KR101701179B1 - Angiotesin I-converting enzyme inhibitory C-terminal tryptophan peptides for treatment of hypertension, and use thereof - Google Patents

Abstract

The peptide having the C-terminus of the peptide exhibiting an inhibitory effect on the angiotensin converting enzyme (ACE) of the present invention as a tryptophan has a remarkable ACE inhibitory effect than the non-added or unsubstituted peptide, It is useful as an active ingredient of a pharmaceutical composition for preventing or treating cardiovascular diseases.

Description

C-terminal tryptophan peptides for the inhibition of angiotensin I-converting enzyme for the treatment of cardiovascular diseases and their use {Angiotesin I-converting enzyme inhibitory C-terminal tryptophan peptides for treatment of hypertension,

The present invention relates to a method for preventing cardiovascular diseases, including a peptide having an inhibitory activity by replacing the C-terminal residue of an angiotensin I-converting enzyme inhibitory peptide obtained from oyster hydrolyzate with a tryptophan And a therapeutic composition.

Cardiovascular diseases, including arteriosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications arising after cardiovascular arterioplasty, cerebral infarction, cerebral hemorrhage, and stroke are on the increase. The cause of hypertension has been largely unknown, but it is known to occur as a complex product of family factors, race, salt intake, genetic factors such as insulin resistance and obesity, or environmental factors such as excessive drinking and aging, Among the various factors of elevation, renin-angiotensin-aldosterone ring is known to play an important role in controlling blood pressure and body fluid volume in vivo (Weiss, D. et al (2001) Circulation 103: 448-454).

Renin-angiotensin system is activated by blood flow, sodium reduction and sympathetic activity increase in kidney, and renin secreted from juxtaglomerular cell of renal artery decomposes angiotensin into angiotensin I, The angiotensin converting enzyme (ACE) converts angiotensin II to vasoconstrictive angiotensin converting enzyme (ACE), resulting in angiotensin II

Controls blood pressure through increased nerve regeneration and aldosterone synthesis. ACE also plays a role in breaking down and inactivating bradykinin, which shows vasodilatory action.

Therefore, a substance that inhibits ACE which greatly affects the rise of blood pressure, or an angiotensin converting enzyme inhibitor (ACE inhibitor) is expected to treat or prevent cardiovascular diseases such as hypertension, heart disease, arteriosclerosis or cerebral hemorrhage There have been reported various clinical studies and experiments to confirm that it is possible to effectively reduce chronic kidney disease, arteriosclerosis, heart attack and death due to such diseases.

Angiotensin I-converting enzyme (ACE) cleaves the dipeptide of His-Leu, the C-terminal end of angiotensin molecule I, to produce an octapeptide involved in vasoconstriction, thereby raising blood pressure. Thus, angiotensin I-converting enzyme plays an important role in cardiovascular function and can inhibit angiotensin I-converting enzyme to treat cardiovascular disease.

Angiotensin I-converting enzyme inhibitors do not act directly on the blood vessels or the heart, but rather on the renin-angiotensin-aldosterone, which lowers blood pressure by boosting blood pressure and inhibiting the system of accumulating salt and water.

Renin system inhibition includes an angiotensin I converting enzyme inhibitor and an angiotensin II receptor blocker (Matchar et al., 2008). Inhibition of angiotensin I converting enzyme is most important in the treatment of hypertension, heart attack, myocardial infarction and diabetic nephropathy (Natesh et al., 2003; Acharya et al., 2003). Most inhibitors of angiotensin I converting enzyme can directly interact with zinc in the catalytic region through enzyme subsites S1, S2 and S2 'hydrogen bonding and hydrophobic interactions (Natesh et al., 2003; Schechter and Berger, 1968).

The importance of biologically active peptides as a starting point for the development of drug and drug related compounds is increasing. Among various bioactive peptides, food-origin inhibitors of angiotensin I converting enzyme have been widely known, but the understanding of the relationship between the structure-activity of peptides inhibiting these enzymes is difficult to predict due to differences among researchers (Wu et al. , 2006).

Quantitative structure-activity correlation (QSAR) is an important part of computer drug design when providing information on medicinal chemistry and drug activity. These correlations are mathematical expressions that relate chemical structures to various physical, chemical, and biological properties of a compound. The resulting formula can predict the properties of the new compound. Recently, QSAR, molecular docking and molecular dynamics simulations have been proposed as a tool for the discovery of angiotensin converting enzyme inhibitor peptides and the design of new enzyme inhibitors. Can be confirmed.

