおよび UV検出器を連結 した高速液体クロマトグラフィー(ウォーターズ社製)によってさらに分画した。分画条 件は、 0.1%TFAを含むァセトニトリル 0— 20% (100分)のリニアグラジェント溶出(流 速 lml/分)で、 215nmで検出を行い、ピークごとに分取を行った。各フラクションを濃 縮した後に、それぞれを 25mMのトリス-塩酸バッファー( ^¾.0) 50 1に溶解し、そのう ちの 5 1を用いて実施例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1 と同様に DPPIV阻害率を求めたところ、 DPPIV阻害活性は 6番目と 7番目のピーク部 分に認められ、阻害率は 6番目のピークで 15·9%、 7番目のピークで 19.5%であった。 [0085] Among the fractions fractionated as described above, the 76th fraction having inhibitory activity was subjected to high performance liquid chromatography (Waters) connected with a capsule pack Phenyl UG120 (4.6x250: manufactured by Shiseido Co., Ltd.) column and a UV detector. The product was further fractionated. Fractionation conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and fractionation was performed for each peak. After each fraction was enrichment, respectively 2 5 mM Tris - was dissolved in hydrochloric acid buffer (^ ¾.0) 50 1, subjected to the activity inhibition experiments against DPPIV in the same manner as in Example 1 by using the power sale Chino 5 1 It was. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibitory activity was the sixth and seventh peak portions. The inhibition rate was 15.9% for the 6th peak and 19.5% for the 7th peak.
[0086] DPPIV阻害活性を確認したフラクションを、アトランティス dC18 (4.6x250 :ウォータ ーズ社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィー(ウォーター ズ社製)によってさらに精製した。精製条件は、 0.1%TFAを含むァセトニトリル 0— 20 % (100分)のリニアグラジェント溶出(流速 lml/分)で、 215nmで検出を行い、ピーク 分取を行った。分取した一部を濃縮し、超純水に溶解し、プロテインシーケンサ (アブ ライドバイオシステムズ社製)によりアミノ酸配列を同定した。その結果、活性ペプチド のアミノ酸配列は Gly-Pro-Arg-Gly-Arg-Thr-Gly-Asp-Ala-Gly-Pro-Val (配列番号 8)であることが明らかになった。 [0086] The fraction in which the DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) coupled with an Atlantis dC18 (4.6x250: Waters) column and a UV detector. The purification conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and peak fractionation. A portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (manufactured by Abride Biosystems). As a result, it was revealed that the amino acid sequence of the active peptide was Gly-Pro-Arg-Gly-Arg-Thr-Gly-Asp-Ala-Gly-Pro-Val (SEQ ID NO: 8).
[0087] 標準品として合成された Gly-Pro-Arg-Gly-Arg-Thr-Gly-Asp-Ala-Gly-Pro-Valを 用いて DPPIVの阻害活性を調べた。合成ペプチド Gly-Pro-Arg-Gly-Arg-Thr-Gly- Asp- Ala-Gly- Pro-Valを 25mMのトリス-塩酸バッファー(ρΗ8·0)に 1.0, 2.5, 5.0mMと なるように溶解し、そのうちの 5 1を用いて実施例 1と同様に DPPIVに対する活性阻 害実験を行った。実施例 1と同様に DPPIV阻害率を求めたところ、サンプル終濃度が O. lmMのとき 46.8%、 0.25mMのとき 68.7%、 0.5mMのとき 82.1%であった。また、近似曲 線を引いて DPPIVの活性を 50%阻害するときの濃度を求めたところ、 112.9 Mであ つた(表 1を参照。「GPRGRTGDAGPV」)。 [0087] The inhibitory activity of DPPIV was examined using Gly-Pro-Arg-Gly-Arg-Thr-Gly-Asp-Ala-Gly-Pro-Val synthesized as a standard product. Dissolve the synthetic peptide Gly-Pro-Arg-Gly-Arg-Thr-Gly-Asp-Ala-Gly-Pro-Val in 25 mM Tris-HCl buffer (ρΗ8 · 0) to 1.0, 2.5, 5.0 mM. Of these, 51 were used to conduct an activity inhibition experiment against DPPIV in the same manner as in Example 1. When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 46.8% when the final sample concentration was O.lmM, 68.7% when 0.25 mM, and 82.1% when 0.5 mM. The concentration at which 50% inhibition of DPPIV activity was determined by drawing an approximate curve was 112.9 M (see Table 1, “GPRGRTGDAGPV”).
[0088] (実施例 12) [0088] (Example 12)
実施例 3においてカプセルパック C18UG80カラム (1回目)で分画したフラクションの うち比較的阻害活性の高かった 28番目のフラクションを、カプセルパック C18 UG80 (2 0x250mm:資生堂社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィ 一(島津製作所社製)によってさらに分画した。分画条件は、 0.1%TFAを含むァセト 二トリル 0— 20% (100分)のリニアグラジェント溶出(流速 10ml/分)で、 215nmで検出を 行い、 0分から 1本/分で分取を行った。各フラクションを濃縮した後に、それぞれを 2 5mMのトリス-塩酸バッファー(pH 8.0) 200 μ 1に溶解し、そのうちの 5 μ 1を用いて実施 例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害 率を求めたところ、 DPPIV阻害活性は 82番目のフラクションにのみ認められ、阻害率 は 16.9%であった。 [0089] 前記のように分画したフラクションのうち阻害活性のあった 82番目のフラクションを、 カプセルパック Phenyl UG120 (4.6x250:資生堂社製)カラムおよび UV検出器を連結 した高速液体クロマトグラフィー(ウォーターズ社製)によってさらに分画した。分画条 件は、 0.1%TFAを含むァセトニトリル 0— 20% (100分)のリニアグラジェント溶出(流 速 lml/分)で、 215nmで検出を行い、ピークごとに分取を行った。各フラクションを濃 縮した後に、それぞれを 25mMのトリス-塩酸バッファー(pH 8.0) 50 1に溶解し、その うちの 5 1を用いて実施例 1と同様に DPPIVに対する活性阻害実験を行った。実施 例 1と同様に DPPIV阻害率を求めたところ、 DPPIV阻害活性は 4番目のピーク部分に のみ認められ、阻害率は 33.1%であった。 Among the fractions fractionated in the capsule pack C18UG80 column in Example 3 (first time), the 28th fraction having a relatively high inhibitory activity was treated with a capsule pack C18 UG80 (20 x 250 mm: manufactured by Shiseido) column and a UV detector. Further fractionation was performed with a linked high performance liquid chromatography (manufactured by Shimadzu Corporation). Fractionation conditions were as follows: 0.1% TFA containing acetonitrile 2--20% (100 minutes) linear gradient elution (flow rate 10 ml / min), detection at 215 nm, and fractionation from 0 min to 1 bottle / min. went. After concentrating each fraction, each was dissolved in 25 mM Tris-hydrochloric acid buffer (pH 8.0) 200 μl, and using 5 μl of the same, an activity inhibition experiment on DPPIV was conducted in the same manner as in Example 1. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition activity was observed only in the 82nd fraction, and the inhibition rate was 16.9%. [0089] Among the fractions fractionated as described above, the 82nd fraction having inhibitory activity was subjected to high performance liquid chromatography (Waters) connected to a capsule pack Phenyl UG120 (4.6x250: manufactured by Shiseido Co., Ltd.) column and a UV detector. The product was further fractionated. Fractionation conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and fractionation was performed for each peak. After each fraction was enrichment, respectively 2 5 mM Tris - it was dissolved in hydrochloric acid buffer (pH 8.0) 50 1, was subjected to the activity inhibition experiments against DPPIV in the same manner as in Example 1 using 5 1 of them. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition activity was observed only in the fourth peak portion, and the inhibition rate was 33.1%.
[0090] DPPIV阻害活性を確認したフラクションを、アトランティス dC18 (4.6x250 :ウォータ ーズ社製)カラムおよび UV検出器を連結した高速液体クロマトグラフィー(ウォーター ズ社製)によってさらに精製した。精製条件は、 0.1%TFAを含むァセトニトリル 0— 20 % (100分)のリニアグラジェント溶出(流速 lml/分)で、 215nmで検出を行い、ピーク 分取を行った。分取した一部を濃縮し、超純水に溶解し、プロテインシーケンサ (アブ ライドバイオシステムズ社製)によりアミノ酸配列を同定した。その結果、活性ペプチド のアミノ酸配列は Gly-Pro-Va卜 Gly-Pro (配列番号 9)であることが明らかになった。  [0090] The fraction in which DPPIV inhibitory activity was confirmed was further purified by high performance liquid chromatography (Waters) connected with an Atlantis dC18 (4.6x250: Waters) column and a UV detector. The purification conditions were linear gradient elution of 0-20% (100 min) acetonitrile containing 0.1% TFA (flow rate 1 ml / min), detection at 215 nm, and peak fractionation. A portion of the fraction was concentrated, dissolved in ultrapure water, and the amino acid sequence was identified by a protein sequencer (manufactured by Abride Biosystems). As a result, it was revealed that the amino acid sequence of the active peptide was Gly-Pro-Va 卜 Gly-Pro (SEQ ID NO: 9).
[0091] 標準品として合成された Gly-Pro-Vaト Gly-Proを用レ、て DPPIVの阻害活性を調べ た。合成ペプチド Gly- Pro-Va卜 Gly-Proを 25mMのトリス-塩酸バッファー(pH 8.0)に 1 .0, 2.5, 5.0mMとなるように溶解し、そのうちの 5 1を用いて実施例 1と同様に DPPIV に対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めたところ、サ ンプル終濃度カ^) . lmMのとき 47.8%、 0.25mMのとき 69.2%、 0.5mMのとき 81.5%であった 。また、近似曲線を引いて DPPIVの活性を 50%阻害するときの濃度を求めたところ、 1 08.0〃 Mであった(表 1を参照。「GPVGP」)。  [0091] The inhibitory activity of DPPIV was examined using Gly-Pro-Va and Gly-Pro synthesized as standard products. Synthetic peptide Gly-Pro-Va 卜 Gly-Pro was dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 1.0, 2.5, 5.0 mM, and 51 of them was used as in Example 1. In addition, an activity inhibition experiment against DPPIV was conducted. When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 47.8% at the final sample concentration of ^ .lmM, 69.2% at 0.25 mM, and 81.5% at 0.5 mM. The concentration at which DPPIV activity was inhibited by 50% by drawing an approximate curve was found to be 108.0 1 M (see Table 1, “GPVGP”).
