KR20190033839A - Anti-inflammatory composition comprising macropin peptide as effective component - Google Patents

Abstract

The present invention relates to a health functional food composition and a pharmaceutical composition for preventing or alleviating inflammatory diseases, containing a macropin peptide consisting of an amino acid sequence of SEQ ID NO: 1 as an active ingredient. According to the present invention, the macropin peptide has an excellent anti-inflammatory effect against an inflammatory response and little cytotoxicity.

Description

[0001] The present invention relates to an anti-inflammatory composition comprising macropin peptide as an active ingredient,

The present invention relates to an antiinflammatory composition comprising a macropin peptide as an active ingredient.

Bacterial infections are one of the most common and deadly causes of human disease, and unfortunately the abuse of antibiotics has caused antibiotic resistance in bacteria. In fact, the rate at which bacteria respond to new antibiotics is much faster than the rate at which new antibiotic analogs are developed. For example, enterococcus faecalis ( Enterococcus faecalis), Mycobacterium-to M. tuberculosis (Mycobacterium tuberculosis and Pseudomonas aeruginosa have developed resistance against all known antibiotics.

Tolerance to antibiotics is distinct from resistance to antibiotics, which was first discovered in the 1970s by Pneumococcus sp. And provided important clues to the mechanism of action of penicillin. Resistant species stop growing in the presence of the usual concentration of antibiotics but do not eventually die. Resistance occurs when antibiotics inhibit the cell wall synthetase because autolytic enzymes such as autolysin do not activate. This suggests that penicillin activates the endogenous hydrolytic enzyme By killing bacteria, bacteria also inhibit their activity, resulting in the survival of antibiotics.

It is clinically very important for bacteria to tolerate various antibiotics because of the inability to eradicate resistant bacteria and the effectiveness of antibiotic treatment in clinical infections. In addition, tolerance is considered to be a prerequisite for resistance to antibiotics, because strains that survive antibiotic therapy are developed. These strains acquire new genetic elements that are resistant to antibiotics and continue to grow in the presence of antibiotics. In fact, all resistant bacteria are known to have resistance (Liu and Tomasz, J. Infect. Dis., 1985, 152, 365-372), the development of novel antibiotics capable of killing these antibiotic resistant bacteria Is required.

In terms of mechanism of action, antibiotic resistance consists of two major pathways: the first is phenotypic tolerance that occurs when the growth rate decreases in all bacteria, the second is the genetic variation caused by mutations in certain bacteria It is tolerance. In both cases, the underlying phenomenon is the down regulation of autolysin activity, which is transient in the case of external resistance to external stimuli, It is permanent in the case of genetic resistance in which mutations are caused. The most simple genetic resistance is the loss of autolyzine enzyme, which has not been clinically found to have resistance due to deficiency of the suicide enzyme due to various reasons. By controlling the activity of lysine.

As discussed above, in order to combat bacteria that are resistant to antibiotics, it is necessary to develop new antibiotics and to develop new antibiotics that act independently of autolysin activity. It is also necessary to provide antibiotics for effectively treating such new antibiotics in the treatment of bacterial infections and inflammation.

Bacteria, on the other hand, can synthesize peptides and small organic molecules to kill neighboring bacteria. These bacteriocins are structurally classified into three classes. The first is lantibiotics, the second is nonlantibiotics, and the third is secreted by signal peptides. Animals, including marine organisms, also produce naturally occurring peptide antibiotics, which are structurally divided into three groups. The first is the cysteine-rich β-sheet peptide, the second is the α-helical amphipathic molecule, and the third is the proline-rich peptide. These antimicrobial peptides are known to play an important role in host defense and the innate immune system. Amphiphilic peptides reported so far include magainin 2, MA, buforin 2, and melittin (ME) peptides. Peptides such as non-margarine 2 and butorphine 2 are known as peptides that act by forming pores in the membrane of bacteria, and melittin acts by destroying the membrane of bacteria.

Macropin peptides were first found in bee venom ( Macropis fulvipes ), and macropin has antifungal activity as well as antifungal activity.

