KR20140131077A - Composition containing peptides from spirulina maxima for prevention or treatment of Cardiovascular disorders - Google Patents

Abstract

The present invention relates to a composition containing peptides derived from Spirulina maxima having amino acid sequences of LDAVNR or MMLDF as active ingredients for preventing or treating cardiovascular disorders. The peptides according to the present invention inhibit the generation of various cytokines, cell adhesion molecules, chemotactic factors, protein in signal transduction pathway, and active oxygen species which induce the onset and progression of cardiovascular disorders such as atherosclerosis. Therefore the present invention can be usefully used as a composition for preventing or treating cardiovascular disorders. In particular, the Spirulina maxima can be used long-term without toxicity and side effects since the Spirulina maxima is derived from nature, thereby can also be usefully used as a functional health food.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating cardiovascular diseases comprising, as an active ingredient, a peptide derived from Spirulina maxima,

The present invention relates to a composition for preventing or treating cardiovascular diseases comprising a peptide derived from Spirulina maxima as an active ingredient. More particularly, the present invention relates to a composition for preventing or treating cardiovascular diseases comprising a peptide derived from Spirulina maxima, And a peptide having the peptide as an active ingredient. The present invention also relates to a composition for preventing or treating cardiovascular diseases.

In recent years, there have been many cases in which novel bioactive substances derived from marine resources are utilized as various health functional foods or pharmaceutical compositions. Marine organisms are known to biosynthesize substances such as carotenoids, terpenoids, xanthophylls, chlorophyll, vitamins, saturated fatty acids, amino acids, proteins, acetogenin, polyphenols, alkaloids and polysaccharides, Or a report of resources capable of producing secondary metabolites. Over the past several decades, numerous novel compositions have been isolated from marine organisms and have been studied for their excellent biological activity. In particular, marine algae are known to be the best biomass producers, and a variety of natural living materials produced from marine algae can be used for anti-aging, antiviral, antioxidant, antiallergic, anti-cancer, anti- .

Among them, information about the structure, composition and sequence characteristics of marine peptides is becoming known, and attempts are being made to utilize them as bio-materials. Marine peptides can be produced by solvent extraction, enzyme hydrolysis, microbial fermentation, etc. Generally, they are composed of 3 to 20 amino acids. Their biofunctions are determined according to the composition and sequence of the amino acids constituting the peptide Is known to lose. The marine peptides used to extract marine peptides to date include squid, mussel, oyster, tuna, cod, mackerel, pollack, eel, and algae. The extracted marine peptids exhibit antihypertensive or antimicrobial activity Has been reported to reduce the likelihood of cardiovascular disease. Therefore, marine peptides are valuable resources to be studied with health functional foods and pharmaceutical compositions.

Atherosclerosis refers to a combination of atherosclerosis and atherosclerosis. Atherosclerosis is defined as atheroma, which is caused by deposition of cholesterol on the innermost covering of the blood vessels and proliferation of endothelial cells. When atherosclerosis develops, the inner part of the atheroma is diluted like porridge and the surrounding area is surrounded by a hard fibrous membrane, which is surrounded by a hardening half. When the hardening half becomes unstable, the blood vessel ruptures, In case of bleeding into atheroma, the diameter of the inside of the blood vessel becomes suddenly narrowed or the blood vessel becomes completely blocked, resulting in an obstacle to blood circulation in the peripheral blood. In addition, arteriosclerosis is a phenomenon of degeneration in the middle layer of the blood vessels, progression of fibrosis and decrease of elasticity of blood vessels. When arteriosclerosis develops, systolic hypertension is caused and cardiac hypertrophy occurs in which the heart muscle is thickened.

Atherosclerosis is a type of inflammatory disease. It has an inflammatory tissue called atherosclerotic plaque, such as rheumatoid synovial membrane. The atherosclerotic plaque migrates to blood-derived inflammatory cells and binds to vascular endothelial cells and connective tissue cells , Leading to a chronic inflammatory response.

The present inventors focused on natural substances capable of preventing or treating such atherosclerosis, and found that the peptide derived from Spirulina maxima regulates the expression of various cytokines and chemotactic factors related to atherosclerosis, The present invention can be utilized for preventing or treating atherosclerosis.

Korean Patent Publication No. 10-2009-0066995

It is therefore an object of the present invention to provide a composition for the prevention and treatment of cardiovascular diseases comprising a peptide derived from Spirulina maxima as an active ingredient.

It is still another object of the present invention to provide a health functional food for preventing and improving cardiovascular diseases comprising the novel peptide as an active ingredient.

To this end, the present invention provides a composition for preventing or treating cardiovascular diseases, comprising the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

In one embodiment of the present invention, the cardiovascular disease may be atherosclerosis.

In one embodiment of the present invention, the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 may be derived from Spirulina maxima.

In one embodiment of the present invention, the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 may exhibit the preventive or therapeutic effect of cardiovascular disease by inhibiting the expression of IL-6 or IL-8.

In one embodiment of the present invention, the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 may inhibit the production of PGE2, LTB4 or MCP-1, thereby preventing or treating cardiovascular diseases.

In one embodiment of the present invention, the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 may inhibit the production of PGE2, LTB4 or MCP-1 by inhibiting the expression of COX-2, 5-LO or MCP- .

In one embodiment of the present invention, the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 inhibits the production or expression of P-selectin or E-selectin, thereby preventing or treating cardiovascular diseases Lt; / RTI >

In one embodiment of the present invention, the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 may inhibit the expression of TLR4, thereby exhibiting a preventive or therapeutic effect of cardiovascular disease.

In one embodiment of the present invention, the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 may be effective in preventing or treating cardiovascular diseases by inhibiting the ERK signaling pathway.