Concerning the substitution of amino acid residues or functional groups of the peptide inhibitors of angiotensin I-converting enzyme, three-dimensional QSAR analysis provided useful information for ACE inhibitors with potent activity towards the C-domain of ACE. Substitution of groups containing phenyl rings and carbon chains in the propionyl and sulfone groups of captopril is essential for improving ACE inhibition (San Juan and Cho, 2005). While low molecular weight amino acids with hydrophobic side chains are preferred at the N-terminus, amino acids with bulky side chains at the C-terminus are preferred (Undenigwe et al., 2012). Starting at the C-terminus, the C-1 position is the most significant position in the pentapeptide model. While C-1 prefers aliphatic and small moieties, the potent ACE inhibitory peptide apparently favored sulfamic and bulky hydrophobic amino acids at C-4 (Sagardia et al., 2013). Tripeptide ACE inhibitory activity is greater than dipeptide, with leucine residues at the N-terminus and proline residues at the C-terminus (Byun and Kim, 2002). The active site of ACE indicates that the substrate for the binding of the inhibitor to the optimal carboxy-terminal amino acid sequence or enzyme is Phe-Ala-Pro. The side chains of these three amino acid residues are presumed to specifically interact with subsite or pocket in the active site of the ACE, called S1, S'2, respectively (Cushman and Ondetti, 1999). Van der Waal's volume, validated charge index and hydrophobic index of the side chain are indicators of the useful structural properties of the peptide QSAR assay (Long et al., 2010).

Molecular docking and kinetic simulation studies are likely to be an area for the selective screening of peptides for ACE (inhibitory peptides) theoretical inhibitory activity (Jalkute et al., 2013). Norri et al (2012) tested the efficacy of AutoDock Vina and LIGPLOT assays with a small intestinal stability model for in silico identification of potent ACE-inhibiting peptides.

Therefore, the present inventors have made efforts to further improve the inhibitory activity of the potent ACE inhibitory peptide obtained from the protein hydrolyzate against the angiotensin I-converting enzyme. As a result, it has been found that when the C-terminal of the ACE inhibitory peptide is replaced with tryptophan, , And the interaction between the angiotensin I-converting enzyme and the inhibitory peptide was investigated through QSAR, molecular docking and molecular dynamics simulation to confirm that the inhibitory effect was remarkable. Thus, the present invention was completed .

An object of the present invention is to provide a peptide having any one of amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 5 and having an angiotensin I-converting enzyme (ACE) inhibiting ability.

Also, the object of the present invention is to provide a pharmaceutical composition for preventing or treating cardiovascular diseases, which contains any one or two or more peptides composed of any one of amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 5 as an active ingredient, A health functional food or a blood pressure lowering agent or a health functional food for lowering blood pressure.

In addition, an object of the present invention is to provide a method for producing a peptide for lowering blood pressure, which comprises the step of adding or substituting tryptophan at the C-terminal in the amino acid sequence of a peptide having an ACE inhibitory activity derived from an oyster hydrolyzate .

In order to achieve the above object, the present invention provides a peptide having any one of amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 5 and having an angiotensin I-converting enzyme (ACE) inhibitory activity .

The present invention also provides a pharmaceutical composition for preventing or treating cardiovascular diseases, which contains any one or two or more peptides composed of any one of amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 5 as an active ingredient, Provide food.

In addition, the present invention provides a blood pressure-lowering agent or a health functional food for lowering blood pressure, which contains any one or two or more peptides composed of any one of amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 5 as an active ingredient.

Further, the present invention provides a method for producing a peptide for lowering blood pressure comprising the step of adding or replacing tryptophan at the C-terminus in the amino acid sequence of a peptide having an ACE inhibitory activity derived from oyster hydrolyzate .

The peptide having the C-terminus of the peptide exhibiting an inhibitory effect on the angiotensin converting enzyme (ACE) of the present invention as a tryptophan has a remarkable ACE inhibitory effect than the non-added or unsubstituted peptide, It is useful as an active ingredient of a pharmaceutical composition for preventing or treating cardiovascular diseases.

Brief Description of Drawings Fig. 1 is a view showing a retention time and a separation form of synthetic peptides (A, TAW; B, YAW; C, VKW; D, KYW; E, AFW) by liquid chromatography.
FIG. 2 is a graph showing the masses of synthetic peptides (A, TAW; B, YAW; C, VKW; D, KYW; E, AFW) obtained by mass spectrometry.
FIG. 3 is a diagram showing discrimination analysis according to ACE inhibition (log IC ₅₀ ) of peptide groups (blue group) and unsubstituted peptides (red group) added with tryptophan at the C-terminus or substituted.
Figure 4 shows a principal component analysis of the key indicators of the peptide on ACE inhibition (log IC ₅₀ ).
FIG. 5 is a diagram showing a PLC (Partial Least Squares) analysis of indicators of a peptide group (blue group) to which tryptophan was added or substituted at the C-terminus.
Figure 6 shows a molecular docking model of an ACE inhibitor and lisinopril.
7 shows a molecular docking model of an ACE inhibitor and VK.
8 shows a molecular docking model of an ACE inhibitor and VKW.
9 is a diagram showing changes in RMSD according to simulation time of ACE / VK / VKW (A) and VK / VKW.

