KR102032945B1 - Peptides having anti-inflammatory activity and composition the same for anti-inflammatory - Google Patents

Abstract

The present invention relates to an alpha-helical peptide having an anti-inflammatory activity and an anti-inflammatory composition comprising the same. More specifically, the lysine and arginine are used as cationic amino acids based on the alpha-helical structure, and isoleucine and tryptophan are hydrophobic amino acids. A new antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9, synthesized using an amphiphilic structure with different hydrophilicity and hydrophobicity A new antimicrobial active peptide synthesized with an amphiphilic structure using hydrophilicity and hydrophobicity, including lysine and arginine, isoleucine and tryptophan as hydrophobic amino acids; Pharmaceutical composition for anti-inflammatory, containing It relates to medical compositions and food additives, the additive composition. Since the anti-inflammatory active peptide of the present invention does not have cytotoxicity and shows excellent anti-inflammatory activity, it may be usefully used as an additive for anti-inflammatory drugs, cosmetics or food preservatives that are safe for human body.

Description

Peptides showing anti-inflammatory activity and anti-inflammatory composition containing the same as an active ingredient {Peptides having anti-inflammatory activity and composition the same for anti-inflammatory}

The present invention relates to a peptide exhibiting anti-inflammatory activity and an anti-inflammatory composition comprising the same as an active ingredient, and more particularly, to nitrogen monoxide (NO) and tumor necrosis factor-α (TNF-α ) Has an anti-inflammatory effect that inhibits the production, has no cytotoxicity, and has very excellent bioavailability, and an anti-inflammatory pharmaceutical composition, cosmetic composition, or food composition comprising the peptide as an active ingredient.

The proportion of elderly people continues to increase due to the recent economic development and the development of medical technology and the prolongation of the average life span. Chronic inflammatory diseases such as atopy and asthma are caused by chronic inflammatory reactions due to immune system abnormalities caused by environmental pollution and increased stress. It is an increasing trend (Chang et al. , 1994, Korean J. Gastroentrol., 26:907-918.; Heinzemann and Daser, 2002, Int. Arch.Allergy Immunol. 127:170-180.; Song et al. , 1998, Korean J. Intern. Med., 55:158-168.; Sung et al. , 2012, J. Ethnopharmacol. 144:94-100.).

In general, the inflammatory response is a defense mechanism of living tissues against external stimuli such as bacterial infection or internal stimuli such as metabolites in the body, and various cytokines such as TNF-α, IL-1β, IL-6, etc. It occurs as cytokines and nitric oxide (NO) are produced. In addition, lipid polysaccharide known as endotoxin (lipopolysaccharide, LPS) is induced by the activation of the outer membrane of gram-negative bacteria present in the cells, the NF- κ B (nuclear facter- κ B ) within a transfer element cells in macrophages or monocytes It induces gene expression of inflammatory cytokines, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), and produces inflammatory mediators.

Therefore, in order to control the inflammatory response and is a key factor in regulating the secretion of iNOS, COX-2, or expression, and cytokine and nitric oxide of NF- κ B, this modulates the activity of the factor, such materials are inflammatory diseases (Kim et al. , 2013b, J. Korean Med. Ophthalmol. Otolaryngol. Dermatol., 26:54-64.)

Substances currently used for anti-inflammatory purposes include non-steroidal flufenamic acid, ibuprofen, benzydamine, indomethacin, and the like; Steroids include prednisolone, dexamethasone, hydrocortisone, and betamethasone, but these substances are highly toxic and cause serious side effects such as liver damage, cancer, and stroke. There are restrictions. In addition, there may be a problem that causes severe immunosuppression due to inability to selectively act on substances that cause inflammation. Accordingly, the development of anti-inflammatory drugs using natural products that are safe for the living body and have the advantage of being easy to take for a long time compared to conventional medicines is being made. Since it has to be done, there is a problem such as a high production cost.

In order to improve the above problems, a new concept of anti-inflammatory drugs is being developed as an alternative to conventional chemo-inflammatory drugs or inflammatory drugs using natural products, and in particular, many studies have been conducted on the synthesis of peptides having anti-inflammatory activity having anti-inflammatory activity. Is losing.

However, the synthesized peptide has very good anti-inflammatory activity in in vitro experiments and does not exhibit cytotoxicity, but in reality, in in vivo experiments, the anti-inflammatory effect is often insignificant. This is because there are various reasons, but mainly because the physiological and anatomical conditions in vivo are very different from the experimental conditions in vitro. First, if the physiological condition of salt is present, the activity of the peptide with a low positive charge is significantly inhibited. Second, because peptides have a small molecular weight and size, they are almost absorbed by the kidneys and released into the body. Third, it is easily cut off by protein and peptide degrading enzymes (protease and peptidase) present in all tissues, cells, body fluids, and blood in a living body and loses its activity.

Accordingly, the inventors of the present invention solved the above problems and while continuing research on substances exhibiting excellent anti-inflammatory activity, developed a peptide that can be economically mass-produced using only 10 to 14 common amino acid residues, and the peptide is By confirming that it does not exhibit cytotoxicity and exhibits excellent anti-inflammatory activity, the present invention has been completed.

Accordingly, one object of the present invention is to provide a peptide that has no cytotoxicity and exhibits excellent anti-inflammatory activity.

