KR102224707B1 - Composition for preventing or treating hyperlipidemia comprising bioactive peptides - Google Patents

Abstract

The present invention relates to a composition for preventing or treating hyperlipidemia containing a peptide isolated from a casein hydrolyzate as an active ingredient, wherein the peptide isolated from the casein hydrolyzate increases the expression level of the protein ABCG5 that controls cholesterol excretion and As it was confirmed that the effect of reducing the blood cholesterol level of the intra-cholesterol and hyperlipidemia animal model was confirmed, the composition containing the peptide isolated from the casein hydrolyzate as an active ingredient may be provided as a hyperlipidemia therapeutic agent or a health food for improvement.

Description

Composition for preventing or treating hyperlipidemia containing bioactive peptides as an active ingredient {Composition for preventing or treating hyperlipidemia comprising bioactive peptides}

The present invention is to provide a composition for preventing or treating hyperlipidemia containing a physiologically active peptide isolated from a casein hydrolyzate as an active ingredient.

The number of hyperlipidemia patients due to excessive intake of fat and cholesterol with recent dietary changes is increasing remarkably. Hyperlipidemia refers to a condition in which the lipid component in the blood is more than normal. The main components of serum lipids are composed of lipid-soluble substances such as cholesterol, triglycerides, phospholipids and free fatty acids, and are called hypercholesterolemia, hypertriglyceridemia, etc., depending on which of these lipid substances is mainly increased.

Increased serum cholesterol and triglycerides are reported to be the most common cause of hyperlipidemia.The accumulation of excess fat can cause blood circulation disorders and microcirculation failure, leading to atherosclerosis, ischemic heart disease, cerebral infarction, high blood pressure, obesity, and diabetes. .

Cholesterol is an essential constituent for maintaining the normal functioning of the human body. It is an essential nutrient for making cells. It is a component of the biological membrane, and at the same time, it is a material that makes various hormones and bile such as adrenal cortical hormone, male hormone, and female hormone.

However, it is known that excessive cholesterol intake causes heart disease by accumulating in blood vessels. As a method of reducing the blood lipid concentration, diet, exercise therapy, and drug therapy to reduce the intake of foods containing a lot of cholesterol or fat are used, but strict management and implementation of diet or exercise therapy is difficult. There is also a limit to the therapeutic effect.

Drugs that reduce lipid levels developed so far include bile acid binding resins, drugs that lower cholesterol such as HMG-CoA reductase inhibitors, which are important enzymes in cholesterol biosynthesis, and triglycerides such as fibric acid derivatives and nicoric acid. Drugs that lower it have been developed, but these drugs have reported side effects such as hepatotoxicity, gastrointestinal disorders and carcinogenicity.

Accordingly, there is an urgent need to develop a drug that can be used for the treatment of hyperlipidemia and related diseases by effectively lowering the blood lipid concentration while solving the problem of side effects of drugs.

Republic of Korea Patent Publication No. 10-2015-0135647 (published on December 3, 2015)

An object of the present invention is to provide a peptide isolated from a casein hydrolyzate to solve the side effects of a conventional hyperlipidemia therapeutic agent, and to provide it as an agent for improving or treating hyperlipidemia that effectively releases cholesterol.

The present invention provides a pharmaceutical composition for preventing or treating hyperlipidemia containing a casein peptide consisting of an amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO: 12 as an active ingredient.

The present invention provides a health food for preventing or improving hyperlipidemia containing a casein peptide consisting of an amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO: 12 as an active ingredient.

In addition, the present invention provides a health food for weight loss containing a casein peptide consisting of an amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO: 12 as an active ingredient.

According to the present invention, as it was confirmed that the peptide isolated from the casein hydrolyzate increased the expression level of the protein ABCG5, which regulates cholesterol excretion, and showed the effect of reducing the cholesterol level in the blood in the animal model of cholesterol and hyperlipidemia in the small intestine cells. , The composition containing the peptide isolated from the casein hydrolyzate as an active ingredient may be provided as a health food for treating or improving hyperlipidemia.