Hereinafter, the present invention will be described in detail.

The present invention provides a peptide having any one of amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 5 and having an angiotensin I-converting enzyme (ACE) inhibitory activity.

The sequence of the peptide is any one or more from the group consisting of VKW (SEQ ID NO: 1), TAW (SEQ ID NO: 2), YAW (SEQ ID NO: 3), KYW (SEQ ID NO: 4) and AFW .

The peptide is preferably separated from the oyster hydrolyzate, but is not limited thereto, and any peptide derived from or synthesized from other substances may be used.

As the separation method, it is preferable to use a separation method customary in the art such as ultrafiltration and chromatography. More specifically, it is preferable to use a chromatography method, more specifically, It is most preferable to use anion exchange chromatography, size exclusion chromatography, reaverse-phase chromatography, or high performance liquid chromatography (HPLC) But is not limited thereto.

As a method for the above synthesis, it is preferable to synthesize by a method of chemical synthesis (WH Freeman and Co., Proteins, structures and molecular principles, 1983) of a conventional peptide in the art. Specifically, the peptide is synthesized by Solution Phase Peptide synthesis ), Solid-phase peptide syntheses, fractional condensation, and F-moc or T-BOC chemical methods are more preferable. More specifically, synthesis by Fmoc solid phase method is most preferable. However, It is not limited. After the synthesis, it is preferable to purify to a purity of 95% or more by reverse phase column chromatography.

The enzyme is preferably a conventional protease in the art per protein, and more preferably protamex or neutrase, but is not limited thereto. The addition of the enzyme is preferably a sequential addition in which the protease is inactivated after the reaction and then the Nutraceutical agent is added, but not limited thereto, and the protease and the Nutraceutical agent can be added at the same time. The addition amount of the enzyme is preferably 0.1-10% of the protein concentration of the enzyme, and the inactivation of the enzyme is preferably inactivated by reacting at 20-100 ° C for 10-120 minutes, but not limited thereto. And the like.

In a specific example of the present invention, the present inventors measured the inhibitory activity on the angiotensin converting enzyme (ACE) of the synthesized peptides, and found that when the tryptophan residue was added or substituted, (See Table 1). As a result, it was confirmed that the peptide synthesized did not show toxicity to the normal hepatocyte Chang cells (Table 2 and Fig. 4). As a result of examining the components of the synthesized peptides, it was confirmed that the linear discriminant function was 0.3041 × 1 - 0.0852 × 2, the misclassification ratio was 0/10 and the hit ratio was 100% (see FIG. 3) And 2 were found to contribute to the value of log IC ₅₀ as KYW>VKW>AFW>YAW> TAW, in which the tryptophan-containing peptide at the C-terminal residue was significantly higher in ACE inhibition than the peptide of the non-tryptophan residue (See Table 3, Figs. 4 and 5). I checked whether the molecular docking of peptides, Captopril, enalaprilate and Phe-Ala-Pro similar is water three ACE inhibitors are captopril in the in the active site catalyst of ACE Zn ^{2 +} is a thiol (thiol) groups, enalaprilate and lisinopril are the carboxyl group and direct interaction was confirmed that the chelate Zn ^{+ 2} (see Table 4, Figs. 6, 7 and 8). Molecular dynamics simulations of the peptides revealed that RMSD for the complex between the ACE and the inhibitors represents the structure of the paralleled complex system (see FIG. 9). When the tryptophan was added to the C-terminus of VK, the interaction between ACE and VKW was greatly changed compared with that of VK, and the interaction with Zn was weakened. As a result, the amino acid residues of ACE and tryptophan It was confirmed that the interaction of hydrophobic residues contributes to the improvement of ACE inhibitory activity by inducing an increase in total interaction energy (see Table 5).

In addition, the peptides of the present invention can be produced by a genetic engineering method as described below. 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, DNA sequences may be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer (e.g., Biosearch or Applied iosystems). The DNA sequence is operatively linked to insert into a vector comprising one or more expression control sequences (e.g., promoters, enhancers, etc.) that regulate the expression of the DNA sequence, and the host cell is transformed with the recombinant expression vector formed therefrom Subsequently, the resulting transformant is cultured under conditions and under conditions suitable for expression of the DNA sequence, and the substantially pure peptide encoded by the DNA sequence is isolated from the culture by a method known in the art , Chromatography). By "substantially pure peptide" is meant that the peptide according to the invention is substantially free of any other proteins derived from the host. Genetic engineering methods for peptide synthesis of the present invention can be found in the following references: Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor Laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., Second (1998) and Third (2000) Edition; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif., 1991; And Hitzeman et al., J. Biol. Chem., 255: 12073-12080,1990.