Another object of the present invention is to provide an anti-inflammatory composition comprising the anti-inflammatory active peptide as an active ingredient.

Another object of the present invention is to provide an anti-inflammatory pharmaceutical composition, cosmetic composition or food composition comprising the composition.

In one aspect, the present invention provides an anti-inflammatory active peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

The term "anti-inflammatory" as used herein means preventing, treating or improving inflammation. Here, the inflammation refers to a disease caused by infection, wounds, surgery, burns, frostbite, electrical stimulation or chemical substances caused by external infectious agents (bacteria, fungi, viruses, various kinds of allergens, etc.), and the disease is Dermatitis, inflammatory bowel disease, gastric ulcer, colitis, cystitis, rhinitis, tonsillitis or asthma, etc., but is not particularly limited thereto.

The anti-inflammatory active peptide of the present invention is a peptide having an alpha-helical structure, wherein the hydrophilic sequence is applied as a cationic amino acid lysine (Lysine, K) or arginine (Arginine, R), and the hydrophobic sequence is applied as a hydrophobic amino acid, Isoleucine. ) Or tryptophan, which is an amphipathic peptide having 10 or 14 amino acids.

In addition, the anti-inflammatory peptide of the present invention is a peptide having an alpha-helical structure, wherein the hydrophilic sequence is applied to the cationic amino acid lysine (Lysine, K) or arginine (Arginine, R), and the hydrophobic sequence is applied to the hydrophobic amino acid isoleucine ( Isoleucine) or tryptophan is applied, and it is an amphipathic peptide having 14 amino acids containing glutamic acid, an anionic amino acid.

As a specific embodiment, the peptides of the present invention have the amino acid sequence described in SEQ ID NO: 1 to 10, these are "KIW-10 (SEQ ID NO: 1), KWI-10 (SEQ ID NO: 2), WIK-10 (SEQ ID NO: 3), RIW-10 (SEQ ID NO: 4), RWI-10 (SEQ ID NO: 5), WIR-10 (SEQ ID NO: 6), KIW-14 (SEQ ID NO: 7), KWI-14 (SEQ ID NO: 8), WIK-14 (SEQ ID NO: No. 9) and WIKE-14 (SEQ ID No. 10) peptide". The amino acid sequences of these peptides are shown in Table 1 below.

Peptide order KIW-10 (SEQ ID NO: 1) KKIIKKIWKW KWI-10 (SEQ ID NO: 2) KKIWKKWIKI WIK-10 (SEQ ID NO: 3) WIKKIWKKIK RIW-10 (SEQ ID NO: 4) RRIIRRIWRW RWI-10 (SEQ ID NO: 5) RRIWRRWIRI WIR-10 (SEQ ID NO: 6) WIRRIWRRIR KIW-14 (SEQ ID NO: 7) KKIIKKIIKKIWKW KWI-14 (SEQ ID NO: 8) KKIWKKWIKKIIKI WIK-14 (SEQ ID NO: 9) WIKKIWKKIIKKIK WIKE-14 (SEQ ID NO: 10) WIKKIWKKIIKEIK

The anti-inflammatory active peptide of the present invention does not show cytotoxicity, and it has been confirmed that it inhibits the production of nitrogen monoxide (NO) and TNF-α, an inflammatory modulator, in cells invaded by external infectious agents. Therefore, the anti-inflammatory active peptide of the present invention can be usefully used for preventing, treating or improving inflammation.

According to one embodiment of the present invention, the present invention has 85% or more homology with a peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. And all peptides having an amino acid sequence exhibiting the same effect may be included. Anti-inflammatory active peptide disclosed in the present invention is a peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or fragments thereof and one or two amino acids are changed. It may include a peptide.

In the present invention, the terms "homology" and "sequence identity" are used interchangeably, which refers to the degree of overlap of the sequence between two amino acids (or similar nucleic acids).

Specifically, in the present invention, in the peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, each amino acid is substituted with another amino acid having a similar structure, or an amino terminal It may include an anti-inflammatory active peptide obtained by substituting the amino group of or the carboxyl group at the carboxy terminal with another functional group. For example, among the synthesized peptides, the peptide in which isoleucine or tryptophan is substituted with another hydrophobic amino acid is an anti-inflammatory activity consisting of the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Like peptides, it has no cytotoxicity and can maintain excellent anti-inflammatory activity. At this time, the hydrophobic amino acids that can be substituted for isoleucine among the amino acid sequences described in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 are tryptophan, alanine, methionine, phenylalanine, proline, valine, and leucine. Among the amino acid sequences described in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, hydrophobic amino acids that may be substituted for tryptophan include isoleucine, alanine, methionine, phenylalanine, tryptophan, proline , Valine and leucine.

According to one embodiment of the present invention, the amino acid sequence having at least 85% homology is characterized in that it has the following sequence.

(i) Isoleucine among the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 is hydrophobic selected from the group consisting of alanine, phenylalanine, methionine, tryptophan, proline, valine, and leucine An amino acid sequence substituted with an amino acid;

(ii) Among the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, tryptophan is hydrophobic selected from the group consisting of alanine, isoleucine, methionine, phenylalanine, proline, valine, and leucine An amino acid sequence substituted with an amino acid; or

(iii) Isoleucine is hydrophobic selected from the group consisting of alanine, phenylalanine, methionine, tryptophan, proline, valine, and leucine among the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 An amino acid sequence in which tryptophan is substituted with an amino acid, and at the same time tryptophan is substituted with a hydrophobic amino acid selected from the group consisting of alanine, isoleucine, methionine, phenylalanine, proline, valine, and leucine.