In one embodiment of the present invention, the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 may be effective for preventing or treating cardiovascular diseases by inhibiting PKC activation.

In one embodiment of the present invention, the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 may exhibit the effect of preventing or treating cardiovascular diseases by inhibiting the production of reactive oxygen species.

The present invention also provides a health functional food for preventing or ameliorating cardiovascular diseases comprising the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

The peptides of SEQ ID NO: 1 or SEQ ID NO: 2 derived from Spirulina maxima according to the present invention inhibit the production of cytokines, adhesion molecules, chemotactic factors, proteins of the signal transduction pathway and reactive oxygen species and thereby prevent the development of cardiovascular diseases such as atherosclerosis Prevention or treatment of cardiovascular diseases. If properly utilized, the composition can be used as a composition for preventing or treating cardiovascular diseases. In addition, the peptide derived from Spirulina maxima is a naturally derived substance and can be used as a health functional food because it can be used safely for a long period of time without toxicity and side effects.

Fig. 1 shows the result of analysis of the amino acid sequence of the peptide (P1) derived from Spirulina maxima.
Figure 2 shows the result of analysis of the amino acid sequence of another peptide (P2) derived from Spirulina maxima.
FIG. 3 shows the results of ELISA analysis of production of IL-6 (A) and IL-8 (B) according to the peptide treatment according to the present invention in histamine-stimulated epithelial cells.
FIG. 4 shows the results of ELISA analysis of production yields of PGE2 (A) and LTB4 (B) according to the peptide treatment according to the present invention in histamine-stimulated epithelial cells.
FIG. 5 shows the results of ELISA analysis of the amount of MCP-1 produced by the peptide treatment according to the present invention in histamine-stimulated epithelial cells.
FIG. 6 shows the amounts of protein expression (A) and mRNA expression (B) of IL-6 and IL-8 according to the peptide treatment according to the present invention in histamine-stimulated epithelial cells by Western blotting and RT-PCR, respectively The results are analyzed.
FIG. 7 shows the amounts of protein expression (A) and mRNA expression (B) of COX-2 and 5-LO according to the present invention in histamine-stimulated epithelial cells by Western blotting and RT-PCR, respectively The results are analyzed.
FIG. 8 shows the results of Western blotting and RT-PCR analysis of protein expression amount (A) and mRNA expression amount (B) of MCP-1 according to the peptide treatment according to the present invention in histamine-stimulated epithelial cells .
FIG. 9 shows the results of fluorescence microscopy for the effect of histidine-stimulated epithelial cells on the formation of E-selectin (A) and P-selectin (B) according to the peptide treatment according to the present invention.
FIG. 10 shows the amount of protein expression (A) and mRNA expression (B) of E-selectin and P-selectin according to the peptide treatment according to the present invention in histamine-stimulated epithelial cells by Western blotting and RT-PCR, respectively The results are analyzed.
FIG. 11 is a photograph of the degree of mononuclear leukocyte recruitment by epithelial cells stimulated by histamine according to the present invention by fluorescence microscopy.
12 shows the results of Western blotting and RT-PCR analysis of protein expression amount (A) and mRNA expression amount (B) of TLR-4 according to the peptide treatment according to the present invention in histamine-stimulated epithelial cells .
FIG. 13 shows the results of Western blotting and RT-PCR analysis of protein expression amount (A) and mRNA expression amount (Eg) of Egr-1 according to the peptide treatment according to the present invention in histamine-stimulated epithelial cells .
14 shows the results of Western blot analysis of the amount of c-fos protein expressed by histamine-treated epithelial cells treated with the peptide according to the present invention.
FIG. 15 shows the results of Western blotting analysis of the inhibitory effect of MAPK on the ERK activity inhibitor (A) and the peptide (B) treatment according to the present invention in histamine-stimulated epithelial cells.
FIG. 16 shows the results of Western blotting analysis of the inhibitory effect of PKC on peptides treated with histamine in epithelial cells stimulated by histamine according to the present invention.
FIG. 17 shows the results of observation of ROS scavenging activity of histamine-stimulated epithelial cells according to the present invention with FACs (A) and fluorescent images (B).
18 shows the blocking effect of the histamine receptor on histamine-stimulated epithelial cells treated with the peptide according to the present invention using FACs.

When histamine binds to its receptor, the production of proinflammatory cytokines such as IL-6, IL-8, p-selectin, VCAM-1 and ICAM-1 is induced from endothelial cells and production of these factors And expression have been reported to be able to induce an initial response of atherosclerosis. Therefore, a substance capable of inhibiting the production and expression of these factors may be used for the prevention or treatment of atherosclerosis.

The present inventors confirmed that peptides (P1, P2) derived from the enzymatic hydrolysates of Spirulina maxima effectively inhibited histamine-induced production of IL-6 and IL-8 in EA.hy926 endothelial cells, Predicted that the peptides (P1, P2) could be used for the prevention or treatment of atherosclerosis and further experiments were conducted to confirm the mechanism.

Prostaglandin E2 (PGE2) and leucotriene B4 (LTB4) are proteins produced by the COX-2 and 5-LO enzymes and are known to play important roles in the progression of atherosclerosis. PGE2 is known to be involved in the instability of arteriosclerotic plaques. LTB4 is a potential chemotactic factor of mononuclear leukocytes, neutrophil granulocytes, and T lymphocytes. It promotes the adhesion of leukocytes to the endothelial cell layer, increases vascular permeability, Lt; RTI ID = 0.0 > (VSMCs). ≪ / RTI > In addition, monocyte chemotactic factor-1 (MCP-1) is detected in the endothelial cells, macrophages, and smooth muscle cells of the atherosclerotic artery of patients with bypass blood vessel regeneration, and monocytes are collected into the subendothelial cell layer, It is known to contribute to deterioration.

