WO2014030977A1 - Pharmaceutical composition comprising, as active ingredients, peptides which exhibit inhibitory activity against angiotensin-i converting enzyme for preventing or treating cardiovascular diseases - Google Patents

Pharmaceutical composition comprising, as active ingredients, peptides which exhibit inhibitory activity against angiotensin-i converting enzyme for preventing or treating cardiovascular diseases Download PDF

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Publication number
WO2014030977A1
WO2014030977A1 PCT/KR2013/007598 KR2013007598W WO2014030977A1 WO 2014030977 A1 WO2014030977 A1 WO 2014030977A1 KR 2013007598 W KR2013007598 W KR 2013007598W WO 2014030977 A1 WO2014030977 A1 WO 2014030977A1
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Prior art keywords
peptide
peptides
amino acid
preventing
seq
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PCT/KR2013/007598
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French (fr)
Korean (ko)
Inventor
정세영
최영준
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경희대학교 산학협력단
경상대학교 산학협력단
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Application filed by 경희대학교 산학협력단, 경상대학교 산학협력단 filed Critical 경희대학교 산학협력단
Priority to CN201380054488.XA priority Critical patent/CN104736552B/en
Priority claimed from KR1020130100232A external-priority patent/KR101533308B1/en
Publication of WO2014030977A1 publication Critical patent/WO2014030977A1/en
Priority to US14/627,642 priority patent/US10323062B2/en
Priority to US15/856,926 priority patent/US10308681B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052—Val-amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
    • C07K5/06069—Ser-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
    • C07K5/0823—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp and Pro-amino acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • composition for the prevention or treatment of cardiovascular diseases comprising a peptide showing angiotensin-I converting enzyme inhibitory activity as an active ingredient
  • the present invention is a pharmaceutical for preventing or treating cardiovascular diseases comprising a peptide showing the inhibitory activity against angiotensin converting enzyme (ACE) isolated from a fraction of oyster enzyme hydrate and the peptide as an active ingredient To a red composition.
  • ACE angiotensin converting enzyme
  • cardiovascular disease including atherosclerosis, hypertension, angina, myocardial infarction, ischemic heart disease, heart failure, complications after angioplasty, cerebral infarction, cerebral hemorrhage, and stroke.
  • cardiovascular disease is the second largest cause of death in Korea, ranking second only to malignant tumors.
  • Mortality rates have been reported to increase significantly.
  • Hypertension a representative cardiovascular disease, is becoming a global problem that accounts for 15 to 15% of the adult disease. Higher blood pressure is a chronic disease that requires a lifetime of treatment, and the social and economic losses are enormous.
  • Renin-Angiotensin System is known to play an important role in essential hypertension, which accounts for 80% of hypertension, and decreases blood flow, sodium, and sympathy in the kidney through general physiological activity.
  • Increasing nervous system activation activates the renin-angiotensin system, and the renin secreted from the juxtaglomerular cells of the renal arteries breaks down angiotensin into angiotensin I, which is then vascularized by angiotensin converting enzyme (ACE). It is converted to angiotensin ⁇ which contracts, and consequently, angiotensin ⁇ regulates blood pressure through neuroregulation and increased aldosterone synthesis.
  • ACE also breaks down and inactivates bradykinin, which exhibits vasorelaxation.
  • ACE inhibitors or angiotensin converting enzyme inhibitors which have a significant effect on blood pressure rise, are expected to treat or prevent cardiovascular diseases such as hypertension, heart disease, arteriosclerosis, or cerebral hemorrhage.
  • cardiovascular diseases such as hypertension, heart disease, arteriosclerosis, or cerebral hemorrhage.
  • Many studies have been conducted, and the results of various clinical studies and experiments have been reported to confirm that they can effectively reduce chronic kidney disease, arteriosclerosis, heart attack and death.
  • angiotensin converting enzyme inhibitors such as these have been used for the treatment of commercial hypertension.
  • these compounds are easily decomposed in pharmaceutical dosage forms, which not only reduces their stability, but also act on other cells, causing problems with side effects such as weakness in the whole body, vomiting, coughing, headache, anorexia and taste disorders. Reported to be (Lim SD. Et. Al (2008) Korean J. Food Sci. Ani. Resour, 28 (5): 587-595).
  • Oysters are known as health foods that are as healthy as the milk of the sea.
  • Nutritional ingredients include glycogen, taurine, proteins and vitamins, as well as various minerals and are widely known as ingredients for dietary supplements.
  • it is effective in strengthening the function of the heart and liver, and preventing hypertension, arteriosclerosis and heart disease by reducing cholesterol, and because it contains a large amount of selenium, it not only shows heavy metal detoxification function but also functions of organs such as heart, liver and pancreas. Height is known as tonic and stamina food.
  • Korean Unexamined Patent Publication No. 10-2012-0092735 discloses an ACE inhibitor or antihypertensive composition containing Mesaeng as an active ingredient as an active ingredient.
  • 1275766 discloses ACE inhibitory or antihypertensive compositions comprising abalone viscera protein extracts or fractions thereof.
  • peptides showing ACE inhibitory activity have been reported from S-hydrolysates of natural substances.
  • valine-tyrosine a peptide derived from sardine protein
  • Korean Patent No. 10-1106303 discloses the ability to inhibit the ACE activity of the peptide prepared from oyster through the enzyme treatment.
  • ACE from enzymatic hydrolysates of natural substances can Efforts to develop peptides showing activity inhibitory activity resulted in the extraction, isolation and purification of oyster enzyme hydrates to obtain novel peptides showing the ability to inhibit ACE activity. And since the anti-hypertensive effect, the peptide isolated from the oyster enzyme hydrolyzate of the present invention has been completed by confirming that it can be usefully used as an active ingredient in the pharmaceutical composition for the prevention or treatment of cardiovascular diseases.
  • An object of the present invention is to provide a step tide having an inhibitory ability against angiotensin converting enzyme (ACE) consisting of amino acid sequences described in SEQ ID NO: 1 to 12.
  • ACE angiotensin converting enzyme
  • Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating cardiovascular disease, comprising a fraction of oyster enzyme hydrate containing the peptide as an active ingredient.
  • Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating cardiovascular diseases containing any one or more of the peptides as an active ingredient.
  • Still another object of the present invention is to provide a health food for preventing or improving cardiovascular disease, which contains a fraction of the oyster enzyme hydrolyzate including the peptide as an active ingredient.
  • Another object of the present invention to provide a health food for preventing or improving cardiovascular disease containing any one or more of the peptides as an active ingredient.
  • Still another object of the present invention is to provide a method for preventing cardiovascular disease, comprising administering to a subject a complex of the oyster enzyme hydrolyzate including the tempide.
  • Still another object of the present invention is to provide a method for preventing cardiovascular disease, including the step of administering any one or more of the peptides.
  • Another object of the present invention is to provide a use of a fraction of the oyster enzyme hydrolyzate comprising the peptide for use as a pharmaceutical composition for the prevention or treatment of cardiovascular diseases.
  • Another object of the present invention to provide a use of any one or more of the above peptides for use as a pharmaceutical composition for the prevention or treatment of cardiovascular diseases.
  • Still another object of the present invention is to provide a use of a fraction of an oyster enzyme hydrolyzate comprising the peptide for use as a health food for preventing or improving cardiovascular disease.
  • Still another object of the present invention is to provide a use of any one or more of the above peptides for use as a health food for preventing or improving cardiovascular disease.
  • the present invention provides a peptide having an inhibitory ability against angiotensin converting enzyme (ACE) consisting of the amino acid sequence described in SEQ ID NO: 1 to 12.
  • ACE angiotensin converting enzyme
  • the present invention also provides a pharmaceutical composition for the prevention or treatment of cardiovascular diseases containing a fraction of oyster enzyme hydrate containing the peptide as an active ingredient.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of cardiovascular diseases containing any one or more of the 3 ⁇ 4 Tide as an active ingredient.
  • the present invention provides a health food for preventing or improving cardiovascular disease, which contains a fraction of the oyster enzyme hydrolyzate including the peptide as an active ingredient.
  • the present invention provides a health food for preventing or improving cardiovascular disease containing any one or more of the peptide symptoms as an active ingredient.
  • the present invention also provides a method of treating cardiovascular diseases comprising administering a fraction of the oyster enzyme hydrolyzate comprising the peptide to a subject having a cardiovascular disease.
  • the present invention provides a method for treating cardiovascular diseases comprising the step of administering any one or more of the 3 ⁇ 4 tide to a subject having a cardiovascular disease.
  • the present invention also provides a method for preventing cardiovascular disease comprising administering to a subject a fraction of an oyster enzyme hydrolyzate comprising the peptide.
  • the present invention provides a method of preventing cardiovascular diseases comprising the step of administering any one or more of the 3 ⁇ 4 Tide.
  • the present invention provides the use of a fraction of the oyster enzyme hydrolyzate comprising the 3 ⁇ 4 tide for use as a pharmaceutical composition for the prevention or treatment of cardiovascular diseases.
  • the present invention is to provide a use of any one or more of the above peptides for use as a pharmaceutical composition for the prevention or treatment of cardiovascular diseases.
  • the present invention also provides the use of a fraction of oyster enzyme hydrolysate comprising the peptide for use as a health food for preventing or improving cardiovascular disease.
  • the present invention also provides the use of any one or more of the above peptide for use as a health food for preventing or improving cardiovascular disease.
  • Peptides exhibiting inhibitory activity against angiotensin converting enzyme (ACE) isolated from a fraction of the oyster enzyme hydrate of the present invention exhibits an effective blood pressure control effect and a high blood pressure preventing effect, Fractions of the degradation products or peptides isolated therefrom can be usefully used as active ingredients in pharmaceutical compositions for the prevention or treatment of cardiovascular diseases.
  • ACE angiotensin converting enzyme
  • FIG. 1 is a view showing a process for purifying oyster enzyme hydrolyzate.
  • A represents anion exchange chromatography
  • B represents purification of size exclusion chromatography of fraction 4 of the anion exchange chromatography
  • C represents reverse-phase chromatography. It is a figure which shows a tablet.
  • Fig. 2 shows the analysis of the mass and amino acid sequence of peptides isolated from oyster enzyme hydrolysates.
  • FIG. 3 is a diagram showing the effect of blood pressure regulation in vivo by a single administration of oyster enzyme hydrolyzate.
  • Figure 4 is a diagram showing the effect of blood pressure regulation in vivo by a single administration of the functional peptide.
  • FIG. 5 is a diagram showing the effect of blood pressure regulation in vivo by repeated administration of oyster enzyme hydrolyzate and functional peptide.
  • FIG. 6 is a diagram showing the effect of inhibiting the concentration of angiotensin converting enzyme (ACE) in the oyster enzyme hydrolyzate and functional peptide.
  • 7 is a diagram showing the effect of inhibiting the concentration of angiotensin -I (angiotensin-n) in the oyster enzyme hydrolyzate and functional peptide.
  • ACE angiotensin converting enzyme
  • the present invention provides a peptide having an inhibitory activity against angiotensin converting enzyme (ACE) consisting of the amino acid sequence set forth in SEQ ID NOs: 1-12.
  • ACE angiotensin converting enzyme
  • the peptide is preferably separated from the oyster enzyme hydrolyzate, but is not limited thereto. Any peptide derived from another substance or synthesized may be used.
  • a conventional separation method such as ultrafiltration, chromatography, and more preferably, a chromatographic method, more specifically, It is recommended to use anion exchange chromatography, size exclusion chromatography, reverse se- phase chromatography, or high performance liquid chromatography (HPLC). Most preferably, but not limited to.
  • the method for the synthesis it is preferable to synthesize by conventional chemical synthesis method of the peptide (WH Freeman and Co., Proteins; structures and molecular principles, 1983), specifically, liquid phase peptide synthesis method (Solution Phase Peptide synthesis) ), Solid-phase peptide syntheses, fragment condensation and F-moc or T-B0C chemistry are more preferable, and more specifically, it is most preferably synthesized by solid-phase peptide synthesis. It is not limited.
  • the enzyme is a conventional protease of the protein art, and specifically, it is protarax or neutrase. More preferred but not limited thereto.
  • the addition of the enzyme is preferably a sequential addition of inactivation after the reaction of protamex, followed by the addition of neutrase, but is not limited thereto. Protamex and neutrase may be added at the same time.
  • the protein concentration of the enzyme hydrolyzate is 0.1 to 10%, and the inactivation of the enzyme is preferably inactivated by reacting for 10 to 120 minutes at 20 to 100 ° C, but is not limited thereto. Can be adjusted appropriately.
  • the present inventors hydrolyzed oyster proteins using proteolytic enzymes to prepare oyster enzyme hydrolysates, and purified functional peptides having ACE activity inhibitory activity from the oyster enzyme hydrolysates.
  • seven samples showing ACE activity inhibitory activity were selected (see FIG. 1 and Tables 1 and 2).
  • the present inventors selected the peptide consisting of the amino acid sequence of SEQ ID NOS: 1 to 12 as a result of confirming the mass and amino acid sequence of the peptide in order to screen the functional peptide showing the ACE activity inhibitory activity in the sample (Fig.
  • the peptide of the present invention does not exhibit cytotoxicity against ACE, and thus shows a significant activity inhibitory ability, and thus may be usefully used as an ACE activity inhibitor.
  • the peptide of the present invention can be prepared by the following genetic engineering method. First, a DNA sequence encoding the peptide is constructed according to a conventional method. DNA sequences can be prepared by PCR amplification using appropriate primers. Alternatively by standard methods known in the art, for example, DNA sequences may also be synthesized using an automated DNA synthesizer (eg, from Biosearch or Applied iosystems).
  • the DNA sequence is inserted into a vector comprising one or more expression control sequences (eg, promoters, enhancers, etc.) that are operably linked to regulate expression of the DNA sequence, and the host cell is formed with a recombinant expression vector formed therefrom.
  • expression control sequences eg, promoters, enhancers, etc.
  • the resulting transformants are cultivated under medium and conditions appropriate for the DNA sequence to be expressed, thereby allowing the substantially pure peptides encoded by the DNA sequences from the culture to be known in the art. For example, by chromatography).
  • substantially pure peptide it is meant that the peptide according to the invention is substantially free of any other protein derived from the host.
  • the peptides of the present invention can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical formulations.
  • Parenteral administration may refer to administration via a route other than oral such as rectal, intravenous, peritoneal, intramuscular, arterial, transdermal, nasal, inhalation, ocular and subcutaneous.
  • the peptide of the present invention can be administered in various parenteral formulations, and when formulated, it is prepared by using the usual fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. .
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories.
  • non-aqueous solvents and suspensions propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.
  • As a base of suppositories witepsol, macrogol, tween 61, cacao butter, uririnji, glycerogelatin and the like can be used.
  • the depth of the present invention can be used in combination with a number of carriers (pharmaceutically acceptable carrier) such as physiological saline or organic solvents, carbohydrates such as glucose, sucrose or dextran, to increase stability or absorption
  • carriers such as physiological saline or organic solvents, carbohydrates such as glucose, sucrose or dextran, to increase stability or absorption
  • Antioxidants such as ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers may be used as medicaments.
  • the effective dose of the peptide of the present invention is 0.01 to 100 mg / kg, preferably 0.1 to 10 mg / kg, and may be administered once to three times a day.
  • the total effective amount of the peptide of the present invention can be administered to the patient in bolus form or in a single dose by infusion for a relatively short period of time, and multiple doses can be It may be administered by a fractionated treatment protocol to be administered. Since the concentration is determined in consideration of various factors such as the age and health status of the patient as well as the route and frequency of treatment of the drug, in view of this point, the present invention can be used by those skilled in the art. Appropriate effective dosages for the particular use of the novel peptides as pharmaceutical compositions may be determined.
  • the present invention also provides a pharmaceutical composition for preventing or treating cardiovascular disease, comprising a fraction of the oyster enzyme hydrolyzate comprising the peptide consisting of the amino acid sequence of SEQ ID NO: 1 to 12 as an active ingredient.
  • the fraction is preferably 10 kD or less, but is not limited thereto.
  • the cardiovascular disease is preferably one or more selected from the group consisting of hypertension, heart disease, stroke, thrombosis, angina pectoris, heart failure, myocardial infarction, atherosclerosis and arteriosclerosis, but is not limited thereto.
  • the present inventors confirmed the blood pressure control effect by administering the peptide to the hypertension rats once or several times to confirm the blood pressure control effect of the peptide of the present invention in vivo, oyster enzyme singer It was confirmed that the degradation product and the YA peptide showed a significant blood pressure lowering effect (see FIGS. 4 and 5), and the concentrations of blood ACE and angiotensin-iKangiotensin-II were significantly reduced (see FIGS. 6 and 7). ).
  • the fraction of the oyster enzyme hydrolyzate comprising the peptide consisting of the amino acid sequence described in SEQ ID NOS: 1 to 12 of the present invention exhibits an effective blood pressure control effect and a high blood pressure prevention effect, the fraction is to prevent cardiovascular disease or It can be usefully used as an active ingredient in therapeutic pharmaceutical compositions.
  • compositions of the present invention may be in various parenteral formulations.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used.
  • the pharmaceutical composition may further include a nutrient, vitamin, electrolyte, flavoring agent, coloring agent, neutralizing agent, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, and protective colloid thickeners. pH regulators, stabilizers, preservatives.
  • Glycerin, alcohol, carbonation agent used in carbonated beverages, and the like may further contain the carrier, excipient or diluent, lactose, dextrose, sucrose, sorbbi, mannitol, gyrite, erythritol, Malty, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline salulose, polyvinyl pyridone, water, methyl hydroxybenzoate, Propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, physiological saline, but may be one or more selected from the group consisting of, but is not limited to conventional carriers Either excipients or diluents may be used.
  • the components may be added independently or in combination with the peptides of the present invention.
  • compositions of the present invention are preferably formulated using suitable methods known in the art or using methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, EastonPA. Can be.
  • composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, the effective dose level is the type of disease, severity, drug Activity, sensitivity to drug, time of administration, route of administration and rate of administration, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts. It may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutics, and may be administered in a single or multiple doses. It is important to administer the amount, which can be readily determined by one skilled in the art.
  • the dosage of the composition of the present invention varies depending on the weight of the patient, age, sex, health status, diet, time of administration, administration method, excretion rate and the severity of the disease, the daily dosage is based on the amount of extract 300 seconds 0.01 to 1000 mg / kg, preferably 30 to 500 mg / kg, more preferably 50 to 300 mg / kg, and may be administered 1 to 6 times a day.
  • the dosage may be increased or decreased depending on the route of administration, the increase in obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention by any method.
  • composition of the present invention alone or surgery, radiation therapy, hormone therapy, It can be used in combination with methods using chemotherapeutic and biological response modifiers.
  • present invention provides a pharmaceutical composition for preventing or treating cardiovascular diseases containing any one or more of the peptides consisting of the amino acid sequence of SEQ ID NO: 1 to 12 as an active ingredient.
  • any one or more of the peptides may be usefully used as an active ingredient of a pharmaceutical composition for preventing or treating cardiovascular diseases.
  • the present invention provides a health food for preventing or improving cardiovascular diseases, comprising as an active ingredient a fraction of an oyster enzyme hydrolyzate comprising a tempide composed of the amino acid sequence of SEQ ID NO: 1 to 12.
  • the present invention provides a health food for preventing or improving cardiovascular disease containing any one or more of the peptides as an active ingredient.
  • Fractions of the oyster enzyme hydrolyzate of the present invention or peptides isolated therefrom exhibits significant activity inhibitory activity against ACE, and exhibits effective blood pressure control and hypertension prevention effects in the body.