In the present invention, the term "amino acid" includes naturally 22 standard amino acids as well as D-isomers and modified amino acids. Accordingly, in one aspect of the present invention, the anti-inflammatory active peptide may be a peptide containing D-amino acid. Meanwhile, in another aspect of the present invention, the anti-inflammatory active peptide may include a post-translational modified non-standard amino acid or the like. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (e.g., including acetylation, myristoylation and palmitoylation), alkylation. ), carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (e.g., beta-removal deimidation) , Deamidation) and structural changes (eg, formation of disulfide bridges). In addition, it includes changes in amino acids, such as changes in amino groups, carboxyl groups, or side chains, caused by chemical reactions occurring in the process of bonding with crosslinkers to form peptide conjugates.

The anti-inflammatory active peptide consisting of the amino acid sequences described in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 of the present invention exhibits high anti-inflammatory effect without losing activity even in the presence of physiological salts. , It is not toxic to human cells, has strong anti-inflammatory properties, and has excellent bioavailability.

As another aspect, the present invention comprises an anti-inflammatory active peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 as an active ingredient. It provides an anti-inflammatory composition.

The term “prevention” as used herein refers to suppressing the occurrence of a disease or disease in an individual who has not been diagnosed with a disease or disease, but is prone to such a disease or disease.

As used herein, the terms “treatment” and “improvement” mean (a) inhibition of the development of inflammatory diseases (b) alleviation and (c) elimination of inflammatory diseases in an individual.

The term “individual” used in the present invention refers to monkeys, cattle, horses, pigs, sheep, dogs, cats, rats, mice, including humans with diseases that can improve symptoms by administering the anti-inflammatory composition of the present invention. , Means a mammal such as a chimpanzee.

The anti-inflammatory composition further comprises an active ingredient having an anti-inflammatory effect together with an anti-inflammatory active peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. It may contain more than one species.

The anti-inflammatory active peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 contained as an active ingredient in the anti-inflammatory composition is based on the total weight of the total composition. It may be included in an amount of 0.001 to 50% by weight, preferably 0.01 to 30% by weight, more preferably 0.01 to 10% by weight. When the content of the anti-inflammatory active peptide is less than 0.001% by weight, the anti-inflammatory effect is weak, and when it exceeds 50% by weight, the increase in the effect according to the content increase is not proportional and may be inefficient, and the stability of the formulation is not secured. There is no problem.

As one specific use, the composition of the present invention can be used as a pharmaceutical composition for preventing or treating inflammation.

When the composition of the present invention is used as a pharmaceutical composition, in addition to the anti-inflammatory active peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 as an active ingredient It may further include an acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and carbohydrate compounds (e.g., lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, etc.) , Gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, gum arabic, vegetable oil (e.g. corn oil, cotton seed oil , Soy milk, olive oil, coconut oil), polyethylene glycol, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil, but are not limited thereto.

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition may be formulated and used in the form of oral dosage forms such as powders, granules, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions according to a conventional method.

The pharmaceutical composition may prevent or treat inflammation, and the treatment method includes administering the pharmaceutical composition in a pharmaceutically effective amount to mammals such as mice, mice, livestock, and humans by various routes. The administration method may be performed in any manner, for example, oral, rectal or intravenous injection, intraperitoneal administration, intramuscular administration, subcutaneous administration, or topical administration by application.

The dosage of the pharmaceutical composition may vary depending on factors such as formulation method, administration method, age, weight, sex, and pathological condition of the patient. The dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg/kg on an adult basis. However, the dosage does not limit the scope of the present invention in any way.

As one specific use, the composition of the present invention can be used as a cosmetic composition for preventing or improving inflammation.

When the composition of the present invention is used as a cosmetic composition, in addition to the anti-inflammatory active peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 as an active ingredient Ingredients commonly used may additionally be included. For example, it may include antioxidants, stabilizers, solubilizers, conventional adjuvants such as vitamins, pigments and perfumes, and carriers.

The cosmetic composition may be prepared in any formulation conventionally prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils , Powder foundation, emulsion foundation, wax foundation, and spray may be formulated, but are not limited thereto. In more detail, it may be prepared in a formulation such as a nourishing cream, an astringent lotion, a soft lotion, a lotion, an essence, a nourishing gel or a massage cream.

When the formulation of the cosmetic composition is a paste, cream or gel, as a carrier component, animal oil, vegetable oil, wax, paraffin, starch, gum tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. Can be used.

When the formulation of the cosmetic composition is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, Propellants such as propane/butane or dimethyl ether.

When the formulation of the cosmetic composition is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the cosmetic composition is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, micro Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, and the like may be used.

When the formulation of the cosmetic composition is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid Amide ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters may be used.

As one specific use, the composition of the present invention can be used as a food composition for preventing or improving inflammation.

