KR101983679B1 - Alpha-helical peptide having antimicrobial actvity against drug-resistant bacteria and biofilm and antimicrobial composition comprising the same - Google Patents

Abstract

The present invention relates to an alpha-helical peptide having antimicrobial activity against drug-resistant bacteria and an antimicrobial composition containing the same. More particularly, the present invention relates to an alpha-helical peptide having an antifungal activity, 1, 2, 3, 4, 5, 6, 7, 8 or 9 synthesized in an amphipathic structure existing on the surface having different hydrophilicity and hydrophobicity by using as a hydrophobic amino acid a novel antimicrobial active peptide , Lysine and arginine as the cationic amino acids of SEQ ID NO: 10, isoleucine and tryptophan as hydrophobic amino acids, and glutamic acid as an anionic amino acid, and a new antibacterial composition synthesized with an amphipathic structure having hydrophilic and hydrophobic faces Active peptides and the compounds containing them It relates to compositions for. Since the antimicrobial active peptide of the present invention has no cytotoxicity and exhibits excellent antimicrobial activity, it can be effectively used as an additive for antimicrobial drugs, cosmetics or food preservatives which are safe for human body, and is useful as a composition for preventing or inhibiting biofilms formed by drug- Can be usefully used.

Description

[0001] The present invention relates to an alpha-helical peptide having antimicrobial activity against drug-resistant bacteria and a biofilm, and an antimicrobial composition containing the same.

The present invention relates to an alpha-helical peptide having an antibacterial activity against drug-resistant bacteria and an antibacterial composition containing the same, and more particularly to a novel alpha-helical peptide having a novel antibacterial activity An alpha-helical peptide for antibiotics, a pharmaceutical composition for antibacterial use containing the same, a cosmetic additive composition and a food additive composition.

Resistance to bacterial diseases appeared shortly after the first use of antibiotics in the 1940s. Especially, all types of antibiotics resistant to all types of antibiotics (MRSA: methicillin resistant Staphylococcus aureus, VRE: vancomycin resistant Enterococcus, imipenem resistant Pseudomonas aeruginosa, penicillin resistant Streptococcus pneumoniae) There is a growing concern that the

The US Centers for Disease Control (CDC) reported in 2005 that antimicrobial resistance (AMR) hospital antibiotics reports that 2 million bacterial infections occur every year, of which 90,000 die. In Japan, 231 cases of Ashinobacterium spp. Were detected in 2007 and 274 cases were detected in 2009, indicating an increasing trend. NDM-1 (New Delhi metallo-β-lactamase) is a new type of β-lactamase occurring in New Delhi, India. It is a carbapenem-resistant carbapenem such as imipenem (Carbapenemase). Up to now, bacteria in NDM-1 have been found in 355 people in 16 countries, including India and Pakistan. As a result of the abuse of antibiotics in hospitals, which are the places where these resistant strains are cultured and cultured, more resistant strains have become more frequent and there are no more treatments for these "Super-bacteria". The topic of the WHO World Health Day 2011 was "antibiotic resistance" in 2011 and announced that it is more resistant to antibiotic resistance than diseases such as obesity, diabetes and HIV. According to WHO, 96.4%, 84.9%, 80.7%, Japan 78.9%, Korea 77.7%, France 46.1%, Spain 43.6 and the USA 35.4%, respectively. It is seriously high. In addition, recent indicators such as the frequency of antibiotic-resistant pneumococcal infections in the United States, the number of antibiotics in OECD countries, and the antibiotic prescription rate of 55% in the OECD countries were 55%, indicating the seriousness of abuse of antibiotics in Korea .

In Korea at the end of 2010, drug resistant bacteria such as vallomycin-resistant Staphylococcus aureus (VRSA), methacyline-resistant yellow porphyritis (MRSA), carbapenem-resistant Enterococcus ( CRE), vancomycin resistant enterococci (VRE) Multi-drug resistant Pseudomonas aeruginosa , MDR PA), and multi-drug resistant Acinetobacter (MDR AA) were designated as super-bacterial infectious diseases, suggesting that the Korean super-bacteria war has already begun.

On the other hand, a biofilm refers to an environment in which a plurality of bacteria coexist in a viscous substance secreted by bacteria or the like. The biofilm is an extracellular polymeric substance (EPS) in which the bacteria themselves are formed by secretion of polysaccharides, proteins and DNA. Since not only germs but also fungi form biofilms and form clumps and microbes attached to the biofilm can cause infection, and biofilm bacteria are difficult to remove by antimicrobial treatment, biofilms are recognized as causing major problems in modern infectious diseases There is growing interest in this. Biofilms are especially present in otitis media, prostatitis, cystic fibrosis, and dental plaques. The biofilms formed in surgical instruments are also responsible for most chronic infections. There are about 170,000 new biofilm infections every year in the United States, and it continues to increase despite antibiotic treatment. In addition, the formation of biofilm has been reported to increase the minimum inhibitory concentration (MIC) by 100-1000 times over antibiotic treatment (Antimicrob Agents Chemother 47: 3407-3414, 2003).

Although it is said that multinational pharmaceutical companies can treat resistant bacteria with new antibiotics, antibiotic resistance is mainly caused by the endotoxin secreted during the bacterial killing process due to the presence of septic shock ) Can lead to death. In this reality, a new concept of antibiotics has been developed as an alternative to conventional chemical antibiotics, and in particular, peptides having antimicrobial activity have been actively studied.