  • the isolated peptide may be usefully used as an active ingredient of a health food for preventing or improving cardiovascular disease. There is no particular limitation on the kind of food.
  • Examples of foods to which the substance may be added include dairy products, formulated milk products, including drinks, meat, sausages, bread, biscuits, rice cakes, chocolate, candy, snacks, sweets, pizza, ramen, other noodles, gums, ice cream, Special dietary foods such as infant foods, processed meats, fish products, tofu, jelly, health supplements, soy sauce, miso, seasoned foods such as red pepper paste or mixed soy sauce, sauces, other processed foods, pickles such as kimchi or pickles, various soups , Beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, etc. It includes all dietary supplements in the sense.
  • the food, drink or food additives may be prepared by a conventional manufacturing method.
  • the term "health food” refers to physiological defense rhythm control or disease prevention of a food group or a food composition which has added value to the food by using physical, biochemical or biotechnological techniques to act and express the function of the food for a specific purpose. And it means a food processed and designed to express the gynecological function in relation to the living body, and preferably, the health food of the present invention is sufficient for the bodily ganglia function for the prevention or improvement of cardiovascular diseases It means the food which can express.
  • the health food may include a food supplement acceptable food supplement additive, and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of health foods.
  • Fractions of the oyster enzyme hydrolyzate of the present invention or peptides separated therefrom can be added to food as is or used with other foods or food ingredients, and can be suitably used according to conventional methods. It may be appropriately determined depending on the purpose (prevention or improvement).
  • the amount of the fraction or peptide in the health food can be added in an amount of 0.1 to 90 parts by weight of the total food weight.
  • the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.
  • the functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the fractions or peptides as essential ingredients in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks.
  • natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose dung; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xyl, sorbitol and erythritol.
  • Natural flavors can be advantageously used.
  • the proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 compositions of the present invention.
  • fractions of the oyster enzyme hydrolyzate of the present invention or the peptide isolated therefrom are various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors such as flavoring agents, colorants and neutralizing agents (cheese, chocolate, etc.) ), Pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonated drinks used in carbonated beverages.
  • the fraction of the oyster enzyme hydrolyzate of the present invention or peptide isolated therefrom may contain pulp for the production of natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination.
  • the proportion of such additives is not so critical but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the fraction of the oyster enzyme hydrolyzate of the present invention or peptide isolated therefrom.
  • the present invention also provides a method for treating cardiovascular disease, comprising administering to a subject with a cardiovascular disease a fraction of an oyster enzyme hydrolyzate comprising a peptide consisting of the amino acid sequences set forth in SEQ ID NOs: 1-12. do.
  • the present invention also provides a method of treating cardiovascular diseases comprising administering any one or more of the peptides to a subject having a cardiovascular disease.
  • Fractions of the oyster enzyme hydrolyzate of the present invention or peptides isolated therefrom exhibits significant activity inhibitory activity against ACE, and exhibits effective blood pressure control and hypertension prevention effects in the body.
  • the isolated peptides can be usefully used in the treatment of cardiovascular diseases, including administering to a subject having a cardiovascular disease.
  • the present invention also provides a method for preventing cardiovascular disease, comprising administering to a subject an aliquot of the oyster enzyme hydrolyzate comprising a peptide consisting of the amino acid sequences set forth in SEQ ID NOs: 1-12.
  • the present invention provides a method for preventing cardiovascular disease, including the step of administering any one or more of the peptides.
  • Fractions of the oyster enzyme hydrolyzate of the present invention or peptides isolated therefrom exhibits significant activity inhibitory activity against ACE, and exhibits effective blood pressure control and hypertension prevention effects in the body.
  • the isolated peptide may be usefully used for a method for preventing cardiovascular disease, comprising administering to a subject.
  • the present invention also provides the use of a fraction of an oyster enzyme hydrolyzate comprising a peptide consisting of the amino acid sequence set forth in SEQ ID NOs: 1 to 12 for use as a pharmaceutical composition for the prevention or treatment of cardiovascular diseases.
  • the present invention also provides the use of any one or more of the above peptides for use as a pharmaceutical composition for the prevention or treatment of cardiovascular diseases.
  • the present invention also provides the use of a fraction of oyster enzyme hydrolysate comprising the peptide for use as a health food for preventing or improving cardiovascular disease.
  • the present invention is for preventing or improving cardiovascular disease. It provides the use of any one or more of the peptides for use as a health food.
  • Fractions of the oyster enzyme hydrolyzate of the present invention or peptides isolated therefrom exhibits significant activity inhibitory activity against ACE, and shows effective blood pressure control and hypertension prevention effect in the body
  • the isolated peptide may be usefully used as a pharmaceutical composition for preventing or treating cardiovascular diseases, or as an active ingredient of a health food for preventing or improving cardiovascular diseases.
  • the present invention will be described in detail by examples and production examples.
  • the oyster protein was hydrolyzed using the enzyme.
  • Protamex a protase derived from Bacillus, was further added. Reaction was carried out for 1 hour at 40 ° C. to prepare a primary oyster enzyme hydrolyzate. Then, the primary oyster enzyme hydrolyzate was treated for 1 hour at 100 ° C to inactivate protamex, and then added Neutrase (Neutrase; Novozyme-Korea, Korea) for 1 hour at 50 ° C. The reaction was performed to prepare a secondary oyster enzyme hydrolyzate, and again treated at 100 ° C for 1 hour to inactivate neutrase.
  • Neutrase Neutrase
  • Anion exchange chromatography was performed to purify a functional peptide having an ACE activity inhibitor from an oyster enzyme hydrolyzate.
  • FIG. 1 a fraction including peptide * isolated from the oyster enzyme hydrolyzate was obtained (FIG. 1).
  • HAo HA concentration of negative control
  • HA HA concentration of the sample
  • Example ⁇ 2-2> the samples of fractions 2, 3 and 4 selected in Example ⁇ 2-2> were concentrated in a speed vacuum concentrator (Scan Vacuum 40, Rapjin Aps, Denmark), respectively, and then Superdex. After injection of the concentrated sample 200 ⁇ into a peptide column (superdex peptide column; 10X300 mm; GE Healthcare Co., Korea) and performing size exclusion chromatography according to the molecular weight under the conditions of [Table 1], each sample Fractions were obtained to perform the same method as in Example ⁇ 2-2> to confirm the ACE activity inhibitory ability.
  • a speed vacuum concentrator Scan Vacuum 40, Rapjin Aps, Denmark
  • Example ⁇ 2-3> fraction samples selected in Example ⁇ 2-3>, respectively, source 513 ⁇ 4 ⁇ column ( 30111 6513 ⁇ 4 51 " (; Lee 1 " 1; 4.6> ⁇ 150 ⁇ ; GE Scare Inc., Korea) was injected to the sample and subjected to reverse phase chromatography according to the molecular weight under the conditions of [Table 1], and then Fractions were obtained for the samples, and the same method as in Example ⁇ 2-2> was performed to confirm ACE activity inhibitory ability.
  • source 513 ⁇ 4 ⁇ column 30111 6513 ⁇ 4 51 " (; Lee 1 " 1; 4.6> ⁇ 150 ⁇ ; GE Scare Inc., Korea) was injected to the sample and subjected to reverse phase chromatography according to the molecular weight under the conditions of [Table 1], and then Fractions were obtained for the samples, and the same method as in Example ⁇ 2-2> was performed to confirm ACE activity inhibitory ability.
  • Example 3 Identification of Functional Peptide Mass and Sequence Having ACE Activity Inhibition
  • the mass and amino acid sequences of the peptides were edman digested.
  • Maldi / Tope Maldi / Tope (Matrix-Assisted Laser Desorption Ionization / Time-0f-Fight Mass Spectroscopy; MALDI / TOF).
  • Example ⁇ 2-4> seven samples having the ACE inhibitory activity selected in Example ⁇ 2-4> were completely dried in a vacuum concentrated centrifuge, respectively, and dissolved completely by adding 0. TFA aqueous solution 20 /. Then, activating with 50% acetonitrile and loading the dissolved sample in a ZipTip C18 column (Pierce, No. 87782) parallel with 0.13 ⁇ 4> TFA solution, 0.13 ⁇ 4> TFA After washing 2-3 times with an aqueous solution, the peptide was eluted using 70% acetonitrile containing 0.1% TFA to remove other salts.
  • the eluted peptide solution 10 ⁇ was loaded into a micro-filter pretreated with biobrene (AB system, USA), and then the filter was dried using argon gas.
  • the dried filter is mounted on a cartridge and functional peptide using a pulsed-liquid method using an ABI482 automated protein sequencer (ABI482 automated protein sequencer; Applied Biosystems, USA). The sequence of amino acids constituting the was confirmed.
  • Example ⁇ 2-4> the seven samples having the ACE inhibitory activity selected in Example ⁇ 2-4> by the nano- Q-TOF2 (Micromass, UK) which is a mass spectrometer using the electrospray ionization (ESI) method ESI
  • ESI electrospray ionization
  • the C-terminus is bound to the resin, the N-terminus is protected by Fmoc, and the residues are trityl (Trt), t-butyloxycarbonyl (t— which can be removed by TFA). Butyloxycarbonyl; Boc) or t-butyl (t-butyl; t-Bu)
  • Trt trityl
  • Boc t-butyloxycarbonyl
  • t-butyl t-butyl
  • t-Bu The amino acid protected by the protecting group was prepared as a material for amino acid synthesis constituting the sequence of the peptide identified in ⁇ Example 3>.
  • the prepared amino acid was 2- (1H-benzotriazol-1-yl) -1, 1,3,3-tetramethylruronium-nuclear flurophosphate) ((2- (lH-Benzotirazloe-l-yl)- l, l, 3,3-t etramethyluroniura hexaf luorophate; HBTU), hydroxybenzotriazole (HOBt) and
  • %, 2.5%, 2.5% (v / v) were added to the peptides synthesized and mixed to remove the protecting groups of the C-terminal resin and residues.
  • reversed phase HPLC using Vydac Everest C18 column (22 250 mm, 10 / ⁇ ; Grace, USA) was performed under conditions of linear gradient with 40% acetonitrile solution containing 0.1% TFA.
  • the peptide was purified and isolated. Purified peptides were identified by LC / MS using Agilent HP1100 series (Agilent, USA).
  • Example ⁇ 4-1> was confirmed by performing the same method as Example ⁇ 2-2> to inhibit ACE activity.
  • HepG2 cells which are liver cancer cells, were inoculated with MEM medium containing antibiotics for 10% fetal bovine serum (FBS) and cultured to maintain 5> C02 at 37 ° C. It was. When the cells cover about 80% of the dish, the cells were washed with phosphate-buffered saline-ethylenediaminetetraacetic acid (PBS-EDTA) and passaged with trypsin, and the medium was changed every 48 hours.
  • PBS-EDTA phosphate-buffered saline-ethylenediaminetetraacetic acid
  • Example ⁇ 4-1> was dissolved in a MEM medium containing 0.2% PBS at a concentration of 10 /, 50 ug / mi, 100 zg / ml or 200 g / m to prepare a sample. After that, the prepared cells were treated and incubated for 24 hours. After incubation, washed twice with PBS solution, treated with ⁇ reagent, and further incubated for 2 hours, the absorbance was measured at 490 nm with an ELISA plate reader.
  • the oyster enzyme hydrolyzate was separated by more than 10 kD and below.
  • the oyster enzyme hydrolyzate prepared in ⁇ Example 1> was used as a BioVax 10 (Biomax 10; Millipore, USA) ultrafiltration membrane and Pelicon XL (pellicon XL; Millipore, USA) Were filtered in an ultrafilter (Labscale TFF system, Millipore, USA).
  • the peptide was administered to the hypertensive rats once and the blood pressure regulation effect was confirmed.
  • Spontaneiusly Hypertensive Rat SHR
  • Wistar Rat Wistar Rat
  • the experimental group and the control group is divided into the conditions of the following [Table 5] oyster enzyme hydrolyzate prepared in ⁇ Example 1>, more than 10 kD and less than the oyster enzyme hydrolyzate in Example ⁇ 5-1>, Peptides or captopril (Example: Boryeong Pharmaceutical Co., Ltd.) prepared in Example ⁇ 4-1> were orally or intraperitoneally administered, and then 42 ° at 0, 3, 6, 9, 12 and 24 hours after the start of administration. After fixing for 20 minutes to a rat temperature control unit (rat temperature control unit) maintained at C, the tail blood vessels were fully expanded, and the systolic blood pressure was measured to confirm antihypertensive activity.
  • rat temperature control unit rat temperature control unit
  • a dose of the sample corresponding to mg / kg was administered to physiological saline 2 as a dose for the positive control group and the experimental group.
  • the positive control group administered captopril which is a commonly used blood pressure lowering agent, exhibited 18 to 10,000 blood pressure strengthening effects as compared to the negative control hypertensive rats.
  • the hydrolyzate When the hydrolyzate was administered, it showed 33 to 38% of blood pressure strengthening effect compared to the negative control group.
  • the oyster enzyme hydrolyzate below 10 kD had a significant effect of reducing the blood pressure of the experimental group to a level similar to that of the normal control group by 12 hours. It confirmed that it represents (FIG. 3).
  • the experimental group administered the functional peptide of the present invention confirmed a significant level of blood pressure strengthening effect compared to the hypertensive rats of the negative control group, in particular, peptide YA showed a 33.9% blood pressure strengthening effect after 6 hours after the most effective It was confirmed (FIG. 4).
  • the peptide was repeatedly administered to hypertensive rats and the blood pressure regulation effect was confirmed.
  • Example ⁇ 5-2> the same rats as in Example ⁇ 5-2> were purchased and bred in the same environment, and the 10 kD prepared in Example ⁇ 5-1> was divided into experimental groups and control groups according to the conditions shown in Table 6 below.
  • Oyster enzyme hydrolyzate was orally administered at 10 o'clock every morning for 4 weeks for valine-tyrosine (Val-Tyr) ol, a blood pressure lowering substance isolated from the YA peptide or sardine hydrolyzate prepared in Example 4-1, Systolic blood pressure was measured in the same manner as in Example ⁇ 5-2> at 0, 1, 2, 3 and 4 weeks.
  • angiotensin-II angiotensin-II concentration of the stored serum were confirmed according to the protocol according to the manufacturer of the commercial ELISA kit (ELISA KIT; USCN Life Sciences, China).
  • the peptide was administered to hypertensive rats and the antihypertensive effect was confirmed.
  • male toaster rats 12 to 16 weeks of age were purchased 22 ° 3 ° C, humidity 50 ° 5%, and 12 hours of light and dark cycle, feed and drinking water is sufficient to freely intake, stabilized for 1 week, then randomly 8 dogs [according to the conditions in the table] It was divided into experimental group and control group. Then, daily intravenous injection of 40 mg of nitro-L-arginine methyl ester L-NAMA per 1 kg of rat weight to induce hypertension in the experimental, negative and positive controls.
  • systolic blood pressure was measured in the same manner as in Example ⁇ 5-2>, and then the control and experimental groups Mean systolic blood pressure was calculated.

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Abstract

The present invention relates to peptides which are separated from the fractions of oyster enzyme hydrolysate and which exhibit inhibitory activity against angiotensin-converting enzyme (ACE), and to a pharmaceutical composition comprising the peptides as active ingredients for preventing or treating cardiovascular diseases. More particularly, the peptides of the present invention separated from the fractions of oyster enzyme hydrolysate may significantly inhibit the activity of ACE, and may exhibit the effects of blood pressure regulation and hypertension prevention. Therefore, the fractions of oyster enzyme hydrolysate or peptides separated therefrom can be effectively used as active ingredients for a pharmaceutical composition for preventing or treating cardiovascular diseases.

Description

[명세서】  [Specification】
[발명의 명칭]  [Name of invention]
안지오텐신 -I 전환 효소 저해능을 나타내는 펩타이드를 유효성분으로 포함하는 심혈관계 질환 예방또는 치료용 약학적 조성물  Pharmaceutical composition for the prevention or treatment of cardiovascular diseases comprising a peptide showing angiotensin-I converting enzyme inhibitory activity as an active ingredient
【기술분야】 Technical Field
본 발명은 굴 효소 가수분해물 (oyster enzyme hydrate)의 분획물로부터 분리된 안지오텐신 전환 효소 (angiotensin converting enzyme; ACE)에 대해 저해능을 나타내는 펩타이드 및 상기 펩타이드를 유효성분으로 포함하는 심혈관계 질환 예방또는 치료용 약학적 조성물에 관한 것이다.  The present invention is a pharmaceutical for preventing or treating cardiovascular diseases comprising a peptide showing the inhibitory activity against angiotensin converting enzyme (ACE) isolated from a fraction of oyster enzyme hydrate and the peptide as an active ingredient To a red composition.
【배경기술】 Background Art
급속한 경제발전과 더불어 질병양상도 크게 변하여 동맥경화증， 고혈압， 협심증, 심근경색， 허혈성 심장질환, 심부전， 경혈관 동맥 성형술 후 발생하는 합병증， 뇌경색， 뇌출혈， 및 뇌졸중올 포함하는 심혈관계 질환이 증가하는 추세에 있다. 2010년 통계청에서 발표한 사망원인 통계에 따르면， 심혈관계 질환은 우리나라 사망원인의 2위로서， 악성 종양 다음으로 높은 순위를 차지하고 있으며， 남성은 55세 이상, 여성은 65세 이상에서 심혈관계 질환의 사망률이 크게 증가하는 것으로 보고되었다. 대표적인 심혈관계 질환인 고혈압은 성인병 비율의 15 내지 를 차지할 만큼 세계적인 문제로 대두되고 있으며， 비교적 증상은 없는 편이지만 고혈압 환자에게서 동맥 경화, 뇌졸중， 심근 경색 및 말기 신장병과 같은 다른 심혈관계 질환의 합병증의 위험도가 증가하는 것으로 알려져 있어 보다 적극적인 관리와 치료가 요구된다ᅳ 그러나， 고혈압은 만성 질환으로 평생 동안 치료가 필요하므로사회적 및 경제적 손실 또한 막대하다.  In addition to rapid economic development, the disease pattern changes significantly, increasing cardiovascular disease including atherosclerosis, hypertension, angina, myocardial infarction, ischemic heart disease, heart failure, complications after angioplasty, cerebral infarction, cerebral hemorrhage, and stroke. There is a trend. According to statistics on the cause of death published by the National Statistical Office in 2010, cardiovascular disease is the second largest cause of death in Korea, ranking second only to malignant tumors. Mortality rates have been reported to increase significantly. Hypertension, a representative cardiovascular disease, is becoming a global problem that accounts for 15 to 15% of the adult disease. Higher blood pressure is a chronic disease that requires a lifetime of treatment, and the social and economic losses are enormous.
고혈압의 원인은 대부분 밝혀진 바 없으나， 가족력， 인종, 염분의 섭취량， 인슐린 저항성 및 비만과 같은 유전적 인자 또는 과도한 음주 및 노화와 같은 환경적 인자의 복합적인 산물로서 발생하는 것으로 알려져 있으며， 혈압 상승의 다양한 요인 가운데 레닌—안지오텐신 -알도스테론 (renin-angiotensin-aldosterone) 고리가 생체 내에서 혈압 및 체액량을 조절하는 데 증요한 역할을 하는 것으로 알려져 있다 (Weiss, D. et . al (2001) Circulation 103： 448-454). The cause of hypertension is largely unknown, but family history, race, salt intake, genetic factors such as insulin resistance and obesity, or excessive drinking and aging It is known to occur as a complex product of environmental factors, and the renin-angiotensin-aldosterone ring plays an important role in regulating blood pressure and fluid volume in vivo. Known (Weiss, D. et. Al (2001) Circulation 103: 448-454).