The peptide of the present invention can be administered parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation. Parenteral administration may mean administration through an administration route other than oral, such as rectal, intravenous, peritoneal, muscular, arterial, transdermal, nasal, inhalation, ocular and subcutaneous. That is, the peptide of the present invention can be administered in various forms of parenteral administration. In the case of formulation, the peptide is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant and the like. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. As a suppository base, witepsol, macrogol, tween 61, cacao paper, Liu ling, glycerogelatin and the like can be used.

In addition, the peptide of the present invention can be used in combination with various carriers permitted as pharmaceutical agents, such as physiological saline or an organic solvent, and can be used in combination with a carbohydrate such as glucose, sucrose or dextran, Antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as pharmaceuticals. 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 can be administered once to three times a day.

The total effective amount of the peptides of the invention can be administered to a patient in a single dose, such as by bolus form or by infusion for a relatively short period of time, And administered by a fractionated treatment protocol. Since the concentration of the effective dose of the patient is determined in consideration of various factors such as the route of administration and the number of treatments as well as the age and health condition of the patient, in view of this point, Lt; RTI ID = 0.0 > of < / RTI > novel peptide as a pharmaceutical composition.

Also provided is a pharmaceutical composition for preventing or treating cardiovascular diseases or a blood pressure lowering agent comprising any one or two or more peptides composed of any one of amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 5 as an active ingredient .

Wherein the cardiovascular disease is selected from the group consisting of hypertension, heart disease, stroke, thrombosis, angina pectoris, heart failure, myocardial infarction, cerebral infarction, cerebral hemorrhage, ischemic heart disease, complications arising after perianal artery arteriopathy, atherosclerosis and arteriosclerosis But is not limited thereto.

Accordingly, the peptide comprising the amino acid sequence of SEQ ID NOS: 1 to 5 of the present invention can be effectively used as an active ingredient of a pharmaceutical composition for the prevention or treatment of cardiovascular diseases.

The compositions of the present invention may be of various parenteral formulations. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used.

More specifically, the pharmaceutical composition may further contain, in addition to the active ingredient, a nutritional agent, a vitamin, an electrolyte, a flavoring agent, a coloring agent, a stabilizer, a pectic acid and a salt thereof, an alginic acid and a salt thereof, an organic acid, a protective colloid thickening agent, A preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated drink, and the like. The carrier, excipient or diluent may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Selected from the group consisting of water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, physiological saline One or more, but is not limited thereto, and any conventional carrier, excipient or diluent may be used. The components can be added to the peptides of the invention either independently or in combination.

Further, the pharmaceutical compositions of the present invention may be formulated using any of the known methods known in the art or by methods disclosed in Remington ' s Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA) .

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, and the effective dose level will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, and the daily dose is the amount of Saururus chinensis extract The dose is 0.01 to 1000 mg / kg, preferably 30 to 500 mg / kg, more preferably 50 to 300 mg / kg, and can be administered 1 to 6 times a day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

Also, the present invention provides a health functional food for prevention or improvement of cardiovascular diseases or a blood pressure lowering health food containing any one or two or more peptides composed of any one of amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 5 as an active ingredient do.

Wherein the cardiovascular disease is selected from the group consisting of hypertension, heart disease, stroke, thrombosis, angina pectoris, heart failure, myocardial infarction, cerebral infarction, cerebral hemorrhage, ischemic heart disease, complications arising after perianal artery arteriopathy, atherosclerosis and arteriosclerosis But is not limited thereto.

Therefore, the peptide consisting of the amino acid sequence of SEQ ID NOS: 1 to 5 of the present invention can be effectively used as an active ingredient of a health functional food for preventing or ameliorating cardiovascular diseases.

In the present invention, the term " health functional food " means a food group imparted with added value to function and express the function of the relevant food by physical, biochemical or biotechnological techniques, Prevention and recovery of the body. The health functional food of the present invention preferably has a function of controlling the body in preventing or improving cardiovascular diseases. Means foods that can be fully expressed. The health functional food may include food-acceptable food supplementary additives, and may further include suitable carriers, excipients and diluents conventionally used in the production of health functional foods.

The peptides of the present invention can be added directly to the food or can be used together with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of the peptide in the health food may be 0.1 to 90 parts by weight of the total food. However, in the case of long-term ingestion intended for health and hygiene purposes or health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount in the above range.

The health functional beverage composition of the present invention is not particularly limited to the other ingredients other than those containing the peptide as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 of the composition of the present invention.

In addition to the above, the peptide of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the peptides of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the peptide of the present invention.

In addition, the present invention provides a method for producing a peptide for lowering blood pressure comprising the step of adding or substituting tryptophan at the C-terminal in the amino acid sequence of a peptide having an ACE inhibitory activity derived from oyster hydrolyzate .

The peptide having ACE inhibitory activity is TAY, VK, KY, YA and AFY.

Therefore, the present invention can be effectively used for the prevention or treatment of cardiovascular diseases by using a peptide preparation method in which a C-terminal is added or substituted with tryptophan.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

It should be noted, however, that the following examples and experimental examples are illustrative of the present invention, and the present invention is not limited to the following examples and experimental examples.