[0092] (実施例 13)  [0092] (Example 13)
コラーゲンペプチド HACP-01に含まれると予想される 3残基のペプチド Gly-Pro-Me t、 Gly-Pro-Ala, Gly-Pro_Ser、 Gly-Pro-Hypの合成品を標準品として用いて、 HPLC による保持時間を元に HACP-01中からこれらの 3残基のペプチドの単離を試みた。 それぞれ単一ピークまで精製した後にプロテインシーケンサにより、配列が目的のぺ プチドと一致していることを確認した。すなわち、予想された 3残基のペプチドが HAC P-01中に含まれていることが明らかとなった。これらのペプチドにも DPPIVの阻害活 性があることが予想されたので、それぞれのペプチドの DPPIV阻害活性を調べた。 Collagen peptide HACP-01 is expected to be included in the 3-residue peptide Gly-Pro-Met, Gly-Pro-Ala, Gly-Pro_Ser, and Gly-Pro-Hyp as standard products. Based on the retention time by, we attempted to isolate these 3-residue peptides from HACP-01. After purification to a single peak for each, the sequencer is targeted by the protein sequencer. Confirmed to match with peptide. In other words, it was revealed that the expected 3-residue peptide was included in HAC P-01. Since these peptides were also expected to have DPPIV inhibitory activity, the DPPIV inhibitory activity of each peptide was examined.
[0093] 標準品である合成ペプチド Gly-Pro-Metを 25mMのトリス-塩酸バッファー(pH 8.0) に 2.5, 5.0, 10mMとなるように溶解し、そのうちの 5 1を用いて実施例 1と同様に DPPI Vに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めたところ、 サンプル終濃度が 0.25mMのとき 26.0%、 0.5mMのとき 48.0%、 l.OmMのとき 61.0%であつ た。また、近似曲線を引いて DPPIVの活性を 50%阻害するときの濃度を求めたところ 、 609.5 Mであった(表 1を参照。「GPM」)。  [0093] The standard synthetic peptide Gly-Pro-Met was dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 2.5, 5.0, 10 mM, and 51 of them was used as in Example 1. In addition, an activity inhibition experiment against DPPI V was performed. When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 26.0% when the final sample concentration was 0.25 mM, 48.0% when 0.5 mM, and 61.0% when l.OmM. The concentration at which 50% inhibition of DPPIV activity was determined by drawing an approximate curve was 609.5 M (see Table 1, “GPM”).
[0094] 標準品である合成ペプチド Gly-Pro-Alaを 25mMのトリス-塩酸バッファー(pH 8.0) に 2.5, 5.0, 10mMとなるように溶解し、そのうちの 5 1を用いて実施例 1と同様に DPPI Vに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めたところ、 サンプル終濃度が 0.25mMのとき 23.5%、 0.5mMのとき 38.2%、 l.OmMのとき 52.3%であつ た。また、近似曲線を引いて DPPIVの活性を 50%阻害するときの濃度を求めたところ 、 890.7 Mであった(表 1を参照。「GPA」)。  [0094] The standard synthetic peptide Gly-Pro-Ala was dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 2.5, 5.0, 10 mM, and 51 of them was used as in Example 1. In addition, an activity inhibition experiment against DPPI V was performed. When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 23.5% when the final sample concentration was 0.25 mM, 38.2% when 0.5 mM, and 52.3% when l.OmM. The concentration at which 50% inhibition of DPPIV activity was determined by drawing an approximate curve was 890.7 M (see Table 1, “GPA”).
[0095] 標準品である合成ペプチド Gly-Pro-Serを 25mMのトリス-塩酸バッファー(pH 8.0) に 5.0, 10, 20mMとなるように溶解し、そのうちの 5 1を用いて実施例 1と同様に DPPI Vに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めたところ、 サンプル終濃度力 S0.5mMのとき 27.0%、 l.OmMのとき 42.7%、 2.0mMのとき 60.5%であつ た。また、近似曲線を引いて DPPIVの活性を 50%阻害するときの濃度を求めたところ 、 1314.1 Mであった(表 1を参照。「GPS」)。  [0095] The standard synthetic peptide Gly-Pro-Ser was dissolved in 25 mM Tris-HCl buffer (pH 8.0) to 5.0, 10, 20 mM, and 51 of them was used as in Example 1. In addition, an activity inhibition experiment against DPPI V was performed. When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 27.0% at the sample final concentration force S0.5 mM, 42.7% at l.OmM, and 60.5% at 2.0 mM. The concentration at which 50% inhibition of DPPIV activity was determined by drawing an approximate curve was 1314.1 M (see Table 1, “GPS”).
[0096] 標準品である合成ペプチド Gly-Pro-Hypを 25mMのトリス-塩酸バッファー(pH 8.0) に lOOmMとなるように溶解し、そのうちの 5 1、 12.5 1、 25 1を用いて実施例 1と同様 に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めた ところ、サンプル終濃度が 10mMのとき 28.2%、 25mMのとき 57.8%、 50mMのとき 83.0%で あった。また、近似曲線を引いて DPPIVの活性を 50%阻害するときの濃度を求めたと ころ、 19259.5 Mであった(表 1を参照。「GPO」)。  [0096] The standard synthetic peptide Gly-Pro-Hyp was dissolved in 25 mM Tris-hydrochloric acid buffer (pH 8.0) to give lOOmM, and 5 1, 12.5 1, 25 1 of them were used in Example 1. In the same way, an activity inhibition experiment against DPPIV was conducted. When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 28.2% when the final sample concentration was 10 mM, 57.8% when 25 mM, and 83.0% when 50 mM. The concentration at which DPPIV activity was inhibited by 50% by drawing an approximate curve was 19259.5 M (see Table 1, “GPO”).
[0097] [表 1] 【表 1】 [0097] [Table 1] 【table 1】
ペプチド DPPIV活性を 50%阻害する濃度 ( M)  Concentration that inhibits peptide DPPIV activity by 50% (M)
GPM 609.5  GPM 609.5
GPA 890.7  GPA 890.7
GPS 1314.1  GPS 1314.1
GPO 19259.5  GPO 19259.5
GPR 283.1 GPR 283.1
GPSGNA 135.9  GPSGNA 135.9
GPAGPA 1 1 1.3  GPAGPA 1 1 1.3
GPVGAR 163.0  GPVGAR 163.0
GPVGPA 60.8  GPVGPA 60.8
GPIGSA 107.1  GPIGSA 107.1
GPSGERGPO 190.5  GPSGERGPO 190.5
GPRGRTGDAGPV 1 12.9  GPRGRTGDAGPV 1 12.9
GPVGP 108.0  GPVGP 108.0
GLAGPO 21 1.1  GLAGPO 21 1.1
[0098] (実施例 14) [Example 14]
糖尿病モデルラットを用いた糖負荷試験を行った。実験動物は Zucker Fattyラット を用いた。飼育環境は恒温恒湿、 12時間明 /12時間暗のサイクルの実験動物飼育 室で、市販の固型飼料 (CRF-1、オリエンタル酵母工業社製)を用い、自由に水を摂 取させた。一般状態を 5日以上毎日観察し、健康状態が良好であることを確認し、入 荷時、力二ユレーシヨン揷入時および試験物質投与時に体重測定を行った。採血の ための留置力二ユレーシヨン揷入手術は、糖負荷試験の前日に行った。群分けは試 験物質投与前に体重層別化無作為抽出法により行った。  A glucose tolerance test was conducted using diabetic model rats. As experimental animals, Zucker Fatty rats were used. The breeding environment was a laboratory animal breeding room with a constant temperature and humidity, 12 hours light / 12 hours dark cycle, and water was freely ingested using a commercially available solid feed (CRF-1, manufactured by Oriental Yeast Co., Ltd.). . The general condition was observed every day for 5 days or more, and it was confirmed that the health condition was good. Body weight was measured at the time of arrival, at the time of ingestion of force and at the time of test substance administration. Surgery for indwelling force for blood collection was performed the day before the glucose tolerance test. Grouping was performed by randomized stratification by weight stratification prior to test substance administration.
[0099] 前日力も 18時間以上絶食したラットから採血を行った後、コラーゲンペプチド HACP -01 (ゼライス社製、 400mg/ml)、コラーゲンペプチド SCP_5000 (新田ゼラチン社製、 4 OOmg/ml)あるいはコントロールとして市販の注射用水(大塚製薬工場社製)をそれぞ れ 10ml/kg経口投与し、その 30分後に 10%グルコースをそれぞれ 10ml/kg経口投与 した。グルコース投与直前および投与後 5, 10, 15,30分後に採血を行った。採血後、血 漿を分離し、以下の実験に用いた。 [0100] DPPIVの活性を阻害した条件で糖負荷を行ったときに、濃度および活性変化を受 けると考えられる血中パラメータとして、インスリン、 GLP-1と DPPIV活性がある。本実 験ではこれらを経時的に測定することによって、コラーゲンペプチドの有効性評価を 試みた。 [0099] After collecting blood from rats that had been fasted for 18 hours or more on the previous day, collagen peptide HACP-01 (400 mg / ml, Zerais Co., Ltd.), collagen peptide SCP_5000 (Nitta Gelatin Co., 4 OOmg / ml) or control Commercially available water for injection (manufactured by Otsuka Pharmaceutical Factory) was orally administered at 10 ml / kg, and 30 minutes later, 10% glucose was orally administered at 10 ml / kg. Blood was collected immediately before glucose administration and 5, 10, 15, 30 minutes after administration. After blood collection, plasma was separated and used for the following experiments. [0100] The blood parameters that are considered to undergo changes in concentration and activity when glucose is loaded under conditions that inhibit DPPIV activity include insulin, GLP-1 and DPPIV activity. In this experiment, we attempted to evaluate the effectiveness of collagen peptides by measuring them over time.