특히, 고혈압의 80%에 해당하는 본태성 고혈압의 원인으로 레닌-안지오텐신계 (Renin-Angiotensin System, RAS)가 증요한 역할올 담당하는 것으로 알려져 있으며 , 일반적인 생리활성으로 신장에서 혈류량, 나트륨 감소， 교감신경계 활성증가에 의해 레닌-안지오텐신계가 활성화되고， 신장동맥의 방사구체세포 (juxtaglomerular cell)에서 분비된 레닌이 안지오텐신을 안지오텐신 I으로 분해하며 , 이는 다시 안지오텐신 전환효소 (angiotensin converting enzyme, ACE)에 의해 혈관 수축작용을 하는 안지오텐신 Π로 전환되어 결과적으로 안지오텐신 Π가 신경조절 및 알도스테론 합성 증가를 통하여 혈압을 조절한다. ACE는 또한 혈관이완작용을 나타내는 브래디키닌 (bradykinin)을 분해하여 불활성화하는 역할을 한다.  In particular, Renin-Angiotensin System (RAS) is known to play an important role in essential hypertension, which accounts for 80% of hypertension, and decreases blood flow, sodium, and sympathy in the kidney through general physiological activity. Increasing nervous system activation activates the renin-angiotensin system, and the renin secreted from the juxtaglomerular cells of the renal arteries breaks down angiotensin into angiotensin I, which is then vascularized by angiotensin converting enzyme (ACE). It is converted to angiotensin Π which contracts, and consequently, angiotensin Π regulates blood pressure through neuroregulation and increased aldosterone synthesis. ACE also breaks down and inactivates bradykinin, which exhibits vasorelaxation.
따라서， 혈압의 상승에 큰 영향을 미치는 ACE를 저해하는 물질 또는 안지오텐신 전환효소 저해제 (ACE inhibitor)는 고혈압， 심장병 , 동맥경화 또는 뇌출혈 등의 심혈관계 질환 등을 치료 또는 예방할 수 있는 것으로 기대되어， 이에 대한 많은 연구가 진행되고 있어， 구체적으로 만성신장병， 동맥경화， 심장발작 및 이로 인한 사망 등을 효과적으로 감소시킬 수 있음을 확인하기 위한 다양한 임상 연구 및 실험의 결과가보고되고 있다.  Therefore, ACE inhibitors or angiotensin converting enzyme inhibitors (ACE inhibitors), which have a significant effect on blood pressure rise, are expected to treat or prevent cardiovascular diseases such as hypertension, heart disease, arteriosclerosis, or cerebral hemorrhage. Many studies have been conducted, and the results of various clinical studies and experiments have been reported to confirm that they can effectively reduce chronic kidney disease, arteriosclerosis, heart attack and death.
현재 이러한 결과에 기초하여 , 라미프릴 (ramipril), 캐토프릴 (captopr i 1 ) , 에날라프릴 (enarapil), 리시노프릴 (r isinopr i 1 )， 포시노프릴 (fosinori 1)이나 스피라프릴 (spirapril) 등과 같은 화학적으로 합성된 안지오텐신 전환효소 저해제가 상업적인 고혈압 치료제로 사용되고 있다. 그러나 상기 화합물들은 약학적 투여 형태에서 쉽게 분해되므로 안정성이 떨어질 뿐만 아니라， 다른 세포에도 작용하여 전신에 힘이 빠지거나， 구토， 기침， 두통， 식욕부진 및 미각이상을 일으키는 등 부작용과 관련된 문제가 있는 것으로 보고되고 있다 (Lim SD. et. al (2008) Korean J. Food Sci . Ani. Resour , 28(5)： 587-595) . 따라서， 안정성을 가지는 동시에， 인체에 부작용을 나타내지 않는 안전성 또한 가지는 천연 물질 유래의 ACE 저해제의 개발이 요구되고 있다. 굴은 바다의 우유라 할 만큼 몸에 좋은 건강식품으로 알려져 있으며， 날 것을 식용하지 않는 서양에서도 생굴은 널리 식용되고 있다. 영양 성분으로는 글리코겐， 타우린， 단백질 및 비타민은 물론 다양한 미네탈을 함유하고 있어 건강기능 식품의 소재로 널리 알려져 있다. 또한 심장 및 간장의 기능 강화 및， 콜레스테를 감소에 의한 고혈압， 동맥경화 및 심장병 예방 등에 효과가 있으며， 셀레늄을 다량 함유하고 있어 중금속 해독 기능올 나타낼 뿐만 아니라 심장， 간장, 췌장 등 장기의 기능올 높이는 강장 및 스테미너 식품으로 알려져 있다. 현재까지 알려진 천연 물질 유래의 ACE 저해제로서， 대한민국 공개특허 10-2012-0092735호에는 메생이 효소 처리 추출물을 유효성분으로 함유하는 ACE 저해용 또는 항고혈압 조성물에 대하여 개시하고 있으며， 대한민국 등록특허 제 10- 1275766호에는 전복 내장 단백질 추출물 또는 이의 분획물을 포함하는 ACE 저해 또는 항고혈압조성물에 대하고 개시하고 있다. Based on these results, ramipril, captopr i 1, enarapil, r isinopr i 1, fosinori 1 or spirapril Chemically synthesized angiotensin converting enzyme inhibitors such as these have been used for the treatment of commercial hypertension. However, these compounds are easily decomposed in pharmaceutical dosage forms, which not only reduces their stability, but also act on other cells, causing problems with side effects such as weakness in the whole body, vomiting, coughing, headache, anorexia and taste disorders. Reported to be (Lim SD. Et. Al (2008) Korean J. Food Sci. Ani. Resour, 28 (5): 587-595). Therefore, there is a demand for the development of an ACE inhibitor derived from a natural substance which has stability and also has safety that does not cause adverse effects on the human body. Oysters are known as health foods that are as healthy as the milk of the sea. Nutritional ingredients include glycogen, taurine, proteins and vitamins, as well as various minerals and are widely known as ingredients for dietary supplements. In addition, it is effective in strengthening the function of the heart and liver, and preventing hypertension, arteriosclerosis and heart disease by reducing cholesterol, and because it contains a large amount of selenium, it not only shows heavy metal detoxification function but also functions of organs such as heart, liver and pancreas. Height is known as tonic and stamina food. As an ACE inhibitor derived from a natural substance known to date, Korean Unexamined Patent Publication No. 10-2012-0092735 discloses an ACE inhibitor or antihypertensive composition containing Mesaeng as an active ingredient as an active ingredient. 1275766 discloses ACE inhibitory or antihypertensive compositions comprising abalone viscera protein extracts or fractions thereof.
또한, 천연 물질의 S소 가수분해물로부터 ACE 저해 활성을 나타내는 펩타이드가 보고된 바 있다. 이 중 정어리 단백질 유래의 펩타이드인 발린-티로신 (Valine-tyrosine)은 경증고혈압 환자에 대해 강압효과를 나타내는 것이 보고되어 (Shimizu, M (1994) Melbourne Sept, 18)， 현재 대한민국 제 1호의 개별 인정성 건강식품으로 인정받았으며， 대한민국 등록특허 10-1106303호에서는 효소처리를 통하여 굴로부터 제조한 펩타이드의 ACE 활성 저해능에 관하여 개시하고 있다. 그러나, 천연 물질 유래의 기능성 펩타이드의 개발에 대하여는 여전히 그 요구가 증가하고 있다ᅳ 따라서， 본 발명의 발명자들은 천연 물질의 효소 가수분해물로부터 ACE 활성 저해능을 나타내는 펩타이드를 개발하기 위해 노력한 결과， 굴 효소 가수분해물 (oyster enzyme hydrate)로부터 추출 , 분리 및 정 제하여 ACE 활성 저해능을 나타내는 신규한 펩타이드를 수득하였고， 상기 펩타이드는 효과적 인 ' 혈압 조절 효과 및 고혈압 예방 효과를 나타내므로， 본 발명의 굴 효소 가수분해물로부터 분리한 펩타이드는 심혈관계 질환의 예방 또는 치료용 약학적 조성물의 유효 성분으로 유용하게 사용될 수 있음을 확인함으로써 본 발명을 완성하였다 . In addition, peptides showing ACE inhibitory activity have been reported from S-hydrolysates of natural substances. Among these, valine-tyrosine, a peptide derived from sardine protein, has been reported to have a coercive effect in patients with mild hypertension (Shimizu, M (1994) Melbourne Sept, 18). Recognized as a health food, Korean Patent No. 10-1106303 discloses the ability to inhibit the ACE activity of the peptide prepared from oyster through the enzyme treatment. However, there is still a growing demand for the development of functional peptides derived from natural substances. Therefore, the inventors of the present invention have found that ACE from enzymatic hydrolysates of natural substances can Efforts to develop peptides showing activity inhibitory activity resulted in the extraction, isolation and purification of oyster enzyme hydrates to obtain novel peptides showing the ability to inhibit ACE activity. And since the anti-hypertensive effect, the peptide isolated from the oyster enzyme hydrolyzate of the present invention has been completed by confirming that it can be usefully used as an active ingredient in the pharmaceutical composition for the prevention or treatment of cardiovascular diseases.
【발명의 상세한 설명】 [Detailed Description of the Invention]
【기술적 과제】  [Technical problem]
본 발명의 목적은 서 열번호 1 내지 12로 기 재되는 아미노산 서 열로 구성되는 안지오텐신 전환효소 (angiotens in convert ing enzyme ; ACE)에 대하여 저해능을 갖는 텝타이드를 제공하는 것 이다.  An object of the present invention is to provide a step tide having an inhibitory ability against angiotensin converting enzyme (ACE) consisting of amino acid sequences described in SEQ ID NO: 1 to 12.
본 발명의 또 다른 목적은 상기 펩타이드를 포함하는 굴 효소 가수분해물 (oyster enzyme hydrate)의 분획물을 유효성분으로 함유하는 심 혈관계 질환 예방 또는 치료용 약학적 조성물을 제공하는 것이다 .  Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating cardiovascular disease, comprising a fraction of oyster enzyme hydrate containing the peptide as an active ingredient.
본 발명의 또 다른 목적은 상기 펩타이드 중 어느 하나 이상을 유효성분으로 함유하는 심혈관계 질환 예방 또는 치료용 약학적 조성물을 제공하는 것 이다.  Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating cardiovascular diseases containing any one or more of the peptides as an active ingredient.
본 발명의 또 다른 목적은 상기 펩타이드를 포함하는 굴 효소 가수분해물의 분획물을 유효성분으로 함유하는 심 혈관계 질환 예방 또는 개선용 건강 식품을 제공하는 것 이다 .  Still another object of the present invention is to provide a health food for preventing or improving cardiovascular disease, which contains a fraction of the oyster enzyme hydrolyzate including the peptide as an active ingredient.
본 발명의 또 다른 목적은 상기 펩타이드 중 어느 하나 이상을 유효성분으로 함유하는 심혈관계 질환 예방 또는 개선용 건강 식품을 제공하는 것이다.  Another object of the present invention to provide a health food for preventing or improving cardiovascular disease containing any one or more of the peptides as an active ingredient.
본 발명의 또 다른 목적은 상기 펩타이드를 포함하는 굴 효소 가수분해물의 분획물을 심 혈관계 질환을 가지는 개체에 투여하는 단계를 포함하는 심 혈관계 질환의 치료 방법올 제공하는 것이다 . 본 발명의 또 다른 목적은 상기 펩타이드 중 어느 하나 이상을 심혈관계 질환을 가지는 개체에 투여하는 단계를 포함하는 심혈관계 질환의 치료 방법을 제공하는 것 이다 . It is another object of the present invention to provide a method for treating cardiovascular diseases comprising administering a fraction of the oyster enzyme hydrolyzate comprising the peptide to a subject having a cardiovascular disease. Still another object of the present invention is to provide a method for treating cardiovascular diseases comprising administering any one or more of the peptides to a subject having a cardiovascular disease.
본 발명의 또 다른 목적은 상기 템타이드를 포함하는 굴 효소 가수분해물의 붓획물을 개체에 투여하는 단계를 포함하는 심 혈관계 질환의 예방 방법을 제공하는 것이다 .  Still another object of the present invention is to provide a method for preventing cardiovascular disease, comprising administering to a subject a complex of the oyster enzyme hydrolyzate including the tempide.
본 발명의 또 다른 목적은 상기 펩타이드 중 어느 하나 이상을 투여하는 단계를 포함하는 심혈관계 질환의 예방 방법올 제공하는 것이다 .  Still another object of the present invention is to provide a method for preventing cardiovascular disease, including the step of administering any one or more of the peptides.
본 발명의 또 다른 목적은 심혈관계 질환 예방 또는 치료용 약학적 조성물로 사용하기 위한 상기 펩타이드를 포함하는 굴 효소 가수분해물의 분획물의 용도를 제공하는 것이다 .  Another object of the present invention is to provide a use of a fraction of the oyster enzyme hydrolyzate comprising the peptide for use as a pharmaceutical composition for the prevention or treatment of cardiovascular diseases.
본 발명 의 또 다른 목적은 심혈관계 질환 예방 또는 치료용 약학적 조성물로 사용하기 위한 상기 펩타이드 중 어느 하나 이상의 용도를 제공하는 것이 다ᅳ  Another object of the present invention to provide a use of any one or more of the above peptides for use as a pharmaceutical composition for the prevention or treatment of cardiovascular diseases.
본 발명의 또 다른 목적은 심 혈관계 질환 예방 또는 개선용 건강 식품으로 사용하기 위 한 상기 펩타이드를 포함하는 굴 효소 가수분해물의 분획물의 용도를 제공하는 것 이다 .  Still another object of the present invention is to provide a use of a fraction of an oyster enzyme hydrolyzate comprising the peptide for use as a health food for preventing or improving cardiovascular disease.
본 발명의 또 다른 목적은 심 혈관계 질환 예방 또는 개선용 건강 식품으로 사용하기 위 한 상기 펩타이드 중 어느 하나 이상의 용도를 제공하는 것이다 .  Still another object of the present invention is to provide a use of any one or more of the above peptides for use as a health food for preventing or improving cardiovascular disease.
【기술적 해결방법】 Technical Solution
상기 목적을 달성하기 위하여， 본 발명은 서열번호 1 내지 12로 기재되는 아미노산 서 열로 구성되는 안지오텐신 전환효소 (angiotensin convert ing enzyme ; ACE)에 대하여 저해능을 갖는 펩타이드를 제공한다 .  In order to achieve the above object, the present invention provides a peptide having an inhibitory ability against angiotensin converting enzyme (ACE) consisting of the amino acid sequence described in SEQ ID NO: 1 to 12.
또한， 본 발명은 상기 펩타이드를 포함하는 굴 효소 가수분해물 (oyster enzyme hydrate)의 분획물을 유효성분으로 함유하는 심혈관계 질환 예방 또는 치료용 약학적 조성물을 제공한다ᅳ 또한， 본 발명은 상기 ¾타이드 중 어느 하나 이상을 유효성분으로 함유하는 심혈관계 질환 예방 또는 치료용 약학적 조성물을 제공한다 . The present invention also provides a pharmaceutical composition for the prevention or treatment of cardiovascular diseases containing a fraction of oyster enzyme hydrate containing the peptide as an active ingredient. In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cardiovascular diseases containing any one or more of the ¾ Tide as an active ingredient.
또한， 본 발명은 상기 펩타이드를 포함하는 굴 효소 가수분해물의 분획물을 유효성분으로 함유하는 심혈관계 질환 예방 또는 개선용 건강 식품을 제공한다 .  In addition, the present invention provides a health food for preventing or improving cardiovascular disease, which contains a fraction of the oyster enzyme hydrolyzate including the peptide as an active ingredient.
또한， 본 발명은 상기 펩타이드 증 어느 하나 이상을 유효성분으로 함유하는 심 혈관계 질환 예방 또는 개선용 건강 식품을 제공한다 .  In addition, the present invention provides a health food for preventing or improving cardiovascular disease containing any one or more of the peptide symptoms as an active ingredient.
또한， 본 발명은 상기 펩타이드를 포함하는 굴 효소 가수분해물의 분획물을 심혈관계 질환을 가지는 개체에 투여하는 단계를 포함하는 심혈관계 질환의 치료 방법을 제공한다 .  The present invention also provides a method of treating cardiovascular diseases comprising administering a fraction of the oyster enzyme hydrolyzate comprising the peptide to a subject having a cardiovascular disease.
또한， 본 발명은 상기 ¾타이드 중 어느 하나 이상을 심혈관계 질환을 가지는 개체에 투여하는 단계를 포함하는 심혈관계 질환의 치료 방법을 제공하는 것이다 .  In addition, the present invention provides a method for treating cardiovascular diseases comprising the step of administering any one or more of the ¾ tide to a subject having a cardiovascular disease.
또한 , 본 발명은 상기 펩타이드를 포함하는 굴 효소 가수분해물의 분획물을 개체에 투여하는 단계를 포함하는 심 혈관계 질환의 예방 방법을 제공한다 .  The present invention also provides a method for preventing cardiovascular disease comprising administering to a subject a fraction of an oyster enzyme hydrolyzate comprising the peptide.
또한 본 발명은 상기 ¾타이드 중 어느 하나 이상을 투여 하는 단계를 포함하는 심혈관계 질환의 예방 방법을 제공한다 .  In another aspect, the present invention provides a method of preventing cardiovascular diseases comprising the step of administering any one or more of the ¾ Tide.
또한， 본 발명은 심혈관계 질환 예방 또는 치료용 약학적 조성물로 사용하기 위 한 상기 ¾타이드를 포함하는 굴 효소 가수분해물의 분획물의 용도를 제공한다 .  In addition, the present invention provides the use of a fraction of the oyster enzyme hydrolyzate comprising the ¾ tide for use as a pharmaceutical composition for the prevention or treatment of cardiovascular diseases.
또한， 본 발명은 심혈관계 질환 예방 또는 치료용 약학적 조성물로 사용하기 위한 상기 펩타이드 중 어느 하나 이상의 용도를 제공하는 것 이다 . 또한， 본 발명은 심혈관계 질환 예방 또는 개선용 건강 식품으로 사용하기 위한 상기 펩타이드를 포함하는 굴 효소 가수분해물의 분획물의 용도를 제공한다.  In addition, the present invention is to provide a use of any one or more of the above peptides for use as a pharmaceutical composition for the prevention or treatment of cardiovascular diseases. The present invention also provides the use of a fraction of oyster enzyme hydrolysate comprising the peptide for use as a health food for preventing or improving cardiovascular disease.
