KR101756362B1 - Composition having antihypotensive effect and preperation method of the same - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for hypotensive, a food composition and a method for producing the same, which have a peptide derived from hydrocracked hydrocracker, which comprises a peptide having an amino acid sequence of Leu-Trp-His-Thr- A composition having a reducing activity can be provided.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for lowering blood pressure and a method for manufacturing the same,

The present invention relates to a pharmaceutical composition for lowering blood pressure, a food composition and a preparation method thereof, and relates to the hypotensive pharmacological activity of a peptide derived from hydrocracked hydrocracked material.

[Background Art] [0002] In recent years, efforts have been made to obtain a substance having antihypertensive effect from natural substances in hypertension formulations, which have been produced through conventional chemical synthesis methods, due to problems such as side effects. In addition, in order to search for new substances that can be used in diverse applications such as pharmaceuticals and food materials such as enzymes and health supplements, researches on natural living organisms, especially those that are capable of mass culture and breeding, .

Marine organisms have a unique metabolic process and unique environment that are not found in terrestrial organisms, and thus have a wide range of novel bioactive substances. In addition, there has been a lot of research on terrestrial life, but marine life has not been sufficiently studied and it is also a field of high expectation for the development of new useful natural materials.

Among them, Styela clava is used only for food in the world, and it is important for the biomaterial industry because it is unique in that it is easy to mass-produce and has a very high added value as a material. For example, in the Journal of the Korean Society of Food Science and Nutrition, Vol. 35, No. 3, describes the antioxidant and anticancer effects according to the processing methods and solvents of healdock. In the Journal of the Korean Society of Food Science and Nutrition, Vol. 36, No. 10, And the protection effect against leukocyte and liver damage. The Korean Society of Food Science and Nutrition 37 (12) describes the antioxidant activity of each part of hederock.

However, given the potential of Midduck, which is capable of mass culture and contains a variety of physiologically active ingredients, there is a continuing need for research to obtain a composition that can enhance the health of the human body, such as medicines or functional foods, It is true.

Korean Patent No. 10-1150425

An object of the present invention is to provide a pharmaceutical composition, a food composition, and a method for producing the same which have peptides derived from hydrocracked hydrocrackers.

In order to achieve the above object, the pharmaceutical composition for lowering blood pressure according to an embodiment of the present invention comprises a peptide having an amino acid sequence of Leu-Trp-His-Thr-His as an active ingredient.

The food composition according to another embodiment of the present invention contains a peptide having an amino acid sequence of Leu-Trp-His-Thr-His as an active ingredient.

An angiotensin I converting enzyme (ACE) inhibitor according to another embodiment of the present invention includes a peptide having an amino acid sequence of Leu-Trp-His-Thr-His.

A method for producing a composition comprising an amino acid sequence of Leu-Trp-His-Thr-His according to another embodiment of the present invention and comprising a peptide as an active ingredient, comprises hydrolyzing the helix using a digestive enzyme, ; A separation step of separating the hydrocracked hydrocracked product by protein size and separating an active fraction having an inhibitory effect on angiotensin I converting enzyme (ACE) from the separated fraction; And a composition comprising the peptide having the amino acid sequence of Leu-Trp-His-Thr-His from the active fraction as an active ingredient is prepared.

Hereinafter, the present invention will be described in more detail.

The pharmaceutical composition for lowering blood pressure according to an embodiment of the present invention includes a peptide having an amino acid sequence of Leu-Trp-His-Thr-His as an active ingredient.

The peptide having the amino acid sequence of Leu-Trp-His-Thr-His (LWHTH) is a peptide derived from pepsin hydrolyzate of Styela clava . As a result of the oral administration test using congenital hypertensive rat model, Has pharmacological activity equivalent to or superior to that of captopril.

In addition, the peptide (LWHTH) is stable to the digestive enzyme even when orally administered, and the structure or amino acid sequence of the peptide may not be changed by digestive enzymes.