On the other hand, Korean Patent No. 1171821 discloses " papyriloxine 1 peptide isolated from larva of Tiger butterfly, its use as antibacterial and anti-inflammatory agent ", Korean Patent No. 1599587 discloses " antibacterial, antifungal, A prothyatomycin antibiotic peptide derivative showing antiinflammatory activity and its use ', but the composition for antiinflammation containing the macropin peptide of the present invention as an active ingredient has not been disclosed.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a macropin peptide which is excellent in anti-inflammatory effect against inflammation caused by bacteria, .

In order to solve the above problems, the present invention provides a health functional food composition comprising an amino acid sequence of SEQ ID NO: 1 and containing macropin peptide as an active ingredient for preventing or ameliorating inflammatory diseases.

The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease containing the macropin peptide as an active ingredient.

The present invention also provides an anti-inflammatory composition comprising the macropin peptide as an active ingredient.

The present invention also provides a cosmetic composition for preventing or ameliorating an inflammatory disease containing the macropin peptide as an active ingredient.

The macropin peptide of the present invention is excellent in anti-inflammatory effect against the inflammatory reaction caused by gram-positive bacteria, and thus can be effectively used as an effective ingredient of a health functional food composition, a pharmaceutical composition, a cosmetic composition and the like for preventing or treating inflammatory diseases have.

Fig. 1 shows the result of confirming the formation of a secondary structure of macropin peptide. In Fig. 1, the symbol indicates a sodium phosphate solution (SP buffer), the symbol SD denotes a SDS solution, and the symbol □ denotes a trifluoroethanol (TFE) solution.
FIG. 2 shows the results of examining the effect of macropin peptide on the bacterial membrane through PI staining after treatment of Staphylococcus aureus and Pseudomonas aeruginosa membrane with macropin peptide.
FIG. 3 shows the results of confirming the ability of macropin peptide to destroy Staphylococcus aureus and Pseudomonas aeruginosa through a low-vacuum scanning electron microscope (SEM).
Fig. 4 shows the result of treating macrophiligenic cells with inflammation induced by Staphylococcus aureus and confirming expression of intracellular cytokines.
Fig. 5 shows the antimicrobial effect of macrophine peptides in inflammation-induced mice induced by Staphylococcus aureus.

In order to achieve the object of the present invention, the present invention provides a health functional food composition comprising the amino acid sequence of SEQ ID NO: 1 and containing macropin peptide as an active ingredient for preventing or improving inflammatory diseases.

The macropin peptide having the amino acid sequence represented by SEQ ID NO: 1 is a peptide having antimicrobial activity derived from bee ( Macropis fulvipes ), and can be produced by a conventional peptide synthesis method known in the art, and is not particularly limited to the production method Do not.

The term " inflammation " includes without limitation inflammatory reactions in the body, including acute and / or chronic inflammatory diseases; And inflammatory diseases.

The inflammation according to the present invention may be caused by bacteria, preferably Gram-negative bacteria, Gram-positive bacteria or antibiotic-resistant bacteria, and more preferably the Gram-negative bacteria are Escherichia coli or Pseudomonas aeruginosa aeruginosa , Gram-positive bacteria are Listeria monocytogenes or Staphylococcus aureus , and the antibiotic-resistant bacteria are Staphylococcus aureus or Pseudomonas aeruginosa having antibiotic resistance, But is not limited to, Pseudomonas aeruginosa .

In one embodiment of the present invention, the effect of inhibiting the expression of inflammatory cytokines was confirmed by treating macrophage peptides in inflammation-induced cells with Staphylococcus aureus , a Gram-positive bacterium.

In one embodiment of the present invention, the composition inhibits the expression of cytokines or inflammation-related proteins, and preferably inhibits expression of TNF-alpha or IL-8 (Interleukin 8) But is not limited thereto.

The food is not particularly limited as long as it can be ingested to reduce inflammation.

In the health functional food composition according to one embodiment of the present invention, the inflammatory disease is selected from the group consisting of dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Sjogren's syndrome, multiple sclerosis and other conditions, such as ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder periitis, tendinitis, Acute or chronic inflammatory diseases, but the present invention is not limited thereto.

When the macropin peptide of the present invention is used as a food additive, the macropin peptide can be directly added or used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the macropin peptide of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight based on the raw material, in the production of food or beverage. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the food to which the peptide can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.