< Example  1> Peptides  Confirmation of synthesis and purity measurement

Ten peptides that did not replace the C-terminus of the peptide (ACE inhibitor) that inhibits angiotensin I-converting enzyme with tryptophan were used as data. The in vitro inhibition activity IC ₅₀ values were used as log substitutions to reduce the heterosceductivity of the data (Table 1). ACE (5 units from rabbit lung), hippuryl-histidyl-leucine (HHL), captopril and lisinopril were purchased from Sigma (St. Louis, MO, USA) and the peptides TAY, VK, KY, YA , AFY, TAW, VKW, KYW, YAW and AFW were synthesized by GL Biochem (Shanghai, China). HPLC grade acetonitrile and methanol were used, and reagent grade was used for all other reagents. A Waterchers 120 ODS-AP (5 μm, 4.6 × 250 mm) C18 column was purchased from Daiso (Tokyo, Japan) for analysis.

The synthetic peptides were synthesized by Fmoc solid phase method and purified by reverse phase column chromatography to a purity of 95% or more. The mass of the purified peptide was confirmed by an electron spray ion mass spectrometer

Peptides MF MW LogP IP BP MP WS IC ₅₀ ,
uM LogIC ₅₀ TAY C16H23N3O6 353.38 -0.58 6.02 676.71 332.75 2880 16.7 1.223 VK C11H23N3O3 245.32 -0.43 9.69 471.14 302.74 2435 29.0 1.462 KY C15H23N3O4 309.37 0.10 9.30 573.92 317.74 2131 51.5 1.712 YA C12H16N2O4 252.27 0.33 6.02 508.34 308.17 4546 93.9 1.973 AFY C21H25N3O5 399.45 1.48 6.02 716.55 338.56 973.6 75.6 1.879 TAW C18H24N4O5 376.41 -0.52 6.02 724.66 339.75 188.9 0.63 -0.201 VKW C22H33N5O4 451.53 0.49 9.69 762.14 345.22 266.6 0.02 -1.699 KYW C26H33N5O5 495.57 1.02 9.30 864.92 349.84 212.5 0.43 -0.367 YAW C23H26N4O5 438.48 1.25 6.02 799.34 349.84 491.9 0.49 -0.310 AFW C23H26N4O4 422.20 1.53 6.02 764.50 345.56 63.41 1.55 0.190

MF, molecular formula; MW, molecular weight; IP, isoelectric point; BP, boiling point; WS, water solubility at 25 ° C (mg / L), MP, melting point

LogP is calculated by Hyperchem version 8.0.

The isoelectric point is calculated at http://www.genscript.com/sni-bin2/peptide_calculation.cgi .

The boiling point, melting point and solubility are calculated by the EPI Suite.

As a result, as shown in Fig. 1, among the peptides having effective ACE inhibitory activity obtained from the oyster hydrolyzate, the dipeptide has the C-terminal amino acid residues and the last residue at the C-terminal in the case of the tripeptide The tryptophan-containing peptides synthesized by replacing tryptophan with a tryptophan showed a distinct peak but a minor peak due to the impurity in the reversed phase column of C18 (Fig. 1). The purity of the peptides calculated for each peak area was found to be greater than 95%. [M + H] +, which is the mass of each peptide synthesized and purified, was 376.72, 438.96, 432.15, 496.15 and 422.94, respectively, and the theoretical masses 376.41, 438.48, 432.15, 495.57, 422.20 and 1 Da (Fig. 2). Especially, 1 Da error occurred in VKW, but it was judged that there was no big problem when compared with the purification by peak on HPLC chromatogram.

< Experimental Example  1> Of peptide Angiotensin  I-converting enzyme ( angiotensin  converting enzyme; ACE) &lt; / RTI &gt;

In order to determine whether the peptide synthesized in Example 1 had inhibitory activity against angiotensin I-converting enzyme, the following experiment was conducted.

Specifically, the ACE inhibitory activity was determined by the method of Wanasundara et al. (2002) using HHL as a substrate and the content of hippuric acid (HA) isolated from high performance liquid chromatography (HPLC system, Shimadzu, Kotyo, Japan) Were measured. The buffer for reagent preparation was 50 mM tris-HCl (pH 8.3). 50 μl of 3.0 mM HHL, 50 μl of the enzyme solution (50 μl) and 50 μl of the sample solution were added to the test tube, allowed to stand in a constant temperature water bath at 37 ° C for 30 minutes, allowed to react, and reacted for 30 minutes while shaking. 150 μl of glacial acetic acid was added to stop the reaction, and the content of hippuric acid was measured by HPLC. For the quantification of HA, 10 μl of a reaction solution equipped with a Watchers C18 reversed phase column was injected and a peak was detected at 228 nm while eluting with 12.5% (v / v) acetonitrile solution (pH 3.0). As a control, 50 μl of buffer was used instead of the sample, and the inhibition of enzyme activity was calculated by the following equation. The ACE inhibitory activity of each sample was measured, and the IC ₅₀ was defined as the concentration required to inhibit 50% of the ACE activity after linear regression analysis of inhibitory activity by concentration.