[0101] 非特許文献(J.Med.Chem. 2003 Jun 19;46(13):2774_89·)に従って、血漿中の DPPI V活性を経時的に測定した。具体的には得られた血漿 15 1、試験用バッファー(25m Mへぺス、 140mM塩化ナトリウム、 80mM塩化マグネシウム、 1% BSA: pH7.8) 15〃 1を 混合して室温で 5分間放置した。試験用バッファーで 100 Mとなるように希釈した基 質溶液(Gly-Pro-MCA O lを添加し、室温で 20分間放置した。検出は、 96ゥエル プレート対応蛍光検出器 (フルォロスキャンアセント:サーモエレクトロン社製)で、 DP PIVによって遊離される AMC量を測定した。なお、励起波長は 390nm、測定波長は 46 0匪で行った。活性は 20分の反応によって生じた蛍光値から、基質のみを含む場合 に示す蛍光値を差し引いたものとした。  [0101] DPPI V activity in plasma was measured over time according to non-patent literature (J. Med. Chem. 2003 Jun 19; 46 (13): 2774_89). Specifically, the obtained plasma 151 and test buffer (25 mM hepes, 140 mM sodium chloride, 80 mM magnesium chloride, 1% BSA: pH 7.8) 15 to 1 were mixed and left at room temperature for 5 minutes. . Substrate solution diluted to 100 M with test buffer (Gly-Pro-MCA O 1 was added and left at room temperature for 20 minutes. Detection was performed using a 96-well plate fluorescence detector (Fluoroscan Ascent: The amount of AMC released by DP PIV was measured using Thermo Electron Co., Ltd. The excitation wavelength was 390 nm and the measurement wavelength was 460 mm, and the activity was determined from the fluorescence value generated by the reaction for 20 minutes. The fluorescence value shown in the case of containing only was subtracted.
[0102] コラーゲンペプチドによる DPPIV阻害効果の評価は、各個体の被験物質投与前 (-3 0分)の血漿中 DPPIV活性を通常時の活性とし、この活性に対して各血漿での活性の 差を算出し、その比を取ることによって阻害率とした。すなわち、各個体の被験物質 投与前の阻害率は 0%となる。その結果、それぞれの実験区における DPPIV阻害率は 、 HACP-01ではグルコース投与直前(0分)で 37%、投与後 5分で 38%、 10分で 36% 、 15分で 35%、 30分で 31%であった。 SCP-5000ではグルコース投与直前(0分)で 12 %、投与後 5分で 15%、 10分で 20%、 15分で 10%、 30分で 20%であった。注射用水 ではグルコース投与直前(0分)で- 3%、投与後 5分で- 10%、 10分では 4%、 15分で は- 14%、 30分では- 7%であった(図 3を参照)。これらの結果から、 HACP-01摂取群 ではグルコース投与後 10分を除いて、注射用水摂取群に対して DPPIV活性が有意 に阻害されていることが確認できた。すなわち、コラーゲンペプチド HACP-01を摂取 することによって、血中の DPPIVの活性が阻害されることが明らかとなった。  [0102] Evaluation of DPPIV inhibitory effect by collagen peptides is based on the normal activity of DPPIV in plasma before test substance administration (-30 minutes) in each individual. Was calculated, and the ratio was taken as the inhibition rate. That is, the inhibition rate of each individual before administration of the test substance is 0%. As a result, the DPPIV inhibition rate in each experimental group was 37% immediately before glucose administration (0 minutes) in HACP-01, 38% 5 minutes after administration, 36% in 10 minutes, 35% in 15 minutes, 30 minutes It was 31%. In SCP-5000, it was 12% immediately before glucose administration (0 minutes), 15% 5 minutes after administration, 20% 10 minutes, 10% 15 minutes, and 20% 30 minutes. For water for injection, it was -3% at just before glucose administration (0 minutes), -10% at 5 minutes after administration, 4% at 10 minutes, -14% at 15 minutes, and -7% at 30 minutes (Figure 3). See). From these results, it was confirmed that DPPIV activity was significantly inhibited in the HACP-01 ingestion group compared to the water for injection group except for 10 minutes after glucose administration. That is, it was clarified that the activity of DPPIV in blood was inhibited by ingesting the collagen peptide HACP-01.
[0103] GLP-1検出キット(リンコリサーチ社製)を用いて、血漿中の GLP-1濃度の測定を行 つた。 96ゥエルプレートに固定化された GLP-1抗体に各血漿 50 1を添加して、 4°Cで 20時間から 24時間放置した。固定化された GLP-1以外のものを洗浄することによって 除去し、固定化された GLP-1に結合する、アルカリフォスファターゼを有する抗体を 添加し、室温 2時間放置した。洗浄によって非特異的に結合した抗体を除去後、 50 g/mlに調製した基質溶液(4-メチルゥンベリフェリルフォスフェート) 200 ,1 1を添加して 酵素反応を開始させた。室温で 50分間放置後、停止液 50 ^ 1添加して反応を停止さ せ、反応によって得られる蛍光物質ゥンベリフエロンを 96ゥヱルプレート対応蛍光検 出器 (フルォロスキャンアセント:サーモエレクトロン社製)で測定した。なお検出にお いては、励起波長は 355nm、測定波長 460nmにて行った。濃度既知の GLP-1を測定 することによって得た検量線から、各血漿中の GLP-1濃度を決定した。 [0103] GLP-1 concentration in plasma was measured using a GLP-1 detection kit (manufactured by Rinco Research). Each plasma 50 1 was added to the GLP-1 antibody immobilized on a 96-well plate and left at 4 ° C for 20 to 24 hours. By washing anything other than immobilized GLP-1 After removing, an antibody having alkaline phosphatase that binds to immobilized GLP-1 was added, and left at room temperature for 2 hours. After removing non-specifically bound antibody by washing, 200,11 substrate solution (4-methylumbelliferyl phosphate) prepared at 50 g / ml was added to start the enzyme reaction. After leaving at room temperature for 50 minutes, the reaction was stopped by adding stop solution 50 ^ 1 and the fluorescent substance umbelliferone obtained by the reaction was measured with a 96-wall plate fluorescence detector (Fluoroscan Ascent: manufactured by Thermo Electron). . For detection, the excitation wavelength was 355 nm and the measurement wavelength was 460 nm. The concentration of GLP-1 in each plasma was determined from a calibration curve obtained by measuring GLP-1 at known concentrations.
[0104] コラーゲンペプチドによる血中 GLP-1濃度上昇効果の評価は、各個体の被験物質 投与前 (-30分)の血漿中 GLP-1濃度を通常時の濃度 (100%)とし、この濃度に対する 各血漿での濃度の割合を算出した。その結果、それぞれの実験区における GLP-1濃 度上昇率は、 HACP-01ではグルコース投与直前(0分)で 120%、投与後 5分で 122% 、 10分で 117%、 15分で 112%、 30分で 116%であった。 SCP-5000ではグルコース投 与直前(0分)で 111 %、投与後 5分で 115%、 10分で 104%、 15分で 103%、 30分で 98 %であった。注射用水ではグルコース投与直前 (0分)で 106%、投与後 5分で 110%、 10分では 100%、 15分では 97%、 30分では 97%であった(図 4を参照)。これらの結果 から、 HACP-01摂取群では注射用水摂取群に対してグルコース投与後 10分、 15分、 30分で GLP-1濃度が有意に高くなつていることが確認できた。すなわち、コラーゲン ペプチド HACP-01を摂取することによって、血中の GLP-1濃度が上昇することが明ら 力、となった。 [0104] The effect of collagen peptide on the increase in blood GLP-1 concentration was determined by setting the plasma GLP-1 concentration before administration of the test substance (-30 minutes) to the normal concentration (100%) for each individual. The ratio of the concentration in each plasma relative to was calculated. As a result, the rate of increase in GLP-1 concentration in each experimental group was 120% immediately before glucose administration (0 minutes), 122% 5 minutes after administration, 117% at 10 minutes, and 112% at 15 minutes in HACP-01. It was 116% in 30 minutes. In SCP-5000, 111% immediately before glucose administration (0 minutes), 115% at 5 minutes after administration, 104% at 10 minutes, 103% at 15 minutes, and 98% at 30 minutes. Water for injection was 106% immediately before glucose administration (0 minutes), 110% 5 minutes after administration, 100% at 10 minutes, 97% at 15 minutes, and 97% at 30 minutes (see Figure 4). From these results, it was confirmed that the GLP-1 concentration was significantly higher in the HACP-01 intake group than in the injection water intake group at 10, 15, and 30 minutes after glucose administration. In other words, it became clear that ingesting the collagen peptide HACP-01 increased the concentration of GLP-1 in the blood.
[0105] インスリン測定キット (森永生科学研究所社製)を用いて、血漿中のインスリン濃度 の測定を行った。 96ゥヱルプレートに固定化されたインスリン抗体に検体希釈液 2で 調整したモルモット抗インスリン血清 50 1、検体希釈液 2を 45 1、各血漿 5 μ 1を添カロ して、 4°Cで 16時間から 20時間静置した。固定化されたインスリン以外のものを洗浄す ることによって除去し、固定化されたインスリンに結合する、酵素標識抗モルモット IgG 抗体を添加し、室温 3時間静置した。洗浄によって非特異的に結合した抗体を除去 後、酵素基質溶液 100 ^ 1を添加して酵素反応を開始させた。遮光下室温で 30分間 静置後、反応停止液 50 1添加して反応を停止させ、マイクロプレートリーダー(バイ ォラッド社製)にて 490nm (副波長 630nm)で吸光度を測定した。濃度既知のインスリン を測定することによって得た検量線から、各血漿中のインスリン濃度を決定した。 [0105] The insulin concentration in plasma was measured using an insulin measurement kit (manufactured by Morinaga Bioscience Institute). Guinea pig anti-insulin serum 50 1 prepared with sample diluent 2 and insulin diluted on 96-well plate, 45 1 sample diluent 2 and 5 μl of each plasma are added, starting at 4 ° C for 16 hours. Let stand for 20 hours. The non-immobilized insulin was removed by washing, and enzyme-labeled anti-guinea pig IgG antibody that binds to the immobilized insulin was added and allowed to stand at room temperature for 3 hours. After removing non-specifically bound antibody by washing, an enzyme substrate solution 100 ^ 1 was added to start the enzyme reaction. Allow to stand at room temperature for 30 minutes in the dark, then stop the reaction by adding Reaction Stop Solution 50 1 and microplate reader Absorbance was measured at 490 nm (sub-wavelength: 630 nm) by Olad. The insulin concentration in each plasma was determined from a calibration curve obtained by measuring insulin with known concentrations.