In addition to the above, the macropin peptide of the present invention may be used as a nutritional supplement, a vitamin, an electrolyte, a flavoring agent, a coloring agent, a pectic acid and its salt, an alginic acid and its salt, an organic acid, a protective colloid thickener, a pH adjusting agent, a stabilizer, Alcohols, carbonating agents used in carbonated drinks, and the like.

The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease comprising an amino acid sequence of SEQ ID NO: 1 and containing macropin peptide as an active ingredient.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the macropin peptides of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in suitable aggregates.

The pharmaceutical composition comprising the macropin peptide according to the present invention can be administered orally or parenterally in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, oral preparation, suppository or sterile injection solution Can be used. Examples of the carrier, excipient and diluent which can be contained in the pharmaceutical composition including the macropin peptide include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. have. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The preferred dosage of the macropin peptide of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for the desired effect, the macropin peptide of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg, on a daily basis. The administration may be carried out once a day or divided into several times. The dose does not in any way limit the scope of the invention.

The macropin peptide of the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention also provides an anti-inflammatory composition comprising the macropin peptide as an active ingredient. The macropin peptide of the present invention has excellent anti-inflammatory effect and low cytotoxicity, and thus can act as an active ingredient of excellent anti-inflammatory composition.

The present invention also provides a cosmetic composition for preventing or ameliorating an inflammatory disease containing the above macropin peptide as an active ingredient.

The cosmetic composition of the present invention can be used as a cosmetic composition for external use such as ointment for skin, cream, softener, nutrition lotion, pack, essence, hair tonic, shampoo, rinse, hair conditioner, hair treatment, skin lotion, skin softener, A lotion, a moisturizing lotion, a nutritional lotion, a massage cream, a nutritional cream, a moisturizing cream, a hand cream, a foundation, a nutrition essence, a soap, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion and a body cleanser. The composition of each of these formulations may contain various kinds of bases and additives necessary for formulation of the formulation, and the kinds and amounts of these ingredients can be easily selected by those skilled in the art.

When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

In the case of the solution or emulsion of the present invention, a solvent, a solvent or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

When the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include 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 acid diethanolamides, vegetable oils, linolenic derivatives or ethoxylated glycerol fatty acid esters.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1. Synthesis and separation of peptides Purification

The inventors synthesized according to Merrifield, RB., J. Am. Chem. Soc., 85, 2149, 196, Merrifield.

Specifically, the peptide of which the carboxyl terminal of the peptide designed in the present invention is in the form of NH ₂ was used as a starting material for Rink Amide MBHA-Resin, and the peptide having an OH-type carboxyl terminal was Fmoc ( 9-fluorenylmethoxycarbonyl) -amino acid-Wang Resin was used as a starting material.

The extension of the peptide chain by coupling of Fmoc-amino acid was performed by DCC (N-hydroxybenzo trizole (HOBt) -dicyclo-hexycarbodiimide) method. After the Fmoc-amino acid of the amino terminal of each peptide was coupled, the Fmoc group was removed with NMP (20% piperidine / N-methyl pyrolidone) solution, washed several times with NMP and DCM (dichoromethane), and then dried with nitrogen gas. A solution of trifluoroacetic acid (TFA), phenol, thioanisole, H ₂ O and triisopropylsilane in a ratio of 85: 5: 5: 2.5: 2.5 (v / v) , And the mixture was reacted for 2 to 3 hours to remove the protecting group and to separate the peptide from the resin, followed by precipitation of the peptide with diethylether. The crude peptide obtained was purified on a purified reverse phase (RP) -HPLC column (Delta Pak, C18300 Å, 15,19.0 mm × 30 cm) in an acetonitrile gradient containing 0.05% TFA , Waters, USA). The synthetic peptide was hydrolyzed with 6N hydrochloric acid at 110 ° C, and the residue was concentrated under reduced pressure. The residue was dissolved in 0.02N hydrochloric acid and the amino acid composition was determined with an amino acid analyzer (Hitachi 8500 A). To confirm the purity and molecular weight of the peptide, MALDI mass spectrometry (Hill, et al., Rapid Commun. Mass Spectrometry, 5: 395, 1991).

As a result, peptides were synthesized with a purity of 95% or more as shown in Table 1, and the molecular weights thereof were confirmed to be the same molecular weights as expected.