The peptide (LWHTH) may bind to ACE to form an ACE-LWHTH complex.

ACE (Angiotensin I converting enzyme) is an enzyme that converts di-peptide (angiotensin, His-Leu) from angiotensin I, which is a decapeptide, into angiotensin II with vasoconstrictive action. An increase in angiotensin II promotes hypertension by promoting strong hypertension and secretion of the antidiuretic hormone aldosterone, inhibiting the excretion of water and sodium, and increasing circulating blood volume. ACE also degrades and inactivates vascular relaxant bradykinin, which in turn results in elevated blood pressure.

Therefore, ACE inhibitors can inhibit vasoconstriction by inhibiting the action of ACE, thereby exhibiting hypotensive action, thus improving hypertension.

The peptide (LWHTH) binds to ACE to form an ACE-LWHTH complex, and a hydrogen bond, a pi bond, and an electrostatic bond may be contained in the complex. Specifically, Arg522, which is an amino acid of ACE, is capable of hydrogen bonding with Tryptophan and Threonine, Glu403 with Leucine and hydrogen bonding, and Lys118 with pi-bonding with benzene ring of Tryptophan. Furthermore, Arg522 is an amino acid which constitutes the active site of ACE. The peptide (LWHTH) directly binds to the active site of ACE and inhibits ACE activity.

Histidine, located at the C-terminal of the active peptide, plays an important role in binding to the active site of the enzyme. Histidine located at the C-terminal of the LWHTH peptide directly blocks the enzyme active site, and LWHTH peptide binds to the native substrate To inhibit the activity of ACE.

The blood pressure lowering effect caused by the peptide (LWHTH) may alleviate essential hypertension and / or secondary hypertension.

The blood pressure lowering effect of the peptide (LWHTH) can lower both the systolic blood pressure and the diastolic blood pressure, and particularly has a rapid and excellent blood pressure lowering effect.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier or excipient. For example, the pharmaceutical compositions may be formulated as tablets, capsules, elixirs, suspensions, syrups, and the like, mixed with conventional pharmaceutical carriers and excipients, , ≪ / RTI > and / or wafers, and the like.

The pharmaceutical composition may comprise, but is not limited to, about 0.1 to 90% by weight, more usually about 10 to 30%, of active compound.

The pharmaceutical composition may be in the form of corn starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, and / or alginic acid lt; RTI ID = 0.0 > alginic < / RTI > acid, and excipients. Croscarmellose, microcrystalline cellulose, corn starch, sodium starch glycolate, and / or alginic acid may be used as the disintegrant commonly used in the pharmaceutical composition, but are not limited thereto.

In general, the liquid composition may optionally be mixed with a non-aqueous solvent, such as, for example, ethanol, glycerin, sorbitol, and polyethylene glycol, together with a suspending agent, a solubilizer (such as cyclodextrin), a preservative, a surfactant, a wetting agent, But may not be limited to, suspensions or solutions in suitable liquid carriers such as oils or water. On the other hand, liquid preparations can be prepared from reconstitutable powders. For example, powders containing active compounds, suspensions, sucrose and sweeteners may be reconstituted to form suspensions with water, and syrups may be prepared from powders containing the active ingredient, sucrose and sweetener, .

Compositions in tablet form may be prepared using any pharmaceutical carrier conventionally used in preparing solid compositions. Examples of such carriers may include, but are not limited to, binders such as magnesium stearate, starch, lactose, sucrose, microcrystalline cellulose and polyvinylpyrrolidone. Tablets may also include a color film coating, or may include, but are not limited to, color as a part of the carrier. Additionally, the active compound may be, but is not limited to, a tablet containing a hydrophilic or hydrophobic matrix and being formulated in a controlled release dosage form.

Pharmaceutical compositions in the form of capsules may be prepared, for example, by incorporating the active compounds and excipients into hard gelatine capsules using conventional encapsulation procedures, but are not limited thereto. Alternatively, semisolid matrices of active compounds and high molecular weight polyethylene glycols can be prepared and filled into hard gelatine capsules, or solutions or edible oils of the active compounds in polyethylene glycol, for example liquid paraffin or fractionated a suspension of fractionated coconut oil may be prepared and filled into soft gelatine capsules, but this is not so limited.