아울러 ᅳ 본 발명은 심혈관계 질환 예방 또는 개선용 건강 식품으로 사용하기 위한 상기 펩타이드 증 어느 하나 이상의 용도를 제공한다 . 【유리한 효과】 ᅳ The present invention also provides the use of any one or more of the above peptide for use as a health food for preventing or improving cardiovascular disease. Advantageous Effects
본 발명의 굴 효소 가수분해물 (oyster enzyme hydrate)의 분획물로부터 분리된 안지오텐신 전환 효소 (angiotensin converting enzyme； ACE)에 대해 저해능을 나타내는 펩타이드는 효과적인 혈압 조절 효과 및 고혈압 예방 효과를 나타내므로， 상기 굴 효소 가수분해물의 분획물 또는 이로부터 분리된 펩타이드는 심혈관계 질환의 예방 또는 치료용 약학적 조성물의 유효 성분으로 유용하게 사용될 수 있다. 【도면의 간단한 설명】  Peptides exhibiting inhibitory activity against angiotensin converting enzyme (ACE) isolated from a fraction of the oyster enzyme hydrate of the present invention exhibits an effective blood pressure control effect and a high blood pressure preventing effect, Fractions of the degradation products or peptides isolated therefrom can be usefully used as active ingredients in pharmaceutical compositions for the prevention or treatment of cardiovascular diseases. [Brief Description of Drawings]
도 1은 굴 효소 가수분해물을 정제하는 과정을 나타내는 도이다. A는 음이온 교환크로마토그래피 (anion exchange chromatography)정제를 나타내며， B는 상기 음이온 교환 크로마토그래피의 분획 4의 크기 배제 크로마토그래피 (size exclusion chromatography) 정제를 나타내고， C는 역상 크로마토그래피 (reaverse-phase chromatography) 정제를 나타내는 도이다. 도 2는 굴 효소 가수분해물로부터 분리한 펩타이드의 질량 및 아미노산 서열의 분석을 나타내는 도아다.  1 is a view showing a process for purifying oyster enzyme hydrolyzate. A represents anion exchange chromatography, B represents purification of size exclusion chromatography of fraction 4 of the anion exchange chromatography, and C represents reverse-phase chromatography. It is a figure which shows a tablet. Fig. 2 shows the analysis of the mass and amino acid sequence of peptides isolated from oyster enzyme hydrolysates.
도 3은 굴 효소 가수분해물의 단회 투여에 의한 생체 내 혈압 조절 효과를 나타내는 도이다.  3 is a diagram showing the effect of blood pressure regulation in vivo by a single administration of oyster enzyme hydrolyzate.
도 4은 기능성 펩타이드의 단회 투여에 의한 생체 내 혈압 조절 효과를 나타내는 도이다.  Figure 4 is a diagram showing the effect of blood pressure regulation in vivo by a single administration of the functional peptide.
도 5은 굴 효소 가수분해물 및 기능성 펩타이드의 반복 투여에 의한 생체 내 혈압 조절 효과를 나타내는 도이다.  5 is a diagram showing the effect of blood pressure regulation in vivo by repeated administration of oyster enzyme hydrolyzate and functional peptide.
도 6는 굴 효소 가수분해물 및 기능성 펩타이드의 혈중 안지오텐신 전환 효소 ( Angiotensin converting enzyme; ACE)농도 억제 효과를 나타내는 도이다. 도 7은 굴 효소 가수분해물 및 기능성 펩타이드의 혈중 안지오텐신 -Il(angiotensin-n)농도 억제 효과를 나타내는 도이다.  6 is a diagram showing the effect of inhibiting the concentration of angiotensin converting enzyme (ACE) in the oyster enzyme hydrolyzate and functional peptide. 7 is a diagram showing the effect of inhibiting the concentration of angiotensin -I (angiotensin-n) in the oyster enzyme hydrolyzate and functional peptide.
도 8은 굴 효소 가수분해물 및 기능성 펩타이드의 생체 내 고혈압 예방 효과를 나타내는 도이다. 8 shows in vivo hypertension prevention of oyster enzyme hydrolyzate and functional peptide Fig. Showing the effect.
【발명의 실시를위한 최선의 형태】 【Best Mode for Implementation of the Invention】
이하， 본 발명을 상세하게 설명한다. 본 발명은 서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 안지오텐신 전환효소 (angiotensin converting enzyme； ACE)에 대하여 저해능을 갖는 펩타이드를 제공한다.  Hereinafter, the present invention will be described in detail. The present invention provides a peptide having an inhibitory activity against angiotensin converting enzyme (ACE) consisting of the amino acid sequence set forth in SEQ ID NOs: 1-12.
상기 펩타이드는 굴 효소 가수분해물로부터 분리된 것이 바람직하나， 이에 한정되지 않으며， 다른 물질로부터 유래한 것 또는 합성한 것 모두 사용할 수 있다.  The peptide is preferably separated from the oyster enzyme hydrolyzate, but is not limited thereto. Any peptide derived from another substance or synthesized may be used.
상기 분리를 위한 방법으로 한외여과 (ultrafiltration), 크로마토그래피 (chromatography)와 같은 당업계의 통상적인 분리 방법을 사용하는 것이 바람직하며, 구체적으로는 크로마토그래피 방법을 사용하는 것이 보다 바람직하고， 더욱 구체적으로는 음이은교환 크로마토그래피 (anion exchange chromatography) , 크기배제 크로마토그래피 (size exclusion chromatography) , 역상 크로마토그래피 (reaver se— phase chromatography) 또는 고성능 액체 크로마토그래피 (high performance liquid chromatography; HPLC) 방법을사용하는 것이 가장 바람직하나, 이에 .한정되지 않는다.  As a method for the separation, it is preferable to use a conventional separation method such as ultrafiltration, chromatography, and more preferably, a chromatographic method, more specifically, It is recommended to use anion exchange chromatography, size exclusion chromatography, reverse se- phase chromatography, or high performance liquid chromatography (HPLC). Most preferably, but not limited to.
상기 합성을 위한 방법으로 당업계의 통상적인 펩타이드의 화학적 합성 방법 (W. H. Freeman and Co. , Proteins; structures and molecular principles, 1983)으로 합성하는 것이 바람직하며， 구체적으로는 액상 펩타이드 합성법 (Solution Phase Peptide synthesis), 고상 펩타이드 합성법 (solid-phase peptide syntheses), 단편 응축법 및 F-moc 또는 T-B0C 화학법으로 합성하는 것이 보다 바람직하고, 더욱 구체적으로는 고상 펩타이드 합성법으로 합성하는 것이 가장 바람직하나, 이에 한정되지 않는다.  As the method for the synthesis, it is preferable to synthesize by conventional chemical synthesis method of the peptide (WH Freeman and Co., Proteins; structures and molecular principles, 1983), specifically, liquid phase peptide synthesis method (Solution Phase Peptide synthesis) ), Solid-phase peptide syntheses, fragment condensation and F-moc or T-B0C chemistry are more preferable, and more specifically, it is most preferably synthesized by solid-phase peptide synthesis. It is not limited.
상기 효소는 단백질 당업계의 통상적인 가수분해효소 (protease)인 것이 바람직하고，구체적으로 프로타멕스 (protaraex)또는 뉴트라제 (neutrase)인 것이 보다 바람직하나 이에 한정되지 않는다. 상기 효소의 첨가는 프로타멕스의 반웅 후 불활성화한 다음 뉴트라제를 첨가하는 순차적 첨가인 것이 바람직하나 이에 한정되지 않으며，프로타멕스 및 뉴트라제를 동시에 첨가할 수 있다.상기 효소의 첨가량은 굴 효소 가수분해물의 단백질 농도의 0.1 내지 10%인 것이 바람직하고， 효소의 불활성화는 20 내지 100°C에서 10 내지 120 분간 반응하여 불활성화하는 것이 바람직하나 이에 한정되지 않으며， 처리하는 효소의 종류에 의해 적절하게 조절될 수 있다. 본 발명의 구체적인 실시예에 있어서， 본 발명자들은 굴 효소 가수분해물을 제조하기 위하여 단백질 가수분해효소를 사용하여 굴 단백질을 가수분해하였으며， 상기 굴 효소 가수분해물로부터 ACE 활성 저해능을 가지는 기능성 펩타이드를 정제하기 위하여 크로마토그래피를 수행한 결과， ACE 활성 저해능을 나타내는 7 개의 시료를 선별하였다 (도 1 및， 표 1 및 표 2 참조). 또한, 본 발명자들은 상기 시료에서 ACE 활성 저해능을 나타내는 기능성 펩타이드를 스크리닝하기 위하여 펩타이드의 질량 및 아미노산 서열올 확인한 결과， 서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 펩타이드를 선별하였으며 (도 2 및 표 3 참조)， 상기 펩타이드를 화학적으로 합성하여 ACE 활성 저해능 및 세포 독성을 확인한 결과， 본 발명의 펩타이드 모두가 세포에 대한 독성을 나타내지 않으면서 ACE 활성 저해능을 나타내는 것을 확인하였다 (표 4 참조). It is preferable that the enzyme is a conventional protease of the protein art, and specifically, it is protarax or neutrase. More preferred but not limited thereto. The addition of the enzyme is preferably a sequential addition of inactivation after the reaction of protamex, followed by the addition of neutrase, but is not limited thereto. Protamex and neutrase may be added at the same time. It is preferable that the protein concentration of the enzyme hydrolyzate is 0.1 to 10%, and the inactivation of the enzyme is preferably inactivated by reacting for 10 to 120 minutes at 20 to 100 ° C, but is not limited thereto. Can be adjusted appropriately. In a specific embodiment of the present invention, the present inventors hydrolyzed oyster proteins using proteolytic enzymes to prepare oyster enzyme hydrolysates, and purified functional peptides having ACE activity inhibitory activity from the oyster enzyme hydrolysates. As a result of chromatographic analysis, seven samples showing ACE activity inhibitory activity were selected (see FIG. 1 and Tables 1 and 2). In addition, the present inventors selected the peptide consisting of the amino acid sequence of SEQ ID NOS: 1 to 12 as a result of confirming the mass and amino acid sequence of the peptide in order to screen the functional peptide showing the ACE activity inhibitory activity in the sample (Fig. 2 and Table 3), as a result of chemically synthesizing the peptides to confirm the ACE activity inhibitory activity and cytotoxicity, it was confirmed that all of the peptides of the present invention exhibits the ability to inhibit ACE activity without showing toxicity to cells (see Table 4).
따라서， 본 발명의 펩타이드는 ACE에 대하여 세포 독성을 나타내지 않으면서 유의적인 활성 저해능을 나타내므로， ACE 활성 저해제로 유용하게 사용될 수 있다. 또한, 본 발명의 펩타이드는 하기와 같은 유전공학적 방법에 의해 제조될 수 있다. 우선, 통상적인 방법에 따라 상기 펩타이드를 코딩하는 DNA 서열을 작제한다. DNA 서열은 적절한 프라이머를 사용하여 PCR 증폭함으로써 제작할 수 있다. 다른 방법으로 당업계에 공지된 표준 방법에 의해, 예컨대， 자동 DNA 합성기 (예를 들면， Biosearch 또는 Applied iosystems사의 제품)를 사용하여 DNA 서열을 합성할 수도 있다. 상기 DNA 서열은 이쎄 작동가능하게 연결되어 DNA 서열의 발현을 조절하는 하나 또는 그 이상의 발현 조절 서열 (예: 프로모터， 인핸서 등)을 포함하는 백터에 삽입하고, 이로부터 형성된 재조합 발현 백터로 숙주세포를 형질전환한 다음， 생성된 형질전환체를 상기 DNA 서열이 발현되도록 하기에 적절한 배지 및 조건 하에서 배양하여, 배양물로부터 상기 DNA 서열에 의해 코딩된 실질적으로 순수한 펩타이드를 당¾계에 공지된 방법 (예컨대， 크로마토그래피)을 이용하여 회수한다. 상기 '실질적으로 순수한 펩타이드'라 함은 본 발명에 따른 펩타이드가 숙주로부터 유래된 어떠한 다른 단백질도 실질적으로 포함하지 않는 것을 의미한다. 본 발명의 펩타이드 합성을 위한 유전공학적 방법은 다음의 문헌올 참고할 수 있다: Man i at is et al., MolecularCloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982； Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, N.Y. , Second (1998) and Third(2000) Edition; Gene Expression Technology, Method in Enzymo 1 ogy , Genetics and MolecularTherefore, the peptide of the present invention does not exhibit cytotoxicity against ACE, and thus shows a significant activity inhibitory ability, and thus may be usefully used as an ACE activity inhibitor. In addition, the peptide of the present invention can be prepared by the following genetic engineering method. First, a DNA sequence encoding the peptide is constructed according to a conventional method. DNA sequences can be prepared by PCR amplification using appropriate primers. Alternatively by standard methods known in the art, for example, DNA sequences may also be synthesized using an automated DNA synthesizer (eg, from Biosearch or Applied iosystems). The DNA sequence is inserted into a vector comprising one or more expression control sequences (eg, promoters, enhancers, etc.) that are operably linked to regulate expression of the DNA sequence, and the host cell is formed with a recombinant expression vector formed therefrom. After transformation, the resulting transformants are cultivated under medium and conditions appropriate for the DNA sequence to be expressed, thereby allowing the substantially pure peptides encoded by the DNA sequences from the culture to be known in the art. For example, by chromatography). By "substantially pure peptide" it is meant that the peptide according to the invention is substantially free of any other protein derived from the host. Genetic engineering methods for peptide synthesis of the present invention may be referred to the following literature: Man i at is et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, Second (1998) and Third (2000) Edition; Gene Expression Technology, Method in Enzymo 1 ogy, Genetics and Molecular
Biology, Method in Enzymo 1 ogy , Guthrie & Fink (eds.), Academic Press, SanBiology, Method in Enzymo 1 ogy, Guthrie & Fink (eds.), Academic Press, San
Diego, Calif, 1991；및 Hitzeman et al . , J. Biol . Cheni. , 255:12073-12080, 1990. 본 발명의 펩타이드는 임상투여시 비경구로 투여가 가능하며 일반적인 의약품 제제의 형태로사용될 수 있다. 비경구 투여는 직장， 정맥， 복막, 근육, 동맥， 경피， 비강 (nasal), 흡입， 안구 및 피하와 같은 경구 이외의 투여경로를 통한투여를 의미할 수 있다. Diego, Calif, 1991; and Hitzeman et al. , J. Biol. Cheni. , 255: 12073-12080, 1990. The peptides of the present invention can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical formulations. Parenteral administration may refer to administration via a route other than oral such as rectal, intravenous, peritoneal, intramuscular, arterial, transdermal, nasal, inhalation, ocular and subcutaneous.
즉， 본 발명의 펩타이드는 실제로 비경구의 여러 가지 제형으로 투여될 수 있는데， 제제화할 경우에는 보통사용하는 충진제， 증량제， 결합제， 습윤제， 붕해제, 계면활성제 등의 회석제 또는 부형제를 사용하여 조제된다. 비경구투여를 위한 제제에는 멸균된 수용액， 비수성용제, 현탁제， 유제， 동결건조제제， 좌제가 포함된다. 비수성용제， 현탁용제로는 프로필렌글리콜 (Propylene glycol), 폴리에틸렌 글리콜， 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 둥이 사용될 수 있다. 좌제의 기제로는 위텝솔 (witepsol), 마크로골， 트원 (tween) 61, 카카오지， 리우린지, 글리세로제라틴 등이 사용될 수 있다. That is, the peptide of the present invention can be administered in various parenteral formulations, and when formulated, it is prepared by using the usual fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As a base of suppositories, witepsol, macrogol, tween 61, cacao butter, uririnji, glycerogelatin and the like can be used.
또한， 본 발명의 뎁타이드는 생리식염수 또는 유기용매와 같이 약제로 허용된 여러 전달체 (carrier)와 흔합하여 사용될 수 있고， 안정성이나 흡수성을 증가시키기 위하여 글루코스， 수크로스 또는 덱스트란과 같은 카보하이드레이트， 아스코르브 산 (ascorbic acid) 또는 글루타치온과 같은 항산화제 (antioxidants), 킬레이트화제 (chelating agents)， 저분자 단백질 또는 다른 안정화제 (stabilizers)들이 약제로 사용될 수 있다.  In addition, the depth of the present invention can be used in combination with a number of carriers (pharmaceutically acceptable carrier) such as physiological saline or organic solvents, carbohydrates such as glucose, sucrose or dextran, to increase stability or absorption Antioxidants such as ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers may be used as medicaments.
본 발명의 펩타이드의 유효용량은 0.01 내지 100 mg/kg이고， 바람직하게는 0.1 내지 10 mg/kg 이며, 하루 1회 내지 3회 투여될 수 있다. 본 발명의 펩타이드의 총 유효량은 볼루스 (bolus) 형태 혹은 상대적으로 짧은 기간 동안 주입 (infusion) 등에 의해 단일 투여량 (single does)으로 환자에게 투여될 수 있으며， 다증 투여량 (multiple does)이 장기간 투여되는 분할 치료 방법 (fractionated treatment protocol)에 의해 투여될 수 있다. 상기 농도는 약의 투여 경로 및 치료 횟수뿐만 아니라 환자의 나이 및 건강상태 등 다양한 요인들을 고려하여 환자의 유효 투여량이 결정되는 것이므로 이러한 점을 고려할 때， 이 분야의 통상적인 지식을 가진 자라면 본 발명의 신규한 펩타이드의 약학적 조성물로서의 특정한 용도에 따른 적절한 유효 투여량을 결정할 수 있을 것이다. 또한， 본 발명은 서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 펩타이드를 포함하는 굴 효소 가수분해물의 분획물을 유효성분으로 함유하는 심혈관계 질환 예방 또는 치료용 약학적 조성물을 제공한다.  The effective dose of the peptide of the present invention is 0.01 to 100 mg / kg, preferably 0.1 to 10 mg / kg, and may be administered once to three times a day. The total effective amount of the peptide of the present invention can be administered to the patient in bolus form or in a single dose by infusion for a relatively short period of time, and multiple doses can be It may be administered by a fractionated treatment protocol to be administered. Since the concentration is determined in consideration of various factors such as the age and health status of the patient as well as the route and frequency of treatment of the drug, in view of this point, the present invention can be used by those skilled in the art. Appropriate effective dosages for the particular use of the novel peptides as pharmaceutical compositions may be determined. The present invention also provides a pharmaceutical composition for preventing or treating cardiovascular disease, comprising a fraction of the oyster enzyme hydrolyzate comprising the peptide consisting of the amino acid sequence of SEQ ID NO: 1 to 12 as an active ingredient.
상기 분획물은 10 kD 이하인 것이 바람직하나， 이에 한정되지 않는다. 상기 심혈관계 질환은 고혈압， 심장병， 뇌졸증， 혈전증， 협심증, 심부전， 심근경색, 죽상경화증 및 동맥경화로 이루어진 군으로부터 선택된 하나 또는 그 이상인 것이 바람직하나， 이에 한정되지 않는다. 본 발명의 또 다른 구체적인 실시예에 있어서， 본 발명자들은 본 발명의 펩타이드가 생체 내에서 나타내는 혈압 조절 효과를 확인하기 위해 고혈압 쥐에 펩타이드를 단회 또는 수회 투여하여 혈압 조절 효과를 확인한 과, 굴 효소 가수분해물 및 YA 펩타이드가 유의적인 혈압 강하효과를 나타내는 것을 확인하였으며 (도 4 및 도 5 참조)， 혈중 ACE 및 안지오텐신 -iKangiotensin-II)의 농도가 현저하게 감소하는 것을 확인하였다 (도 6 및 도 7 참조). The fraction is preferably 10 kD or less, but is not limited thereto. The cardiovascular disease is preferably one or more selected from the group consisting of hypertension, heart disease, stroke, thrombosis, angina pectoris, heart failure, myocardial infarction, atherosclerosis and arteriosclerosis, but is not limited thereto. In another specific embodiment of the present invention, the present inventors confirmed the blood pressure control effect by administering the peptide to the hypertension rats once or several times to confirm the blood pressure control effect of the peptide of the present invention in vivo, oyster enzyme singer It was confirmed that the degradation product and the YA peptide showed a significant blood pressure lowering effect (see FIGS. 4 and 5), and the concentrations of blood ACE and angiotensin-iKangiotensin-II were significantly reduced (see FIGS. 6 and 7). ).