Thus, such molecules have been regarded as therapeutic targets that can reduce the progression of atherosclerosis. The present inventors have found that the peptides (P1, P2) according to the present invention can inhibit the production of these molecules As a result, it was confirmed that the peptides according to the present invention inhibit histamine-induced production of PGE2, LTB4 and MCP-1 in a concentration-dependent manner in endothelial cells. In addition, it was further confirmed that such a phenomenon is caused by down-regulation of expression of COX-2, 5-LO, and MCP-1 at mRNA and protein levels. Therefore, the peptide according to the present invention can inhibit the expression of COX-2, 5-LO and MCP-1, thereby reducing the production of PGE2, LTB4 and MCP-1, and as a result, can be utilized for the prevention or treatment of atherosclerosis And it was found.

On the other hand, vascular adhesion molecules are known to play an important role in the development and progression of atherosclerosis, and inhibition of the production of these adhesion molecules may prevent progression of atherosclerosis. From this point of view, the present inventors have observed that the peptides according to the present invention have an effect on the production and expression of P-selectin and E-selectin in histamine-stimulated endothelial cells, selectin expression, indicating that it is possible to reduce the adhesion of monocytes to histamine-stimulated endothelial cells.

On the other hand, the increased expression of TLR4 is known to increase the development of atherosclerosis. Therefore, the present inventors have found that the peptide (P1, P2) according to the present invention has no effect on the expression of TLR4 in histamine-stimulated endothelial cells As a result of checking, EA. It was confirmed that the expression of TLR4 increased by histamine in Hy926 cells was effectively inhibited by the peptide according to the present invention.

Egr-1 (Eraly grwoth respons factor 1) is known as a major modulator in the development of atherosclerosis. In other words, Egr-1, a zinc finger protein, acts as a transcription factor and regulator of various genes involved in the development of thrombosis, restenosis, and atherosclerosis. The present inventors further confirmed that the expression of Egr-1 increased by histamine in the endothelial cells was effectively inhibited by the peptides (P1, P2) according to the present invention because the peptide according to the present invention regulates Egr-1 expression The expression of Egr-1 was inhibited by inhibiting the expression of c-fos, a transcription factor of the present invention.

In this regard, recent publications disclose that the expression of c-fos and Egr-1 is regulated by the ERK signaling pathway, and the present inventors have found that the peptides (P1, P2) I also tested the effect. As a result, it was confirmed that the peptides (P1, P2) according to the present invention inhibited phosphorylation of ERK and also inhibited phosphorylation of p38 and JNK. ERK phosphorylation is carried out by activation of PKC (protein kinase C). The present inventors have further confirmed the effect of the peptide according to the present invention on the activation of PKC. When the peptide (P1, P2) according to the present invention is treated Phosphorylation of PKC was also effectively inhibited.

On the other hand, active oxygen species (ROS) are known to be important factors associated with the pathological signs of endothelial cells in relation to atherosclerosis, and the present inventors have found that the peptides according to the present invention can inhibit production of reactive oxygen species I checked. As a result, it was found that the peptide according to the present invention effectively blocked the production of reactive oxygen species in histamine-stimulated endothelial cells.

Finally, the present inventors have confirmed the effect of the peptides according to the present invention on the expression of histamine receptors. It is known that histamine, together with its receptor, induces gene expression of cytokines, lipid metabolites, chemotactic factors and adhesion molecules in endothelial cells, and these gene expressions are known to promote inflammation and atherosclerosis Because. As a result of the experiment, the present inventors confirmed that the peptide according to the present invention effectively blocks the histamine receptor H1, thereby hindering the activity of the subsequent signal transduction pathway.

Through the above experimental results, the inventors of the present invention have found that the peptides (P1, P2) according to the present invention can be effectively used as a composition for preventing or treating cardiovascular diseases including atherosclerosis.

Accordingly, the present invention provides a composition for preventing or treating cardiovascular diseases comprising the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

In the present invention, "treatment ", as used herein, unless otherwise indicated, refers to reversing, alleviating, inhibiting, or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or disorder , And the term "treating ", as used herein, refers to the act of treating when" treatment "is defined as above.

Thus, "treatment" or "therapy " of a cardiovascular-related disorder in a mammal may include one or more of the following:

(1) inhibiting the development of cardiovascular-related diseases,

(2) prevent the spread of cardiovascular-related diseases,

(3) relieving cardiovascular disease,

(4) prevent recurrence of cardiovascular-related diseases and

(5) palliating symptoms of cardiovascular disease

The peptides purified from Spirulina maxima according to the present invention can be obtained by hydrolysis, extraction and separation from nature using hydrolysis, extraction and separation methods known in the art. In the present invention, For example, crude extract, polar solvent-soluble extract or non-polar solvent-soluble extract. However, synthetic peptides not extracted from Spirulina maxima can also be utilized as a functional material having an antioxidative effect, and peptides separated and purified without using any additional solvent may be used as the functional material of the present invention.

The composition of the present invention may be a pharmaceutical composition or a food composition.

The pharmaceutical composition of the present invention can be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A lubricant or a flavoring agent can be used.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile solutions suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, the functional peptide of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention may be contained in the composition at a concentration of 10 to 200 μM, and may be included in the composition in an amount of 0.1 to 95% by weight based on the total weight of the composition.

The composition of the present invention may also be a food composition. In addition to containing the functional peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, which is an active ingredient, the food composition may contain various flavors or natural Carbohydrates and the like as additional components.

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. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).

The food composition of the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .

In addition, the food composition may contain, in addition to the functional peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, an effective ingredient, various flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, (Cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, . In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

The peptide derived from Spirulina maxima, which is an active ingredient of the present invention, is a natural substance and has almost no side effects such as chemical agents. Therefore, it can be safely used for long-term use for the prevention or treatment of cardiovascular diseases.