The sequence, molecular weight and retention time of the peptides synthesized in the present invention Peptides Amino acid sequence Molecular weight (Da) Retention time (minutes) Macropin GFGMALKLLKKVL-NH ₂ (SEQ ID NO: 1) 1416.9 28.946

Example 2. Measurement of antimicrobial activity

In order to compare the antimicrobial activity of the peptides prepared by the method of Example 1, the present inventors measured the minimum inhibitory concentration (MIC) value, which is the minimum concentration of peptides in which the cells are not cleaved.

Specifically, the strains described in the following Table 2 were purchased and cultured in a mid-log phase in a medium having a composition suitable for each strain, diluted to a concentration of 2 x 10 ⁵ cells / 100 μl, Were prepared on a titration plate (Nunc, USA). Then, the macropin peptide synthesized in Example 1 was added by serial dilution by ½ times to each well, followed by incubation at 37 ° C. for 18 hours. The microplate plate reader (Merck Elisa reader, Germany ) Was used to measure the absorbance at a wavelength of 600 nm to determine the MIC value for each strain.

The bacterial strain used in the present invention and its source division Strain name source Accession number Gram
Negative bacteria Escherichia coli US Cell Line Bank ATCC 25922 Pseudomonas aeruginosa
( Pseudomonas aeruginosa ) US Cell Line Bank ATCC 27853 Gram
Positive bacteria Staphylococcus aureus
( Staphylococcus aureus ) US Cell Line Bank ATCC 25923 Listeria monocytogenes
( Listeria monocytogenes ) Korean Cell Line Bank KCTC 3710



Antibiotic
Resistant bacteria


Staphylococcus aureus
( Staphylococcus aureus ) Separator 95085 Separator 691054 Separator 254348 Separator 254422 Separator 547582 Separator 771687 Separator 949987

Pseudomonas aeruginosa
( Pseudomonas aeruginosa ) Separator 3320 Separator 3318 Isolation 1034 Separator 3241 Separator 1162 Separator 3399

As a result, as shown in Table 3, the macropin peptide showed almost similar antibacterial activities in both Gram-negative bacteria, Gram-positive bacteria and antibiotic-resistant bacteria.

Antimicrobial activity of macropin peptide division Strain name Minimum inhibitory concentration (μM) Gram
Negative bacteria Escherichia coli 6.25 P. Erusinosa 6.25 Gram
Positive bacteria S. Aureus 3.13 L. monocytogenes 25


Antibiotic
Resistant bacteria S. Aureus 95085 3.13 S. Aureus 691054 3.13 S. Aureus 254348 3.13 S. Aureus 254422 3.13 S. Aureus 547582 3.13 S. Aureus 771687 3.13 S. Aureus 949987 3.13 P. Erusinosa 3320 3.13 P. Erusinosa 3318 3.13 P. ERUJINO 1034 3.13 P. Erusinosa 3241 1.56 P. Erusinosa 1162 3.13 P. Erusinosa 3399 3.13

Example  3. Antifibrotic membrane  Active measurement

In order to compare the antimicrobial activity of the peptides prepared by the method of Example 1, the present inventors measured the biofilm inhibition concentration of peptides in which the cells were not cleaved.

Specifically, microbes in which the biofilm is well formed among the strains described in Table 2 were cultured in each medium to the mid-log phase, and then diluted to a cell concentration of 5 × 10 ⁵ cells / And inoculated on a titration plate (Nunc). Then, the macropin peptide synthesized in Example 1 was diluted with phosphate buffer solution (PBS) at a ratio of 1/10 to each well, added with 10 μl, and cultured at 37 ° C for 24 hours. The supernatant was completely removed, fixed with 100% methanol for 15 minutes, stained with crystal violet staining solution for 2 hours, washed 3 times, dissolved with 95% ethanol, and absorbance at a wavelength of 595 nm using a microtiter plate reader And the minimum inhibitory concentration of biofilm for each strain was determined.

As a result, it was confirmed that the macropin peptide showed similar or strong biofilm inhibiting activity in all bacterial strains as shown in Table 4 below.