또한， 본 발명자들은 본 발명의 펩타이드가 생체 내에서 나타내는 고혈압 예방 효과를 확인하기 위해, 고혈압을 유발한 쥐에 본 발명의 펩타이드를 투여하고 혈압 상승 억제 효과를 확인한 결과， 쥐의 혈압 상승 효과를 유의적으로 감소하는 것을 확인하였다 (도 8 참조).  In addition, the inventors of the present invention, in order to confirm the antihypertensive effect of the peptide of the present invention in vivo, as a result of administering the peptide of the present invention to a rat inducing hypertension and confirming the effect of inhibiting blood pressure increase, It was confirmed that the decrease by (see Figure 8).
따라서， 본 발명의 서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 펩타이드를 포함하는 굴 효소 가수분해물의 분획물은 효과적인 혈압 조절 효과 및 고혈압 예방 효과를 나타내므로， 상기 분획물은 심혈관계 질환의 예방또는 치료용 약학적 조성물의 유효 성분으로 유용하게 사용될 수 있다.  Therefore, the fraction of the oyster enzyme hydrolyzate comprising the peptide consisting of the amino acid sequence described in SEQ ID NOS: 1 to 12 of the present invention exhibits an effective blood pressure control effect and a high blood pressure prevention effect, the fraction is to prevent cardiovascular disease or It can be usefully used as an active ingredient in therapeutic pharmaceutical compositions.
본 발명의 조성물은 비경구의 여러 가지 제형일 수 있다. 상기 조성물을 제제화할 경우에는 보통 사용하는 충진제, 증량제， 결합제, 습윤제， 붕해제， 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. The compositions of the present invention may be in various parenteral formulations. In formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used.
보다 상세하게는 상기 약학 조성물은 상기 유효성분 외에 추가로 영양제， 비타민,전해질,풍미제，착색제，중진제，펙트산 및 그의 염，알긴산 및 그의 염， 유기산， 보호성 콜로이드 증점제. pH 조절제， 안정화제, 방부제. 글리세린， 알코올， 탄산 음료에 사용되는 탄산화제 등을 추가로 함유할 수 있다ᅳ 또한, 상기 담체， 부형제 또는 회석제는 락토스， 덱스트로스， 수크로스， 솔비를, 만니틀，자이리틀, 에리스리틀， 말티를, 전분， 아카시아고무, 알지네이트， 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트， 셀를로오스， 메틸 셀를로오스, 미정질 샐루로오스， 폴리비닐 피를리돈， 물， 메틸하이드록시벤조에이트， 프로필하이드록시벤조에아트， 탈크， 마그네슘 스테아레이트 및 광물유, 덱스트린, 칼슘카보네이드， 프로필렌글리콜， 리퀴드 파라핀， 생리식염수로 이루어진 군에서 선택된 하나 또는 그 이상 일 수 있으나, 이에 한정되는 것은 아니며 통상의 담체， 부형제 또는 희석제 모두 사용가능하다. 상기 성분들은 본 발명의 펩타이드에 독립적으로 또는 조합하여 추가될 수 있다. In more detail, the pharmaceutical composition may further include a nutrient, vitamin, electrolyte, flavoring agent, coloring agent, neutralizing agent, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, and protective colloid thickeners. pH regulators, stabilizers, preservatives. Glycerin, alcohol, carbonation agent used in carbonated beverages, and the like, may further contain the carrier, excipient or diluent, lactose, dextrose, sucrose, sorbbi, mannitol, gyrite, erythritol, Malty, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline salulose, polyvinyl pyridone, water, methyl hydroxybenzoate, Propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, physiological saline, but may be one or more selected from the group consisting of, but is not limited to conventional carriers Either excipients or diluents may be used. The components may be added independently or in combination with the peptides of the present invention.
더 나아가 본 발명의 약학 조성물은 당해 기술 분야의 공지된 적절한 방법을 사용하여 또는 레밍턴의 문헌 (Remington's Pharmaceutical Science (최근판)， Mack Publishing Company, EastonPA)에 개시되어 있는 방법올 이용하여 바람직하게 제형화될 수 있다.  Furthermore, the pharmaceutical compositions of the present invention are preferably formulated using suitable methods known in the art or using methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, EastonPA. Can be.
본 발명의 조성물은 약제학적으로 유효한 양으로 투여한다. 본 발명에 있어서， "약제학적으로 유효한 양''은 의학적 치료에 적용 가능한 합리적인 수혜 /위험 비율로 질환을 치료하기에 충분한 양을 의미하며， 유효용량 수준은 환자의 질환의 종류， 증증도, 약물의 활성， 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율， 치료기간，동시 사용되는 약물을 포함한요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며， 단일 또는 다증 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며， 이는 당업자에 의해 용이하게 결정될 수 있다.  The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, the effective dose level is the type of disease, severity, drug Activity, sensitivity to drug, time of administration, route of administration and rate of administration, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts. It may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutics, and may be administered in a single or multiple doses. It is important to administer the amount, which can be readily determined by one skilled in the art.
본 발명의 조성물의 투여량은 환자의 체중, 연령 성별， 건강상태， 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도에 따라 그 범위가 다양하며， 일일 투여량은 삼백초 추출물의 양을 기준으로 0.01 내지 1000 mg/kg이고， 바람직하게는 30내지 500 mg/kg이고， 더욱 바람직하게는 50내지 300 mg/kg이며， 하루 1 ~6회 투여될 수 있다. 그러나 투여 경로， 비만의 증증도, 성별， 체증， 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.  The dosage of the composition of the present invention varies depending on the weight of the patient, age, sex, health status, diet, time of administration, administration method, excretion rate and the severity of the disease, the daily dosage is based on the amount of extract 300 seconds 0.01 to 1000 mg / kg, preferably 30 to 500 mg / kg, more preferably 50 to 300 mg / kg, and may be administered 1 to 6 times a day. However, the dosage may be increased or decreased depending on the route of administration, the increase in obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention by any method.
본 발명의 조성물은 단독으로， 또는 수술, 방사선 치료， 호르몬 치료， 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다. 또한， 본 발명은 서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 펩타이드 중 어느 하나 이상올 유효성분으로 함유하는 심혈관계 질환 예방 또는 치료용 약학적 조성물을 제공한다. The composition of the present invention alone or surgery, radiation therapy, hormone therapy, It can be used in combination with methods using chemotherapeutic and biological response modifiers. In addition, the present invention provides a pharmaceutical composition for preventing or treating cardiovascular diseases containing any one or more of the peptides consisting of the amino acid sequence of SEQ ID NO: 1 to 12 as an active ingredient.
본 발명의 펩타이드는 효과적인 혈압 조절 효과 및 고혈압 예방 효과를 나타내므로， 상기 펩타이드 중 어느 하나 이상는 심혈관계 질환의 예방 또는 치료용 약학적 조성물의 유효 성분으로 유용하게 사용돨수 있다. 또한， 본 발명은 서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 템타이드를 포함하는 굴 효소 가수분해물의 분획물을 유효성분으로 함유하는 심혈관계 질환 예방 또는 개선용 건강 식품을 제공한다.  Since the peptide of the present invention exhibits an effective blood pressure control effect and a high blood pressure preventing effect, any one or more of the peptides may be usefully used as an active ingredient of a pharmaceutical composition for preventing or treating cardiovascular diseases. In addition, the present invention provides a health food for preventing or improving cardiovascular diseases, comprising as an active ingredient a fraction of an oyster enzyme hydrolyzate comprising a tempide composed of the amino acid sequence of SEQ ID NO: 1 to 12.
또한， 본 발명은 상기 펩타이드 중 어느 하나 이상을 유효성분으로 함유하는 심혈관계 질환 예방또는 개선용 건강 식품을 제공한다.  In addition, the present invention provides a health food for preventing or improving cardiovascular disease containing any one or more of the peptides as an active ingredient.
본 발명의 굴 효소 가수분해물의 분획물 또는 이로부터 분리된 펩타이드는 ACE에 대하여 유의적인 활성 저해능을 나타내며， 체내에서 효과적인 혈압 조절 효과 및 고혈압 예방 효과를 나타내므로， 상기 굴 효소 가수분해물의 분획물 및 이로부터 분리된 펩타이드는 심혈관계 질환의 예방 또는 개선용 건강 식품의 유효 성분으로 유용하게 사용될 수 있다. 상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 드링크제， 육류， 소시지， 빵, 비스킷， 떡， 초콜릿， 캔디류， 스낵류,과자류,피자，라면，기타 면류，껌류，아이스크림류를 포함한 낙농제품， 조제유류 또는 영유아식과 같은 특수영양식품， 식육가공품, 어육제품, 두부류， 묵류， 건강보조식품, 간장，된장,고추장 또는 혼합장과 같은 조미식품， 소스류, 기타 가공식품， 김치 또는 장아찌와 같은 절임식품， 각종 스프， 음료수, 알코올 음료 및 비타민 복합제， 유제품 및 유가공 제품 등이 있으며, 통상적인 의미에서의 건강기능식품을 모두 포함한다. 상기 식품， 음료 또는 식품첨가제는 통상의 제조방법으로 제조될 수 있다. Fractions of the oyster enzyme hydrolyzate of the present invention or peptides isolated therefrom exhibits significant activity inhibitory activity against ACE, and exhibits effective blood pressure control and hypertension prevention effects in the body. The isolated peptide may be usefully used as an active ingredient of a health food for preventing or improving cardiovascular disease. There is no particular limitation on the kind of food. Examples of foods to which the substance may be added include dairy products, formulated milk products, including drinks, meat, sausages, bread, biscuits, rice cakes, chocolate, candy, snacks, sweets, pizza, ramen, other noodles, gums, ice cream, Special dietary foods such as infant foods, processed meats, fish products, tofu, jelly, health supplements, soy sauce, miso, seasoned foods such as red pepper paste or mixed soy sauce, sauces, other processed foods, pickles such as kimchi or pickles, various soups ， Beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, etc. It includes all dietary supplements in the sense. The food, drink or food additives may be prepared by a conventional manufacturing method.
본 발명에서 "건강 식품" 이란 식품에 물리적， 생화학적 또는 생불공학적 수법 등을 이용하여 해당 식품의 기능을 특정 목적에 작용 및 발현하도록 부가가치를 부여한 식품군이나 식품 조성이 갖는 생체 방어 리듬 조절 또는 질병방지 및 회복 등에 관한 체조절기능을 생체에 대하여 층분히 발현하도록 설계하여 가공한 식품을 의미하며， 바람직하게는 본 발명의 건강 식품은 심혈관계 질환의 예방 또는 개선에 관한 체조절기능을 생체에 대하여 충분히 발현할 수 있는 식품을 의미한다. 상기 건강 식품에는 식품학작으로 허용 가능한 식품 보조 첨가제를 포함할 수 있으며, 건강 식품의 제조에 통상적으로 사용되는 적절한 담체， 부형제 및 회석제를 더욱 포함할 수 있다. 본 발명의 굴 효소 가수분해물의 분획물 또는 이로부터 분리된 펩타이드는 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고， 통상적인 방법에 따라 적절하게 사용될 수 있다ᅳ 유효 성분의 혼합량은 그의 사용 목적 (예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로， 건강식품 중의 상기 분획물 또는 펩타이드의 양은 전체 식품 중량의 0.1 내지 90 증량부로 가할 수 있다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며， 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도사용될 수 있다.  In the present invention, the term "health food" refers to physiological defense rhythm control or disease prevention of a food group or a food composition which has added value to the food by using physical, biochemical or biotechnological techniques to act and express the function of the food for a specific purpose. And it means a food processed and designed to express the gynecological function in relation to the living body, and preferably, the health food of the present invention is sufficient for the bodily ganglia function for the prevention or improvement of cardiovascular diseases It means the food which can express. The health food may include a food supplement acceptable food supplement additive, and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of health foods. Fractions of the oyster enzyme hydrolyzate of the present invention or peptides separated therefrom can be added to food as is or used with other foods or food ingredients, and can be suitably used according to conventional methods. It may be appropriately determined depending on the purpose (prevention or improvement). In general, the amount of the fraction or peptide in the health food can be added in an amount of 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.
본 발명의 건강 기능성 음료 조성물은 지시된 비율로 필수 성분으로서 상기 분획물 또는 펩타이드를 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드， 예를 들어， 포도당， 과당등; 디사카라이드， 예를 들어 말토스, 슈크로스 둥; 및 폴리사카라이드, 예를 들어 덱스트린， 시클로덱스트린 등과 같은 통상적인 당， 및 자일리를, 소르비를, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제 (타우마틴， 스테비아 추출물 (예를 들어 레바우디오시드 A, 글리시르히진등) 및 합성 향미제 (사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 당 일반적으로 약 1 내지 20 g, 바람직하게는 약 5 내지 12 g이다. The functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the fractions or peptides as essential ingredients in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. Examples of the above described natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose dung; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xyl, sorbitol and erythritol. Natural flavors (tauumatin, stevia extract (for example rebaudioside) as flavoring agents other than those mentioned above A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 compositions of the present invention.
상기 외에 본 발명의 굴 효소 가수분해물의 분획물 또는 이로부터 분리된 펩타이드는 여러 가지 영양제, 비타민， 광물 (전해질)， 합성 풍미제 및 천연 풍미제 등의 풍미제， 착색제 및 중진제 (치즈， 초콜릿 등)， 펙트산 및 그의 염, 알긴산 및 그의 염，유기산, 보호성 콜로이드 증점제， pH조절제， 안정화제， 방부제， 글리세린, 알코올， 탄산음료에 사용되는 탄산화제 둥을 함유할 수 있다. 그 밖에 본 발명의 굴 효소 가수분해물의 분획물 또는 이로부터 분리된 펩타이드는 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 굴 효소 가수분해물의 분획물 또는 이로부터 분리된 펩타이드 100 중량부 당 0.1 내지 약 20 증량부의 범위에서 선택되는 것이 일반적이다. 또한， 본 발명은 서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 펩타이드를 포함하는 굴 효소 가수분해물의 분획물을 심혈관계 질환을 가지는 개체에 투여하는 단계를 포함하는 심혈관계 질환의 치료 방법을 제공한다.  In addition to the fractions of the oyster enzyme hydrolyzate of the present invention or the peptide isolated therefrom are various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors such as flavoring agents, colorants and neutralizing agents (cheese, chocolate, etc.) ), Pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonated drinks used in carbonated beverages. In addition, the fraction of the oyster enzyme hydrolyzate of the present invention or peptide isolated therefrom may contain pulp for the production of natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the fraction of the oyster enzyme hydrolyzate of the present invention or peptide isolated therefrom. The present invention also provides a method for treating cardiovascular disease, comprising administering to a subject with a cardiovascular disease a fraction of an oyster enzyme hydrolyzate comprising a peptide consisting of the amino acid sequences set forth in SEQ ID NOs: 1-12. do.
또한， 본 발명은 상기 펩타이드 중 어느 하나 이상을 심혈관계 질환올 가지는 개체에 투여하는 단계를 포함하는 심혈관계 질환의 치료 방법을 제공한다.  The present invention also provides a method of treating cardiovascular diseases comprising administering any one or more of the peptides to a subject having a cardiovascular disease.
본 발명의 굴 효소 가수분해물의 분획물 또는 이로부터 분리된 펩타이드는 ACE에 대하여 유의적인 활성 저해능을 나타내며， 체내에서 효과적인 혈압 조절 효과 및 고혈압 예방 효과를 나타내므로， 상기 굴 효소 가수분해물의 분획물 및 이로부터 분리된 펩타이드는 심혈관계 질환을 가지는 개체에 투여하는 단계를 포함하는 심혈관계 질환의 치료 방법에 유용하게 사용될 수 있다ᅳ 또한， 본 발명은 서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 펩타이드를 포함하는 굴 효소 가수분해물의 분-확물을 개체에 투여하는 단계를 포함하는 심혈관계 질환의 예방 방법을 제공한다. Fractions of the oyster enzyme hydrolyzate of the present invention or peptides isolated therefrom exhibits significant activity inhibitory activity against ACE, and exhibits effective blood pressure control and hypertension prevention effects in the body. The isolated peptides can be usefully used in the treatment of cardiovascular diseases, including administering to a subject having a cardiovascular disease. The present invention also provides a method for preventing cardiovascular disease, comprising administering to a subject an aliquot of the oyster enzyme hydrolyzate comprising a peptide consisting of the amino acid sequences set forth in SEQ ID NOs: 1-12.
또한， 본 발명은 상기 펩타이드 중 어느 하나 이상올 투여하는 단계를 포함하는 심혈관계 질환의 예방 방법올 제공한다.  In addition, the present invention provides a method for preventing cardiovascular disease, including the step of administering any one or more of the peptides.
본 발명의 굴 효소 가수분해물의 분획물 또는 이로부터 분리된 펩타이드는 ACE에 대하여 유의적인 활성 저해능을 나타내며， 체내에서 효과적인 혈압 조절 효과 및 고혈압 예방 효과를 나타내므로， 상기 굴 효소 가수분해물의 분획물 및 이로부터 분리된 펩타이드는 개체에 투여하는 단계를 포함하는 심혈관계 질환의 예방 방법에 유용하게 사용될 수 있다. 또한, 본 발명은 심혈관계 질환 예방 또는 치료용 약학적 조성물로 사용하기 위한 서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 펩타이드를 포함하는 굴 효소 가수분해물의 분획물의 용도를 제공한다.  Fractions of the oyster enzyme hydrolyzate of the present invention or peptides isolated therefrom exhibits significant activity inhibitory activity against ACE, and exhibits effective blood pressure control and hypertension prevention effects in the body. The isolated peptide may be usefully used for a method for preventing cardiovascular disease, comprising administering to a subject. The present invention also provides the use of a fraction of an oyster enzyme hydrolyzate comprising a peptide consisting of the amino acid sequence set forth in SEQ ID NOs: 1 to 12 for use as a pharmaceutical composition for the prevention or treatment of cardiovascular diseases.
또한, 본 발명은 심혈관계 질환 예방 또는 치료용 약학적 조성물로 사용하기 위한상기 펩타이드 중 어느 하나 이상의 용도를 제공한다.  The present invention also provides the use of any one or more of the above peptides for use as a pharmaceutical composition for the prevention or treatment of cardiovascular diseases.
또한， 본 발명은 심혈관계 질환 예방 또는 개선용 건강 식품으로 사용하기 위한 상기 펩타이드를 포함하는 굴 효소 가수분해물의 분획물의 용도를 제공한다.  The present invention also provides the use of a fraction of oyster enzyme hydrolysate comprising the peptide for use as a health food for preventing or improving cardiovascular disease.
아울러， 본 발명은 심혈관계 질환 예방 또는 개선용. 건강 식품으로 사용하기 위한상기 펩타이드 중 어느 하나 이상의 용도를 제공한다.  In addition, the present invention is for preventing or improving cardiovascular disease. It provides the use of any one or more of the peptides for use as a health food.