Tablet binders include, but are not limited to, acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylmethylcellulose, sucrose, starch and / or ethylcellulose. Lubricants that may be used include, but are not limited to, magnesium stearate or other metal stearate, stearic acid, silicone fluids, talc, waxes, oils and / or colloidal silicas. have. Fragrance such as peppermint, oil of wintergreen, cherry flavor, and the like may also be used, but may not be limited thereto. In addition, a coloring agent may be added to render the dosage form more apparently visible or to help identify the product.

The pharmaceutical composition may optionally be freeze-dried, and then reconstituted with a suitable solvent immediately prior to administration, but may not be limited thereto.

The pharmaceutical composition may be in powder or solution form.

The pharmaceutical composition may be oral, intraperitoneal, intravenous, intramuscular, and / or subcutaneous.

The food composition according to another embodiment of the present invention includes a peptide having an amino acid sequence of Leu-Trp-His-Thr-His as an active ingredient and has a hypobarbital lowering function. Descriptions of the peptides and their functionalities are redundant, so the description thereof is omitted.

The food composition may be used in the form of a health functional food, a nutritional supplement, a health food, or a food additive, and may be variously used for foods and beverages. Examples of the food composition include various foods such as beverages, gums, tea, vitamin complexes, and health supplement foods, and they can be used in the form of pills, powders, granules, infusions, tablets, capsules or beverages. At this time, the amount of the extract in the food or beverage may generally be from 0.05 to 30% by weight of the total food weight of the health food composition of the present invention, and 0.05 to 30 g based on 100 ml of the health beverage composition have.

The food supplementary additives that can be used in the food composition may include food additives customary in the art, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers and the like, and various flavoring agents Natural carbohydrates and the like as additional components. Examples of the natural carbohydrate include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, etc .; Polysaccharides such as dextrin, cyclodextrin and the like; And sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tautatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavors (saccharin, aspartame, etc.) can be used as flavors other than those described above.

The food composition may be in the form of a flavoring agent such as a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and fillers (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, Colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.

The food composition may be administered parenterally or orally, and may be administered in an amount of 0.001 to 5 g per 1 kg of body weight per day, divided into 1 to several times. The dosage level for a particular patient may vary depending on the patient's body weight, age, sex, health condition, diet, time of administration, method of administration, rate of absorption, severity of the disease,

An angiotensin I converting enzyme (ACE) inhibitor according to another embodiment of the present invention includes a peptide having an amino acid sequence of Leu-Trp-His-Thr-His as an active ingredient. Since the content of the ACE and the peptide (LWHTH) are overlapped with those described above, the description thereof is omitted.

A method of preparing a composition according to another embodiment of the present invention includes: a hydrolysis step of hydrolyzing hydrolysis using a digestive enzyme to obtain hydrolyzate hydrolyzate; And separating the hydrolyzate hydrolyzate according to protein size and separating an active fraction having an inhibitory effect on angiotensin I converting enzyme (ACE) from the separated fraction, wherein Leu-Trp-His-Thr -His and having a peptide as an active ingredient.

The midduck may be natural or extruded, and does not specifically limit the area or time of harvest. In addition, the supernatant may be freeze-dried and pulverized for ease of storage and ease of hydrolysis at a later stage. At this time, the meat meat and / or the skin portion can be used.