[0106] コラーゲンペプチドによる血中インスリン濃度上昇効果の評価は、各個体の被験物 質投与前 (-30分)の血漿中インスリン濃度を通常時の濃度 (100%)とし、この濃度に対 する各血漿での濃度の割合を算出した。その結果、それぞれの実験区におけるイン スリン濃度上昇率は、 HACP-01ではグルコース投与直前(0分)で 222%、投与後 5分 で 189%、 10分で 210%、 15分で 230%、 30分で 233%であった。 SCP-5000ではダルコ ース投与直前(0分)で 128%、投与後 5分で 131 %、 10分で 155%、 15分で 203%、 30 分で 198%であった。注射用水ではグルコース投与直前 (0分)で 96%、投与後 5分で 114%、 10分では 110%、 15分では 94%、 30分では 81 %であった(図 5を参照)。これら の結果から、 HACP-01摂取群では注射用水摂取群に対してグルコース投与直前 (0 分)、グルコース投与後 15分、 30分でインスリン濃度が有意に高くなつていることが確 認できた。すなわち、コラーゲンペプチド HACP-01を摂取することによって、血中のィ ンスリン濃度が上昇することが明らかとなった。 [0106] The effect of increasing the insulin concentration in blood by collagen peptides was evaluated by setting the plasma insulin concentration before each test substance administration (-30 minutes) to the normal concentration (100%) for each individual. The concentration ratio in each plasma was calculated. As a result, the rate of increase in insulin concentration in each experimental group was 222% immediately before glucose administration (0 minutes), 189% 5 minutes after administration, 210% 10 minutes, 230% 15 minutes in HACP-01, It was 233% in 30 minutes. In SCP-5000, it was 128% immediately before administration of dalcose (0 minutes), 131% 5 minutes after administration, 155% at 10 minutes, 203% at 15 minutes, and 198% at 30 minutes. Water for injection was 96% immediately before glucose administration (0 minutes), 114% 5 minutes after administration, 110% at 10 minutes, 94% at 15 minutes, and 81% at 30 minutes (see Figure 5). From these results, it was confirmed that the insulin concentration was significantly higher in the HACP-01 intake group immediately before glucose administration (0 min), 15 min after glucose administration, and 30 min than in the water intake group for injection. . In other words, it was clarified that ingesting the collagen peptide HACP-01 increases the blood insulin concentration.
[0107] また、上記の実験期間中、被検体であるラットには、機能障害等の副作用はみられ なかった。  [0107] Further, during the experimental period described above, no adverse effects such as dysfunction were observed in the test rat.
[0108] (実施例 15)  [Example 15]
糖尿病モデルラットを用いた長期投与試験を行った。実験動物は Zucker Fattyラッ トを用いた。飼育環境は恒温恒湿、 12時間明 /12時間暗のサイクルの実験動物飼育 室で、飼料は群分け前の 1週間 (馴化期間)は、糖尿病 ·肥満研究用高脂肪飼料(「Q uick Fat」、 日本クレア社製)を用い、群分け後 6週間は試験物質(コラーゲンペプチド : HACP-01)あるいは対照物質(アミノ酸混合物)を 10%添加した Quick Fatを用い、 自由に水を摂取させた。なお、対照物質のアミノ酸混合物は試験物質のコラーゲン ペプチドのアミノ酸組成と同じになるように混合した。一般状態を 5日以上毎日観察し 、健康状態が良好であることを確認した。群分けは試験物質投与前に体重層別化無 作為抽出法により行なった。  A long-term administration test was conducted using diabetic model rats. The experimental animals used were Zucker Fatty rats. The breeding environment is a laboratory animal breeding room with a constant temperature and humidity, 12 hours light / 12 hours dark cycle, and the diet is a high fat diet for diabetes and obesity research (“Quick Fat”) for one week before the grouping (acclimation period). (Manufactured by Clea Japan Co., Ltd.), and 6 weeks after grouping, water was freely ingested using Quick Fat supplemented with 10% test substance (collagen peptide: HACP-01) or control substance (amino acid mixture). The amino acid mixture of the control substance was mixed so as to have the same amino acid composition as the test substance collagen peptide. The general condition was observed every day for 5 days or more to confirm that the health condition was good. Grouping was performed by randomized stratification by weight stratification prior to test substance administration.
[0109] 体重は入荷時、群分け時、試験物質投与期間中は 1週間に 1度、測定を行った (表 [0109] Body weight was measured at the time of arrival, at the time of grouping, and once a week during the test substance administration period (Table 1).
2を参照)。摂餌量 '摂水量は 1週間に 2度測定を行なった。採血は群分け時、試験物 質投与 3週間後および 6週間後は空腹時(18時間以上の絶食)、その他は通常時に 週 1度、尾動静脈より行った。採血後、血漿を分離し、以下の実験に用いた。 2). Food intake 'Water intake was measured twice a week. Collect blood at the time of grouping. After 3 weeks and 6 weeks, the animals were fasted (fasted for 18 hours or more), and the others were performed once a week at normal times via the caudal vein. After blood collection, plasma was separated and used for the following experiments.
[表 2]  [Table 2]
【表 2】 [Table 2]
体重 (g)  Weight (g)
試験物質投与期間 試験物質摂取群 対照物質摂取群  Test substance administration period Test substance intake group Control substance intake group
0週間 290.9±9.1 288.8±8.0  0 weeks 290.9 ± 9.1 288.8 ± 8.0
3週間 449.8± 20.3 453.9± 19.7  3 weeks 449.8 ± 20.3 453.9 ± 19.7
6週間 562.0±41.0 564.4±39.3  6 weeks 562.0 ± 41.0 564.4 ± 39.3
※測定は空腹時  * Measured on an empty stomach
[0111] グルコース CII テストヮコー(和光純薬工業社製)を用いて、血漿中のグルコース 濃度を求めた。その結果、試験物質摂取群では対照物質摂取群に対して試験物質 投与 1週間後および 3週間後でグルコース濃度が有意に低くなつていることが確認で きた (図 6を参照)。すなわち、コラーゲンペプチド HACP-01を摂取することによって、 血中のグルコース濃度が低下することが明ら力、となった。 [0111] The glucose concentration in plasma was determined using a glucose CII test KOKO (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, it was confirmed that the glucose concentration in the test substance intake group was significantly lower at 1 and 3 weeks after the test substance administration compared to the control substance intake group (see Fig. 6). That is, it became clear that ingesting the collagen peptide HACP-01 lowered the blood glucose concentration.
[0112] トリグリセライド E テストヮコー(和光純薬工業社製)を用いて、血漿中のトリダリセラ イド濃度を求めた。その結果、試験物質摂取群では対照物質摂取群に対して試験物 質投与 2週間後、 3週間後、 4週間後および 5週間後でトリダリセライド濃度が有意に低 くなつていることが確認できた (図 7を参照)。すなわち、コラーゲンペプチド HACP-01 を摂取することによって、血中のトリダリセライド濃度が低下することが明らかとなった。  [0112] The triglyceride E test concentration was obtained using Wako Pure Chemical Industries, Ltd., and the tridallyceride concentration in plasma was determined. As a result, it was confirmed that in the test substance intake group, the tridalylide concentration was significantly lower after 2, 3, 4 and 5 weeks after the test substance administration compared to the control substance intake group. (See Figure 7.) That is, it has been clarified that the intake of collagen peptide HACP-01 decreases the blood tridalylide concentration.
[0113] 尿酸 C テストヮコー(和光純薬工業社製)を用いて、血漿中の尿酸濃度を求めた 。その結果、試験物質摂取群では対照物質摂取群に対して試験物質投与 4週間後 および 6週間後で尿酸濃度が有意に低くなつていることが確認できた (図 8を参照)。す なわち、コラーゲンペプチド HACP-01を摂取することによって、血中の尿酸濃度が低 下することが明らかとなった。  [0113] The uric acid concentration in plasma was determined using a uric acid C test kit (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, it was confirmed that the uric acid concentration was significantly lower in the test substance intake group 4 weeks and 6 weeks after the test substance administration than in the control substance intake group (see FIG. 8). In other words, it was clarified that the intake of collagen peptide HACP-01 reduces the uric acid concentration in the blood.
[0114] 試験物質投与 3週間後および 6週間後に採尿を行った。 24時間蓄尿を採取し、尿 酸 C テストヮコー(和光純薬工業社製)を用いて尿中の尿酸濃度を測定し、一日当 たりの尿酸排泄量を求めた。その結果、試験物質摂取群では対照物質摂取群に対 して、試験物質投与 3週間後では 15.2%、試験物質投与 6週間後では 22.7%、尿酸 排泄量が減少していることが確認できた (図 9を参照)。すなわち、コラーゲンペプチド HACP-01を摂取することによって、尿酸排泄量が低下することが明ら力、となった。ま た、コラーゲンペプチド HACP-01を摂取することによって、血中の尿酸濃度も低下 することから、尿酸合成が抑制されて!/、ると考えられる。 [0114] Urine was collected 3 weeks and 6 weeks after administration of the test substance. A 24-hour urine collection was collected, and the uric acid concentration in the urine was measured using a uric acid C test kit (manufactured by Wako Pure Chemical Industries, Ltd.) to determine the amount of uric acid excretion per day. As a result, the test substance intake group compared to the control substance intake group. Thus, it was confirmed that uric acid excretion decreased by 15.2% after 3 weeks of test substance administration and by 22.7% after 6 weeks of test substance administration (see Fig. 9). That is, it became clear that the amount of uric acid excretion decreased by ingesting the collagen peptide HACP-01. In addition, the intake of the collagen peptide HACP-01 also reduces the uric acid concentration in the blood, which is thought to suppress uric acid synthesis! /.
[0115] 最終採血終了後の剖検時に、腎臓周囲、精巣周囲および腸間膜の腹腔内脂肪と 、背部皮下の褐色脂肪を摘出し、重量を測定した。その結果、試験物質摂取群では 対照物質摂取群に対して、体重当たりの腎臓周囲、精巣周囲、腹腔内脂肪の合計、 背部皮下および腹腔内脂肪と褐色脂肪の合計の脂肪重量の割合が有意に低くなつ ていることが確認できた (表 3を参照)。すなわち、コラーゲンペプチド HACP-01を摂取 することによって、内臓脂肪量が低下することが明らかとなった。  [0115] At the time of necropsy after the end of the final blood collection, peritoneal, testicular, and mesenteric abdominal fat and brown fat in the back were removed and weighed. As a result, in the test substance intake group, the ratio of the peri-kidney, peri-testis, and intra-abdominal fat per body weight, and the total fat weight ratio of the back subcutaneous and intra-abdominal fat and brown fat per body weight was significantly higher than the control substance intake group. It was confirmed that it was lower (see Table 3). That is, it was clarified that the visceral fat amount decreased by ingesting the collagen peptide HACP-01.