The present invention provides a health functional food for preventing or ameliorating cardiovascular-related diseases comprising, as an active ingredient, a functional peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 derived from Spirulina maxima.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and rings for the purpose of prevention and improvement of cardiovascular-related diseases.

In the present invention, the term "health functional food" refers to a food prepared and processed by using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods, and the nutritional control on the structure and function of the human body Or for the purpose of obtaining a beneficial effect for health use such as physiological action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the food items included in the above food additives include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, a health functional food in the form of a tablet may be prepared by mixing a functional peptide consisting of the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2 derived from Spirulina maxima, an active ingredient of the present invention, with excipients, binders, disintegrants and other additives Granulation may be carried out by a conventional method, followed by compression molding with a lubricant or the like, or the mixture may be directly compression molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.

The hard capsule of the capsule-type health functional food can be prepared by filling a normal hard capsule with a mixture of the functional peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, which is an active ingredient of the present invention, with an additive such as an excipient , And the soft capsule can be prepared by filling a mixture obtained by mixing an Acupuncture with an excipient such as an excipient into a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

In the ring-shaped health functional food, a mixture of a functional peptide consisting of the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2 derived from Spirulina maxima, an active ingredient of the present invention, and an excipient, a binder and a disintegrant is mixed by a conventionally known method It can be prepared by molding, and if necessary, it can be coated with white sugar or other agent, or the surface can be coated with a substance such as starch or talc.

The granular health functional food may be prepared by mixing a functional peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 derived from Spirulina maxima, an active ingredient of the present invention, together with excipients, binders and disintegrants, by a conventionally known method Granules, and may contain a flavoring agent, a compatibilizing agent and the like as necessary.

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

The present invention also provides the use of a composition comprising, as an active ingredient, a peptide derived from Spirulina maxima for producing a medicament or food for the prevention or treatment of cardiovascular-related diseases. The composition of the present invention containing the purified peptide as an active ingredient can be used for the manufacture of medicines or foods for the prevention or treatment of cardiovascular diseases.

The invention also provides a method of preventing or treating a cardiovascular-related disorder comprising administering to a mammal a peptide of SEQ ID NO: 1 or SEQ ID NO: 2.

The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. In the treatment method of the present invention, in the case of an adult, it is preferable to administer the peptide of the present invention at a dose of 0.01 mg / kg to 250 mg / kg once or several times a day.

In the therapeutic method of the present invention, the composition comprising the peptide according to the present invention as an active ingredient may be administered orally, rectally, intravenously, intraarterally, intraperitoneally, intramuscularly, intrasternally, transdermally, topically, Lt; / RTI >

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

1-1. Material preparation

Spirulina maxima was obtained from Marine Instute, Ansan, Korea. Pronase E, trypsin, alpha-chymotrypsin and pepsin were purchased from Sigma-Aldrich (St. Louis, Mo., USA). An ultrafiltration membrane for the hydrolyzate fraction based on molecular weight was purchased from Millipore Co. (Bedford, USA). DMEM medium, bovine serum (FBS) and antibiotics were purchased from Gibco BRL (Gaithersburg, MD, USA). Enzyme immunoassay reagents for cytokine analysis were purchased from R & D Systems (Minneapolis, MN, USA). Dimethyl sulfoxide, o-phthalaldehyde, dinitrophenyl-bovine serum albumin (DNP-BSA), dinitrophenyl-specific immunoglobulin E, 3- (4,5-dimethyl-2-yl) -2,5-diphenyltetrazolium bromide (MTT) and other reagents used in the present invention were purchased from Sigma-Aldrich (St. Louis, Mo., USA).

BODIPY ^® FL histamine were purchased from Invitrogen, Molecular Probe (Eugene, OR , USA), histamine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Oligo (dT) 15 primer, M-MLV reverse transcriptase and GoTaq DNA polymerase were purchased from Promega (Madison, Wis., USA) for cytokine analysis and purchased from R & D Systems Respectively. Antigen-specific antibodies for Western blotting were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and Cell Signaling Technology Inc. (Danvers, MA , USA). All primers used in this experiment were synthesized in Bioneer (Taejon, Daeduk-gu, Seoul). All other reagents were purchased from Sigma-Aldrich (St. Louis, Mo., USA).

1 -2. Cell culture

Human umbilical vein endothelial cells (EA. Hy926) and human mononuclear leukemic lymphoma cells (U937) were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells are 10% heat-inactivated FBS, 2mM L- glutamine, 10 mM HEPES buffer, 100 U / ml penicillin G, 100 mg / ml 5% by using a DMEM culture medium containing streptomycin CO _2, 37 ℃ And cultured under wet conditions.

1-3. Statistical analysis

The statistical difference between the groups was analyzed by Duncan's multi-range ANOVA. The difference was considered to be significant when p <0.05. Statistical software was SAS v9.1 (SAS Institute Inc., Cary, NC, USA).

< Example  2>

Spolina  Maxima Spirulina maxima ) Origin Of peptide  Produce

2-1. enzyme Hydrolyzate  Produce

The dried S. spirulina maxima were dissolved in 0.1 M sodium phosphate buffer and stored at -20 &lt; 0 &gt; C for 12 hours. After frozen at -20 ° C and thawed at 37 ° C four times, the mixture was sonicated for 1 hour and heat-treated at 70 ° C for 1 hour. Thereafter, the mixture was centrifuged at 3000 rpm for 10 minutes, and the blue supernatant was recovered and lyophilized. Protein extracts were hydrolyzed with either pronase E or digestive enzymes trypsin, alpha-chymotrypsin and pepsin. The hydrolyzate of proanase E (SM1) was obtained by hydrolysis in a 0.1M sodium phosphate buffer using PRONAYA E at a temperature of 50 ° C and pH 8 for 8 hours, and hydrolyzate by digestion enzyme (SM2) was hydrolyzed with trypsin and alpha-chymotrypsin in 0.1M sodium phosphate buffer at 37 ° C and pH 8 for 4 hours and then treated with pepsin at 37 ° C and pH 2 &Lt; / RTI &gt; by stirring for a further hour. The reaction was terminated by heat treatment in a boiling water bath for 10 minutes and the reduced-pressure lyophilized hydrolyzate was stored at -80 ° C until use.