본 발명의 굴 효소 가수분해물의 분획물 또는 이로부터 분리된 펩타이드는 ACE에 대하여 유의적인 활성 저해능을 나타내며， 체내에서 효과적인 혈압 조절 효과 및 고혈압 예방 효과를 나타내므로， 상기 굴 효소 가수분해물의 분획물 및 이로부터 분리된 펩타이드는 심혈관계 질환 예방 또는 치료용 약학적 조성물, 또는 심혈관계 질환 예방 또는 개선용 건강 식품의 유효성분으로 유용하게 사용될 수 있다. 이하， 본 발명을 실시예 및 제조예에 의해 상세히 설명한다. Fractions of the oyster enzyme hydrolyzate of the present invention or peptides isolated therefrom exhibits significant activity inhibitory activity against ACE, and shows effective blood pressure control and hypertension prevention effect in the body, The isolated peptide may be usefully used as a pharmaceutical composition for preventing or treating cardiovascular diseases, or as an active ingredient of a health food for preventing or improving cardiovascular diseases. Hereinafter, the present invention will be described in detail by examples and production examples.
단， 하기 실시예 및 제조예는 본 발명을 예시하는 것일 뿐， 본 발명의 내용이 하기 실시예 및 제조예에 의해 한정되는 것은 아니다.  However, the following Examples and Preparation Examples are merely illustrative of the present invention, the contents of the present invention is not limited by the following Examples and Preparation Examples.
<실시예 1>굴 효소 가수분해물 (oyster enzyme hydrate)의 제조 Example 1 Preparation of Oyster Enzyme Hydrohydrate
본 발명의 굴 효소 가수분해물을 제조하기 위하여 , 효소를 이용하여 굴 단백질을 가수분해하였다.  In order to prepare the oyster enzyme hydrolyzate of the present invention, the oyster protein was hydrolyzed using the enzyme.
구체적으로， 양식산 굴 (통영시 소재 수하조합에서 구입) 3 kg을 끓는 물에서 3분 동안 데치고， 채를 이용하여 물기를 제거한 다음，굴 무게의 2배에 해당하는 물을 첨가한 후 분쇄기 (M-12S, 한국 후지 기계 주식회사)를 이용하여 3000 rpm에서 2 분간 마쇄 (grinding)하였다. 그런 다음， 5 L 용량의 자 발효기 (Jar fermenter; 한국 발효기 사， 한국)에 상기 마쇄한 굴 및 트랜스글루타미나제 (Transglutaminase; TGase; 아지노모토 사， 일본)를 첨가하여 150 rpm으로 교반하면서 30°C에서 1시간 동안 반응한후 100°C에서 1 시간 동안 불활성화고， 바실러스 (Bacillus) 유래의 단백질 가수분해효소 (protase)인 프로타멕스 (Protamex; 노보자임—코리아 사， 한국)를 추가로 첨가하여 40°C에서 1 시간 동안 반응해 1차 굴 효소 가수분해물을 제조하였다. 그런 다음， 상기 1차 굴 효소 가수분해물을 100°C에서 1시간 동안 처리하여 프로타멕스를 불활성화한 뒤，뉴트라제 (Neutrase;노보자임-코리아사 한국)를 첨가하여 50°C에서 1 시간 동안 반응하여 2차 굴 효소 가수분해물을 제조하고， 다시 100°C에서 1 시간 동안 처리하여 뉴트라제를 불활성화하였다. 그런 다음， 2차 굴 효소 가수분해물을 원심분리하여 상층액을 회수하였고， 4°C에서 최종 농도가 60%(v/v)가 되도록 에탄을을 첨가하여 1 시간 동안 반응하면서 에탄을 불용성 물질 및 잔여 단백질을 침전한 다음， 8000 xg에서 25 분간 원심분리하여 상층액 만을 수득하였다.수득한 상층액은 0.45 의 막으로 여과하고, 40°C 이하의 온도로 설정한 희전진공증발기 (Rotary vacuum Evaporator； N— 1 형， EYELA 사, 일본)에서 에탄을을 증발하여 굴 효소 가수분해물을수득하였고, 동결건조 또는 분무건조하여 보관하였다. Specifically, boil 3 kg of aquaculture oysters (purchased from a water-based combination in Tongyeong) for 3 minutes in boiling water, remove water with a pole, add water equal to twice the weight of the oyster, and then grinder (M- 12S, Korea Fuji Machine Co., Ltd.) was ground for 2 minutes at 3000 rpm. Then, a 5 L capacity chair fermenter (Jar fermenter; Korea fermenter Inc., Korea), oyster and trans articles the polish-pulverization in the rutile Minato claim by the addition of (Transglutaminase;; TGase Ajinomoto Co., Japan) with stirring at 150 rpm 30 ° After 1 hour of reaction at C, inactivated for 1 hour at 100 ° C. Protamex, a protase derived from Bacillus, was further added. Reaction was carried out for 1 hour at 40 ° C. to prepare a primary oyster enzyme hydrolyzate. Then, the primary oyster enzyme hydrolyzate was treated for 1 hour at 100 ° C to inactivate protamex, and then added Neutrase (Neutrase; Novozyme-Korea, Korea) for 1 hour at 50 ° C. The reaction was performed to prepare a secondary oyster enzyme hydrolyzate, and again treated at 100 ° C for 1 hour to inactivate neutrase. Then remove and then centrifuged a second oyster enzymatic hydrolyzate was recovered and the supernatant, was added to the ethane reacted for one hour so as to have a final concentration of 60% (v / v) at 4 ° C the insoluble ethane material and The remaining protein was precipitated and then centrifuged at 8000 xg for 25 minutes to obtain only the supernatant. The obtained supernatant was filtered with a membrane of 0.45 and a rotary vacuum evaporator set to a temperature of 40 ° C or less. Oyster enzyme by evaporating ethane from N— type 1, EYELA, Japan) Hydrolysates were obtained and stored lyophilized or spray dried.
<실시예 2> 안지오텐신 -I 전환 효소 (Angiotensin-I converting enzyme; ACE) 저해능을 가지는 기능성 펩타이드 (peptide)의 정제 Example 2 Purification of Functional Peptides with Inhibitory Activity of Angiotensin-I Converting Enzyme (ACE)
<2-1>음이은교환크로마토그래피 (anion exchange chromatography) 정제 굴 효소 가수분해물로부터 ACE 활성 저해능올 가지는 기능성 펩타이드를 정제하기 위하여 , 음이은교환크로마토그래피를 수행하였다.  <2-1> Anion exchange chromatography Purification Anion exchange chromatography was performed to purify a functional peptide having an ACE activity inhibitor from an oyster enzyme hydrolyzate.
구체적으로， 상기 <실시예 1>에서 제조한 굴 효소 가수분해물 0.5g을 pH 7.5의 20 mM 트리스—염산 (Tris-HCl) 완층용액 12 ι ^에 녹여 Q-세파로오즈 컬럼 (Q-Sepharose column; 16X100匪; GE헬스케어 사， 한국)에 주입하여 하기 [표 1]의 조건으로 음이온교환 크로마토그래피를 수행한 후, 굴 효소 가수분해물의 분획 (fraction)을 2 ra£씩 나누어 수득하고， 검출된 펩타이드를 확인하였다.  Specifically, 0.5 g of the oyster enzyme hydrolyzate prepared in Example 1 was dissolved in a 12 mM solution of pH 7.5 in 20 mM Tris-HCl (Tris-HCl), a Q-Sepharose column. 16X100 匪; GE Healthcare Inc., Korea) and subjected to anion exchange chromatography under the conditions of the following [Table 1], and fractions of oyster enzyme hydrolysates were obtained by dividing fractions by 2 ra £, and then detected. Identified peptides.
그 결과， 도 1에서 나타난 바와 같이 굴 효소 가수분해물로부터 분리된 펩타이드 *를 포함하는 분획을 수득하였다 (도 1).  As a result, as shown in FIG. 1, a fraction including peptide * isolated from the oyster enzyme hydrolyzate was obtained (FIG. 1).
【표 1】  Table 1
기능성 펩타이드 정제를 위한 크로마토그래피 조건  Chromatography Conditions for Purification of Functional Peptides
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000021_0001
Figure imgf000022_0001
a크기배제 크로마토그래피 (size exclusion chromatography)  a size exclusion chromatography
b 역상크로미 "토그래피 (reaverse一 phase chromatography)  b reverse phase chromography
c TFA: 트리플루오로아세트산0^^ 01"0^^ acid) c TFA: trifluoroacetic acid 0 ^^ 01 " 0 ^^ acid)
d ACN: 아세토니트릴 (acetonitrile)  d ACN: acetonitrile
* 용매의 농도 구배를 위해 100 ιη^/100 분의 0 내지 100% B 용매 구배 조건에서 수행하였다.  * Conducted at 0-100% B solvent gradient conditions of 100 η ^ / 100 min for concentration gradient of solvent.
** 용매의 농도 구배를 위해 90 m«/90 분의 구배 조건에서 선형 구배 (linear gradation)을 수행하였다. <2-2> ACE활성 저해능 확인  ** A linear gradient was performed at a gradient condition of 90 m «/ 90 minutes for the concentration gradient of the solvent. <2-2> ACE activity inhibition
상기 실시예 <2-1>에서 수득한 분획의 ACE 활성 저해능을 확인하기 위하여 , Wu 등의 방법 (Wu et al. , 2002； J Chromatography A 95으ᅳ 125-130)을 웅용하여 ACE의 활성을 측정하였다.  In order to confirm the inhibitory ability of ACE activity of the fraction obtained in Example <2-1>, the activity of ACE was determined by using Wu et al. (Wu et al., 2002; J Chromatography A 95 125-130). Measured.
구체적으로, 0.3 M 염화 나트름, 5 mM N-벤조일 -글리신-히스티딘 -류신 (N— benzoyl— Gly—His-Leu, HHL; 시그마 사; 제품번호 H1635) 및 0.25 mUnit ACE (시그마 화학사， 미국)를 포함하는 0.1 M 붕산 (borate) 완층용액 (pH 8.3)을 반웅 용액으로 제조하였다. 그런 다음, 상기 실시예 <2— 1>에서 제조한 분획을 시료로 하여 40/z«를 상기 반응 용액 150^와 혼합하여 37°C 항온 수조에서 30 분간 교반하면서 반웅하였다. 반응 후， ACE 반응을 중지하기 위하여 1 M 염산 (HC1) 150 ^를 가하고 10,000rpm에서 10 분 동안 원심분리 (제품명 5415C; 에펜도르프 사， 함부르크， 독일)하여 상둥액을 수득하였다ᅳ 그런 다음， 역상 컬럼 (Watchers 120 0DS— AP, 4.6X250 廳， 5 ； 다이소 사， 일본)을 장착한 고성능 액쎄 크로마토그래피 (high performance liquid chromatography; HPLC)에 상기 상등액 20 ^를 주입하여 HHL로부터 ACE에 의해 유리된 히푸르산 (hippuric acid; HA)의 함량을 측정하였다. HPLC를 위해， A 용매로 0.1% TFA 수용액을 사용하였고 B 용매로 0.1% TFA를 포함하는 아세토니트릴을 사용하여， 5 내지 60% B 용매 /20 분의 조건에서 선형 구배 하였고, 1 /분의 유속으로 용출하면서 228 ran 파장에서 흡광도를 측정하였다ᅳ 그런 다음， 하기 [수학식 1]을 사용하여 ACE 저해 활성을 계산하고， 시료의 농도에 따른 ACE 저해 활성을 선형 회귀분석 (JMP 통계 package ver. 7， SAS Institute, Cary, NC, USA)하여 ACE의 활성을 5OT 저해하는 시료의 농도를 IC50으로 정의하여 ACE 활성 저해능을 확인하였다. 음성 대조군으로는 시료를 대신하여 O. TFA수용액 20 ^을 사용하여， 상기와 동일한 방법으로 ACE활성 저해능을 확인하였다. Specifically, 0.3 M sodium chloride, 5 mM N-benzoyl-glycine-histidine-leucine (N— benzoyl— Gly—His-Leu, HHL; Sigma; product no.H1635) and 0.25 mUnit ACE (Sigma Chemicals, USA) 0.1 M borate complete solution containing a pH (pH 8.3) was prepared as a reaction solution. Then, 40 / z «was mixed with the reaction solution 150 ^ using the fraction prepared in Example <2-1> as a sample and reacted with stirring in a 37 ° C constant temperature water bath for 30 minutes. After the reaction, 150 ^ 1 M hydrochloric acid (HC1) was added to stop the ACE reaction and centrifuged at 10,000 rpm for 10 minutes (product name 5415C; Eppendorf, Hamburg, Germany) to obtain a supernatant. The supernatant 20 ^ was injected into high performance liquid chromatography (HPLC) equipped with a column (Watchers 120 0DS—AP, 4.6X250 Hz, 5; Daiso, Japan) to release the supernatant by ACE from HHL. The content of hipuric acid (HA) was measured. For HPLC, using a 0.1% TFA aqueous solution as solvent A and acetonitrile with 0.1% TFA as B solvent, linear gradient at conditions of 5 to 60% B solvent / 20 minutes The absorbance was measured at 228 ran wavelength while eluting at a flow rate of 1 / min. Then, ACE inhibitory activity was calculated using Equation 1 below, and linear regression analysis of ACE inhibitory activity according to the concentration of the sample was performed. (JMP statistics package ver. 7, SAS Institute, Cary, NC, USA) to determine the concentration of the sample that inhibits the activity of ACE 5OT by IC50 to confirm the ability to inhibit ACE activity. As a negative control, O. TFA aqueous solution 20 ^ was used in place of the sample, and ACE activity inhibition was confirmed in the same manner as above.
【수학식 1】  [Equation 1]
ACE저해활성 (0/0) = i^스 X100 ACE inhibitory activity ( 0/0 ) = i ^ X X100
HAo  Hao
HAo: 음성 대조군의 HA농도  HAo: HA concentration of negative control
HA: 시료의 HA농도 그 결과， 하기 [표 2]에서 나타난 바와 같이 2， 3 및 4번 분획의 시료에서 ACE 활성 저해능을 나타내는 것을 확인하였다 (표 2).  HA: HA concentration of the sample As a result, it was confirmed that showing the inhibitory activity of the ACE activity in the samples of fractions 2, 3 and 4 as shown in Table 2 (Table 2).
<2-3>크기 배제 크로마토그래피 정제 <2-3> size exclusion chromatography purification
굴 효소 가수분해물로부터 ACE 활성 저해능을 가지는 기능성 펩타이드를 정제하기 위하여, 크기 배제 크로마토그래피를 수행하였다.  In order to purify the functional peptide having ACE activity inhibitory activity from the oyster enzyme hydrolyzate, size exclusion chromatography was performed.
구체적으로， 상기 실시예 <2-2>에서 선별한 2, 3 및 4번 분획의 시료를 각각 미서卜원심진공기 (Speed Vacuum Concentrator; 스캔 스피드 40， 랩진 Aps, 덴마크)에서 농축한 후 수퍼덱스 펩티드 컬럼 (superdex peptide column; 10X300 mm; GE헬스케어 사, 한국)에 상기 농축한 시료 200 ^를 주입하여 상기 [표 1]의 조건으로 분자량에 따른 크기 배제 크로마토그래피를 수행한 후, 각각의 시료에 대한 분획을 수득하여 상기 실시예 <2-2〉와 동일한 방법을 수행하여 ACE 활성 저해능을 확인하였다.  Specifically, the samples of fractions 2, 3 and 4 selected in Example <2-2> were concentrated in a speed vacuum concentrator (Scan Vacuum 40, Rapjin Aps, Denmark), respectively, and then Superdex. After injection of the concentrated sample 200 ^ into a peptide column (superdex peptide column; 10X300 mm; GE Healthcare Co., Korea) and performing size exclusion chromatography according to the molecular weight under the conditions of [Table 1], each sample Fractions were obtained to perform the same method as in Example <2-2> to confirm the ACE activity inhibitory ability.
그 결과, 도 1 및 하기 [표 2]에서 나타난 바와 같이 크기 배제 크로마토그래피를 통해 펩타이드를 포함하는 분획인 2-1， 2-2, 2-3， 3-1, 3-2， 4-1및 4-2분획을 선별하였으며 (도 1)， 상기 선별한 분획이 ACE활성 저해능을 나타내는 것을 확인하였다 (표 2). As a result, 2-1, 2-2, 2-3, 3-1, 3-2, a fraction containing peptides through size exclusion chromatography, as shown in FIG. 1 and Table 2 below. 4-1 and 4-2 fractions were selected (FIG. 1), and the selected fractions showed ACE activity inhibitory activity (Table 2).
<2-4> 역상크로마토그래피 정제 <2-4> Reversed phase chromatography purification
굴 효소 가수분해물로부터 ACE 활성 저해능을 가지는 기능성 펩타이드를 정제하기 위하여， 역상 HPLC를 수행하였다.  In order to purify the functional peptide having ACE activity inhibitory activity from oyster enzyme hydrolyzate, reverse phase HPLC was performed.
구체적으로,상기 실시예 <2-3>에서 선별한 2-1, 2-2, 2-3, 3-1， 3-2, 4-1 및 4-2분획 시료를 각각 소스 51¾ ^컬럼(30111 651¾ 51"(；이1腿1; 4.6><150麵； GE 스케어 사， 한국)에 상기 시료를 주입하여 상기 [표 1]의 조건으로 분자량에 따른 역상 크로마토그래피를 수행한 후, 각각의 시료에 대한 분획을 수득하여 상기 실시예 <2-2>와 동일한 방법을 수행하여 ACE 활성 저해능을 확인하였다. Specifically, 2-1, 2-2, 2-3, 3-1, 3-2, 4-1 and 4-2 fraction samples selected in Example <2-3>, respectively, source 51¾ ^ column ( 30111 651¾ 51 " (; Lee 1 " 1; 4.6><150麵; GE Scare Inc., Korea) was injected to the sample and subjected to reverse phase chromatography according to the molecular weight under the conditions of [Table 1], and then Fractions were obtained for the samples, and the same method as in Example <2-2> was performed to confirm ACE activity inhibitory ability.
그 결과， 도 1 및 하기 [표 2]에서 나타난 바와 같이 역상 크로마토그래피를 통해 펩타이드를 포함하는 분획을 선별하였으며 (도 1)， 상기 선별한 분획이 ACE활성 저해능올 나타내는 것을 확인하였다 (표 2).  As a result, as shown in FIG. 1 and Table 2, fractions containing peptides were selected through reverse phase chromatography (FIG. 1), and the selected fractions showed ACE activity inhibitory activity (Table 2). .
【표 2】 Table 2
굴 효소 가수분해물의 정제 단계에 따른 분획명， 분획 번호， ACE 활성 저해능 및 펩타이드 서열  Fraction Name, Fraction Number, ACE Inhibitory Activity and Peptide Sequence
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000024_0001
Figure imgf000025_0001
<실시예 3> ACE활성 저해능을 가지는 기능성 펩타이드 질량 및 서열의 확인 굴 효소 가수분해물로부터 분리한 ACE 활성 저해능을 가지는 기능성 펩타이드를 스크리닝하기 위하여， 상기 펩타이드의 질량 및 아미노산 서열을 에드만 (edman) 분해법 및 말디 /토프 (Matrix-Assisted Laser Desorption Ionization/ Time-0f-Fl ight Mass Spectroscopy; MALDI/TOF)를 수행하여 확인하였다. Example 3 Identification of Functional Peptide Mass and Sequence Having ACE Activity Inhibition In order to screen functional peptides having ACE activity inhibitory activity isolated from oyster enzyme hydrolyzate, the mass and amino acid sequences of the peptides were edman digested. And Maldi / Tope (Matrix-Assisted Laser Desorption Ionization / Time-0f-Fight Mass Spectroscopy; MALDI / TOF).