     ACE inhibition (%) = (HA of the control HA sample) / HA x 100 of the control

As a result, as shown in Table 1, the logarithm of the partition coefficient of octanol to water of the tripeptide added or substituted with tryptophan was all increased, and the result showed that the tryptophan residue The hydrophobicity of the &lt; / RTI &gt; On the other hand, the melting point increased slightly while the breaking point increased, but the isoelectric point did not change. In vitro ACE inhibition increased 27-1450 fold with the addition or replacement of tryptophan residues. In particular VKW increases 1450-fold as compared to the VK exhibited IC ₅₀ value of 0.02 uM corresponding to 1/20 compared to the IC50 value of 0.0011 uM captopril being used as new drugs for the treatment of hypertension. These results suggest that tryptophan addition or substitution of C-terminal residues is effective for improving ACE inhibition of peptides.

< Experimental Example  2> Of peptide  Cytotoxicity measurement

The following experiment was conducted to examine whether the peptides synthesized in Example 1 were toxic to cells.

Specifically, the cells were cultured in DMEM medium containing 10% FBS at 37 ° C to maintain 5% CO ₂ . At this time, antibiotics for medium were used to suppress microbial contamination and lunch. After the cells were covered with 80% of the dish, they were washed with phosphated-buffered saline-EDTA (PBS-EDTA) and trypsinized to subculture. Cells were cultured by changing the medium every 48 hours. Samples were dissolved in DMEM (FBS free) medium in 0.2% PBS solution. The final concentration of the sample was tested in the concentration range of 1 ~ 200 ㎍ / ㎖. To determine the cytotoxicity using MTT assay, 100 μl of HepG2 was dispensed in a 96-well plate at a cell concentration of 1 × 10 ⁵ cells / ml, cultured for 24 hours for cell attachment, replaced with FBS free medium supplemented with the sample, Lt; / RTI &gt; After washing twice with PBS, the plate was treated with MTT reagent and absorbance was measured with a microplate reader (VICTOR X multilabel plate readers, Perkin Elmer 1420, Waltham, MA, USA) at 490 nm.

As a result, as shown in Table 2 and FIG. 4, toxicity to Chang cells, which is a normal hepatocyte, was not observed in all of the ACE inhibitory peptides used in the experiment, and the number of cells was slightly decreased along with the increase in the peptide KYW , And there was no significant difference from the control group (Table 2, p <0.5). These results show that the addition or replacement of tryptophan residues does not induce cytotoxicity. However, it is necessary to confirm in vivo (in viv o) which is capable of ensuring the safety of the same.

< Experimental Example  3> Of peptide  Discrimination, principal component and PLS  analysis

In order to investigate the components of the peptides synthesized in Example 1, the following experiment was conducted.

Specifically, in the case of a potent ACE inhibitory dipeptide obtained from an oyster hydrolyzate, the addition of a tryptophan at the C-terminus and the substitution of a tryptophan residue at the C-terminal amino acid in the case of a tripeptide The effect on in vitro ACE inhibitory activity was determined.

Between each index obtained as a result of docking between each peptide and ACE by SYBYL (Table 3) and Hellberg et al. (1987) performed principal component analysis according to the indices describing the variation of the amino acid sequence in the peptide by three main components z1, z2 and z3 at various amino acid positions. The discriminant analysis and principal component analysis were linearly discriminated by the JMP package (ver. 10, SAS Institute, Cary, NC, USA). To determine whether the addition or substitution of tryptophan residues contributes to the enhancement of the ACE inhibition of the peptide, the canonical 1 (x1) was used as the log IC ₅₀ value and the canonical 2 (x2) was used by Hellberg et al. The discrimination analysis was performed by applying z2 and z3 according to the amino acid position of the proposed tripeptide to the terminal residues of the amino acid to be tested. The regression tables and observations containing the group mean were located at the opposite sites to the group containing the tryptophan residues and the group not including the tryptophan residues to correctly discriminate the peptides within the experimental range.

As a result, as shown in FIG. 3, the linear discriminant function was 0.3041 × 1 - 0.0852 × 2. The false classification rate was 0/10 = 0% and the accuracy rate was 100%. However, this result is related to the 10 peptides used in this study, and it may be necessary to test the misclassification rate by using a large number of data to obtain a more accurate discriminant function (FIG. 3).

Further, log IC ₅₀ On the other hand, principal component analysis was performed regardless of the measurement unit by using the sample correlation correlation index for each peptide index obtained through molecular docking for the setting of the index contributing to the value.