[0116] [表 3]  [0116] [Table 3]
【表 3】 [Table 3]
体重当たりの脂肪重量の割合(%)  Percentage of fat weight per body weight (%)
測定部位 試験物質摂取群 対照物質摂取群  Measurement site Test substance intake group Control substance intake group
腎臓周囲 4.36±0.38 4.77 ±0.39  Around the kidney 4.36 ± 0.38 4.77 ± 0.39
精巣周囲 3.41 ±0.19 3.70±0.23  Testicular circumference 3.41 ± 0.19 3.70 ± 0.23
腸間膜 2.65±0.33 2.78±0.35  Mesenterium 2.65 ± 0.33 2.78 ± 0.35
腹腔内脂肪の合計 10.42 ±0.46 1 1.25±0.61  Total intra-abdominal fat 10.42 ± 0.46 1 1.25 ± 0.61
背部皮下 (褐色脂肪) 0.25 ±0.06 0.33±0.07  Subcutaneous (brown fat) 0.25 ± 0.06 0.33 ± 0.07
全合計 10.67 ±0.47 1 1.59±0.63  Total 10.67 ± 0.47 1 1.59 ± 0.63
[0117] なお、上記の実験期間中、被検体であるラットには、機能障害等の副作用はみられ なかった。 [0117] It should be noted that during the experimental period described above, there were no side effects such as dysfunction in the subject rat.
[0118] (実施例 16) [Example 16]
10名の社内ボランティアによる血糖値測定試験をクロスオーバーにて行った。ヘル シンキ宣言の精神に従い、被験者に対しては本試験の主旨を十分説明したうえで文 書による同意を得て試験を実施した。被験者は、前日から 9時間以上絶食した状態で 、血糖自己測定器 (ワンタッチウルトラ:ジョンソン 'エンド ' ·ジョンソン社製)により血糖 値を測定後、コラーゲンペプチド(HACP-Ol) lOgと水、卵白ペプチド 10gと水、あるい は水のみのいずれかを摂取した。その 30分後に再び血糖値を測定した後、経口糖 忍容カ試験用糖溶液(トレーラン G75 :味の素社製)を摂取した。その後、被験者は糖 溶液摂取後 10, 20, 30, 45, 60, 90, 120分に各自で血糖値を測定した。 8名の被験者 力 Sコラーゲンペプチド '·卵白ペプチド '·水のみの 3日間、 2名の被験者がコラーゲンぺ プチド '水のみの 2日間の試験に参加した。なお、被験者にはゥォッシュアゥト期間と して最低 2日間を設けた。 A blood glucose measurement test with 10 in-house volunteers was conducted at the crossover. In accordance with the spirit of the Declaration of Helsinki, the subjects were fully explained to the subject of the study, and the study was conducted with written consent. The subject was fasted for more than 9 hours from the previous day, and after measuring the blood glucose level with a blood glucose self-monitoring device (One Touch Ultra: Johnson 'End' Johnson), collagen peptide (HACP-Ol) lOg and water, egg white peptide 10g and water Took either water only. Thirty minutes later, the blood glucose level was measured again, and an oral glucose tolerance test sugar solution (Traillan G75: Ajinomoto Co., Inc.) was ingested. Thereafter, each subject measured blood glucose level at 10, 20, 30, 45, 60, 90, and 120 minutes after ingesting the sugar solution. Eight subjects Force S collagen peptide '· egg white peptide' · water only for 3 days, 2 subjects participated in the collagen peptide 'water only 2 day study. The subjects were given a minimum of 2 days for the washout period.
[0119] その結果、それぞれの摂取群における血糖 は、コラーゲンペプチド摂取群ではコ ラーゲンペプチド摂取前 (-30分)で 87.5mg/dl、糖溶液摂取直前 (0分)で 92. lmg/dl、 摂取後 10分で 115.6mg/dl、 20分で 130.1mg/dl、 30分で 130.0mg/dl、 45分で 113.6mg/ dl、 60分で 101.6mg/dl、 90分で 117.4mg/dl、 120分で 107. lmg/dlであった。卵白ぺプ チド摂取群では卵白ペプチド摂取前 (-30分)で 90.4mg/dl、糖溶液摂取直前 (0分) で 89.4mg/dl、摂取後 10分で 116.6mg/dl、 20分で 140.3mg/dl、 30分で 140. lmg/dl、 45 分で 135.4mg/dl、 60分で 122.0mg/dl、 90分で 106.9mg/dl、 120分で 100.9mg/dlであつ た。コントロール群では水摂取前(-30分)で 89.7mg/dl、糖溶液摂取直前(0分)で 87. 4mg/dl、摂取後 10分で 115.4mg/dl、 20分で 137.7mg/dl、 30分で 145.1mg/dl、 45分で 140.0mg/dl、 60分で 129.5mg/dl、 90分で 111.8mg/dl、 120分で 106.0mg/dlであった。 ( 図 10を参照)。これらの結果から、コラーゲンペプチド摂取群ではコントロール群に対 して糖溶液摂取後 45分、 60分で血糖 が有意に低くなつていることが確認できた。す なわち、コラーゲンペプチド HACP-01を摂取することによって、血糖 の上昇を抑制 することが明らかとなった。なお、 ldlは 100mlである。なお、 90分以降は被験者体内 の生体内恒常性の作用によりコラーゲンペプチド摂取群と他の 2群との差は小さくな つた。 [0119] As a result, blood glucose in each intake group was 87.5 mg / dl before collagen peptide intake (-30 minutes) and 92. lmg / dl immediately before sugar solution intake (0 minutes) in the collagen peptide intake group. 115.6 mg / dl after 10 minutes, 130.1 mg / dl at 20 minutes, 130.0 mg / dl at 30 minutes, 113.6 mg / dl at 45 minutes, 101.6 mg / dl at 60 minutes, 117.4 mg / dl at 90 minutes, It was 107. lmg / dl in 120 minutes. In the egg white peptide intake group, 90.4 mg / dl before ingestion of egg white peptide (-30 minutes), 89.4 mg / dl immediately before ingestion of sugar solution (0 minutes), 116.6 mg / dl at 10 minutes after ingestion, 140.3 at 20 minutes mg / dl, 140.lmg / dl at 30 minutes, 135.4mg / dl at 45 minutes, 122.0mg / dl at 60 minutes, 106.9mg / dl at 90 minutes, 100.9mg / dl at 120 minutes. In the control group, 89.7 mg / dl before water intake (-30 minutes), 87.4 mg / dl immediately before sugar solution intake (0 minutes), 115.4 mg / dl 10 minutes after intake, 137.7 mg / dl after 20 minutes, It was 145.1 mg / dl at 30 minutes, 140.0 mg / dl at 45 minutes, 129.5 mg / dl at 60 minutes, 111.8 mg / dl at 90 minutes, and 106.0 mg / dl at 120 minutes. (See Figure 10). From these results, it was confirmed that the blood glucose level was significantly lower in the collagen peptide ingestion group 45 and 60 minutes after ingesting the sugar solution than in the control group. In other words, it was clarified that the intake of the collagen peptide HACP-01 suppressed the increase in blood glucose. Ldl is 100ml. After 90 minutes, the difference between the collagen peptide intake group and the other two groups became smaller due to the effect of homeostasis in the body of the subject.
[0120] (実施例 17)  [0120] (Example 17)
コラーゲンペプチド HACP-01 (ゼライス社製)を合成吸着剤 DIAION HP20 (三菱化 学社製)を用いて分画した。合成吸着剤 100gとコラーゲンペプチド 10gを水中で混合 し、 1時間撹拌した後、静置して上清をろ過および回収した。その後、 100mlの 10%ェ タノール水溶液を加えて、 10分間撹拌した後、静置して上清をろ過および回収した。 同様の操作を 20%、 30%、 40%、 50%、 75%のエタノール水溶液および 100%のエタ ノール溶液にっレ、ても行い、ろ液を回収した。 Collagen peptide HACP-01 (manufactured by Zelice) was fractionated using a synthetic adsorbent DIAION HP20 (manufactured by Mitsubishi Chemical Corporation). 100 g of the synthetic adsorbent and 10 g of collagen peptide were mixed in water, stirred for 1 hour, allowed to stand, and the supernatant was filtered and collected. Thereafter, 100 ml of a 10% aqueous ethanol solution was added, stirred for 10 minutes, and allowed to stand, and the supernatant was filtered and collected. Repeat the same operation for 20%, 30%, 40%, 50%, 75% aqueous ethanol and 100% ethanol. The filtrate was recovered even after the addition of the sol solution.
[0121] 回収した 8画分のろ液の吸光度を分光光度計(日本分光社製)により測定した。 215 nmの測定はろ液を 1000倍希釈して行った。 280nmの測定はろ液をそのまま用いて行 つた。その結果、 215nmの吸光度はエタノール濃度 0%溶出画分で 0.205、 10%溶出 画分で 0.087、 20%溶出画分で 0.163、 30%溶出画分で 0.243、 40%溶出画分で 0.21 9、 50%溶出画分で 0.179、 75%溶出画分で 0.117、 100%溶出画分で 0.027であった( 図 11を参照)。また、 280nmの吸光度はエタノール濃度 0%溶出画分で 0.288、 10%溶 出画分で 0.187、 20%溶出画分で 0.351、 30%溶出画分で 0.552、 40%溶出画分で 1. 075、 50%溶出画分で 1.573、 75%溶出画分で 1.592、 100%溶出画分で 1.053であつ た。 280nmにおける吸収は、 HACP-01に混入している夾雑物によるものと思われる。 これらの各画分について官能検査を行ったところ、エタノール濃度力 %、 10%、 20 %の溶出画分では、 30%以上の溶出画分に比べて不快臭(コラーゲン臭)が顕著に 低減されていた。 [0121] The absorbance of the collected 8 fraction filtrates was measured with a spectrophotometer (manufactured by JASCO Corporation). The measurement at 215 nm was performed by diluting the filtrate 1000 times. The measurement at 280 nm was performed using the filtrate as it was. As a result, the absorbance at 215 nm was 0.205 for the ethanol concentration fraction at 0%, 0.087 for the 10% elution fraction, 0.163 for the 20% elution fraction, 0.243 for the 30% elution fraction, 0.219 for the 40% elution fraction, It was 0.179 for the 50% elution fraction, 0.117 for the 75% elution fraction, and 0.027 for the 100% elution fraction (see Figure 11). The absorbance at 280 nm is 0.288 for the ethanol concentration 0% elution fraction, 0.187 for the 10% elution fraction, 0.351 for the 20% elution fraction, 0.552 for the 30% elution fraction, and 1.075 for the 40% elution fraction. It was 1.573 for the 50% elution fraction, 1.592 for the 75% elution fraction, and 1.053 for the 100% elution fraction. Absorption at 280nm is probably due to contaminants in HACP-01. When sensory tests were performed on each of these fractions, the unpleasant odor (collagen odor) was significantly reduced in the elution fractions with ethanol concentration of 10%, 20%, and 30% or more. It was.