When the composition of the present invention is used as a food composition, in addition to the anti-inflammatory active peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 as an active ingredient, when preparing food Ingredients conventionally added to, for example, proteins, carbohydrates, fats, nutrients, flavoring and flavoring agents may further be included.

Examples of the carbohydrates include monosaccharides (eg, glucose, fructose, etc.); They are conventional sugars such as disaccharides (eg maltose, sucrose, oligosaccharides, etc.) and polysaccharides (eg, dextrins, cyclodextrins, etc.) and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents such as taumatin, stevia extract (eg, rebaudioside A, glysirhizin) and the like, and synthetic flavoring agents such as saccharin and aspartame may be used.

There is no particular limitation on the type of the food. Examples of foods to which the composition of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, There are drinks, alcoholic beverages, and vitamin complexes, and all health foods in the usual sense are included.

When the food composition of the present invention is prepared as a drink, citric acid, liquid, in addition to the anti-inflammatory active peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 as an active ingredient It may further contain fructose, sugar, glucose, acetic acid, malic acid, and fruit juice.

In addition to the above-described ingredients, the food composition includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols. , Carbonating agents used in carbonated beverages, and the like may be contained. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, beverages and vegetable beverages. These components may be used independently or in combination.

As described above, the anti-inflammatory active peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the present invention is nitrogen monoxide (NO) in cells invaded by external infectious agents. And it shows an anti-inflammatory effect that inhibits the production of TNF-α, and is a material that is not only harmless and has very excellent bioavailability without cytotoxicity, so it is used in pharmaceutical compositions, cosmetic compositions, or food compositions for preventing, treating or improving inflammation. It can be applied safely.

1 is a diagram illustrating the design of an α-helical anti-inflammatory peptide having an α-helical structure based on a helical wheel diagram using arginine, lysine, or glutamic acid as hydrophilic amino acids and isoleucine and tryptophan as hydrophobic amino acids. Is shown.
2 is a graph measuring the amount of nitrogen monoxide (NO) produced by an anti-inflammatory active peptide in macrophages (RAW 264.7) stimulated with lipopolysaccharide (LPS).
3 is a graph measuring the amount of tumor necrosis factor-α (TNF-α) produced by an anti-inflammatory active peptide in macrophages (RAW 264.7) stimulated with lipopolysaccharide (LPS).
Figure 4 is a housekeeping gene by anti-inflammatory active peptide in macrophages (RAW 264.7) stimulated with lipopolysaccharide (LPS), glyceraldehyde 3-phosphate dehydrogenase (Glyceraldehyde 3-phosphate dehydrogenase, GAPDH). ), the amount of mRNA production of the cytokine Interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) was subjected to Reverse Transcription Polymerase Chain Reaction (RT-PCR). It is the result.
5 is a result of measuring a lipid polysaccharide (LPS) to which a fluorescent dye, Fluorescein isothiocyanate (FITC) is attached, and macrophages (RAW 264.7) stimulated with an anti-inflammatory active peptide with a flow cytometer. .
6 is a photograph of fluorescein isothiocyanate (FITC)-adhered lipopolysaccharide (LPS) and macrophages (RAW 264.7) stimulated with anti-inflammatory active peptides observed with a fluorescence microscope. .
7 is a graph showing the ability to destroy mouse red blood cells (%) of the anti-inflammatory active peptide according to the present invention.
8 is a graph showing the results of a cytotoxicity experiment on mouse skin fibroblasts (NIH3T3) of the anti-inflammatory active peptide according to the present invention.

Hereinafter, the content of the present invention will be described in more detail through examples and preparation examples. The following examples and formulation examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples and formulation examples.

Example 1: Preparation of peptide derivatives

Using lysine or arginine as a cationic amino acid and isoleucine or tryptophan as a hydrophobic amino acid, it shows an amphiphilic alpha-helix structure in which hydrophilicity and hydrophobicity exist on different sides, and SEQ ID NOs: 1, 2, 3, 4 , 5, 6, 7, 8 and 9 to prepare an anti-inflammatory active peptide consisting of the amino acid sequence described.

Lysine or arginine is used as a cationic amino acid, isoleucine or tryptophan is used as a hydrophobic amino acid, and the anionic amino acid glutamic acid is used to show an amphiphilic alpha-helix structure in which hydrophilicity and hydrophobicity exist on different sides, and SEQ ID NO: An anti-inflammatory active peptide consisting of the amino acid sequence described in 10 was prepared.

The prepared anti-inflammatory active peptides were respectively KIW-10 (SEQ ID NO: 1), KWI-10 (SEQ ID NO: 2), WIK-10 (SEQ ID NO: 3), RIW-10 (SEQ ID NO: 4), RWI-10 (SEQ ID NO: Number 5), WIR-10 (SEQ ID NO: 6), KIW-14 (SEQ ID NO: 7), KWI-14 (SEQ ID NO: 8), WIK-14 (SEQ ID NO: 9) or WIKE-14 (SEQ ID NO: 10) peptide Named it.

The anti-inflammatory active peptide was synthesized by a Fmoc (9-fluorenylmethoxycarbonyl) solid phase method using a microwave peptide synthesizer (CEM Co., Matthews, NC).