As a result, as shown in Table 3, the main components 1 and 2 can represent 76.1% with respect to the LogIC ₅₀ value, and the cumulative contribution ratio of the main components 1, 2, 3 and 4 was 96.9%. The first main component because it means a log IC ₅₀ value is the degree of delayed in the principal component score of the main component 1 and 2 contribute to the log IC ₅₀ value KYW>VKW>AFW>YAW> as TAW is the order of the IC ₅₀ value The tryptophan residue at the C-terminus significantly contributes to the enhancement of ACE inhibition, as the peptide having tryptophan as the C-terminal residue is much higher than the peptide of the viptorptane residue. PLS analysis showed that the C-terminal residue, the N-terminal residue, and the amino acid sequence as the middle residue of the peptide had the greatest effect on the log IC ₅₀ value, and the peptide D score (score)> G score> CHEM score. Therefore, log IC ₅₀ = -0.1346 (p3z3) +0.1179 (p3z1) -0.1059 (p3z2) +0.1068 (D score) was 97.8%.

< Experimental Example  4> Confirmation of molecular docking

In order to confirm whether or not the peptide of the present invention is docked, the following experiment was conducted.

Specifically, the human ACE structure was obtained from a protein data bank (PDB Id, IO86), and all substrates and Cl ions were removed with the SYBYL software package (Tripos International, St. Louis, MI, USA) (Fig. 1), energy minimization was performed for each molecule with Tripos force field using Powell conjugate optimization algorithm with a convergence criterion of 0.05 kcal / mol with SYBYL software. A docking program of the same program was used to perform a surflex-dock for each peptide molecule. During docking, lisinopril obtained from the IO86 protein complex was used as a ligand and the top 20 structures based on the dock score values (chem score, C score, D score, G score, PMF score, total score) Respectively. The highest dock score among the 20 structures was selected and the interaction between the protein residues and the peptides was analyzed with Discovery Studio 3.5.

As a result, as shown in Table 4 and FIG. 6 to FIG. 8, Lisinopril contained an additional lysine and phenyl group as compared with captopril as an extremely effective ACE inhibitor. The carboxyl group of the lisinopril chelates the zinc of the ACE (Table 4, Fig. 6). Molecular docking results of this study are mutually directly with Captopril, enalaprilate and Phe-Ala-Pro similar is water three ACE inhibitors are captopril in the in the active site catalyst of ACE Zn ^{2 +} is a thiol (thiol) groups, enalaprilate and lisinopril is carboxyl And Zn ^{2 +} was chelated by the action of Zn ²⁺ .

Subtle differences are observed in other areas of the bond pocket. The detailed description of the molecule provided by the ACE-inhibitor structure is expected to improve the combination and design of detailed new and more potent ACE domain-specific inhibitors that can be provided as a new generation of antihypertensive drugs (Natesh et al. 2004). In the C-ACE complex, the second molecule, the isoxazole pehnyl group, produces a strong pi-pi stacking interaction with the aminobenzyl group of the first molecule that docked them in the 'hand-shake' structure. The abnormal architecture and flexibility of the C-ACE active site can be used in the structural-based design of novel C-ACE or vasopeptidase inhibitors (Schwager et al., 2011). VKW with tryptophan at the C-terminal compared to the ACE inhibitory peptide VK has an additional hydrogen bond with GLN 281 and Tyr 523 between the ACE and the inhibitor VKW, a pi-pi interaction with LYS 454 and GLY 2000, It is estimated that the ACE inhibitory activity (IC ₅₀ value) is increased by 1450 times (Table 4 and FIG. 8 ) by more tightly binding by Waals interaction.

ACE inhibitors exclusively bind to the S1 and S2 binding pockets of AnCE through ionic and hydrogen bonding interactions (Akif et al., 2010). Although lisW-S appears to have a similar binding pattern to the parent compound lisinopril, P2 'tryptophan takes a different structure from that which can be exerted by other inhibitors with tryptophan residues at this position. Kinetic analysis shows a 258-fold domain selectivity due to the cooperative effect of C-domain specific residues at S2 'subsite. The high affinity and selectivity of these inhibitors make them the leading compounds of good drugs for cardiovascular drug development (Watermeyer et al., 2010).