Antibacterial activity of macropin peptide division Strain name Biofilm Inhibitory Concentration (μM) Gram-negative bacteria P. Erusinosa 12.5 Gram-positive bacteria S. Aureus 12.5


Antibiotic
Resistant bacteria S. Aureus 95085 25 S. Aureus 691054 25 S. Aureus 254348 50 S. Aureus 254422 50 S. Aureus 547582 50 S. Aureus 771687 50 S. Aureus 949987 50 P. Erusinosa 3320 25 P. Erusinosa 3318 25 P. ERUJINO 1034 50 P. Erusinosa 3241 12.5 P. Erusinosa 1162 25 P. Erusinosa 3399 25

Example 4. Measurement of hemolytic activity

To compare the cytotoxicity of the peptides prepared by the method of Example 1, the erythrocyte hemolytic activity of the synthesized peptides was measured.

Specifically, human erythrocytes were diluted with PBS (pH 7.4) to a concentration of 8% and the macropin peptide was treated at a concentration of 1.56, 3.13, 6.25, 12.5, 25.0 and 50.0 μM / Lt; / RTI > Then, the amount of hemoglobin contained in the supernatant obtained by centrifugation at 1,000 x g was measured by measuring the absorbance at a wavelength of 414 nm. The absorbance of the supernatant was measured after treatment with 1% Triton X-100 (Sigma, USA) at 37 ° C for 1 hour, and the absorbance value was measured using a red blood cell hemolytic activity 100 %, The hemolysis of the peptide was calculated using the following equation (1).

[Equation 1]

(%) = (Absorbance A-Absorbance B) / (Absorbance C-Absorbance B) X 100

Absorbance A represents the absorbance of the reaction solution treated with the peptide measured at a wavelength of 414 nm, the absorbance B represents the absorbance of the reaction solution treated with PBS measured at the wavelength of 414 nm, and the absorbance C is 414 Gt; 1-Triton X-100 < / RTI >

As a result, as shown in Table 5 below, it was confirmed that when the 50 μM concentration of the macrophine peptide was treated, the erythrocyte disruption of less than 20% was induced, and it was confirmed that the toxicity to the red blood cells was low.

Hemolytic activity of macropin peptide % Erythropoietin activity (peptide concentration, μM) 50 25 12.5 6.25 3.125 1.56 Macropin 20 5 2 0.3 0 0

Example 5. Cytotoxicity test in normal cell line

To confirm the cytotoxicity of the peptide prepared by the method of Example 1 on the normal cell line, toxicity was measured using a human keratinocyte line (HaCaT cell line, Dr. NE Fusenig, Heidelberg, Germany).

Specifically, HaCaT cells cultured in DMEM medium containing 10% FBS (Fetal Bovine Serum) were dispensed into microtiter plates at 1 × 10 ⁴ cells / well and cultured for 24 hours. Then, macrophine peptides were added at 1.56, 3.125, 6.25, 12.5, 25.0 and 50.0 [mu] M / well and reacted in a 5% CO ₂ incubator for 24 hours. Twenty-four hours later, 20 μl of a reaction solution in which 5 mg / ml MTT (Thiazolyl Blue Tetrazolium Bromide) was dissolved in phosphate buffered saline (PBS) was added to each well, followed by reaction for 4 hours. Then, the supernatant was removed, 200 쨉 l of DMSO (dimethyl sulfoxide) was added to dissolve the formed MTT crystal, and the wavelength was confirmed at 560 nm to confirm cell viability.

As a result, when the macropin peptide of the active range concentration was treated as shown in Table 6 below. HaCaT cells showed cell viability of 70% or more, indicating low cytotoxicity.

Cytotoxicity analysis of macropin peptide % Cell viability (peptide concentration, mu M) 50 25 12.5 6.25 3.125 1.56 Macropin 50 70 97 98 100 100

Example 6. Measurement of original color spectrum

The peptide prepared by the method of Example 1 was used to measure the circular dichroism method to determine whether the secondary structure, the? -Helical structure, was induced.

Specifically, a macropin peptide was added to a solution of 10 mM sodium phosphate (pH 7.4), 50% trifluoroethanol (TFE), or 30 mM sodium dodecyl sulfate (SDS) 50 μM, added to 0.1 cm long path-length cells, and fixed at 25 ° C. in a jasco 810 spectrophotometer to measure the original colorimetric spectrum.

As a result, it was confirmed that when the peptide was added to the sodium phosphate solution, the macropin peptide did not form a structure, whereas when the peptide was added to the SDS or TFE solution, the α-spiral structure was formed as a secondary structure , It was found that the antibiotic peptide of the present invention forms an a-helical structure on SDS or TFE similar to a bacterial membrane as a microorganism (Fig. 1).