Specifically, the amidated peptide was synthesized using Rink Amide 4-methylbenzhydrylamine Resin (Novagiochem, 0.55 mmol/g). Thereafter, the imidized peptide was precipitated and extracted with ether and cut from the resin to obtain a crude peptide. Purified by zorbax C18 column (21.2 X 250 mm, 300Å, 7-μm) using 0-60% acetonitrile dissolved in 0.1% trifluoroacetic acid as a gradient. I did. The purified peptide was measured for purity using a Shimadzu analytical HPLC system, and the molecular weight of the peptide was measured using a Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry method. The results are shown in Table 2 below.

Peptide order Molecular Weight KIW-10 (SEQ ID NO: 1) KKIIKKIWKW 1369.8 KWI-10 (SEQ ID NO: 2) KKIWKKWIKI 1369.8 WIK-10 (SEQ ID NO: 3) WIKKIWKKIK 1369.8 RIW-10 (SEQ ID NO: 4) RRIIRRIWRW 1509.9 RWI-10 (SEQ ID NO: 5) RRIWRRWIRI 1509.9 WIR-10 (SEQ ID NO: 6) WIRRIWRRIR 1509.9 KIW-14 (SEQ ID NO: 7) KKIIKKIIKKIWKW 1852.5 KWI-14 (SEQ ID NO: 8) KKIWKKWIKKIIKI 1852.5 WIK-14 (SEQ ID NO: 9) WIKKIWKKIIKKIK 1852.5 WIKE-14 (SEQ ID NO: 10) WIKKIWKKIIKEIK 1740.2

Anti-inflammatory active peptides having a purity of 95% or more, such as those of the amino acid sequence shown in Table 2, were synthesized. The measured molecular weight was consistent with the molecular weight obtained by calculating from the amino acid sequence, and the anti-inflammatory active peptide consisting of the amino acid sequences described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 was accurately synthesized I could confirm.

Test Example 1: Measurement of the inhibitory activity of anti-inflammatory active peptides to produce nitric oxide and TNF-α

In order to measure the anti-inflammatory activity of the anti-inflammatory activity peptides prepared in Example 1 (SEQ ID NOs: 1 to 10), the inhibitory activity of nitrogen monoxide and TNF-α production was measured.

RAW 264.7 cells, a mouse macrophage cell line distributed from Korea Cell Line Bank (KCLB, Seoul, Korea), were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum; Gibco, USA), and then in a 24-well plate. Dispense so as to be 1×10 ⁶ cells/mL and incubated for 24 hours in _{a CO 2} incubator at 37°C and 5% concentration. Then, each well was treated with 1 μg/mL of LPS (Lipopolysaccaride), and after 1 hour, the peptides prepared in Example 1 were diluted with 4, 8, 16 and 32 μM, respectively, and then added for 24 hours. During incubation. Thereafter, the levels of nitrogen monoxide (nitrite, NO) and TNF-α in the supernatant collected from the cell culture were measured using a grease reagent of an R&D system (minneapolis, MN) and a Quantikine mouse kit, respectively.

As a control, macrophages not treated with Hn-Mc peptide and LPS (RAW 264.7) were used. As a positive control, macrophages treated with only LPS (RAW 264.7) were used, and as a negative control, macrophages treated with LPS and polymycin B (RAW 264.7) were used.

The results obtained in the above experiment were expressed as mean ± standard error (mean ± SE), and the average difference between the control group and each experimental group was analyzed by Student's t-test, and it was determined that there was a statistically significant difference when the p value was less than 0.05. I did. The results are shown in Fig. 2 (production amount of nitrogen monoxide (NO)) and Fig. 3 (production amount of TNF-α).

As shown in Figs. 2 and 3, as a result of the experiment, the anti-inflammatory active peptide consisting of the amino acid sequences described in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 is in macrophages activated with LPS. It has been shown to inhibit the production of nitrogen monoxide and TNF-α in a concentration-dependent manner. In particular, 32 μM of KIW-14 (SEQ ID NO: 7), KWI-14 (SEQ ID NO: 8), WIK-14 (SEQ ID NO: 9) and WIKE-14 (SEQ ID NO: 10) peptides are almost as much nitrogen monoxide as the negative control (polymycin B). And it was confirmed that it inhibits the production of TNF-α.

Test Example 2: Measurement of mRNA production amount of cytokine genes by anti-inflammatory active peptide

In order to measure the anti-inflammatory activity of the anti-inflammatory active peptide prepared in Example 1, the amount of mRNA produced of the cytokine gene was measured.

In this Test Example 2, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 in Test Example 1 is the peptide consisting of the amino acid sequence of SEQ ID NO: 2, and the peptide consisting of the amino acid sequence of SEQ ID NO: 4 is the amino acid of SEQ ID NO: 5 The peptide consisting of the sequence and the peptide consisting of the amino acid sequence shown in SEQ ID NO: 7 were observed to exhibit similar results to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 8, and SEQ ID NOs: 1, 3, 4, 6, 7, 9 and 10 The test was carried out on the peptide consisting of the amino acid sequence described above.