< Experimental Example  5> Molecular dynamic simulation measurement

Molecular dynamics simulations were performed to obtain a more precise alignment of the complex structure obtained through molecular docking, in order to elucidate the ACE-ligand complex structure as well as the side chain sequences. The ACE inhibitor was tested for MD simulation, the crystal structure was started over the orbit and the skeletal RMSD was analyzed to check the ACE structure

Specifically, the complexes obtained from the Docking study were subjected to molecular dynamics simulations at 10 ns using the GROMACS 4.5.3 package with the AMBER03 force field, which can be run on a Linux cluster computer with high performance (Hess et al ., 2008). Phase files for the inhibitors were generated using ACPYPE (AnteChamber Python Parser interface) (Sousa da Silva and Vranken, 2012). The structure was solvated in a dodecahedron of 1 nm in length and generated a TIP3P water model to simulate in an aqueous environment (Berrendesen et al., 1981; Jorgensen et al., 1983). To ensure the overall neutrality of the simulation system, 10 Na ⁺ ions were added to replace the water molecules. The energy minimization process was performed stepwise to the limit of 1000 KJ / mol. Energy-minimized systems were processed for 100 ps in parallel. (Bussi et al., 2007; Parrinello and Rahman, 1981) achieved a V-rescale thermostat and a Parrinello-Rahman barostat at 300 K and 1 bar. Particle mesh Ewald (PME) method (Essmann et al., 1995) was applied to accurately measure long-range electrostatic interactions. The combination of heavy metals and corresponding hydrogen atoms was constrained to their parallel bond length using the LINC21 algorithm (Hess et al., 1997). The time for the simulation was set to 2 fs, and the data in the production phase was written in ps.

As a result, RMSD for the ACE / inhibitor complex showed the structure of a well paralleled composite system after 5 ns of MD simulation, as shown in Fig. The RMSD for the representative ACE / VKW is lower than the ACE / VK, which indicates that the ACE / VKW is more stable than the ACE / VK during the 10 ns md simulation (FIG. 9A). Conversely, the RMSD for VKW is larger than VK, which means that the structure of VKW is much larger than the structure of VK (FIG. 9B).

< Experimental Example  6> Confirmation of energy measurement between ACE and inhibitor

In order to measure the interaction energy between the ACE and the inhibitor, the following experiment was conducted.

Specifically, covalent bonds and other noncovalent interactions do not involve electron sharing, but involve more dispersed variation of electromagnetic interactions within molecules. Non-covalent interactions are not only important in maintaining the three-dimensional structure of large molecules, but they are also involved in numerous biological processes in which large molecules specifically bind. Non-covalent interactions can be generally categorized into four categories: static, π-effect, van der Waals force, and hydrophobic effect. Coulomb effective energy (electrostatic action) and Lennard-Jones (LJ) effective energy van der Waals action in Gromacs. ACE and a total non-covalent interaction energy (E _total) between the inhibitor is between the third section, that is, the Coulomb effective energy between ACE with inhibitors other than the Coulomb (E _coul), ACE with inhibitors excluding Zn LJ effective energy (E _LJ ) And the coulombic energies between Zn and the inhibitor (E _Zn ).

As a result, as shown in Table 5, E _total of ACE / VKW is larger than ACE / VK over 5 ns simulation. The results like Dl indicate that the interaction of ACE and VKW is stronger than VK, contributing to the stability of the complex. According to the total energy composition, the E _Zn of ACE / VK is larger than that of ACE / VKW, but E _coul and E _LJ are smaller. The above results indicate that the interaction between ACE and VK is preferentially E _Zn and the contribution of E _coul , E _LJ and E _Zn to total energy is similar. The addition of tryptophan to the C-terminus of VK significantly altered the interaction between ACE and VKW compared to VK, but the interaction with Zn was weakened, but the hydrophobic effect and the hydrophobic effect of ACE and the hydrophobic residues of tryptophan, Interactions can induce an increase in total interaction energy and contribute to the improvement of ACE inhibitory activity (Table 5).

Claims (10)

A peptide consisting of any one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 5, as an active ingredient.
The method according to claim 1, wherein the cardiovascular disease is selected from the group consisting of arteriosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications arising after cardiovascular artery arthroplasty, cerebral infarction, cerebral hemorrhage, and stroke Or a pharmaceutically acceptable salt thereof.
The pharmaceutical composition for preventing or treating cardiovascular diseases according to claim 1, wherein the peptide is isolated from oyster hydrolyzate.
A health functional food for preventing or ameliorating cardiovascular diseases, which contains, as an active ingredient, any one or two or more peptides consisting of any one of amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 5.
5. The method of claim 4, wherein the cardiovascular disease is selected from the group consisting of arteriosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications arising after cardiovascular artery arthroplasty, cerebral infarction, cerebral hemorrhage, and stroke A health functional food for prevention or improvement of cardiovascular diseases.
5. The health functional food for preventing or improving cardiovascular diseases according to claim 4, wherein the peptide is isolated from an oyster hydrolyzate.
A peptide consisting of any one of amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 5 as an active ingredient.
A health functional food for lowering blood pressure comprising any one or two or more peptides composed of any one of amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 5 as an active ingredient.
A tryptophan may be added to the C-terminus of the dipeptide VK, KY or YA in the amino acid sequence of the peptide having an angiotensin I-converting enzyme (ACE) inhibitory activity derived from an oyster hydrolyzate, or a tripeptide TAY Or replacing the C-terminal tyrosine of AFY with tryptophan.
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