2-2. enzyme Hydrolyzate  Fraction according to molecular weight

The lyophilized enzyme hydrolyzate was diluted in distilled water and fractionated according to molecular weight using a Hollow Fiber Catridge (GE Healthcare Bio-Sciences Corp., Westborough, Mass., USA) at 10, 5 and 3 kDa cutoff. The fractions were hydrolysates of 10 kDa or more that did not pass through the 10 kDa membrane, hydrolysates of 5-10 kDa which passed through the 10 kDa membrane but not the 5 kDa membrane, hydrolysates of 3-5 kDa which passed the 5 kDa membrane but did not pass through the 3 kDa membrane, And each fraction was stored at -20 ° C after lyophilization under reduced pressure, and further experiments were carried out.

As a result of fractionation according to the molecular weight of the enzyme hydrolyzate, it was confirmed that hydrolysates having a molecular weight of 3 kDa or less can most effectively inhibit histamine release and IL-8 production (data not shown).

2-3. Physiological activity Of peptide  refine

2-3-1. Ion exchange chromatography

Purification of the peptides from the enzyme hydrolyzate was carried out using HiPrep 16/10 diethylaminoethyl high-flow rate (DEAE FF) anion-exchange column (FPLC, AKTA, Amersham Biosciences Co., Uppsala, Sweden) × 100 mm, Amersham Biosciences, Piscataway, NJ, USA). The hydrolyzate (40 mg / ml) was loaded (2 ml) on a HiPrep 16/10 DEAE anion exchange column equilibrated with 20 mM sodium acetate buffer (pH 4.0) and linearized with sodium chloride (0-2 M) Gradient at a flow rate of 2 ml / min. Each fraction was monitored at 280 nm, collected in a volume of 4 ml and then concentrated using a rotary evaporator.

The above-mentioned ion-exchange chromatography showed the most effective inhibition of histamine secretion inhibition and IL-8 production inhibition in the second fraction (data not shown), and the fraction was lyophilized under reduced pressure, and further experiments were conducted.

2-3-2. Gel filtration chromatography

The reduced-pressure freeze-dried fraction was purified using a GE Healthcare Superdex ^TM Peptide 10/300 GL gel filtration column (10 × 300mm). The column was eluted using sodium phosphate buffer (50 mM, pH 7) and 1 ml fractions were collected at a flow rate of 1 ml / min. The fractions were monitored at 280 nm and the physiologically active fractions were lyophilized to further test.

The second fraction obtained by the above ion exchange chromatography was further fractionated into 10 fractions to inhibit histamine secretion and inhibit IL-8 production. As a result, it was found that the 8th fraction inhibited histamine secretion and IL-8 production (Data not shown).

2-3-3. High Performance Liquid Chromatography ( HPLC )

The vacuum lyophilized fraction obtained by the gel filtration chromatography was transferred to Primesphere 10 C18 (10 [mu] l) at a flow rate of 1 ml / min with a leading gradient of acetonitrile (0-40%) containing 0.1% trifluoroacetic acid (RP-HPLC, Dionex Korea Ltd., Sunnyvale, Calif., USA) on a column of 200 mm × 250 mm, Phenomenex, Cheshire, England. The elution peak was measured at 215 nm, the active peak was concentrated using a rotary evaporator, and then lyophilized under reduced pressure. The final purified peptide was used for amino acid sequence analysis.

2-4. Amino acid sequence analysis

The exact molecular weight and amino acid sequence of the purified peptide was analyzed using a Q-TOF mass spectrometer (Micromass, Altrincham, UK) coupled with an Electrospray Ionization (ESI) source. The purified peptide was dissolved in methanol / water (1: 1, v / v) and then injected into an electron spray source and the molecular weight was measured in two transversely charged states (M + 2H) ²⁺ . After the measurement of the molecular weight, the peptide was segmented and tandem mass spectroscopy was used to obtain the peptide sequence of LDAVNR (SEQ ID NO: 1) as shown in FIG. 1 or the peptide sequence (SEQ ID NO: 2) such as MMLDF as shown in FIG. there was.

< Example  3>

In histamine-stimulated epithelial cells, The peptide  Analysis of cytokine production

IL-6 and IL-8 are major cytokines involved in the inflammatory response and are one of the main indicators of atherosclerosis. In addition, PGE 2 has been reported to be associated with the instability of atherosclerotic plaques (Gomez-Hernandez et al., 2006), LTB4 is a chemoattractant factor that attaches white blood cells to the endothelial cells, increases vascular permeability, It is a factor that increases expression in atherosclerotic syndrome by promoting proliferation and migration. On the other hand, MCP-1 is known to be closely related to the progression of atherosclerosis by recruiting mononuclear leukocytes in the subepithelial cell layer.

In order to confirm the therapeutic effect of the purified peptides (P1, P2) by hydrolyzing Spirulina maxima, Hy926 cells were divided into 24-well plates (2 x 10 ⁴ cells / ml), treated with various concentrations of physiologically active peptides (50, 100, 200 μM) for 12 hours and then incubated with histamine (final concentration, 10 μM) The production of IL-6, IL-8, PGE ₂ , LTB 4 and MCP-1 was quantitated using sandwich immunoassay according to the protocol of R & D Systems.