RAW 264.7 cells, a mouse macrophage cell line distributed from Korea Cell Line Bank (KCLB, Seoul, Korea), were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum; Gibco, USA), and then in a 6-well plate. Dispense to 5X10 ⁶ _{cells/mL and incubate for 24 hours in a CO 2} incubator at 37°C and 5% concentration, and each well was treated with 1 μg/mL of LPS (Lipopolysaccaride), and each 32 μM after 1 hour. Peptide was further added and incubated for 8 hours. Then, the cells were collected, total RNA was isolated with a TRIzol reagent, and 5'-GCATCTTCTTGTGCAGTGCC-3' (SEQ ID NO: 11) and 5'-TCACACCCATCACAAACATG-3' (sequence) were used as a primer of GAPDH (Glyceraldehyde 3-phosphate dehydrogenase). Number 12) as a primer of the cytokine IL-6, 5'-CACAAGTCCGGAGAGGAGAC-3' (SEQ ID NO: 13) and 5'-CAGAATTGCCATTGCACAAC-3' (SEQ ID NO: 14) as a primer of TNF-α 5'-CGCAGCAGCACATCAACAAGAGC Reverse Transcription Polymerase Chain Reaction (RT-PCR) was performed using -3' (SEQ ID NO: 15) and 5'-TGTCCTCATCCTGGAAGGTCCACG-3' (SEQ ID NO: 16), and then the amount of mRNA produced was agarose. It was confirmed by electrophoresis with (agarose) gel. The results are shown in FIG. 4.

As shown in Figure 4, the experimental results of the anti-inflammatory active peptide consisting of the amino acid sequence described in SEQ ID NOs: 1, 3, 4, 6, 7, 9 and 10 are cytokines IL-6 and TNF in LPS-activated macrophages. It was confirmed to suppress the mRNA production of -α at the same level as that of the control. Therefore, it was found that the peptide has anti-inflammatory activity.

Test Example 3: Mechanism of anti-inflammatory active peptide

In order to confirm the mechanism of the anti-inflammatory active peptide prepared in Example 1, an experiment was conducted as follows.

In Test Example 3, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 in Test Example 1 is the peptide consisting of the amino acid sequence of SEQ ID NO: 2, and the peptide consisting of the amino acid sequence of SEQ ID NO: 4 is the amino acid of SEQ ID NO: 5 The peptide consisting of the sequence and the peptide consisting of the amino acid sequence shown in SEQ ID NO: 7 were observed to exhibit similar results to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 8, and SEQ ID NOs: 1, 3, 4, 6, 7, 9 and 10 The test was carried out on the peptide consisting of the amino acid sequence described above.

RAW 264.7 cells, a mouse macrophage cell line distributed from Korea Cell Line Bank (KCLB, Seoul, Korea), were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum; Gibco, USA), and then in a 24-well plate. Dispense so as to be 1×10 ⁶ cells/mL and incubated for 24 hours in _{a CO 2} incubator at 37°C and 5% concentration. Then, LPS (Lipopolysaccaride) with FITC attached to each well was treated at 1 μg/mL, and after 1 hour, the anti-inflammatory active peptide prepared in Example 1 was diluted to 16 or 32 μM and added to 8 Incubated for hours. After that, the cells were collected, and the amount of FITC-attached LPS attached to the cells was confirmed by flow cytometry (Fluorescence-activated cell sorting, FACS) and a fluorescence microscope. The results are shown in FIGS. 5 and 6.

As shown in Figs. 5 and 6, as a result of the experiment, the fluorescence of FITC-labeled LPS was negative control in cells treated with an anti-inflammatory active peptide consisting of the amino acid sequences described in SEQ ID NOs: 1, 3, 4, 6, 7, 9 and 10. (polymycin B) appeared at the same level, and the anti-inflammatory active peptide of the present invention directly binds to LPS and interferes with the binding of LPS to LPS-binding protein (LBP). It was confirmed that it showed anti-inflammatory activity by preventing signal transduction to the receptor (Toll-like receptor).

Test Example 4: Measurement of cytotoxicity of anti-inflammatory active peptide

In order to confirm whether the anti-inflammatory active peptide prepared in Example 1 (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) exhibits cytotoxicity, red blood cell destruction ability as follows: And toxicity to normal cells were measured.

4-1. Hemolytic activity of anti-inflammatory peptides on red blood cells

Human red blood cells were diluted with a phosphate buffer solution (PBS, pH 7.0) to a concentration of 8%, and the anti-inflammatory active peptides prepared in Example 1 were serially diluted to a concentration of 1/2 to 1 at 37°C. After reacting for a period of time, it was centrifuged at 1,000 g, and the amount of hemoglobin contained in the supernatant was measured by measuring absorbance at a wavelength of 414 nm. To investigate the degree of cell destruction, 0.1% Triton X-100 was added to human red blood cells, and the absorbance of the supernatant was measured, and the cell destruction ability of 0.1% Triton X-100 was set to 100%, which was an anti-inflammatory peptide. It was calculated according to Equation 1 below in comparison with the red blood cell destruction ability of. The results are shown in FIG. 7.

In the above formula,

Absorbance A is the absorbance of the anti-inflammatory active peptide solution treatment group at a wavelength of 414 nm,

Absorbance B is the absorbance of the group treated with only PBS at a wavelength of 414 nm,

Absorbance C represents the absorbance of the treated group of 0.1% Triton X-100 at a wavelength of 414 nm.