Example 7. Flow cytometry measurement

In order to confirm whether or not the peptide prepared by the method of Example 1 acts on the bacterial membrane, it was analyzed using flow cytometry.

Specifically, the macropin peptide synthesized in Example 1 was treated with Staphylococcus aureus and Pseudomonas erupunosa at two times the growth inhibitory concentration (6.25 and 12.5 μM), followed by reaction at 37 ° C for 1 hour . Then, the supernatant was removed using a centrifuge (10,000 rpm), and then stained with Propidium iodide (PI) at a concentration of 10 μg / ml for 30 minutes at 4 ° C. Then, unbound propidium iodide was removed using a centrifuge, and 1 ml of physiological saline (PBS) was added to remove the aggregation of cells. Then, the effect of the peptide on the bacterial membrane was confirmed using a Bechman flow cytometer Respectively.

As a result, it was confirmed that the macrophine peptide damages the bacterial membrane and the fluorescence signal is shifted to the right (FIG. 2).

Example 8. Analysis of effect of macropin peptide on bacterial membrane

In order to confirm the degree of the antimicrobial activity of the macropin peptide of the present invention, the destructive activity of macropin peptide on the membrane of Staphylococcus aureus and Pseudomonas aeruginosa was examined by scanning electron microscopy , SEM).

Specifically, Staphylococcus aureus is cultured in TSB medium and Pseudomonas aeruginosa is grown in NB medium to mid-log phase, and then added to physiological saline solution at a concentration of 2 x 10 < ⁷ > cells / ml And diluted with the cell concentration. The diluted strains were treated with minimal inhibitory concentrations of macropin peptide and incubated at 37 ° C for 30 minutes. Then, the cells were fixed with 2.5% glutaraldehyde at room temperature for 30 minutes, at 4 ° C for 12 hours, washed with a buffer, resolved with osmium (OsO ₄ ), and then dehydrated stepwise with ethanol . After dehydration, platinum coating was performed and observed under low vacuum scanning electron microscopy.

As a result, when the macrophine peptide was treated with Staphylococcus aureus and Pseudomonas aeruginosa as shown in Fig. 3, it was confirmed that the peptide acts on each bacterial membrane and destroys the membrane of the bacteria and shows antibacterial activity.

Example 9. Confirmation of Expression of TNF-a and IL-8 by Real-Time Polymerase Chain Reaction

In order to confirm whether the peptides of the present invention have an anti-inflammatory effect, it was confirmed by real-time PCR that the macropin peptide suppresses the expression of cytokines induced by Staphylococcus aureus.

Specifically, 1 x 10 ⁶ cells of cultured HaCaT cells (Dr. NE. Fusenig, Heidelberg, Germany) were dispensed into a 6-well plate and cultured for 24 hours. Staphylococcus aureus was counted at a concentration of 1x10 ⁸ cells / And the macropin peptide was treated at a concentration of 3.13 μM / well and reacted in a CO ₂ incubator for 6 hours. After that, the culture supernatant was removed, total RNA was isolated from the cells using a triazole (Invitrogen, USA), cDNA was synthesized, and real-time PCR was performed to determine the expression levels of TNF-a and IL-8 Respectively.

As a result, it was confirmed that the expression levels of TNF-a and IL-8, which were increased by Staphylococcus aureus, were reduced due to the treatment of macropin peptide (Fig. 4).

Example  10. Antimicrobial activity confirmed by mouse

Inflammation was induced by infecting Staphylococcus aureus (1 x ^{10 8} cells / ml) after formation of air pouch on the skin of a 6-week old mouse (Balb / c) The peptide was treated with 1 mg / kg of the skin. After the peptide treatment, the control group (inducing inflammation only) and the experimental group (treating the peptide in the inflammation site) mouse tissues were taken out on days 1, 2, 3 and 4 and washed with physiological saline, Respectively. The colonies formed after culturing in an incubator for 24 hours were counted, and the antimicrobial activity of macropin peptide was confirmed in inflammation-induced mice (FIG. 5).

The preparation examples for the composition of the present invention are illustrated below.