As a result, it was confirmed that the amount of cytokine production was significantly reduced by the peptide treatment. That is, in the case of IL-6, the histidine-treated negative control group treated with 23 pg / ml and histamine-treated only and the positive control group treated with no peptide showed a yield of 380 pg / ml, In the case of the peptide of SEQ ID NO: 2, the amount of IL-6 production was significantly reduced to 112 pg / ml in one case (FIG. 3A). In the case of IL-8, the histidine-treated negative control group was 116 pg / ml, histamine-treated only, and the positive control group not treated with peptide showed 1624 pg / ml. However, the peptide of SEQ ID NO: 1 (Fig. 3B), the production of IL-8 was significantly reduced at 626 pg / ml when treated with the peptide of SEQ ID NO: 2 and at 532 pg / ml with the peptide of SEQ ID NO: 2.

The production of PGE2 and LTB4 also showed 135 pg / ml and 86 pg / ml, respectively, in the case of negative control without histamine treatment, and 813 pg / ml and 432 pg in histamine- / ml. However, when the peptide of SEQ ID NO: 1 was treated, 362 pg / ml and 195 pg / ml were produced, respectively, and when the peptide of SEQ ID NO: 2 was treated, the yields were 302 pg / ml and 156 pg / (P1, P2) significantly reduced PGE2 and LTB4 production in histamine-stimulated epithelial cells (Fig. 4).

Finally, in the production of MCP-1, MCP-1 production was 71 pg / ml when histamine was not treated, whereas MCP-1 production by histamine-treated epithelial cell was 477 pg / ml. -1, but the production of MCP-1 at 233 pg / ml and 191 pg / ml, respectively, when the peptides of SEQ ID NO: 1 and SEQ ID NO: 2 were treated respectively, showed that the MCP-1 significantly increased in histamine-stimulated epithelial cells -1 was effectively reduced by the peptide of the present invention (FIG. 5).

< Example  4>

In histamine-stimulated epithelial cells, The peptide  Analysis of effects on cytokine expression

The inventors of the present invention have found that the amount of cytokine production of epithelial cells stimulated with histamine in Example 3 is remarkably inhibited by the peptide according to the present invention and this effect is caused by inhibiting the expression of cytokines of peptides Western blotting and RT-PCR experiments were carried out.

For Western blotting, EA. Hy926 cells were divided into 6-well plates (2 × 10 ⁴ cells / ml) and physiologically active peptides (50, 100, 200 μM) were treated for 12 hours and stimulated with histamine (final concentration 10 μM). Cells were then lysed with RIPA lysis buffer (NP-40, Sigma-Aldrich, USA) and the same amount of protein was electrophoresed on 10% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked with TBS-T buffer (20 mM Tris, pH 7.6, 0.1% Tween 20) containing 5% (w / v) bovine serum albumin and washed with the appropriate primary antibody (1: 1000 dilution) After the reaction, the cells were washed 3 times with TBS-T buffer and reacted with horseradish peroxidase (HRP) -conjugated IgG secondary antibody (1: 5000 dilution) for 1 hour. After that, the cells were washed 3 times with TBS-T buffer, and the band showing immune response was confirmed by electrochemiluminescence (ECL). In the case of nuclear proteins, proteins extracted by using a nuclear extraction reagent (Sigma, St. Louis, MO, USA) according to the manufacturer's method were used for Western blotting.

On the other hand, for RT-PCR, EA. Hy926 cells were divided into 6-well plates (2 × 10 ⁴ cells / ml) and physiologically active peptides (50, 100, 200 μM) were treated for 12 hours and stimulated with histamine (final concentration 10 μM). Or rat IL-4 (10 ng / ml) for 24 hours. After intracellular RNA was extracted with Trizol reagent according to the manufacturer's method, 2 μg of total RNA was added to RNase-free water and oligo (dT) for cDNA synthesis, denatured at 70 ° C for 5 minutes, and immediately cooled. RNA was reverse transcribed using a master mix containing 1 x RT buffer, 1 mM dNTPs, 500 ng oligo (dT), 140 U M-MLV reverse transcriptase, and 40 U RNase inhibitor for 1 hour at 42 ° C. The synthesized cDNA was used for PCR analysis and GAPDH was used as a control. The primer sequences used in the present invention are as follows.

IL-6 sense 5'-AGG-AGA-CTT-GCC-TGG-TGA-AA-

IL-6 antisense 5'-CAG-GGG-TGG-TTA-TTG-CAT-CT-

IL-8 sense 5'-ATG-ACT-TCC-AAG-CTG-GCC-GTG-GCT-

IL-8 antisense 5'-TCT-CAG-CCC-TCT-TCA-AAA-ACT-TCT-

COX-2 sense 5'-TGA-AAC-CCA-CTCCAA-ACA-CA-3 '

COX-2 antisense 5'-GAG-AAG-GCT-TCC-CAG-CTT-TT-

5-LO sense 5'-ACC-ATT-GAG-CAG-ATC-GTG-GAC-ACG-

5-LO antisense 5'-GCA-GTC-CTG-CTC-TGT-GTA-GAA-TGG-

GAPDH sense 5'-GAG-TCA-ACG-GAT-TTG-GTC-GT-3 '

GAPDH antisense 5'-GAC-AAG-CTT-CCC-GTT-CTC-AG-3 '

Amplification conditions were 30 cycles at 95 ° C for 45 seconds (denaturation), 55 ° C for 50 seconds (annealing) and 72 ° C for 60 seconds (extension), and the amplified product was subjected to 1% agarose electrophoresis, ml EtBr and analyzed with UV-assisted AlphaEase® gel image analysis software (Alpha Innotech, San Leandro, CA, USA).