As shown in Figure 7, the hemolysis phenomenon is KIW-14 (SEQ ID NO: 7) peptide is 8.29% at 200 μM, KWI-14 (SEQ ID NO: 8) peptide is 6.84% at 200 μM, WIK-14 (SEQ ID NO: 9) Peptides were found to be 16.17% at 200 μM, KIW-10 (SEQ ID NO: 1), KWI-10 (SEQ ID NO: 2), WIK-10 (SEQ ID NO: 3), RIW-10 (SEQ ID NO: 4), RWI-10 (SEQ ID NO: 5), WIR-10 (SEQ ID NO: 6) and WIKE-14 (SEQ ID NO: 10) peptides showed little hemolysis at 200 μM.

From this, it can be confirmed that the anti-inflammatory active peptide prepared in Example 1 (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) is a stable peptide that hardly induces hemolysis of red blood cells. there was.

4-2. Measurement of cytotoxicity of anti-inflammatory active peptides

MTT assay was performed after the peptide was treated in a mouse fibroblast line (NIH3T3). The NIH3T3 cell line (10,000 cells/well) was incubated in a 96-well plate in DMEM (Dulbecco's modified eagle medium) containing 10% FBS for _{16 hours in the presence of 5% CO 2} , and the cells prepared in Example 1 Anti-inflammatory active peptides consisting of the amino acid sequences described in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 were each treated and cultured for an additional 36 hours, followed by color development through MTT assay and 570 nm Absorbance was measured at to confirm the degree of survival of the cells. At this time, the cell survival rate (Survival (%)) was determined by Equation 2 below. The results are shown in FIG. 8.

In the above formula,

Absorbance A is the absorbance of the group treated with the anti-inflammatory active peptide solution at a wavelength of 570 nm,

Absorbance B is the absorbance of the group not treated with the anti-inflammatory active peptide solution at a wavelength of 570 nm,

Absorbance C represents the absorbance of the group treated with 0.1% Triton X-100 at a wavelength of 570 nm.

As shown in Fig. 8, the survival rate of NIH3T3 cells is 91.42% at 100 μM for KIW-14 (SEQ ID NO: 7) peptide, 92.59% at 100 μM for KWI-14 (SEQ ID NO: 8) peptide, and 100 μM for WIK-14 peptide. 85.06% (SEQ ID NO: 9) in μM, KIW-10 (SEQ ID NO: 1), KWI-10 (SEQ ID NO: 2), WIK-10 (SEQ ID NO: 3), RIW-10 (SEQ ID NO: 4), RWI -10 (SEQ ID NO: 5), WIR-10 (SEQ ID NO: 6) and WIKE-14 (SEQ ID NO: 10) peptides were found to be 100% at 100 μM. It was confirmed that the peptide of the present invention had little cytotoxicity by showing a survival rate of 85% or more at a concentration of 100 μM.

From the above results, the anti-inflammatory active peptide consisting of the amino acid sequences described in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 prepared in Example 1 has no cytotoxicity, so it is safe for the human body. It was found that it could be used as an anti-inflammatory agent.

Formulation Example 1. Preparation of pharmaceutical composition

1-1. Preparation of liquid

20 mg of anti-inflammatory active peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

According to the usual preparation method of liquid preparation, add and dissolve each component in purified water, add an appropriate amount of lemon flavor, mix the above ingredients, add purified water, add purified water, adjust the total to 100 mL, and fill in a brown bottle. It is sterilized to prepare a liquid formulation.

1-2. Preparation of powder

20 mg of anti-inflammatory active peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10

100 mg lactose

10 mg of talc

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

1-3. Preparation of injections

10 mg of anti-inflammatory active peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10

Mannitol 180 mg

2874 mg of sterile distilled water for injection

Na ₂ HPO ₄ 2H ₂ O 26 mg

It is prepared with the above ingredients per ampoule (2 mL) according to a conventional injection preparation method.

1-4. Manufacture of tablets

10 mg of anti-inflammatory active peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10

100 mg corn starch

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet preparation method.

1-5. Preparation of capsules

10 mg of anti-inflammatory active peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10

3 mg of crystalline cellulose

14.8 mg lactose

Magnesium stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare a capsule.

Formulation Example 2. Preparation Example of Cosmetic Composition

2-1. Manufacture of flexible lotion (skin lotion)

0.25% by weight of an anti-inflammatory active peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10

1.0% by weight of glycerin

2.0% by weight of butylene glycol

2.0% by weight of propylene glycol

0.1% by weight of carboxyvinyl polymer

PG-12 nonylphenyl ether 0.2% by weight

Polysorbate 80 0.4% by weight

Ethanol 10.0% by weight

0.1% by weight of triethanolamine

Preservative, color, and fragrance appropriate amount

Remaining amount of purified water (amount to make the total amount 100% by weight)

It is prepared by including each according to a conventional flexible cosmetic preparation method.

2-2. Preparation of nutrient cosmetic water (milk lotion)

0.5% by weight of an anti-inflammatory active peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10

Beeswax 4.0% by weight

1.5% by weight of polysorbate 60

1.5% by weight of sorbitansquioleate

0.5% by weight of liquid paraffin

Caprylic/Capric Triglyceride 5.0% by weight

3.0% by weight of glycerin

Butylene glycol 3.0% by weight

Propylene glycol 3.0% by weight

0.1% by weight of carboxyvinyl polymer

0.2% by weight of triethanolamine

Preservative, color, and fragrance appropriate amount

Remaining amount of purified water (amount to make the total amount 100% by weight)

It is prepared by including each according to a conventional nutrient cosmetic preparation method.