1 is a result of confirming the expression level of ABCG5, a cholesterol excretion regulatory gene, according to the treatment of casein hydrolyzate in the colon cancer cell line Caco-2.
FIG. 2 is a result of HPLC analysis for obtaining a fraction containing a peptide showing an effect of increasing the expression of ABCG5 among casein hydrolysates.
3 is a result of confirming a fraction containing a peptide showing the best effect of increasing the expression of ABCG5 in the colon cancer cell line Caco-2.
4 is a result of confirming the effect of increasing the expression of ABCG5 after treating the isolated 12 peptides to the colon cancer cell line Caco-2 at a concentration of 300 ng/ml, respectively.
5 is a result of confirming the cholesterol accumulation inhibitory effect according to the treatment of peptides 9 and 12 in the colon cancer cell line Caco-2.
6 is a result of orally administered 5 times a week at 10 mg/kg of peptides 9 and 12, respectively, to a hyperlipidemia mouse animal model and confirming the change in body weight for 10 weeks.
7 is a result of confirming the change in the total cholesterol content in the blood of the mouse animal model after oral administration of 10 mg/kg of peptides 9 and 12 in a hyperlipidemia mouse animal model at 10 mg/kg each 5 times a week for 10 weeks.

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

The accumulation of cholesterol in the blood leads to hyperlipidemia and is the most important factor causing cardiovascular disease. In order to control and treat such hyperlipidemia, a statin-based drug has been developed and used as a therapeutic agent, but as it has been confirmed that statin-based drugs have side effects when taken for a long time, the present inventors improved the side effects of conventional cholesterol regulators. The present invention was completed by confirming the effect of the peptide isolated from the casein hydrolyzate on the expression of the protein that regulates cholesterol excretion during studies on drugs that can safely control cholesterol accumulation.

The present invention can provide a pharmaceutical composition for preventing or treating hyperlipidemia containing a peptide consisting of an amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO: 12 as an active ingredient.

The peptide may be isolated from the casein hydrolyzate.

In more detail, the peptide is a product obtained by hydrolyzing a casein protein with an enzyme, and the enzyme may be selected from the group consisting of pepsin, trypsin, chemotrypsin, elastase, and papain, but is not limited thereto.

The peptide may increase the expression of ABCG5 protein, which regulates cholesterol excretion in cells.

These peptides can reduce blood cholesterol levels and body weight.

According to an embodiment of the present invention, in order to determine whether the 9 and 12 casein peptides isolated from the casein hydrolyzate can exhibit a cholesterol excretion inhibitory effect in vivo, C57BL/ which induces hyperlipidemia using a high cholesterol diet. 6 Mice were orally administered with peptide aqueous solutions No. 9 and No. 12 for 5 times/week in proportion to the body weight so that the amount of 10 mg/kg, and then the change in body weight for 10 weeks was confirmed. Body weight was measured on the day of the weekly administration ended. As a result, as shown in FIG. 7, it was confirmed that the weight of the mice administered with the No. 12 casein peptide decreased.

In addition, after collecting blood from C57BL/6 mice orally administered casein peptide 9 and 12 times for 10 weeks in the same process as described above, serum was extracted from the collected blood to confirm the change in total cholesterol level, as shown in FIG. And casein peptide No. 12 effectively reduced the cholesterol content in the blood.

From the above results, it was confirmed that both casein peptides 9 and 12 exhibited a blood cholesterol lowering effect, and in particular, peptide 12 exhibited a very excellent blood cholesterol lowering effect, and it was confirmed that it is very effective in weight loss.

In one embodiment of the present invention, the pharmaceutical composition for preventing or treating hyperlipidemia containing the peptide consisting of the amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO: 12 as an active ingredient is prepared by injection, granule, powder, or tablet according to a conventional method. , Pills, capsules, suppositories, gels, suspensions, emulsions, drops or liquids may be used in any one of the group consisting of a formulation selected from the group consisting of.

In another embodiment of the present invention, suitable carriers, excipients, and disintegrants commonly used in the preparation of a pharmaceutical composition for preventing or treating hyperlipidemia containing a peptide consisting of an amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO: 12 as an active ingredient , A sweetener, a coating agent, an expanding agent, a lubricant, a lubricant, a flavoring agent, an antioxidant, a buffer solution, a bacteriostatic agent, a diluent, a dispersant, a surfactant, a binder, and a lubricant.It may further include one or more additives selected from the group consisting of.

Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, capsules. And the like, and these solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, and the like. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As a base material for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

According to an embodiment of the present invention, the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal It can be administered to the subject in a conventional manner via the route.

The preferred dosage of the peptide may vary depending on the condition and weight of the subject, the type and extent of the disease, the form of the drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to an embodiment of the present invention, although not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or may be divided into several doses, whereby the scope of the present invention is not limited.

In the present invention, the'subject' may be a mammal including a human, but is not limited to these examples.

The present invention can provide a health food for preventing or improving hyperlipidemia containing a peptide consisting of an amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO: 12 as an active ingredient.

In addition, the present invention can provide a health food for weight loss containing a peptide consisting of an amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO: 12 as an active ingredient.

The health food may be used together with other foods or food additives in addition to the peptides, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use, for example, prevention, health or therapeutic treatment.

The effective dose of the compound contained in the health food may be used in accordance with the effective dose of the therapeutic agent, but in the case of long-term intake for the purpose of health and hygiene or health control, it may be less than the above range, and is effective. It is clear that the ingredient can be used in an amount beyond the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of health food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, Drinks, alcoholic beverages, and vitamin complexes.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.

< Example 1> Casein Peptide Screening

Use of casein protein (alpha-s1, alpha-s2, beta, kappa casein mixture, Sigma-aldrich) or pepsin (pepsin, 0.4% w/v pepsin solution) in human colon cancer cell line Caco-2, pH 2.0, 37 2 hours decomposition at ℃ condition) and trypsin (use of trypsin, 0.4% w/v trypsin solution, pH 7.0, 2 hours decomposition at 37°C) were treated with casein hydrolysate at a concentration of 2 mg/ml, respectively, and After 6 hours, the change in mRNA expression was confirmed.

As a result, it was confirmed that the expression of ABCG5, which is known to be responsible for the excretion of cholesterol in the small intestine cells, was increased by the casein treatment, and in particular, it was further increased by the casein hydrolyzate.

Peptide separation was performed using HPLC (C18, Sunfire column) to select a peptide that increases the expression of ABCG5 among many peptides of casein hydrolysis products.

As a result, as shown in Fig. 2, four peptide fractions in which peptides were mixed were obtained.

The fractions obtained by the above process were treated with Caco-2 cells, and the expression of ABCG5 was compared after 6 hours. At this time, the experiment was repeated twice under the same experimental conditions, and the results of each experiment were analyzed.

As a result, an increase in the expression of ABCG5 was confirmed in fraction 1 as shown in FIG. 3.

As a result of analyzing the sequence of the peptide of fraction 1 from the experimental results, a total of 12 peptides as shown in Table 1 were identified.

Twelve peptides as shown in Table 1 were synthesized with a purity of 99%, treated in Caco-2 cells at a concentration of 300 ng/ml, and the expression of mRNA ABCG5 was confirmed after 6 hours.

As a result, it was confirmed that ABCG5 expression was significantly increased in peptides 9 and 12 as shown in FIG. 4.

From the above results, it was confirmed that not all peptides isolated from all casein hydrolysates exhibited the same effect of inhibiting ABCG5 expression.

< Example 2> In vitro Casein Confirmation of the effect of peptides in inhibiting cholesterol excretion

Casein peptides 9 and 12 identified in the previous experiment were treated on Caco-2 cells for 6 hours, cholesterol mixture (100 μM Cholesterol, 3 mM sodium taurocholate, 30 mM monoolein) was treated for 2 hours, and then cholesterol dye (filipin III) was applied. The amount of cholesterol accumulation in the cell was confirmed by using.

As a result, it was confirmed that cholesterol accumulation was decreased in the cell group treated with the peptide as compared to the group treated with cholesterol alone as shown in FIG. 6.

From the above results, it was confirmed that casein peptides 9 and 12 effectively excrete intracellular cholesterol.