As a result, it was confirmed that the expression levels of IL-6 and IL-8 increased by histamine were effectively reduced at both protein and mRNA levels by treating the peptides (P1, P2) according to the present invention (Fig. 6).

On the other hand, when COX-2, 5-LO expression levels involved in the secretion of PGE2 and LTB4 were observed, the amount of COX-2 and 5-LO expressed by histamine increased, (P1, P2), both at the protein and mRNA level (Fig. 7).

In addition, it was confirmed that histamine-enhanced MCP-1 was also effectively reduced at both protein and mRNA levels by treating the peptides (P1, P2) of the present invention (FIG. 8).

< Example  5>

In histamine-stimulated epithelial cells, The peptide  Analysis of the effect on bonding molecule formation

E-selectin and P-selectin are known to bind leukocytes to the blood vessel wall, and when leukocytes bind to vascular endothelial cells and move to endothelial cells, atherosclerosis develops. Therefore, the present inventors conducted an experiment to confirm the effect of the peptides (P1, P2) of the present invention on the formation of E-selectin and P-selectin as the conjugated molecules. For this purpose, EA. hy926 cells were treated with physiologically active peptides for 12 hours and stimulated with histamine (final concentration 10 [mu] M) for 20 hours. Cells were then washed and reacted with anti-E-selectin or anti-P-selectin antibody for 4 hours and then further reacted with FITC-conjugated secondary antibody. Cells were washed, PBS was added, and fluorescence images were observed using a fluorescence microscope (CTR 6000, Leica, Wetzlar, Germany).

As a result, it was confirmed that when the peptide of the present invention was treated, the formation of histamine-stimulated E-selectin and P-selectin, which are conjugated molecules of epithelial cells, was remarkably reduced (FIG. On the other hand, the protein and mRNA expression levels of E-selectin and P-selectin according to the peptide treatment were tested according to the method described in Example 4, and as a result, the expression amounts of these proteins and mRNA were decreased in a concentration- (Fig. 10).

< Example  6>

In histamine-stimulated epithelial cells, The peptide Mononuclear  Analysis of effects on leukocyte recruitment

In order to observe whether peptides (P1, P2) according to the present invention can inhibit histone-stimulated epithelial cell mononuclear leukocyte recruitment, in vitro Bonding experiments were carried out.

For this purpose, the fusion of U937 cells EA. The binding to hy926 cells was measured using fluorescent signals of U937 cells labeled with acetyloxymethyl ester of calcein. To this end, U937 cells (2 x 10 4 cells / ml) were solubilized in a medium containing calcein-AM (final concentration 5 μM) and allowed to react for 60 minutes at room temperature. After that, the cells were washed, counted again, and then released into the medium. At the same time, EA. Hy926 cells (2 x 105 cells / ml) were treated with physiologically active peptides (200 μM) for 12 hours and stimulated with histamine (final concentration 10 μM). Cell monolayers were then washed and calcein-AM-labeled U937 cells were covered for 1 hour. Fluorescence images were observed using a fluorescence microscope (CTR 6000, Leica, Wetzlar, Germany) to remove unbound cells.

As a result, monocytic leukocytes were not observed in epithelial cells not treated with histamine, but monocyte leukocytes were strongly stained by treatment with histamine. However, monocyte leukocytes were not collected in the epithelial cells treated with the peptide of the present invention and histamine (Fig. 11).

< Example  7>

In histamine-stimulated epithelial cells, The peptide TLR4  Analysis of effects on expression

A number of studies have shown that TLR-4 is closely related to the onset and progression of atherosclerosis. Therefore, in order to confirm the therapeutic effect of the peptides according to the present invention in atherosclerosis, the present inventors examined whether the expression of TLR-4 is inhibited by the peptide according to the present invention. For this purpose, EA. Hy92 cells were pretreated with various concentrations of the peptides, histamine was treated for 20 hours, and the amount of protein and mRNA expression of TLR-4 was observed in the same manner as described in Example 4 above.

As a result, it was confirmed that the histamine-stimulated TLR-4 protein and mRNA expression levels decreased in a concentration-dependent manner upon peptide treatment (FIG. 12).

< Example  8>

Histamine-stimulated epithelial cells Egr -1 expression Of peptide  Impact Analysis

In order to confirm the mechanism relating to the effect of the peptide according to the present invention on histamine-induced inflammation-related molecules and the production amount and expression level of the conjugated molecule, the inventors of the present invention found that the peptide of the present invention I examined the effect.

For this purpose, EA. When hy926 cells were treated with various concentrations of peptides and histamine was treated, the protein expression level and mRNA expression level of Egr-1 were observed. As a result, when the peptide of the present invention was treated, the expression level of Egr- mRNA level (Fig. 12).

As a result, it was confirmed that the histamine-stimulated TLR-4 protein and mRNA expression levels decreased in a concentration-dependent manner upon peptide treatment (FIG. 13).

< Example  9>

In histamine-stimulated epithelial cells, The peptide  c- fos  Analysis of effects on expression

The transcription factor c-fos is known to regulate the expression of Egr-1. Therefore, the present inventors conducted Western blotting to confirm whether the inhibitory effect of the peptides (P1, P2) according to the present invention on Egr-1 expression is due to the inhibitory effect of c-fos expression. As a result, it was confirmed that the expression amount of c-fos was decreased in a concentration-dependent manner by the peptide treatment (Fig. 14).

< Example  10>

In histamine-stimulated epithelial cells, The peptide MAPK  Analysis of effect on activity

MAPK is known as an upper signaling molecule of transcription factors c-fos and Egr-1. Therefore, the present inventors have further tested the effect of the peptide according to the present invention on the activity of MAPK.