구체적으로， 상기 실시예 <2-4>에서 선별한 ACE 저해 활성을 가지는 7 개의 시료를 각각 진공 농축 원심분리기에서 완전히 건조하고， 0. TFA수용액 20/ 를 가하여 완전히 용해하였다. 그런 다음， 50% 아세토나이트릴로 활성화 (activation)하고 0.1¾> TFA수용액으로 평행한 집팁 C18컬럼 (ZipTip C18 column; 피어스 사, 제품번호: 87782)에 상기 용해한 시료를 로딩한 후， 0.1¾> TFA 수용액으로 2 내지 3 회 세척하고， 0.1% TFA을 포함하는 70% 아세토나이트릴을 사용하여 펩타이드는 용출하여 이외 염은 제거하였다. 상기 용출한 펩타이드 용액 10 ^를 바이오브렌 (biobrene; AB 시스템 사, 미국)으로 전처리한 마이크로필터 (micro-filter)에 로딩한 후， 아르곤 (Argon) 가스를 사용하여 필터를 건조하였다. 건조가 끝난 필터는 카트리지에 장착하여 아미노산 자동 서열기인 ABI492 자동 단백질 서열기 (ABI482 automated protein sequencer; 어플라이드 바이오시스템 사， 미국)를 사용하는 펄스분사된 -액체 (pulsed-liquid) 방법을 사용하여 기능성 펩타이드를 구성하는 아미노산의 서열을 확인하였다.  Specifically, seven samples having the ACE inhibitory activity selected in Example <2-4> were completely dried in a vacuum concentrated centrifuge, respectively, and dissolved completely by adding 0. TFA aqueous solution 20 /. Then, activating with 50% acetonitrile and loading the dissolved sample in a ZipTip C18 column (Pierce, No. 87782) parallel with 0.1¾> TFA solution, 0.1¾> TFA After washing 2-3 times with an aqueous solution, the peptide was eluted using 70% acetonitrile containing 0.1% TFA to remove other salts. The eluted peptide solution 10 ^ was loaded into a micro-filter pretreated with biobrene (AB system, USA), and then the filter was dried using argon gas. The dried filter is mounted on a cartridge and functional peptide using a pulsed-liquid method using an ABI482 automated protein sequencer (ABI482 automated protein sequencer; Applied Biosystems, USA). The sequence of amino acids constituting the was confirmed.
또한, 상기 실시예 <2-4>에서 선별한 ACE 저해 활성을 가지는 7 개의 시료를 전기분무 이온화 (electrospray ionization; ESI) 방법을 이용하는 질량분석기인 Q-TOF2(마이크로매스 사， 영국)에서 나노 -ESI 인터페이스 (Nano-ESI-interface)로 결과 의존적 이중 질량분석법 (data dependent MS/MS)을 사용하여 기능성 펩타이드의 질량을 확인하였다. In addition, the seven samples having the ACE inhibitory activity selected in Example <2-4> by the nano- Q-TOF2 (Micromass, UK) which is a mass spectrometer using the electrospray ionization (ESI) method ESI The mass of the functional peptide was confirmed using a data dependent MS / MS with a Nano-ESI-interface.
그 결과， 하기 [표 3] 및 도 2에서 나타난 바와 같이， 서열 번호 1내지 12의 아미노산으로 구성되는 총 12개의 펩타이드를 확인하였다 (표 3및 도 2).  As a result, as shown in the following [Table 3] and Figure 2, a total of 12 peptides consisting of amino acids of SEQ ID NO: 1 to 12 was confirmed (Table 3 and Figure 2).
【표 3】 Table 3
ACE 활성 저해능을 나타내는 기능성 펩타이드의 아미노산서열의 확인  Identification of Amino Acid Sequences of Functional Peptides Inhibiting ACE Activity
Figure imgf000026_0001
Figure imgf000026_0001
<실시예 4> ACE 활성 저해능을 나타내는 기능성 펩타이드의 합성 및 ACE 활성 저해능 확인 Example 4 Synthesis of Functional Peptides Showing ACE Activity Inhibition and Confirmation of ACE Activity Inhibition
<4-1> 기능성 펩타이드 합성  <4-1> Functional Peptide Synthesis
굴 효소 가수분해물로부터 분리한 기능성 펩타이드가 실제로 ACE 활성 저해능을 나타내는지 확인하기 위하여， 플루오레닐메틸옥시카보닐 클로라이드 (Fluorenylmethyloxycarbonyl chloride; Fmoc)一 고상 펩타'이드 합성법 (Solid phase peptide syntheisis; SPPS)을 수행하여 합성 펩타이드를 제조하였다. In order to ensure that the one functional peptide isolated from oyster enzymatic hydrolyzate in fact represents the ACE activity inhibitory ability, fluorenyl methyloxy carbonyl chloride (Fluorenylmethyloxycarbonyl chloride; Fmoc)一solid pepta Eid synthesis (Solid phase peptide syntheisis; SPPS) the Synthetic peptides were prepared to carry out.
구체적으로， C-말단이 수지 (resin)에 결합되어 있고， N-말단이 Fmoc에 보호되며， 잔기는 TFA로 제거할 수 있는 트리틸 (trityl; Trt), t-부틸옥시카보닐 (t— Butyloxycarbonyl; Boc) 또는 t-부틸 (t-butyl； t-Bu)의 보호기로 보호되는 아미노산을 상기 <실시예 3>에서 확인한 펩타이드의 서열을 구성하는 아미노산 합성의 재료로서 준비하였다. 준비한 아미노산을 2-( 1H-벤조트리아졸 -1-일) -1， 1， 3， 3- 테트라메틸루로늄-핵사플루로포페이트 )((2-(lH-Benzotirazloe-l-yl)-l,l,3,3-t etramethyluroniura hexaf luorophophate; HBTU) , 하이드록시벤조트리아졸 (Hydroxybenzotriazole; HOBt) 및Specifically, the C-terminus is bound to the resin, the N-terminus is protected by Fmoc, and the residues are trityl (Trt), t-butyloxycarbonyl (t— which can be removed by TFA). Butyloxycarbonyl; Boc) or t-butyl (t-butyl; t-Bu) The amino acid protected by the protecting group was prepared as a material for amino acid synthesis constituting the sequence of the peptide identified in <Example 3>. The prepared amino acid was 2- (1H-benzotriazol-1-yl) -1, 1,3,3-tetramethylruronium-nuclear flurophosphate) ((2- (lH-Benzotirazloe-l-yl)- l, l, 3,3-t etramethyluroniura hexaf luorophophate; HBTU), hydroxybenzotriazole (HOBt) and
N-메틸모폴린 (N-methylmorpholine; 匪 M)을 포함하는 커플링 (couling) 시약에 첨가한 다음，실온에서 자동합성기 (ASP48S;펩트론 사,한국)를 사용하여 2시간 동안 반웅하고 20% 피페리딘 (piperidine)을 포함하는 디메틸폼아마이드 (dimethyl foraaraide; DMF)를 첨가하여 실온에서 5 분간 반응하면서 Fmoc를 제거하여 펩타이드를 합성한 후， 상기 합성 과정을 반복하여 본 발명의 펩타이드를 합성하였다. 그런 다음， TFA, 1,2-에탄디티올 (1,2-ethanedithiol; EDT)， 티아니솔 (Thioanisole)， 트리이소프로필실란 (Triisopropylsi lane; TIS) 및 물을 각각 90%, 2.5%, 2.5%, 2.5%, 2.5%(v/v)의 비율로 흔합하고 상기 합성한 펩타이드에 첨가하여 C-말단의 수지 및 잔기의 보호기를 제거하였다. 제거 후， 비닥 에버레스트 C18 컬럼 (Vydac Everest C18 column; 22 250 mm, 10 /ΛΙΙ; 그레이스 사, 미국)을 사용하는 역상 HPLC를 0.1% TFA를 포함하는 40% 아세토니트릴 용액으로 선형 구배하는 조건으로 수행하여 펩타이드를 정제 및 분리하였다. 정제한 펩타이드는 애질런트 HP1100 시리즈 (애질런트 사， 미국)을 사용하여 LC/MS로 확인하였다. After addition to a coupling reagent containing N-methylmorpholine (NM), the reaction mixture was reacted at room temperature for 2 hours using an autosynthesizer (ASP48S; Dimethyl formamide (piperidine) containing dimethyl foraaraide (DMF) was added and reacted for 5 minutes at room temperature to remove the Fmoc to synthesize the peptide, the synthesis process was repeated to synthesize the peptide of the present invention. Then, TFA, 1,2-ethanedithiol (EDT), thianisole, triisopropylsilane (TIS) and water were added 90%, 2.5%, 2.5, respectively. %, 2.5%, 2.5% (v / v) were added to the peptides synthesized and mixed to remove the protecting groups of the C-terminal resin and residues. After removal, reversed phase HPLC using Vydac Everest C18 column (22 250 mm, 10 / ΛΙΙ; Grace, USA) was performed under conditions of linear gradient with 40% acetonitrile solution containing 0.1% TFA. The peptide was purified and isolated. Purified peptides were identified by LC / MS using Agilent HP1100 series (Agilent, USA).
그 결과， 굴 효소 가수분해물로부터 분리한 기능성 펩타이드와 동일한 아미노산 서열로 구성되는 합성 펩타이드 12 개를 제조하였다.  As a result, 12 synthetic peptides composed of the same amino acid sequence as the functional peptide isolated from the oyster enzyme hydrolyzate were prepared.
<4-2>합성 펩타이드의 ACE활성 저해능 및 세포 독성 (cell viability) 확인 <4-2> Confirmation of ACE activity inhibitory activity and cell viability of synthetic peptide
굴 효소 가수분해물로부터 분리한 기능성 펩타이드가 실제로 ACE 활성 저해능을 나타내는지 확인하기 위하여 , 합성 펩타이드의 ACE 활성 저해능올 확인하였으며， 세포 독성을 확인하기 위해 MTT분석을 수행하였다. Functional Peptides from Oyster Enzyme Hydrolysates Actually ACE Activity In order to confirm the inhibitory activity, the inhibitory activity of the ACE activity of the synthetic peptide was confirmed, and MTT analysis was performed to confirm the cytotoxicity.
구체적으로， 상기 실시예 <4-1>에서 합성한 펩타이드를 상기 실시예 <2-2>와 동일한 방법을 수행하여 ACE 활성 저해능올 확인하였다. 또한， 세포 독성을 확인하기 위하여 간암세포인 HepG2 세포를 배지용 항생제 및 10% 우태아혈청 (Fetal bovine serum; FBS)을 포함하는 MEM 배지쌔 접종하여, 37°C에서 5 > C02가 유지되도록 배양하였다. 세포가 80% 정도 디쉬를 덮으면 인산염 -완충 생리식염수-에틸렌디아민사아세트산 (phosphat ed-buffer ed saline-ethylenediaminetetraacetic acid; PBS-EDTA)으로 세척한 후 트립신 처리하여 계대 배양하였으며， 배지는 48시간마다 교환하면서 세포를 배양한 후 96웰 -플레이트에 IX 105 세포 / 의 농도로 100 i씩 분주하여 24 시간 동안 배양하면서 세포를 플레이트에 부착하고 배지를 제거하여 세포를 준비하였다. 그런 다음, 상기 실시예 <4-1>에서 합성한 펩타이드를 10 / ,， 50 ug/mi, 100 zg/ml 또는 200 g/m의 농도로 0.2% PBS를 포함하는 MEM배지에 녹여 시료를 제조한 후， 상기 준비한 세포에 처리하여 24 시간 동안 배양하였다. 배양 후, PBS 용액으로 2 회 세척하고 Μπ 시약을 처리한 다음, 2 시간 동안 추가로 배양하여 490 nm에서 엘리사 플레이트 리더기 (ELISA plate reader)로 흡광도를 측정하였다. Specifically, the peptide synthesized in Example <4-1> was confirmed by performing the same method as Example <2-2> to inhibit ACE activity. In addition, HepG2 cells, which are liver cancer cells, were inoculated with MEM medium containing antibiotics for 10% fetal bovine serum (FBS) and cultured to maintain 5> C02 at 37 ° C. It was. When the cells cover about 80% of the dish, the cells were washed with phosphate-buffered saline-ethylenediaminetetraacetic acid (PBS-EDTA) and passaged with trypsin, and the medium was changed every 48 hours. After culturing the cells while dispensing 100 i each in a 96-well plate at a concentration of IX 10 5 cells / while incubating for 24 hours while attaching the cells to the plate and remove the medium to prepare the cells. Then, the peptide synthesized in Example <4-1> was dissolved in a MEM medium containing 0.2% PBS at a concentration of 10 /, 50 ug / mi, 100 zg / ml or 200 g / m to prepare a sample. After that, the prepared cells were treated and incubated for 24 hours. After incubation, washed twice with PBS solution, treated with Μπ reagent, and further incubated for 2 hours, the absorbance was measured at 490 nm with an ELISA plate reader.
그 결과, 하기 [표 4]에서 나타난 바와 같이， 12 개의 기능성 펩타이드는 모두 세포에 대한 독성을 나타내지 않으면서 ACE 활성 저해능을 나타내는 것을 확인하였다 (표 4).  As a result, as shown in the following [Table 4], all 12 functional peptides were confirmed to exhibit the ability to inhibit ACE activity without showing toxicity to the cells (Table 4).
【표 4】  Table 4
기능성 펩타이드의 ACE 활성 저해능 및 세포 독성 확인  Inhibition of ACE Activity and Cytotoxicity of Functional Peptides
Figure imgf000028_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000029_0001
<실시예 5> 굴 효소 가수분해물 및 기능성 펩타이드의 생체 내 (in vivo) 혈압 조절 효과 확인 Example 5 Confirmation of In Vivo Blood Pressure Regulatory Effects of Oyster Enzyme Hydrolysates and Functional Peptides
<5-1>분자량에 따른굴 효소 가수분해물의 분리  <5-1> Separation of Oyster Enzyme Hydrolysates According to Molecular Weight
분자량에 따른 굴 효소 가수분해물의 생체 내 혈압 조절 효과를 확인하기 위하여， 굴 효소 가수분해물을 10 kD 이상 및 이하로 분리하였다. 구체적으로， 상기 <실시예 1>에서 제조한 굴 효소 가수분해물을 바이오백스 10(Biomax 10; 밀리포어 사, 미국) 한외여과막 및 펠리콘 XL(pellicon XL; 밀리포어 사， 미국) 한외여과막을 사용하여 한외여과기 (랩스케일 TFF 시스템, 밀리포어 ^아， 미국)에서 여과하였다.  In order to confirm the in vivo blood pressure control effect of the oyster enzyme hydrolyzate according to the molecular weight, the oyster enzyme hydrolyzate was separated by more than 10 kD and below. Specifically, the oyster enzyme hydrolyzate prepared in <Example 1> was used as a BioVax 10 (Biomax 10; Millipore, USA) ultrafiltration membrane and Pelicon XL (pellicon XL; Millipore, USA) Were filtered in an ultrafilter (Labscale TFF system, Millipore, USA).
그 결과， 굴 효소 가수분해물 전체로부터 10 kD 이상 및 10 kD 이하 굴 효소 가수분해물을 수득하였다. <5-2>단회 투여에 의한 생체 내 혈압조절 효과 확인  As a result, the oyster enzyme hydrolyzate of 10 kD or more and 10 kD or less was obtained from the whole oyster enzyme hydrolyzate. <5-2> Confirmation of blood pressure control effect in vivo by single administration
굴 효소 가수분해물로부터 분리한 기능성 펩타이드가 생체 내에서 나타내는 혈압 조절 효과를 확인하기 위하여， 고혈압 쥐에 상기 펩타이드를 단회 투여하고 혈압 조절 효과를 확인하였다.  In order to confirm the blood pressure regulation effect of the functional peptide isolated from the oyster enzyme hydrolyzate in vivo, the peptide was administered to the hypertensive rats once and the blood pressure regulation effect was confirmed.
구체적으로， 11 주령의 수컷 본태성 고혈압 쥐 (Spontaneiusly Hypertensive Rat, SHR; 중앙실험동물) 및 위스터 래트 (Wistar Rat; 중앙실험동물)을 구입하여 온도 22±3°C, 습도 50±5% 및 명암 주기 12 시간의 사육 환경에서 사료 및 식수를 자유롭게 섭취할 수 있도톡 충분하게 공급하며 1주간 안정화한 다음， 경희대학교 약학대학 실험동물윤리위원회 (Institutional Animal Care and Use Co瞧 ittee)의 승인을 통한 동물실험 가이드라인 (Associat ion for Assessment and Accreditation of Laboratory Care International)에 따라 실험군 및 대조군으로 사용하였다. 상기 실험군 및 대조군은 하기 [표 5]의 조건으로 나누어 상기 <실시예 1>에서 제조한 굴 효소 가수분해물， 상기 실시예 <5-1>에서 제초한 10 kD 이상 및 이하 굴 효소 가수분해물， 상기 실시예 <4-1>에서 제조한 펩타이드 또는 캡토프릴 (Captopril; (주)보령제약 제공)을 경구 또는 복강 투여한 다음， 투여 개시 후 0， 3， 6， 9， 12및 24시간에 42°C로 유지되는 래트 온도 조절 유닛 (rat temperature control unit)에 20 분간 고정하여 충분히 꼬리 혈관올 확장한 후 수축기 혈압을 측정하여 항고혈압 활성을 확인하였다. Specifically, 11-week-old male Spontaneiusly Hypertensive Rat (SHR) and Wistar Rat (Wistar Rat) were purchased to obtain a temperature of 22 ± 3 ° C, 50 ± 5% humidity, and After 12 hours of incubation, the animals were stabilized for 1 week while supplying enough feed and drinking water freely in the breeding environment. It was used as an experimental group and a control group according to the Association ion for Assessment and Accreditation of Laboratory Care International. The experimental group and the control group is divided into the conditions of the following [Table 5] oyster enzyme hydrolyzate prepared in <Example 1>, more than 10 kD and less than the oyster enzyme hydrolyzate in Example <5-1>, Peptides or captopril (Example: Boryeong Pharmaceutical Co., Ltd.) prepared in Example <4-1> were orally or intraperitoneally administered, and then 42 ° at 0, 3, 6, 9, 12 and 24 hours after the start of administration. After fixing for 20 minutes to a rat temperature control unit (rat temperature control unit) maintained at C, the tail blood vessels were fully expanded, and the systolic blood pressure was measured to confirm antihypertensive activity.
【표 5】  Table 5
단회 투여에 의한 생체 내 혈압 조절 효과 확인을 위한 실험군 및 대조군의 조건  Conditions of the experimental group and the control group for confirming the effect of blood pressure regulation in vivo by single administration
Figure imgf000030_0001
Figure imgf000030_0001
a 양성 대조군 및 실험군에 대한 투여 용량으로 mg/kg에 해당하는 시료의 양을 생리 식염수 2 에 혼합하여 투여하였다. 그 결과, 도 3 및 도 4에서 나타난 바와 같이 통상적으로 사용되는 혈압강하제인 캡토프릴을 투여한 양성 대조군에서는 음성 대조군인 고혈압 쥐에 비하여 18 내지 1 만의 혈압 강화 효과를 나타내는 것에 비해， 굴 효소 가수분해물올 투여하였을 때 음성 대조군에 .비하여 33 내지 38%의 혈압 강화 효과를 나타내며，특히 10kD이하 굴 효소 가수분해물의 경우 투여 12시간까지 실험군의 혈압을 정상 대조군과 유사한 수준으로 감소시키는 유의적인 효과를 나타내는 것을 확인하였다 (도 3). A dose of the sample corresponding to mg / kg was administered to physiological saline 2 as a dose for the positive control group and the experimental group. As a result, as shown in Figures 3 and 4, the positive control group administered captopril, which is a commonly used blood pressure lowering agent, exhibited 18 to 10,000 blood pressure strengthening effects as compared to the negative control hypertensive rats. When the hydrolyzate was administered, it showed 33 to 38% of blood pressure strengthening effect compared to the negative control group. Especially, the oyster enzyme hydrolyzate below 10 kD had a significant effect of reducing the blood pressure of the experimental group to a level similar to that of the normal control group by 12 hours. It confirmed that it represents (FIG. 3).