The hydrolysis step is a step of hydrolyzing the hydrolyzate with digestive enzymes to obtain hydrolyzed digestive enzymes. In this case, it is preferable to apply pepsin as a digestive enzyme. More specifically, pepsin and Midock lyophilisate are mixed at a weight ratio of 1: 300 to 800, and the reaction is induced for 30 minutes to 2 hours to complete hydrolysis of the hydrolyzate Can proceed. At this time, the termination of the protein hydrolysis reaction proceeds by inactivating the pepsin, which is a digestive enzyme contained in the mixture of the pepsin and the Michigae lyophilisate, for example, by boiling at 100 ° C for about 10 minutes. Thereafter, the solid content of the mixture of pepsin and Midoduck freeze dried product is removed, and filtration under reduced pressure is performed to obtain hydrocracked hydrocracked product.

The separation step includes the step of isolating the various proteins or peptides contained in the hydrocracked hydrocracked product by size and obtaining the peptide with the amino acid sequence of Leu-Trp-His-Thr-His. As a method for obtaining a peptide having the amino acid sequence of Leu-Trp-His-Thr-His, a method of separating fractions having ACE inhibitory activity can be applied.

The separation according to the protein size can be divided into fractions by, for example, column chromatography.

Among these fractions, peptides having an amino acid sequence of Leu-Trp-His-Thr-His can be obtained through structural analysis of fractions which seem to have ACE inhibitory activity. The peptides of the amino acid sequence of Leu-Trp-His-Thr-His thus isolated can be stored after lyophilization and, if necessary, can be liquefied by dissolving in physiological saline or the like.

The pharmaceutical composition, the food composition and the preparation method thereof for lowering blood pressure according to the present invention are characterized by using a peptide having an amino acid sequence of Leu-Trp-His-Thr-His, which can be synthesized and derived from hydrocracking hydrolysates, .

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing mass spectrometry and amino acid sequence analysis results of hydrocracked hydrolyzate fraction (final selected sub-fraction) in Example 2 of the present invention. FIG.
FIG. 2 is a graph showing the results of evaluating the ACE inhibitory effect of the LWHTH peptide in Example 3 of the present invention (*** P <0.001, analysis by DMRT).
3 is a graph showing the results of LC-MS analysis of the stability of LWHTH peptides treated with digestive enzyme solution in Example 4 of the present invention.
FIG. 4 is a diagram showing the three-dimensional structure of ACE protein and LWHTH peptide (ACE is represented by a red ribbon model, ACE is represented by yellow) in Example 5 of the present invention.
FIG. 5 is a graph showing the two-dimensional structure of the ACE protein and the LWHTH peptide binding site (the LWHTH peptide is represented by a gray-red stick model and the amino acid of the active site of ACE is represented by a red dotted line) in Example 5 of the present invention. The figure shows the combined energy value.
6 is a graph showing changes in systolic blood pressure (A) and diastolic blood pressure (B) evaluated using the congenital hypertensive rat model in Example 6 of the present invention (* P <0.05, ** P <0.01)

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Example 1. Preparation of hydrocracked hydrocracked product

Midduk was supplied from the Midway English Association of Masan, Gyeongnam Province and used for the experiment in the lyophilized state. The lipid freeze - dried product was subjected to protein hydrolysis using pepsin, a digestive enzyme. Specifically, the freeze-thawed freeze-dried product was homogenized in a buffer, and pepsin was added thereto at a ratio of enzyme: substrate of 1: 500 (weight ratio), and hydrolyzed at 37 ° C for 1 hour to obtain hydrodehydroxide of midepectin. The end of the hydrolysis reaction was carried out by boiling at 100 ° C for 10 minutes to inactivate the enzyme. The hydrocracked hydrocracked lipid hydrocracked product was filtered under reduced pressure to obtain the hydrocracked hydrocracked product of Example 1, followed by lyophilization and storage.

Example 2: Peptide Isolation and Structural Analysis of Antihypertensive Activity

Gel filtration chromatography and reverse phase-HPLC were used to separate the active peptides from the hydrodegradated hydrolysates prepared in Example 1 above.

Specifically, the hydrolyzed hydrolyzate was injected onto a Sephadex G-25 gel filtration column (2.5.times.75 cm), and separated by protein size. The separated fractions were separated by reverse phase-HPLC on a C18 ODS column (4.6 × 150 mm). The separation conditions were as follows: Linear gradient of acetonitrile (0-30%), Flow rate of 0.8 mL / min.