[0122] 回収した 8画分のろ液を減圧下のロータリーエバポレーターで濃縮し、エタノールを 除去した後、凍結乾燥を行ってペプチド粉末を得た。このようにして得られたぺプチ ドを MHP- 0, 10, 20, 30, 40, 50, 75, 100 (エタノール濃度力 %、 10%、 20%、 30%、 4 0%、 50%、 75%、 100%の画分)と呼ぶこととする。これらのペプチド粉末をそれぞれ 25mMのトリス-塩酸バッファー(pH 8.0)に 10mg/mlとなるように溶解した。これらのサ ンプルを用いて、実施例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1 と同様に DPPIV阻害率を求めたところ、 MHP-0は 36·8%、 MHP-10は 4·7%、 MHP-20は 22.1%, MHP-30は 11·9%、 MHP-40は 3·8%、 MHP-50は 0%、 MHP-75は 10· 1 %、 MHP-1 00は 0 %であった(図 11を参照 )。  [0122] The collected 8 fraction filtrates were concentrated with a rotary evaporator under reduced pressure to remove ethanol, and then lyophilized to obtain peptide powder. The peptides obtained in this way are MHP-0, 10, 20, 30, 40, 50, 75, 100 (ethanol concentration%, 10%, 20%, 30%, 40%, 50%, 75% and 100% fractions). Each of these peptide powders was dissolved in 25 mM Tris-HCl buffer (pH 8.0) so as to be 10 mg / ml. Using these samples, an activity inhibition experiment on DPPIV was conducted in the same manner as in Example 1. When the DPPIV inhibition rate was determined in the same manner as in Example 1, MHP-0 was 36.8%, MHP-10 was 4.7%, MHP-20 was 22.1%, MHP-30 was 11.9%, MHP- 40 was 3 · 8%, MHP-50 was 0%, MHP-75 was 10 · 1%, and MHP-100 was 0% (see Figure 11).
[0123] これらのペプチドのうち、最も DPPIV阻害活性の高かった MHP-0の分子量分布を 調べた。 AKTApurifierおよび Superdex Peptide 10/300 GL (アマシャムバイオサイエ ンス社製)を用いたゲルろ過クロマトグラフィーにより検出を行った。その結果、 MHP- 0には分子量 500以下の低分子のペプチドが多く含まれていることが確認できた(図 1 2を参照)。  [0123] Among these peptides, the molecular weight distribution of MHP-0 having the highest DPPIV inhibitory activity was examined. Detection was performed by gel filtration chromatography using AKTApurifier and Superdex Peptide 10/300 GL (Amersham Bioscience). As a result, it was confirmed that MHP-0 contained many low molecular weight peptides having a molecular weight of 500 or less (see FIG. 12).
[0124] 以上の結果から、水を溶媒として合成吸着剤 DIAION HP20にコラーゲンペプチド H ACP-01を吸着させ、非吸着画分を回収することにより、 DPPIVの阻害活性をさらに 高め、かつ低分子のペプチドを多く含有する画分 MHP-0を得ることができる。すなわ ち、この方法で得られるペプチドは消化管にて吸収されやすいと思われる低分子で あり、 DPPIV阻害活性も高いことから、精製前の HACP-01と比べて摂取量が少なくて 済むと考えられる。また、不快臭(コラーゲン臭)が低減されているため、摂取しやす いと考えられる。 [0124] From the above results, collagen adsorbent H was added to synthetic adsorbent DIAION HP20 using water as a solvent. By adsorbing ACP-01 and collecting the non-adsorbed fraction, it is possible to obtain a fraction MHP-0 that further enhances the DPPIV inhibitory activity and contains a large amount of low-molecular-weight peptides. In other words, the peptide obtained by this method is a small molecule that is likely to be absorbed in the digestive tract and has a high DPPIV inhibitory activity, so it requires less intake compared to HACP-01 before purification. Conceivable. In addition, since the unpleasant odor (collagen odor) is reduced, it is considered easy to take.
[0125] (実施例 18) [0125] (Example 18)
コラーゲンペプチド HACP-01 (ゼライス社製)をエタノールを用いて沈殿法により分 画した。コラーゲンペプチド 1.5gと 70%、 75%、 80%、 85%、 90%および 95%のェタノ ール水溶液 15mlをそれぞれよく混合し、 -20°Cでー晚静置した。上清画分を回収し、 減圧下のロータリーエバポレーターで濃縮し、エタノールを除去した後、凍結乾燥を 行ってペプチド粉末を得た。沈殿画分は水に溶解した後、凍結乾燥を行ってぺプチ ド粉末を得た。  Collagen peptide HACP-01 (manufactured by Zerais) was fractionated by precipitation using ethanol. 1.5 g of collagen peptide and 15 ml of 70%, 75%, 80%, 85%, 90% and 95% ethanol aqueous solution were mixed well and left at -20 ° C. The supernatant fraction was collected and concentrated with a rotary evaporator under reduced pressure to remove ethanol, and then freeze-dried to obtain peptide powder. The precipitate fraction was dissolved in water and then freeze-dried to obtain peptide powder.
[0126] これらのペプチド粉末をそれぞれ 25mMのトリス-塩酸バッファー(pH 8.0)に 20mg/ml となるように溶解した。これらのサンプルを用いて、実施例 1と同様に DPPIVに対する 活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めたところ、サンプル終 濃度が 2.0mg/mlのとき、エタノール濃度 70%の上清画分は 20.4%、沈殿画分は 5.0 %、エタノール濃度 75%の上清画分は 27.0%、沈殿画分は 4.4%、エタノール濃度 80 %の上清画分は 36.5%、沈殿画分は 5.6%、エタノール濃度 85%の上清画分は 41.7 %、沈殿画分は 13.5%、 90%の上清画分は 38.0%、沈殿画分は 15.8%、 95%の上清 画分は 42.0%、沈殿画分は 17.5%であった。なお、分画前の HACP-01の DPPIV阻 害率は 18.9%であった(図 13を参照)。  [0126] These peptide powders were each dissolved in 25 mM Tris-HCl buffer (pH 8.0) to a concentration of 20 mg / ml. Using these samples, an activity inhibition experiment against DPPIV was conducted in the same manner as in Example 1. When the DPPIV inhibition rate was determined in the same manner as in Example 1, when the final sample concentration was 2.0 mg / ml, the supernatant fraction with an ethanol concentration of 70% was 20.4%, the precipitate fraction was 5.0%, and the ethanol concentration was 75%. The supernatant fraction is 27.0%, the precipitation fraction is 4.4%, the supernatant fraction with ethanol concentration of 80% is 36.5%, the precipitation fraction is 5.6%, the supernatant fraction with ethanol concentration of 85% is 41.7%, the precipitation fraction The fraction was 13.5%, the 90% supernatant fraction was 38.0%, the precipitate fraction was 15.8%, the 95% supernatant fraction was 42.0%, and the precipitate fraction was 17.5%. In addition, the DPPIV inhibition rate of HACP-01 before fractionation was 18.9% (see Figure 13).
[0127] (実施例 19)  [Example 19]
実施例 18にて得られたエタノール濃度 85%の上清画分をさらに合成吸着剤 DIAIO N HP20 (三菱化学社製)を用いて分画した。合成吸着剤とエタノール濃度 85%の上 清画分を水中で混合し、 1時間撹拌した後、静置して上清をろ過および回収した。そ の後、 100%のエタノール溶液を加えて 10分間撹拌した後、静置して上清をろ過およ び回収した。それぞれの画分を減圧下のロータリーエバポレーターで濃縮し、凍結 乾燥を行ってペプチド粉末を得た。 The supernatant fraction having an ethanol concentration of 85% obtained in Example 18 was further fractionated using a synthetic adsorbent DIAIO N HP20 (manufactured by Mitsubishi Chemical Corporation). The synthetic adsorbent and the supernatant fraction with an ethanol concentration of 85% were mixed in water, stirred for 1 hour, allowed to stand, and the supernatant was filtered and collected. Thereafter, a 100% ethanol solution was added and stirred for 10 minutes, and then allowed to stand, and the supernatant was filtered and collected. Concentrate each fraction on a rotary evaporator under reduced pressure and freeze. The peptide powder was obtained by drying.
[0128] このようにして得られたペプチドをそれぞれ E85S_0(HP20)および E85S_100(HP20) ( エタノール濃度 85%上清画分を HP20を用いて分画したエタノール濃度 0%、および 10 0%の画分)と呼ぶこととする。これらのペプチド粉末をそれぞれ 25mMのトリス-塩酸 バッファー(pH 8.0)に 20mg/mlとなるように溶解した。これらのサンプルを用いて、実 施例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV阻 害率を求めたところ、 E85S_0(HP20)は 58.1%、 E85S_100(HP20)は 41.2%であった(図 1 4を参照)。  [0128] The peptides thus obtained were E85S_0 (HP20) and E85S_100 (HP20) (the ethanol fraction 85% fraction was fractionated using HP20 and the ethanol concentrations were 0% and 100%, respectively. Minutes). Each of these peptide powders was dissolved in 25 mM Tris-HCl buffer (pH 8.0) so as to be 20 mg / ml. Using these samples, an activity inhibition experiment against DPPIV was conducted in the same manner as in Example 1. When the DPPIV inhibition rate was determined in the same manner as in Example 1, it was 58.1% for E85S_0 (HP20) and 41.2% for E85S_100 (HP20) (see FIG. 14).
[0129] (実施例 20)  [0129] (Example 20)
コラーゲンペプチド HACP-01および E85S_0(HP20)をそれぞれ 25mMのトリス-塩酸 バッファー(pH 8.0)に 20, 50, 100mg/mlおよび 10, 20, 50mg/mlとなるように溶解した Collagen peptides HACP-01 and E85S_0 (HP20) were dissolved in 25 mM Tris-HCl buffer (pH 8.0) to a concentration of 20, 50, 100 mg / ml and 10, 20, 50 mg / ml, respectively.