< Example 3> In vivo (In vivo ) Casein Confirmation of the effect of peptides in inhibiting cholesterol excretion

To confirm whether casein peptides 9 and 12 can exhibit cholesterol excretion inhibitory effects in vivo, C57BL/6 mice that induced hyperlipidemia using a high cholesterol diet were treated with 10 mg/kg of aqueous peptide solution of peptides 9 and 12. After oral administration for 5 times/week in proportion to the body weight so as to be an amount of, the body weight change for 10 weeks was confirmed. Body weight was measured on the day of the weekly administration ended.

As a result, as shown in FIG. 7, it was confirmed that the weight of the mice administered with the No. 12 casein peptide decreased.

In addition, blood was collected from C57BL/6 mice to which casein peptides 9 and 12 were orally administered for 10 weeks in the same process as described above, and then serum was extracted from the collected blood to confirm the change in total cholesterol level.

As a result, as shown in Fig. 8, casein peptides 9 and 12 effectively reduced the cholesterol content in the blood.

From the above results, it was confirmed that both casein peptides 9 and 12 exhibited a blood cholesterol lowering effect, and in particular, peptide 12 exhibited a very excellent blood cholesterol lowering effect, and it was confirmed that it is very effective in weight loss.

As described above, a specific part of the present invention has been described in detail, and for those of ordinary skill in the art, it is obvious that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> Pusan National University Industry-University Cooperation Foundation <120> Composition for preventing or treating hyperlipidemia comprising bioactive peptides <130> ADP-2019-0106 <160> 12 <170> KopatentIn 2.0 <210> 1 <211> 11 <212> PRT <213> alpha-s1 casein <400> 1 His Ile Gln Lys Glu Asp Val Pro Ser Glu Arg 1 5 10 <210> 2 <211> 8 <212> PRT <213> alpha-s1 casein <400> 2 Lys Lys Tyr Lys Val Pro Gln Leu 1 5 <210> 3 <211> 8 <212> PRT <213> alpha-s1 casein <400> 3 Glu Gly Ile His Ala Gln Gln Lys 1 5 <210> 4 <211> 12 <212> PRT <213> alpha-s2 casein <400> 4 Val Lys Ile Thr Val Asp Asp Lys His Tyr Gln Lys 1 5 10 <210> 5 <211> 10 <212> PRT <213> alpha-s2 casein <400> 5 Ile Thr Val Asp Asp Lys His Tyr Gln Lys 1 5 10 <210> 6 <211> 10 <212> PRT <213> alpha-s2 casein <400> 6 Lys Ala Met Lys Pro Trp Ile Gln Pro Lys 1 5 10 <210> 7 <211> 9 <212> PRT <213> beta-casein <400> 7 Lys Ile His Pro Phe Ala Gln Thr Gln 1 5 <210> 8 <211> 8 <212> PRT <213> beta-casein <400> 8 His Lys Glu Met Pro Phe Pro Lys 1 5 <210> 9 <211> 11 <212> PRT <213> beta-casein <400> 9 Ser Gln Ser Lys Val Leu Pro Val Pro Gln Lys 1 5 10 <210> 10 <211> 9 <212> PRT <213> kappa casein <400> 10 Arg Phe Phe Ser Asp Lys Ile Ala Lys 1 5 <210> 11 <211> 7 <212> PRT <213> kappa casein <400> 11 Ser Asn Thr Val Pro Ala Lys 1 5 <210> 12 <211> 8 <212> PRT <213> kappa casein <400> 12 His Pro His Pro His Leu Ser Phe 1 5

Claims (6)

A pharmaceutical composition for preventing or treating hyperlipidemia, comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 9 as an active ingredient, wherein the peptide increases the expression of ABCG5 protein that regulates cholesterol excretion in cells. The pharmaceutical composition for preventing or treating hyperlipidemia according to claim 1, wherein the peptide is isolated from casein hydrolyzate. delete The pharmaceutical composition for preventing or treating hyperlipidemia according to claim 1, wherein the peptide reduces blood cholesterol levels. A health food for preventing or improving hyperlipidemia, comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 9 as an active ingredient, wherein the peptide increases the expression of ABCG5 protein that regulates cholesterol excretion in cells. delete

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