Peptides dissolved in a methanol / water (1: 1, v / v) solvent were analyzed by Q-TOF (Quadruple time-of-flight) ESI mass spectroscopy (Micromass Co ., Altrincham, UK), and the results are shown in FIG. Peptide information was automatically derived by comparison with sequence information obtained by tandem MS analysis.

Referring to FIG. 1, the amino acid sequence of the peptide was confirmed to be Leu-Trp-His-Thr-His and the molecular weight thereof was 692.2 Da.

Example 3: Peptide Separation and Structural Analysis of LWHTH Peptides

The ACE (Angiotensin I converting enzyme) inhibitory effect of the LWHTH peptide was evaluated by colorimetry using ACE kit-WST (Dojindo Molecular Technologies, Inc.), and the results are shown in FIG.

Referring to FIG. 2, the LWHTH peptide inhibited ACE in a concentration-dependent manner in the range of 6 to 24 μM, and the IC ₅₀ value was found to be 16.42 ± 0.45 μM.

Example 4 Confirmation of Stability of LWHTH Peptide for Digestion Enzyme

LWHTH peptides were incubated with three digesting enzymes (pepsin, 20 mM glycine-HCl buffer (pH 2.0), trypsin, and 50 mM sodium phosphate buffer (pH 8.0) for alpha chymotrypsin) (W / w), respectively, to prepare a LWHTH peptide digesting enzyme solution.

Each of these LWHTH peptide-digesting enzyme solutions was reacted at 37 ° C. for 4 hours to prepare LWHTH peptide-digesting enzyme reaction solutions. Each reaction solution was boiled at 100 ° C. for 15 minutes to terminate the reaction. After centrifuging the LWHTH peptide-digesting enzyme reaction solutions, the ACE inhibitory effect of each reaction product was confirmed, and the stability of the LWHTH peptide was confirmed by LC-MS analysis. The stability of the LWHTH peptide was evaluated by LC-MS analysis. The results are shown in FIG. Referring to FIG. 3, it can be seen that the main peak of the LWHTH peptide remained after the reaction with the digestive enzyme.

The stability of the functional peptide is important because the functionalities of the peptide are lost if the structure or amino acid sequence of the functional peptide is changed. In particular, functional peptides as functional food materials are important for stability against digestive enzymes. The stability of the LWHTH peptide against three digestive enzymes was confirmed, and it was confirmed that the LWHTH peptide can be very stable in the body even when ingested orally.

Example 5 Analysis of ACE and Binding Mode of LWHTH Peptides (in silico)

( in PDB: 1O86) and Accelrys Discovery Studio (DS) 3.0 (Accelrys, Inc.) in the protein data bank (PDB, Inc) using the CDOCKER program. This program predicts the docking and binding potential of a protein-ligand complex, and identifies the location of the binding site in the protein and the binding mode of the ligand. Specifically, after securing the three-dimensional structure of the ACE protein and the ligand, designation of a binding site considering the protein structure and the like, and docking of the ligand using the CDOCKER program are carried out in that order. , The two-dimensional structure and the predicted coupling energy value are shown in Fig.

Enzyme inhibition occurs as the ligand docks to the enzyme, so structural studies of ligands and enzymes are crucial in evaluating the enzyme's ability to inhibit the ligand. CDOCKER is a CHARMm-based docking algorithm and it is a program for predicting the docking mode of the ligand bound to the active site of the enzyme in an in silico virtual three-dimensional space.

ACE is represented by a red ribbon model, and the ACE active site is yellow. Referring to FIG. 4, the LWHTH peptide binds to the active site of ACE to form an ACE-LWHTH complex (in FIG. A portion of the a-helix was deleted and marked for further display).