2-3. Manufacture of nourishing cream

0.5% by weight of an anti-inflammatory active peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10

10.0 wt% beeswax

1.5% by weight of polysorbate 60

PEG 60 hydrogenated castor oil 2.0% by weight

0.5% by weight of sorbitansquioleate

Liquid paraffin 10.0% by weight

5.0% by weight of squalane

Caprylic/Capric Triglyceride 5.0% by weight

5.0% by weight of glycerin

Butylene glycol 3.0% by weight

Propylene glycol 3.0% by weight

0.2% by weight of triethanolamine

Preservative, color, and fragrance appropriate amount

Remaining amount of purified water (amount to make the total amount 100% by weight)

It is prepared by including each according to a conventional nutrient cream manufacturing method.

Formulation Example 3. Preparation Example of Food Composition

3-1. Gel type manufacturing

20% by weight of an anti-inflammatory active peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10

60% by weight dextrin

Vitamin B1 0.002% by weight

CAC-Na 0.15% by weight

Remaining amount of purified water (amount to make the total amount 100% by weight)

It is prepared by including each according to a conventional manufacturing method.

3-2. Beverage type manufacturing

12% by weight of an anti-inflammatory active peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10

Vitamin B1 0.002% by weight

Remaining amount of purified water (amount to make the total amount 100% by weight)

It is prepared by including each according to a conventional manufacturing method.

As described above, the anti-inflammatory active peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 according to the present invention does not lose its activity even in the presence of physiological salts and has high anti-inflammatory properties. It exhibits an inflammatory effect, is not toxic to human cells, and exhibits strong anti-inflammatory power against antibiotic endogenous bacteria, as well as excellent bioavailability. Therefore, the anti-inflammatory peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the present invention is useful in anti-inflammatory pharmaceutical compositions, cosmetic compositions, and food compositions Can be used.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION OF SUNCHON NATIONAL UNIVERSITY <120> Alpha-helical peptide having antimicrobial actvity against drug-resistant bacteria and biofilm and antimicrobial composition comprising the same <130> PA-17-0129 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Alpha-helical peptide KIW-10 <400> 1 Lys Lys Ile Trp Lys Lys Trp Ile Lys Ile 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Alpha-helical peptide KWI-10 <400> 2 Lys Lys Ile Trp Lys Lys Trp Ile Lys Ile 1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Alpha-helical peptide WIK-10 <400> 3 Trp Ile Lys Lys Ile Trp Lys Lys Ile Lys 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Alpha-helical peptide RIW-10 <400> 4 Arg Arg Ile Ile Arg Arg Ile Trp Arg Trp 1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Alpha-helical peptide RWI-10 <400> 5 Arg Arg Ile Trp Arg Arg Trp Ile Arg Ile 1 5 10 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Alpha-helical peptide WIR-10 <400> 6 Trp Ile Arg Arg Ile Trp Arg Arg Ile Arg 1 5 10 <210> 7 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Alpha-helical peptide KIW-14 <400> 7 Lys Lys Ile Ile Lys Lys Ile Ile Lys Lys Ile Trp Lys Trp 1 5 10 <210> 8 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Alpha-helical peptide KWI-14 <400> 8 Lys Lys Ile Trp Lys Lys Trp Ile Lys Lys Ile Ile Lys Ile 1 5 10 <210> 9 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Alpha-helical peptide WIK-14 <400> 9 Trp Ile Lys Lys Ile Trp Lys Lys Ile Ile Lys Lys Ile Lys 1 5 10 <210> 10 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Alpha-helical peptide WIKE-14 <400> 10 Trp Ile Lys Lys Ile Trp Lys Lys Ile Ile Lys Glu Ile Lys 1 5 10 <210> 11 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> Primer 1 of Glyceraldehyde 3-phosphate dehydrogenase <400> 11 gcatcttctt gtgcagtgcc 20 <210> 12 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> Primer 2 of Glyceraldehyde 3-phosphate dehydrogenase <400> 12 tcacacccat cacaaacatg 20 <210> 13 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Primer 1 of IL-6 <400> 13 acaagtccgg agaggagac 19 <210> 14 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> Primer 2 of IL-6 <400> 14 cagaattgcc attgcacaac 20 <210> 15 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> Primer 1 of TNF-a <400> 15 cgcagcagca catcaacaag agc 23 <210> 16 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> Primer 2 of TNF-a <400> 16 tgtcctcatc ctggaaggtc cacg 24

Claims (6)

delete delete delete Inflammation preventive or therapeutic pharmaceutical composition comprising an anti-inflammatory active peptide consisting of any one of amino acids of SEQ ID NO: 1 as an active ingredient. Inflammation preventing or improving cosmetic composition comprising an anti-inflammatory active peptide consisting of any one of amino acids of SEQ ID NO: 1 as an active ingredient. Food composition for preventing or improving inflammation comprising an anti-inflammatory active peptide consisting of any one of amino acids of SEQ ID NO: 1 as an active ingredient.

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