Yes
[0130] 実施例 1と同様に DPPIVに対する活性阻害実験を行った。実施例 1と同様に DPPIV 阻害率を求めたところ、 HACP-01の DPPIV阻害率はサンプル終濃度が 2.0mg/mlの とき 20.6%、 5.0mg/mlのとき 40.9%、 10mg/mlのとき 52.8%であった。 E85S_0(HP20)の DP PIV阻害率はサンプル終濃度力 Sl.Omg/mlのとき 41·8%、 2.0mg/mlのとき 59.9%、 5.0mg /mlのとき 77.0%であった。 (図 15を参照)。また、近似曲線を引いて DPPIVの活性を 50 %阻害するときの濃度を求めたところ、 HACP-01は 8.4mg/ml、 E85S_0(HP20)は 1.4m g/mlであった。すなわち、エタノール沈殿法および合成吸着剤を用いた精製法を組 み合わせてコラーゲンペプチドを精製することによって、 DPPIV阻害活性を 6倍高め ること力 Sでさた。  [0130] Activity inhibition experiments on DPPIV were conducted in the same manner as in Example 1. When the DPPIV inhibition rate was determined in the same manner as in Example 1, the DPPIV inhibition rate of HACP-01 was 20.6% when the final sample concentration was 2.0 mg / ml, 40.9% when 5.0 mg / ml, and 52.8 when 10 mg / ml. %Met. The DP PIV inhibition rate of E85S_0 (HP20) was 41.8% at the final sample concentration Sl.Omg / ml, 59.9% at 2.0 mg / ml, and 77.0% at 5.0 mg / ml. (See Figure 15). The concentration at which DPPIV activity was inhibited by 50% by drawing an approximated curve was determined to be 8.4 mg / ml for HACP-01 and 1.4 mg / ml for E85S_0 (HP20). In other words, the ability to increase the DPPIV inhibitory activity 6-fold by purifying the collagen peptide by combining the ethanol precipitation method and the purification method using a synthetic adsorbent was achieved.
[0131] (実施例 21)  [0131] (Example 21)
実施例 18と同様にして得られたエタノール濃度 85%の上清画分をさらに合成吸着 剤 SEPABEADS SP850 (三菱化学社製)を用いて実施例 16と同様に分画した。回収し た各画分のろ液を減圧下のロータリーエバポレーターで濃縮し、エタノールを除去し た後、凍結乾燥を行ってペプチド粉末を得た。このようにして得られたペプチドを E85 S- 0, 10, 20, 30, 40, 50, 100(SP850) (エタノール濃度 85%上清画分を SP850を用いて 分画したエタノール濃度 0%、 10%、 20%、 30%、 40%、 50%、 100%の画分)と呼ぶ こととする。 The supernatant fraction having an ethanol concentration of 85% obtained in the same manner as in Example 18 was further fractionated in the same manner as in Example 16 using a synthetic adsorbent SEPABEADS SP850 (manufactured by Mitsubishi Chemical Corporation). The collected filtrate of each fraction was concentrated with a rotary evaporator under reduced pressure to remove ethanol, and then freeze-dried to obtain peptide powder. The peptide thus obtained was E85 S-0, 10, 20, 30, 40, 50, 100 (SP850) (ethanol concentration 85% supernatant fraction fractionated using SP850, ethanol concentration 0%, 10%, 20%, 30%, 40%, 50%, 100% fraction) I will do it.
[0132] これらのペプチド粉末をそれぞれ 25mMのトリス-塩酸バッファー(pH 8.0)に 20mg/m 1となるように溶解した。これらのサンプルを用いて、実施例 1と同様に DPPIVに対する 活性阻害実験を行った。実施例 1と同様に DPPIV阻害率を求めたところ、 E85S-0は 5 0.6%, E85S-10は 60.8%、 E85S-20は 56.1%、 E85S-30は 53.6%、 E85S-40は 46.2%、 E85S -50は 38.7%、 E85S-100は 26.0%であった(図 16を参照)。  [0132] Each of these peptide powders was dissolved in 25 mM Tris-HCl buffer (pH 8.0) to a concentration of 20 mg / ml. Using these samples, an activity inhibition experiment against DPPIV was conducted in the same manner as in Example 1. The DPPIV inhibition rate was determined in the same manner as in Example 1. E85S-50 was 38.7% and E85S-100 was 26.0% (see Figure 16).
[0133] (実施例 22)  [Example 22]
実施例 18、 19および 21にて得られたペプチドの分子量分布を調べた。 AKTApurifie rおよび Superdex Peptide 10/300 GL (アマシャムバイオサイエンス社製)を用いたゲ ルろ過クロマトグラフィーにより検出を行った。その結果、分子量 1500以下のペプチド 力 ¾85S (エタノール濃度 85%における上清画分)には 72·2%、 E85P (エタノール濃度 85 %における沈殿画分)には 30.9%、 E85S_0(HP20)には 90.7%、 E85S_100(HP20)には 46. 1%、 E85S_0(SP850)には 99.9%、 E85S_10(SP850)には 92.3%、 E85S_20(SP850)には 72. 8%、 E85S_30(SP850)には 55.2%、 E85S_40(SP850)には 41.1%、 E85S_50(SP850)には 32 • 7%、 E85S_100(SP850)には 26.5%含まれていることが確認できた(表 4を参照)。  The molecular weight distribution of the peptides obtained in Examples 18, 19 and 21 was examined. Detection was performed by gel filtration chromatography using AKTApurifier and Superdex Peptide 10/300 GL (Amersham Biosciences). As a result, peptide strength ¾85S (supernatant fraction at an ethanol concentration of 85%) was 72 · 2%, E85P (precipitate fraction at an ethanol concentration of 85%) was 30.9%, and E85S_0 (HP20) was 90.7%, E85S_100 (HP20) 46.1%, E85S_0 (SP850) 99.9%, E85S_10 (SP850) 92.3%, E85S_20 (SP850) 72.8%, E85S_30 (SP850) 55.2 %, E85S_40 (SP850) contained 41.1%, E85S_50 (SP850) contained 32 • 7%, and E85S_100 (SP850) contained 26.5% (see Table 4).
[0134] [表 4] [0134] [Table 4]
【表 4】 [Table 4]
分子量 1500以下の  Molecular weight less than 1500
ペプチドの割合(%)  Peptide ratio (%)
E85S 72.2  E85S 72.2
E85P 30.9  E85P 30.9
E85S-10 (HP20) 90.7  E85S-10 (HP20) 90.7
E85S-100 (HP20) 46.1  E85S-100 (HP20) 46.1
E85S-0(SP850) 99.9  E85S-0 (SP850) 99.9
E85S-10(SP850) 92.3  E85S-10 (SP850) 92.3
E85S-20(SP850) 72.8  E85S-20 (SP850) 72.8
E85S-30(SP850) 55.2  E85S-30 (SP850) 55.2
E85S-40(SP850) 41.1  E85S-40 (SP850) 41.1
E85S-50(SP850) 32.7  E85S-50 (SP850) 32.7
E85S-100(SP850) 26.5 [0135] 以上の結果から、エタノールを用いた沈殿法あるいは合成吸着剤を用いた精製法 によりコラーゲンペプチド HACP-01の DPPIVの阻害活性をさらに高め、かつ低分子 のペプチドを多く含有する画分を得ることができる。すなわち、これらの方法で得られ るペプチドは消化管にて吸収されやすレ、と思われる低分子であり、 DPPIV阻害活性 も高いことから、精製前の HACP-01と比べて摂取量が少なくて済むと考えられる。 産業上の利用可能性 E85S-100 (SP850) 26.5 [0135] From the above results, a fraction containing a large amount of low-molecular-weight peptides and collagen peptide HACP-01 was further enhanced by the precipitation method using ethanol or the purification method using a synthetic adsorbent. Obtainable. In other words, the peptides obtained by these methods are small molecules that are likely to be absorbed in the digestive tract and have high DPPIV inhibitory activity, so the amount of intake is low compared to HACP-01 before purification. It is thought that it will end. Industrial applicability
[0136] 本発明の DPPIV阻害剤は、哺乳動物の糖尿病予防'治療剤として使用できる。また 、本発明の DPPIV阻害剤は、糖尿病に随伴する各種の疾患(例えば、高脂血、症痛 風あるいは高尿酸血症)の改善剤としても使用すること力 Sできる。  [0136] The DPPIV inhibitor of the present invention can be used as an agent for preventing or treating diabetes in mammals. The DPPIV inhibitor of the present invention can also be used as an ameliorating agent for various diseases associated with diabetes (for example, hyperlipidemia, gout or hyperuricemia).
また、本発明のペプチド等は、安全性に優れるため、食品、菓子等に有効量を配 合すること力 Sできる。これらの食品、菓子等を継続的に摂取することで糖尿病の発症 を予防し得る食品、菓子等の開発も可能である。さらには、糖尿病に随伴する各種の 疾患(例えば、高脂血、症痛風あるいは高尿酸血症)の予防用あるいは、これらの疾 患に罹患した患者用の食品、菓子の開発も可能である。  In addition, since the peptides and the like of the present invention are excellent in safety, it is possible to combine an effective amount with food, confectionery and the like. It is also possible to develop foods and confectionery that can prevent the onset of diabetes by continuously ingesting these foods and confectionery. Furthermore, various diseases associated with diabetes (for example, hyperlipidemia, gout or hyperuricemia) can be prevented, or foods and confectionery for patients suffering from these diseases can be developed.
[0137] 本明細書に包含される本発明の多くの利点を上記に述べた力 この開示は、多くの 点で例示に過ぎないことが理解されよう。本発明の範囲を逸脱しなければ、細部にわ たり、特に、部品の形状、大きさ及び配置等の事項について、様々な変更を行うこと が可能である。  [0137] The above mentioned power of the many advantages of the invention encompassed herein will be understood that this disclosure is merely exemplary in many respects. Without departing from the scope of the present invention, it is possible to make various changes in details, particularly in matters such as the shape, size and arrangement of parts.
本発明の範囲が添付の請求の範囲に述べられている文言により限定されることは 勿論である。  Of course, the scope of the invention is limited by the language set forth in the appended claims.

Claims

請求の範囲 The scope of the claims
[1] コラーゲンまたはゼラチン由来のペプチドであって、式(1):  [1] A peptide derived from collagen or gelatin having the formula (1):
Gly-X-Y- (Gly-Z-W) n (1)  Gly-X-Y- (Gly-Z-W) n (1)
(式中、 nは 0〜4の整数、 Xは Proまたは Leu、 Y、 Ζおよび Wはそれぞれ独立して同 一または異なる任意のアミノ酸残基 (ただし、 Glyを除く)を示す。 )  (In the formula, n is an integer of 0 to 4, X is Pro or Leu, Y, Ζ and W are each independently the same or different arbitrary amino acid residues (excluding Gly).)