Referring to FIG. 5 showing the ACE-LWHTH complex binding site in a two-dimensional structure, the ACE-LWHTH complex has a hydrogen bond, a pi bond, and an electrostatic bond. Arg522, which is the amino acid of ACE, is a hydrogen bond with Tryptophan and Threonine, Glu403 is with Leucine and hydrogen bond, and Lys118 is pi bonding with benzene ring of Tryptophan. In particular, since Arg522 is an amino acid constituting the active site of ACE, it can be confirmed that LWHTH peptide binds directly to the active site of ACE.

In addition, Histidine located at the C-terminal of the active peptide plays an important role in binding to the active site of the enzyme, and it can be confirmed that the Histidine located at the C-terminal of the LWHTH peptide directly blocks the enzyme active site. This indicates that LWHTH peptides inhibit ACE activity by preventing ACE from binding to the original substrate.

The stability of ACE-LWHTH complex can be confirmed by CDOCKER interaction energy and binding evergy. The CDOCKER interaction energy is -102.566 kcal / mol and the binding energy is -372.069 kcal / mol, indicating that the ACE-LWHTH complex is very stable.

Example 6: Oral administration of LWHTH peptide (Congenital Hypertensive Rat Model Test)

Congenital hypertensive rats (10-week-old, specific pathogen-free, body weight: 250-300 g, n = 4) with a tail systolic blood pressure of 180 mmHg or more were individually raised in a breeding room at 24 ℃ for 12 hours.

The LWHTH peptide was dissolved in physiological saline at a concentration of 40 mg / kg body weight and orally administered to congenital hypertensive rats. As a positive control, captopril, which is currently used as a treatment for hypertension, was used and saline was used as a negative control.

The blood pressure lowering effect of the LWHTH peptide using the congenital hypertensive rat model was confirmed by the change of the blood pressure of the tail of congenital hypertensive rats over time using a CODA (TM) blood pressure monitor (Kent Scientific Corp., Torrington, USA) Respectively.

Referring to FIG. 6, the systolic blood pressure in the group to which LWHTH peptide and captoprir was administered was clearly decreased, and the blood pressure decreased until 9 hours after administration. The LWHTH peptide-treated group decreased to 89.4% of the initial blood pressure after 3 hours of administration, and this value was lower than the maximum decrease in the capto-pril administration group (90.3% decrease compared with the initial blood pressure at 6 hours after administration). Diastolic blood pressure also showed a pattern similar to systolic blood pressure, and diastolic blood pressure of the LWHTH peptide-treated group decreased to 83.8% of the initial blood pressure after 3 hours of administration, which was the maximum reduction value of the capto-pril administration group 88.1% of the initial blood pressure).

This means that the LWHTH peptide shows superior blood pressure lowering effect more rapidly than capto pril.

* Statistics: All of the above data are expressed as the mean ± standard deviation of three measurements. Statistical analysis of mean values was performed by ANOVA and Duncan's multiple-range test using SPSS (11.5) software. p <0.05 was considered statistically significant.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

<110> Jeju National University Industry-Academic Cooperation Foundation <120> COMPOSITION HAVING ANTIHYPOTENSIVE EFFECT AND PREPERATION METHOD          OF THE SAME <130> DP20150133 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 5 <212> PRT <213> Styela clava <400> 1 Leu Trp His Thr His   1 5

Claims (4)

Leu-Trp-His-Thr-His as an active ingredient. Leu-Trp-His-Thr-His as an active ingredient and has a blood pressure lowering function. Leu-Trp-His-Thr-His as an active ingredient. A hydrolysis step of hydrolyzing the hydrodeoxygenase using a digestive enzyme to obtain hydrocracked hydrolyzate;
A separation step of separating the hydrolyzate hydrolyzate according to protein size and separating an active fraction having an inhibitory effect on angiotensin I converting enzyme (ACE) from the separated fraction; And
Obtaining a peptide consisting of the amino acid sequence of Leu-Trp-His-Thr-His from the active fraction and a peptide comprising the amino acid sequence of Leu-Trp-His-Thr-His as an active ingredient, Lt; / RTI &gt;

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