또한， 본 발명의 기능성 펩타이드를 투여한 실험군에서도 음성 대조군의 고혈압 쥐에 비해 유의한 수준의 혈압 강화 효과를 확인하였으며， 특히 펩타이드 YA가 투여 후 6 시간 후에 33.9%의 혈압 강화 효과를 나타내어 가장 효과적인 것을 확인하였다 (도 4).  In addition, the experimental group administered the functional peptide of the present invention confirmed a significant level of blood pressure strengthening effect compared to the hypertensive rats of the negative control group, in particular, peptide YA showed a 33.9% blood pressure strengthening effect after 6 hours after the most effective It was confirmed (FIG. 4).
<5-3>반복 투여에 의한 생체 내 혈압조절 효과 확인  <5-3> Confirmation of blood pressure regulation effect in vivo by repeated administration
굴 효소 가수분해물로부터 분리한 기능성 펩타이드가 생체 내에서 나타내는 혈압 조절 효과를 확인하기 위하여， 고혈압 쥐에 상기 펩타이드를 반복 투여하고 혈압 조절 효과를 확인하였다.  In order to confirm the blood pressure regulation effect of the functional peptide isolated from the oyster enzyme hydrolyzate in vivo, the peptide was repeatedly administered to hypertensive rats and the blood pressure regulation effect was confirmed.
구체적으로， 상기 실시예 <5-2>와 동일한 쥐를 구입하여 동일한 환경에서 사육하였고， 하기 [표 6]의 조건에 따라 실험군 및 대조군으로 나누어 상기 실시예 <5-1>에서 제조한 10 kD 이하 굴 효소 가수분해물 상기 실시예 <4— 1>에서 제조한 YA 펩타이드 또는 정어리 가수분해물로부터 분리한 혈압 강하 물질인 발린-타이로신 (Val-Tyr)올 4 주 동안 매일 아침 10 시에 경구투여하였고， 0， 1, 2， 3및 4주차에 상기 실시예 <5-2>과 동일한 방법으로 수축기 혈압을 측정하였다. 또한， 투여 개시 4 주 후에 쥐를 회생하여 하대 정맥에서, 혈액을 채취하였고， 채취한 혈액은 3,000 rpm에서 10 분간 원심분리하여 혈청을 수득하여 영하 80°C에서 보관하였다. 상기 보관한 혈청의 혈증 ACE 농도 및 안지오텐신 -Il(angiotensin-II)의 농도를 상업용 엘리사 키트 (ELISA KIT; USCN 라이프 사이언스 사, 중국)의 제조사에 따른 프로토콜에 따라 확인하였다. Specifically, the same rats as in Example <5-2> were purchased and bred in the same environment, and the 10 kD prepared in Example <5-1> was divided into experimental groups and control groups according to the conditions shown in Table 6 below. Oyster enzyme hydrolyzate was orally administered at 10 o'clock every morning for 4 weeks for valine-tyrosine (Val-Tyr) ol, a blood pressure lowering substance isolated from the YA peptide or sardine hydrolyzate prepared in Example 4-1, Systolic blood pressure was measured in the same manner as in Example <5-2> at 0, 1, 2, 3 and 4 weeks. In addition, 4 weeks after the start of administration, the rats were regenerated, blood was collected from the inferior vena cava, and the collected blood was centrifuged at 3,000 rpm for 10 minutes to obtain serum and stored at minus 80 ° C. The serum ACE concentration and angiotensin-II (angiotensin-II) concentration of the stored serum were confirmed according to the protocol according to the manufacturer of the commercial ELISA kit (ELISA KIT; USCN Life Sciences, China).
【표 6】  Table 6
반복 투여에 의한 생체 내 혈압 조절 효과 확인을 위한 실험군 및 대조군의 조건 Conditions of the experimental group and the control group for confirming the effect of blood pressure regulation in vivo by repeated administration
Figure imgf000032_0001
Figure imgf000032_0001
a 양성 대조군 및 실험군에 대한 투여 용량으로 mg/kg에 해당하는 시료의 양을 생리 식 염수 2 m 에 흔합하여 투여하였다 . 그 결과， 도 5 내지 도 7에서 나타난 바와 같이 모든 실험군에서 음성 대조군의 고혈압 쥐에 비해 유의한 수준의 혈압 강화 효과를 나타내어 전체 굴 효소 가수분해물 및 YA 펩타이드의 경우 투여 개시 후 2 주차까지 혈압이 상승하지 않았으며 , 10 kD 이하 굴 효소 가수분해물의 경우 투여 개시 후 2 주차까지 혈압이 소폭 상승하나 3 부터 혈압 강하효과를 나타내어 유의적 인 혈압 강하효과를 나타내는 것을 확인하였다 (도 5) .  a The amount of sample corresponding to mg / kg was administered to 2 m of physiological saline as the dose to the positive control group and the experimental group. As a result, as shown in Figures 5 to 7 showed a significant level of blood pressure strengthening effect compared to the hypertensive rats of the negative control group in all experimental groups, the blood pressure increases until 2 d after the start of the administration of the total oyster hydrolyzate and YA peptide In the case of the oyster enzyme hydrolyzate of 10 kD or less, the blood pressure slightly increased until the second parking after the start of administration, but the blood pressure lowering effect was confirmed from 3 (Fig. 5).
또한， 음성 대조군인 고혈압군에 비해 모든 실험군에서 혈중 ACE의 농도가 감소한 것올 확인하였으며 (도 6) , 혈중 안지오텐신 -I I 농도 역시 모든 실험군에서 음성 대조군에 비해 유의적으로 감소하여， 정어리 가수분해물로부터 분리된 혈압 강화 효과를 나타내는 기능성 펩타이드로 알려 져 있는 발린—타이로신을 투여한 양성 대조군에 비하여 현저한 감소 효과를 나타내고， 특히 10 kD 이하 굴 효소 가수분해물의 경우에 정상 대조군과 비슷한 수준을 나타내는 것을 확인하였다 (도 7) . ᅳ  In addition, it was confirmed that the concentration of blood ACE in all the experimental group compared to the negative control group hypertension group (Fig. 6), blood angiotensin -II concentration was also significantly reduced in all experimental groups compared to the negative control group, isolated from the sardine hydrolyzate Compared to the positive control group, valine-tyrosine, which is known as a functional peptide, which is known to have a blood pressure-enhancing effect, was shown to have a remarkable reduction effect, especially in the case of oyster enzyme hydrolysates of 10 kD or less (similar to the normal control group). 7). ᅳ
<실시예 6> 굴 효소 가수분해물 및 기능성 펩타이드의 생체 내 고혈압 예방 효과 확인 Example 6 Confirmation of In Vivo Antihypertensive Effect of Oyster Enzyme Hydrolysate and Functional Peptide
굴 효소 가수분해물로부터 분리 한 기능성 펩타이드가 생체 내에서 나타내는 고혈압 예방 효과를 확인하기 위하여 , 고혈압 쥐에 상기 펩타이드를 투여하고 혈압 상승 억제 효과를 확인하였다.  In order to confirm the antihypertensive effect of the functional peptide isolated from the oyster enzyme hydrolyzate in vivo, the peptide was administered to hypertensive rats and the antihypertensive effect was confirmed.
구체적으로， 12 내지 16 주령의 수컷 위스터 래트을 구입하여 온도 22土 3°C, 습도 50土 5% 및 명암 주기 12 시간의 사육 환경에서 사료 및 식수를 자유롭게 섭취할수 있도록 충분하게 공급하며 1주간 안정화한 다음，무작위로 8 마리씩 하기 [표 기의 조건에 따라 실험군 및 대조군으로 나누었다. 그런 다음， 실험군, 음성 대조군 및 양성 대조군에 고혈압을 유발하기 위해 쥐 몸무게 1 kg 당 니트로 -L-아르기닌 메틸-에스터 (Nitro-L-arginine methyl ester L-NAMA;풀루카사) 40mg씩 매일 정맥 주사하였고，상기 <실시예 1>에서 제조한 굴 효소 가수분해물， 상기 실시예 <5-1>에서 제조한 10 kD 이하 굴 효소 가수분해물， 상기 실시예 <4-2>에서 제조한 YA 펩타이드， 캡토프릴 또는 발린 -타이로신을 매일 경구 투여한 다음， 투여 개시 0, 1， 2， 3 및 4 주 후에 상기 실시예 <5-2>과 동일한 방법으로 수축기 혈압을 측정한 후, 각각의 대조군 및 실험군의 평균 수축기 혈압을 계산하였다. Specifically, male toaster rats 12 to 16 weeks of age were purchased 22 ° 3 ° C, humidity 50 ° 5%, and 12 hours of light and dark cycle, feed and drinking water is sufficient to freely intake, stabilized for 1 week, then randomly 8 dogs [according to the conditions in the table] It was divided into experimental group and control group. Then, daily intravenous injection of 40 mg of nitro-L-arginine methyl ester L-NAMA per 1 kg of rat weight to induce hypertension in the experimental, negative and positive controls. The oyster enzyme hydrolyzate prepared in <Example 1>, the 10 kD or less oyster enzyme hydrolyzate prepared in Example <5-1>, the YA peptide prepared in Example <4-2>, capto After orally administering prill or valine-tyrosine daily, 0, 1, 2, 3, and 4 weeks after the start of the administration, systolic blood pressure was measured in the same manner as in Example <5-2>, and then the control and experimental groups Mean systolic blood pressure was calculated.
【표 7】  Table 7
굴 효소 가수분해물 및 기능성 펩타이드의 생체 내 고혈압 예방 효과 확인올 위한 실험군 및 대조군의 조건  Conditions of Experimental and Control Groups to Determine the Effect of Oyster Enzyme Hydrolysates and Functional Peptides on the Prevention of Hypertension in Vivo
Figure imgf000033_0001
Figure imgf000033_0001
a 양성 대조군 및 실험군에 대한 투여 용량으로 mg/kg에 해당하는 시료의 양을 생리 식염수 2 에 흔합하여 투여하였다. 그 결과， 도 8에서 나타난 바와 같이 정상 쥐에 L-N層 E을 투여하여 고혈압을 유발하였으며， 양성 대조군에 비해 굴 효소 가수분해물 및 기능성 펩타이드이 쥐의 혈압 상승 효과를 유의적으로 감소하는 것을 확인하였다 (도 8).  a The amount of the sample corresponding to mg / kg was administered in combination with physiological saline 2 as the dose to the positive control group and the experimental group. As a result, as shown in FIG. 8, LN 層 E was administered to normal rats to induce hypertension, and it was confirmed that oyster enzyme hydrolyzate and functional peptide significantly reduced the blood pressure raising effect of rats compared to the positive control group (FIG. 8).

Claims

【청구의 범위】 [Range of request]
【청구항 1】  [Claim 1]
서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 안지오텐신 전환효소 (angiotensin converting enzyme； ACE)에 대하여 저해능을 갖는 펩타이드.  Peptides having an inhibitory activity against angiotensin converting enzyme (ACE) consisting of the amino acid sequence set forth in SEQ ID NO: 1 to 12.
【청구항 2】 [Claim 2]
제 1항에 있어서， 상기 펩타이드는 굴 효소 가수분해물로부터 분리된 것을 특징으로 하는 템타이드.  The tempide of claim 1, wherein the peptide is isolated from an oyster enzyme hydrolyzate.
【청구항 3】 [Claim 3]
제 2항에 있어서， 상기 효소는 단백질 가수분해효소 (protease)인 것을 특징으로 하는 ¾타이드.  3. The ¾ tide of claim 2, wherein the enzyme is a protease.
【청구항 4】 [Claim 4]
서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 펩타이드를 포함하는 굴 효소 가수분해물의 분획물을 유효성분으로 함유하는 심혈관계 질환 예방 또는 치료용 약학적 조성물.  A pharmaceutical composition for preventing or treating cardiovascular diseases, comprising as an active ingredient a fraction of an oyster enzyme hydrolyzate comprising a peptide consisting of the amino acid sequences set forth in SEQ ID NOs: 1-12.
【청구항 5】 [Claim 5]
제 4항에 있어서, 상기 분획물은 10 kD 이하인 것을 특징으로 하는 심혈관 질환계 예방 또는 치료용 약학적 조성물.  The pharmaceutical composition for preventing or treating cardiovascular diseases according to claim 4, wherein the fraction is 10 kD or less.
【청구항 6】 [Claim 6]
제 4항에 있어서，상기 심혈관계 질환은 고혈압 심장병,뇌졸증， 혈전증， 협심증, 심부전， 심근경색, 죽상경화증 및 동맥경화로 이루어진 군으로부터 선택된 하나 또는 그 이상인 것을 특징으로 하는 심혈관계 질환 예방 또는 치료용 약학적 조성물. The method of claim 4, wherein the cardiovascular disease is one or more selected from the group consisting of hypertension heart disease, stroke, thrombosis, angina pectoris, heart failure, myocardial infarction, atherosclerosis, and arteriosclerosis. Pharmaceutical compositions.
【청구항 7】 [Claim 7]
서 열번호 1 내지 12로 기 재되는 아미노산 서 열로 구성되는 ¾타이드 증 어느 하나 이상을 유효성분으로 함유하는 심 혈관계 질환 예방 또는 치료용 약학적 조성물 .  A pharmaceutical composition for preventing or treating cardiovascular diseases comprising at least one of ¾ tide syndrome consisting of amino acid sequences of SEQ ID NOs 1 to 12 as an active ingredient.
【청구항 8】 [Claim 8]
서열번호 1 내지 12로 기 재되는 아미노산 서열로 구성되는 펩타이드를 포함하는 굴 효소 가수분해물의 분획물을 유효성분으로 함유하는 심혈관계 질환 예방 또는 개선용 건강 식품 .  A health food for preventing or improving cardiovascular disease, comprising as an active ingredient a fraction of an oyster enzyme hydrolyzate comprising a peptide consisting of amino acid sequences set forth in SEQ ID NOs: 1-12.
【청구항 9】 [Claim 9]
서 열번호 1 내지 12로 기 재되는 아미노산 서 열로 구성되는 펩타이드 중 어느 하나 이상을 유효성분으로 함유하는 심혈관계 질환 예방 또는 개선용 건강 식품 .  Health food for the prevention or improvement of cardiovascular diseases, containing any one or more of the peptide consisting of the amino acid sequence of SEQ ID NOs 1 to 12 as an active ingredient.
【청구항 10】 [Claim 10]
서열번호 1 내지 12로 기 재되는 아미노산 서 열로 구성되는 펩타이드를 포함하는 굴 효소 가수분해물의 분획물올 심혈관계 질환을 가지는 개체에 투여하는 단계를 포함하는 심 혈관계 질환의 치료 방법 .  A method of treating cardiovascular diseases comprising administering to a subject having a cardiovascular disease a fraction of an oyster enzyme hydrolyzate comprising a peptide consisting of the amino acid sequences set forth in SEQ ID NOs: 1-12.
【청구항 11】 [Claim 11]
서 ¾번호 1 내지 12로 기 재되는 아미노산 서열로 구성되는 펩타이드 중 어느 하나 이상을 심 혈관계 질환을 가지는 개체에 투여하는 단계를 포함하는 심 혈관계 질환의 치료 방법 .  A method for treating a cardiovascular disease comprising administering at least one of the peptides consisting of amino acid sequences described in Nos. 1 to 12 to a subject having a cardiovascular disease.
【청구항 12】 [Claim 12]
서열번호 1 내지 12로 기 재되는 아미노산 서 열로 구성되는 펩타이드를 포함하는 굴 효소 가수분해물의 분획물을 개체에 투여하는 단계를 포함하는 심혈관계 질환의 예방 방법 . Peptide consisting of the amino acid sequence of SEQ ID NO: 1 to 12 A method of preventing cardiovascular disease comprising administering to a subject a fraction of an oyster enzyme hydrolyzate comprising.
[청구항 13】 [Claim 13]
서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 펩타이드 중 어느 하나 이상을 투여하는 단계를 포함하는 심혈관계 질환의 예방 방법 .  A method for preventing a cardiovascular disease comprising administering any one or more of the peptides consisting of amino acid sequences set forth in SEQ ID NOs: 1-12.
【청구항 14】 [Claim 14]
심혈관계 질환 예방 또는 치료용 약학적 조성물로 사용하기 위한 서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 펩타이드를 포함하는 굴 효소 가수분해물와 분획물의 용도.  Use of an oyster enzyme hydrolyzate and fraction comprising a peptide consisting of the amino acid sequence set forth in SEQ ID NOs: 1 to 12 for use as a pharmaceutical composition for the prevention or treatment of cardiovascular diseases.
【청구항 15] [Claim 15]
심혈관계 질환 예방 또는 치료용 약학적 조성물로 사용하기 위한 서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 펩타이드 중 어느 하나 이상의 용도.  Use of any one or more of peptides consisting of the amino acid sequence set forth in SEQ ID NOs: 1 to 12 for use as a pharmaceutical composition for the prevention or treatment of cardiovascular diseases.
【청구항 16】 [Claim 16]
심혈관계 질환 예방 또는 개선용 건강 식품으로 사용하기 위한 서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 펩타이드를 포함하는 굴 효소 가수분해물의 분획물의 용도.  Use of a fraction of an oyster enzyme hydrolyzate comprising a peptide consisting of the amino acid sequence set forth in SEQ ID NOs: 1-12 for use as a health food for the prevention or improvement of cardiovascular disease.
【청구항 17】 [Claim 17]
심혈관계 질환 예방 또는 개선용 건강 식품으로 사용하기 위한 서열번호 1 내지 12로 기재되는 아미노산 서열로 구성되는 펩타이드 중 어느 하나 이상의 용도.  Use of any one or more of peptides consisting of the amino acid sequence set forth in SEQ ID NOs: 1 to 12 for use as a health food for preventing or ameliorating cardiovascular disease.
PCT/KR2013/007598 2012-08-24 2013-08-23 Pharmaceutical composition comprising, as active ingredients, peptides which exhibit inhibitory activity against angiotensin-i converting enzyme for preventing or treating cardiovascular diseases WO2014030977A1 (en)

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CN201380054488.XA CN104736552B (en) 2012-08-24 2013-08-23 For prevent or treat angiocardiopathy, comprising pharmaceutical composition of the peptide as active component that inhibitory activity is shown to angiotensin i-converting enzyme
US14/627,642 US10323062B2 (en) 2012-08-24 2015-02-20 Pharmaceutical composition for the prevention and treatment of cardiovascular disease comprising the peptide having the ability to inhibit angiotensin-1 converting enzyme as an active ingredient
US15/856,926 US10308681B2 (en) 2012-08-24 2017-12-28 Pharmaceutical composition for the prevention and treatment of cardiovascular disease comprising the peptide having the ability to inhibit angiotensin-1 converting enzyme as an active ingredient

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CN113197299A (en) * 2021-05-17 2021-08-03 北京德创未来生物技术有限公司 Preparation method of oyster powder with microcirculation improving effect
CN115838400A (en) * 2022-12-27 2023-03-24 中国农业大学 Two small red bean peptides for targeted prevention or treatment of metabolic syndrome
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