≪ Preparation Example 1 > Preparation of pharmaceutical preparations

<1-1> Preparation of powder

20 mg of the peptide of the present invention

Lactose 20 mg

After mixing the above components, the mixture was packed in an airtight container to prepare a powder.

<1-2> Preparation of tablets

10 mg of the peptide of the present invention

100 mg of corn starch

Lactose 100 mg

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

10 mg of the peptide of the present invention

3 mg of crystalline cellulose

Lactose 14.8 mg

0.2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

<1-4> Production of liquid agent

20 mg of the peptide of the present invention

10g per isomerization

Mannitol 5 g

Purified water quantity

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

<1-5> Preparation of Injection

10 [mu] g / ml of the peptide of the present invention

Diluted hydrochloric acid BP until pH 7.6

Sodium chloride up to 1 ml for injection

The peptide of the present invention was dissolved in an appropriate volume of injectable sodium chloride BP. The pH of the resulting solution was adjusted to pH 7.6 using diluted hydrochloric acid BP, and the volume was adjusted using main sodium chloride BP and mixed thoroughly. The solution was filled in 5 ml type I ampoule made of transparent glass, sealed in the upper lattice of the air by dissolving the glass, and sterilized with a high pressure steam sterilizer at 120 캜 for 15 minutes or longer to prepare an injection solution.

&Lt; Preparation Example 2 > Preparation of cosmetics

<2-1> Flexible longevity (skin)

To prepare the softening longevity water for antimicrobial use containing the peptide of the present invention, it may be formulated as described in Table 7 below and may be manufactured according to a conventional manufacturing method in the field of cosmetics.

Softening longevity composition ingredient Content (% by weight) The peptide of the present invention 0.1 to 30 1,3-butylene glycol 3.0 glycerin 5.0 Polyoxyethylene (60) Hardened castor oil 0.2 ethanol 8.0 Citric acid 0.02 Sodium citrate 0.06 antiseptic a very small amount Spices a very small amount Purified water To 100

<2-2> Nourishing lotion (lotion)

The antifungal nutritional lotion containing the peptide of the present invention can be prepared as described in Table 8 below and can be produced according to a conventional manufacturing method in the field of cosmetics.

Nutritional Lotion Composition ingredient Content (% by weight) The peptide of the present invention 0.1 to 30 Squalane 10.0 Polyoxyethylene sorbitan monooleate 2.0 Nursery oil 0.1 to 30 1,3-butylene glycol 8.0 glycerin 5.0 Polyoxyethylene (60) Hardened castor oil 0.2 ethanol 8.0 Citric acid 0.02 Sodium citrate 0.06 antiseptic a very small amount Spices a very small amount Purified water To 100

<2-3> Essence

To prepare an antimicrobial essence containing the peptide of the present invention, it may be formulated as described in Table 9 below and may be produced according to a conventional method for manufacturing in the field of cosmetics.

Essence composition ingredient Content (% by weight) The peptide of the present invention 0.1 to 30 Sitosterol 1.7 The polyglyceryl 2-oleate 1.5 Ceramide 0.7 Setares-4 1.2 cholesterol 1.5 Dicetyl phosphate 0.4 Concentrated glycerin 5.0 Carboxyvinyl polymer 0.2 Xanthan gum 0.2 antiseptic a very small amount Spices a very small amount Purified water To 100

<2-4> Cleanser (cleansing foam)

To prepare an antimicrobial cleanser (cleansing foam) containing the peptide of the present invention, it may be formulated as described in Table 10 below and may be manufactured according to a conventional manufacturing method in the field of cosmetics.

Composition of cleanser ingredient Content (% by weight) The peptide of the present invention 0.1 to 30 Sodium N-acylglutamate 20.0 glycerin 10.0 PEG-400 15.0 Propylene glycol 10.0 POE (15) oleyl alcohol ether 3.0 Laurin derivative 2.0 Methyl paraben 0.2 EDTA-4Na 0.03 Spices 0.2 Purified water To 100

<2-5> Nourishing Cream

To prepare an antimicrobial nutrition cream containing the peptide of the present invention, the preparation is carried out according to a conventional method in the field of cosmetics as shown in Table 11 below.