As a result of inhibition of protein expression of c-fos and Egr-1 when treated with inhibitor to inhibit phosphorylation of ERK, inhibition of the activity of MAPK including ERK, p38, and JNK when treated with the peptide of the present invention (Fig. 15). Therefore, the inventors of the present invention have concluded from these results that the peptide according to the present invention inhibits the expression of c-fos and Egr-1, which are transcription factors, due to suppression of ERK activity.

< Example  11>

In histamine-stimulated epithelial cells, The peptide PKC  Analysis of effect on activity

The activity of PKC causes activation of MAPK signal transduction, and the present inventors analyzed how the peptides according to the present invention have an effect on PKC activity. As a result, it was confirmed that PKC activity was inhibited in a concentration-dependent manner when the peptide of the present invention was treated (FIG. 16).

< Example  12>

In histamine-stimulated epithelial cells, The peptide ROS  Impact on production

The reactive oxygen species are involved in the deterioration of epithelial cells associated with atherosclerosis and are known to be one of the leading causes of atherosclerosis. Thus, the present inventors have experimented with the scavenging activity of other peptides according to the present invention on the production of ROS in histamine-stimulated epithelial cells.

For FACs analysis, EA. Hy926 cells were treated with physiologically active peptide (200 μM) for 12 hours and reacted with 5 μM of DHE and DCFH-DA at 37 ° C. for 1 hour. Cells were then washed and analyzed by flow cytometry (BD Diagnostic Systems, Cockeysville, MD, USA) at 37 ° C for 1 hour in PBS buffer containing histamine (final concentration 10 μM). The number on the histogram means the percentage of DCF produced by oxidation of DCFH under ROS conditions.

For fluorescence microscopy, EA. Hy926 cells were treated with physiologically active peptide (200 μM) for 12 hours and reacted with 5 μM of DHE and DCFH-DA at 37 ° C. for 1 hour. The cells were then washed with Tyrode buffer and stimulated with histamine (final concentration 10 μM) for 1 h, then the medium was removed, 3% paraformaldehyde was added for 30 min, and fluorescence microscopy (CTR 6000, Leica, Wetzlar, Germany) And fluorescence images were observed.

As a result, the amount of ROS due to histamine treatment increased from 0.12% to 47%, but it was confirmed that by treating the peptide, the amount of ROS decreased by 22% in P1 treatment and 15% in P2 treatment (FIG. 17A). Also, ROS scavenging activity following peptide treatment was observed in ROS observation through fluorescence microscope (Fig. 17B).

< Example  13>

The effect of histamine on the binding of histamine to histamine in histamine-stimulated epithelial cells. Of peptide  Impact Analysis

The present inventors tried to confirm whether the peptide according to the present invention affects the histamine-induced signal transduction system through binding with the histamine receptor.

For this purpose, EA. Hy926 cells were treated with physiologically active peptide (200 μM) for 12 hours and then washed in cold binding buffer (BD Diagnostic Systems, Cockeysville, MD, USA) containing P1 or P2 and FITC-histamine for 4 hours . Cells were then washed with centrifugation and re-infused with cold binding buffer and analyzed using a flow cytometer (BD Diagnostic Systems, Cockeysville, MD, USA). The number on the histogram means the percentage of histamine-positive cells.

As a result, both the H1 and H2 receptor-specific antagonists blocked the histamine receptors of the epithelial cells and lowered the fluorescence intensity from 94% to 9% and 11%, respectively. Meanwhile, when the peptides P1 and P2 according to the present invention were treated, the fluorescence intensity was lowered to 31% and 29%, respectively. Thus, the present inventors have found that the peptide according to the present invention blocks the histamine receptor, (Fig. 18).

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

P1: Peptide of SEQ ID NO: 1
P2: Peptide of SEQ ID NO: 2

<110> Pukyong National University Industry-University Cooperation Foundation <120> Composition containing peptides from spirulina maxima for          prevention or treatment of Cardiovascular disorders <130> PN1301-027 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 6 <212> PRT <213> Spirulina maxima <400> 1 Leu Asp Ala Val Asn Arg   1 5 <210> 2 <211> 5 <212> PRT <213> Spirulina maxima <400> 2 Met Met Leu Asp Phe   1 5

Claims (12)

A composition for preventing or treating cardiovascular diseases, comprising the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient. The method according to claim 1,
Wherein said cardiovascular disease is atherosclerosis. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method according to claim 1,
Wherein the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 is derived from Spirulina maxima. The method according to claim 1,
Wherein the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 has the effect of preventing or treating cardiovascular diseases by inhibiting the expression of IL-6 or IL-8. The method according to claim 1,
Wherein the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 has the effect of preventing or treating cardiovascular diseases by inhibiting the production of PGE2, LTB4 or MCP-1. 6. The method of claim 5,
Wherein the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 inhibits the production of PGE2, LTB4 or MCP-1 by inhibiting the expression of COX-2, 5-LO or MCP-1 Composition. The method according to claim 1,
Wherein the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 inhibits the production or expression of P-selectin or E-selectin, thereby preventing or treating cardiovascular diseases. &Lt; / RTI &gt; The method according to claim 1,
Wherein the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 has the effect of preventing or treating cardiovascular disease by inhibiting the expression of TLR4. The method according to claim 1,
Wherein the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 has the effect of preventing or treating cardiovascular diseases by inhibiting ERK signal transduction pathway. The method according to claim 1,
Wherein the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 has the effect of preventing or treating cardiovascular diseases by inhibiting the activation of PKC. The method according to claim 1,
Wherein the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 is a compound for preventing or treating cardiovascular diseases by inhibiting the production of reactive oxygen species. A health functional food for preventing or ameliorating a cardiovascular disease comprising the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

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