で表されるアミノ酸配列からなるペプチド、前記のアミノ酸配列の末端のアミノ酸残基 w力 個欠失されたアミノ酸配列からなるペプチドあるいはその塩を含有するジぺプ チジルぺプチダーゼ IV阻害剤。  A dipeptidyl peptidase IV inhibitor comprising a peptide comprising the amino acid sequence represented by the above, a peptide comprising the amino acid sequence deleted at the end of the amino acid sequence, or a salt thereof, or a salt thereof.
[2] 前記ペプチドが [2] The peptide is
Giy_Pro_Argゝ  Giy_Pro_Arg ゝ
Giy-Pro-Ser-Giy-Asn-Aia  Giy-Pro-Ser-Giy-Asn-Aia
Gly-Pro-Ala-Gly-Pro-Ala,  Gly-Pro-Ala-Gly-Pro-Ala,
Gly-Pro-Val-Gly-Ala-Arg,  Gly-Pro-Val-Gly-Ala-Arg,
Gly-Pro-Val-Gly-Pro-Ala,  Gly-Pro-Val-Gly-Pro-Ala,
Giy-Pro-Ile-Gly-¾er-Ala  Giy-Pro-Ile-Gly-¾er-Ala
Giy_Pro_Ser_Giy_Lrlu_Arg_Lrly_Pro_Hypゝ  Giy_Pro_Ser_Giy_Lrlu_Arg_Lrly_Pro_Hyp ゝ
Giy-Pro-Arg- ly-Arg- hr_Gly_Asp_Ala_Giy_Pro_Val、  Giy-Pro-Arg- ly-Arg- hr_Gly_Asp_Ala_Giy_Pro_Val,
Gly-Pro-Vaト Gly-Proおよび  Gly-Pro-Va Gly-Pro and
Gly_Leu_Ala_Gly_Pro_Hyp、  Gly_Leu_Ala_Gly_Pro_Hyp,
力、らなる群より選ばれる 1種類以上のアミノ酸配列からなるペプチドである請求項 1記 載のジぺプチジルぺプチダーゼ IV阻害剤。  The dipeptidyl peptidase IV inhibitor according to claim 1, which is a peptide comprising one or more amino acid sequences selected from the group consisting of
[3] 請求項ほたは 2に記載のジぺプチジルぺプチダーゼ IV阻害剤を有効成分として 含有する糖尿病治療 ·予防剤。 [3] A therapeutic / preventive agent for diabetes comprising the dipeptidyl peptidase IV inhibitor according to claim 2 as an active ingredient.
[4] 哺乳動物に対して、請求項ほたは 2に記載のジぺプチジルぺプチダーゼ IV阻害 剤の有効量を投与することを特徴とする糖尿病の予防'治療法。 [4] A method for the prevention and treatment of diabetes comprising administering an effective amount of the dipeptidyl peptidase IV inhibitor according to claim 2 or 2 to a mammal.
[5] コラーゲンまたはゼラチン由来のペプチドであって、式(1): [5] A peptide derived from collagen or gelatin having the formula (1):
Gly-X-Y- (Gly-Z-W) n (1)  Gly-X-Y- (Gly-Z-W) n (1)
(式中、 nは 0〜4の整数、 Xは Proまたは Leu、 Y、 Ζおよび Wはそれぞれ独立して同 一または異なる任意のアミノ酸残基 (ただし、 Glyを除く)を示す。) (Where n is an integer from 0 to 4, X is Pro or Leu, Y, Ζ and W are each independently the same. Any one or different amino acid residues (except Gly) are indicated. )
で表されるアミノ酸配列からなるペプチド、前記のアミノ酸配列の末端のアミノ酸残基 w力 個欠失されたアミノ酸配列からなるペプチドあるいはその塩を含有し、糖尿病 に随伴する高脂血症の改善作用を有するジぺプチジルぺプチダーゼ IV阻害剤。  A peptide comprising an amino acid sequence represented by the above, a peptide comprising an amino acid sequence deleted at the end of the amino acid sequence, or a salt thereof, or a salt thereof, for improving hyperlipidemia associated with diabetes A dipeptidyl peptidase IV inhibitor having the formula:
[6] コラーゲンまたはゼラチン由来のペプチドであって、式(1): [6] A peptide derived from collagen or gelatin having the formula (1):
Gly-X-Y- (Gly-Z-W) n (1)  Gly-X-Y- (Gly-Z-W) n (1)
(式中、 nは 0〜4の整数、 Xは Proまたは Leu、 Y、 Ζおよび Wはそれぞれ独立して同 一または異なる任意のアミノ酸残基 (ただし、 Glyを除く)を示す。 )  (In the formula, n is an integer of 0 to 4, X is Pro or Leu, Y, Ζ and W are each independently the same or different arbitrary amino acid residues (excluding Gly).)
で表されるアミノ酸配列からなるペプチド、前記のアミノ酸配列の末端のアミノ酸残基 w力 個欠失されたアミノ酸配列からなるペプチドあるいはその塩を含有し、糖尿病 に随伴する痛風あるいは高尿酸血症の改善作用を有するジぺプチジルぺプチダー ゼ IV阻害剤。  A peptide comprising the amino acid sequence represented by the above, a peptide comprising the amino acid sequence deleted at the end of the amino acid sequence, or a salt thereof, or gout associated with diabetes or hyperuricemia A dipeptidyl peptidase IV inhibitor having an improving action.
[7] コラーゲンまたはゼラチン由来のペプチドであって、式(1):  [7] A peptide derived from collagen or gelatin having the formula (1):
Gly-X-Y- (Gly-Z-W) n (1)  Gly-X-Y- (Gly-Z-W) n (1)
(式中、 nは 0〜4の整数、 Xは Proまたは Leu、 Y、 Ζおよび Wはそれぞれ独立して同 一または異なる任意のアミノ酸残基 (ただし、 Glyを除く)を示す。 )  (In the formula, n is an integer of 0 to 4, X is Pro or Leu, Y, Ζ and W are each independently the same or different arbitrary amino acid residues (excluding Gly).)
で表されるアミノ酸配列からなるペプチド、前記のアミノ酸配列の末端のアミノ酸残基 w力 個欠失されたアミノ酸配列からなるペプチドあるいはその塩を含有し、分子量 が 1 , 500以下のペプチドを 50%以上含有することを特徴とするジぺプチジルぺプチ ダーゼ IV阻害剤。  50% of peptides having a molecular weight of 1,500 or less, comprising a peptide consisting of the amino acid sequence represented by A dipeptidyl peptidase IV inhibitor characterized by comprising the above.
[8] コラーゲンまたはゼラチン由来のペプチドであって、式(1): [8] A peptide derived from collagen or gelatin having the formula (1):
Gly-X-Y- (Gly-Z-W) n (1)  Gly-X-Y- (Gly-Z-W) n (1)
(式中、 nは 0〜4の整数、 Xは Proまたは Leu、 Y、 Ζおよび Wはそれぞれ独立して同 一または異なる任意のアミノ酸残基 (ただし、 Glyを除く)を示す。 )  (In the formula, n is an integer of 0 to 4, X is Pro or Leu, Y, Ζ and W are each independently the same or different arbitrary amino acid residues (excluding Gly).)
で表されるアミノ酸配列からなるペプチド、前記のアミノ酸配列の末端のアミノ酸残基 w力 個欠失されたアミノ酸配列からなるペプチドあるいはその塩を含有し、分子量 が 1 , 500以下のペプチドを 70%以上含有することを特徴とするジぺプチジルぺプチ ダーゼ IV阻害剤。 70% of peptides having a molecular weight of 1,500 or less, comprising a peptide consisting of the amino acid sequence represented by A dipeptidyl peptidase IV inhibitor characterized by comprising the above.
[9] コラーゲンまたはゼラチン由来のペプチドであって、式(1): [9] A peptide derived from collagen or gelatin having the formula (1):
Gly-X-Y- (Gly-Z-W) n (1)  Gly-X-Y- (Gly-Z-W) n (1)
(式中、 nは 0〜4の整数、 Xは Proまたは Leu、 Y、 Ζおよび Wはそれぞれ独立して同 一または異なる任意のアミノ酸残基 (ただし、 Glyを除く)を示す。 )  (In the formula, n is an integer of 0 to 4, X is Pro or Leu, Y, Ζ and W are each independently the same or different arbitrary amino acid residues (excluding Gly).)
で表されるアミノ酸配列からなるペプチド、前記のアミノ酸配列の末端のアミノ酸残基 w力 個欠失されたアミノ酸配列からなるペプチドあるいはその塩を含有し、分子量 が 1 , 500以下のペプチドを 90%以上含有することを特徴とするジぺプチジルぺプチ ダーゼ IV阻害剤。  90% of peptides having a molecular weight of 1,500 or less, comprising a peptide consisting of the amino acid sequence represented by the following formula: A dipeptidyl peptidase IV inhibitor characterized by comprising the above.
[10] コラゲナーゼ処理されたコラーゲンまたはゼラチンの分解物を、有機溶媒を用いた 沈殿法あるいは樹脂を用いた精製法の!/、ずれ力、もしくは両方を組み合わせた方法に より精製し、式 (1) :  [10] Collagenase-degraded collagen or gelatin degradation products are purified by the precipitation method using an organic solvent or the purification method using a resin! ):
Gly-X-Y- (Gly-Z-W) n (1)  Gly-X-Y- (Gly-Z-W) n (1)
(式中、 nは 0〜4の整数、 Xは Proまたは Leu、 Y、 Ζおよび Wはそれぞれ独立して同 一または異なる任意のアミノ酸残基 (ただし、 Glyを除く)を示す。 )  (In the formula, n is an integer of 0 to 4, X is Pro or Leu, Y, Ζ and W are each independently the same or different arbitrary amino acid residues (excluding Gly).)
で表されるアミノ酸配列からなるペプチド、前記のアミノ酸配列の末端のアミノ酸残基 w力 個欠失されたアミノ酸配列からなるペプチド組成物を得る工程を含む、ぺプチ ド組成物の製造方法。  And a peptide composition comprising a peptide composition comprising the amino acid sequence deleted from the terminal amino acid residue w force of the amino acid sequence.
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