Nutritional cream composition ingredient Content (% by weight) The peptide of the present invention 0.1 to 30 vaseline 7.0 Liquid paraffin 10.0 Wax 2.0 Polysorbate 60 2.5 Sorbitan sesquioleate 1.5 Squalane 3.0 Propylene glycol 6.0 glycerin 4.0 Triethanolamine 0.5 Xanthan gum 0.5 Tocophenylacetate 0.1 Spices, preservatives a very small amount Purified water To 100

<2-6> Massage Cream

In order to prepare antimicrobial massage cream containing the peptide of the present invention, it is prepared according to the conventional manufacturing method in the field of cosmetics as shown in Table 12 below.

Composition of massage cream ingredient Content (% by weight) The peptide of the present invention 0.1 to 30 Propylene glycol 6.0 glycerin 4.0 Triethanolamine 0.5 Wax 2.0 Tocophenylacetate 0.1 Polysorbate 60 3.0 Sorbitan sesquioleate 2.5 Cetearyl alcohol 2.0 Liquid paraffin 30.0 Xanthan gum 0.5 Spices, preservatives a very small amount Purified water To 100

<2-7> Pack

In order to prepare antimicrobial packs containing the peptides of the present invention, the preparation is carried out according to a conventional method in the field of cosmetics as shown in Table 13 below.

ingredient Content (% by weight) The peptide of the present invention 0.1 to 30 Propylene glycol 2.0 glycerin 4.0 Polyvinyl alcohol 10.0 ethanol 7.0 Paiji-40 Hydrogenated Castor Oil 0.8 Triethanolamine 0.3 Spices, preservatives a very small amount Purified water To 100

Although the present invention has been described and illustrated in detail, it is clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation, , It can be used for manufacturing ointments, that is, ointments which can be applied thinly on the human body according to its effect, and this is included in the spirit and scope of the present invention as defined in the appended claims.

<110> Industry-Academic Cooperation Foundation, Chosun University <120> Anti-inflammatory composition comprising macropin peptide as          effective component <130> PN17361 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 13 <212> PRT <213> Macropis fulvipes <400> 1 Gly Phe Gly Met Ala Leu Lys Leu Leu Lys Lys Val Leu   1 5 10

Claims (12)

A health functional food composition for preventing or ameliorating an inflammatory disease comprising an amino acid sequence of SEQ ID NO: 1 and containing macropin peptide as an active ingredient. The health functional food composition according to claim 1, wherein the inflammation is caused by bacteria. The health functional food composition according to claim 2, wherein the bacterium is at least one selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria, and antibiotic-resistant bacteria. 4. The method according to claim 3, wherein the Gram-negative bacterium is Escherichia coli or Pseudomonas aeruginosa , the gram-positive bacterium is Listeria monocytogenes or Staphylococcus aureus , Wherein the antibiotic-resistant microorganism is Staphylococcus aureus or Pseudomonas aeruginosa having antibiotic resistance. 2. The health functional food composition according to claim 1, wherein the antibiotic-resistant microorganism is Staphylococcus aureus or Pseudomonas aeruginosa . 2. The health functional food composition according to claim 1, wherein the composition inhibits TNF-a or IL-8 expression. A pharmaceutical composition for preventing or treating an inflammatory disease comprising an amino acid sequence of SEQ ID NO: 1 and containing macropin peptide as an active ingredient. The pharmaceutical composition according to claim 6, wherein the inflammation is caused by bacteria. The pharmaceutical composition according to claim 7, wherein the bacterium is at least one selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria, and antibiotic-resistant bacteria. 9. The method according to claim 8, wherein the Gram-negative bacterium is Escherichia coli or Pseudomonas aeruginosa , the gram-positive bacterium is Listeria monocytogenes or Staphylococcus aureus , Wherein the antibiotic resistant bacterium is Staphylococcus aureus or Pseudomonas aeruginosa having antibiotic resistance. 2. The pharmaceutical composition according to claim 1, wherein the antibiotic resistant bacterium is Staphylococcus aureus or Pseudomonas aeruginosa . 7. The method of claim 6, wherein the inflammatory disease is selected from the group consisting of dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, , Fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder inflammation, nephritis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis and acute or chronic inflammatory diseases And a pharmaceutically acceptable carrier or excipient. 1. An antiinflammatory composition comprising a macropin peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. A cosmetic composition for preventing or ameliorating an inflammatory disease comprising an amino acid sequence of SEQ ID NO: 1 and containing macropin peptide as an active ingredient.

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