The human body and living organisms are exposed to various microorganisms both inside and outside and are constantly attacked. However, they have an innate immune system that can protect themselves and can sustain life by autoimmunity. One of these innate immune systems is an antimicrobial active peptide, which differs in amino acid type and sequence, but almost all organisms, including viruses, bacteria, protozoa, fungi, plants, and mammals, secrete or contain antimicrobial active peptides. Antibacterial active peptides are small peptides composed of less than 60 amino acids and act as primary defense agents when organisms are infected by pathogens. The antimicrobial active peptides are mainly cationic and have amphipathic α-helical structure and β-fold structure by hydrophobic amino acid residues. Due to such structural characteristics, the lipid component of the cell membrane of the negatively charged pathogenic bacteria , Which binds to bacterial cell membrane lipids and changes the cell membrane potential through a so-called 'barrel-stave', 'carpet' or 'toroidal-pore' mechanism, or breaks the cell membrane by piercing the cell membrane itself. Some of them enter the cell membrane of the bacteria and then attack the target in the cell to paralyze the cell function, thereby exhibiting the antimicrobial activity.

However, the antimicrobial active peptides have very good antimicrobial activity in vitro and do not exhibit cytotoxicity, but in fact, in vivo experiments, the antimicrobial effect is often insignificant. Although there are many reasons for this, mainly the physiological and anatomical conditions in vivo are very different from the experimental conditions in vitro, and the following problems arise. First, the antimicrobial active peptides having a low positive charge significantly inhibit the activity when salt is present in the physiological condition. Second, because peptides have small molecular weight and size, they are almost absorbed in the kidney and excreted in vitro. Third, it is easily lost by protein and peptide decomposing enzyme (protease and peptidase) present in all tissues or cells, body fluids and blood in the living body and loses its activity.

Accordingly, the inventors of the present invention have been studying the antimicrobial active peptides with reduced cytotoxicity and high utilization efficiency in vivo, and it is possible to economically mass-produce them using only 10 common amino acid residues or only 14 amino acids, high bioavailability has been developed antimicrobial peptides active non-toxic for human cells, in particular, physiologically active salt concentration present under test microorganism also does not lose the activity as well as drug resistant strains isolated directly from a patient for (Pseudomonas aeruginosa 5 species and Staphylococcus aureus 5 species), the present inventors have completed the present invention.

Therefore, it is an object of the present invention to provide a peptide which exhibits no cytotoxicity and exhibits excellent antimicrobial activity as a new mechanism of antibiotic for removing bacteria showing resistance to antibiotics.

It is another object of the present invention to provide an antimicrobial composition comprising the above-mentioned antimicrobial active peptide as an active ingredient.

It is still another object of the present invention to provide a composition for preventing or inhibiting biofilms formed by drug-resistant bacteria using the above-mentioned antimicrobial active peptides.

In one embodiment, the present invention provides an antimicrobial active peptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

The antimicrobial active peptide of the present invention is a peptide having an alpha-helical structure, wherein the antimicrobial active peptide is applied as a lysine (K) or arginine (Ar), which is a cationic amino acid, and the hydrophobic sequence is a hydrophobic amino acid Is an amphipathic peptide having 10 or 14 amino acids with isoleucine or tryptophan.

The antimicrobial active peptide of the present invention is a peptide having an alpha-helical structure. The antimicrobial active peptide is applied as a lysine (K) or arginine (Ar), which is a cationic amino acid, It is an amphipathic peptide with 14 amino acids containing glutamic acid, an anionic amino acid, which is a hydrophobic amino acid, isoleucine or tryptophan.

In a specific embodiment, the peptides of the present invention have the amino acid sequences set forth in SEQ ID NOS: 1-10 and are referred to as "KIW-10 (SEQ ID NO: 1), KWI-10 (SEQ ID NO: 2), WIK- 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- No. 9) and WIKE-14 (SEQ ID NO: 10) peptide ", whose amino acid sequences are shown in Table 1 below.

Peptides 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

According to one embodiment of the present invention, the peptide of the present invention has 85% or more homology with the peptide consisting of the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, And all of the peptides exhibiting the same effect can be included. The peptides disclosed in the present invention include peptides composed of the amino acid sequences described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or fragments thereof and peptides in which one or two amino acids have been changed can do.

In the present invention, the terms "homology" and "sequence identity" are used interchangeably, indicating the degree of overlapping of sequences between two amino acids (or similar nucleic acids).

Specifically, in the present invention, in the antimicrobial activity peptide consisting of the amino acid sequence of 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, An antimicrobial active peptide in which the amino group at the amino terminus or the carboxyl group at the carboxy terminus is replaced with another functional group. For example, a peptide obtained by substituting isoleucine or tryptophan with another hydrophobic amino acid among the synthesized peptides is similar to the peptide consisting of the amino acid sequence of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 It has no cytotoxicity and can maintain excellent antimicrobial activity. The hydrophobic amino acids that can be substituted for isoleucine in the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 include tryptophan, alanine, methionine, phenylalanine, proline, valine and leucine And hydrophobic amino acids which may be substituted for tryptophan in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 include isoleucine, alanine, methionine, phenylalanine, tryptophan , Proline, valine and leucine.

According to one embodiment of the present invention, the amino acid sequence having 85% or more homology is characterized by being the following sequence.

(i) the isoleucine in the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 is selected from the group consisting of alanine, phenylalanine, methionine, tryptophan, proline, valine and leucine An amino acid sequence substituted with an amino acid;

(ii) wherein the tryptophan of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 is 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) the isoleucine in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 is selected from the group consisting of alanine, phenylalanine, methionine, tryptophan, proline, valine and leucine Amino acid sequence and wherein the tryptophan is replaced by a hydrophobic amino acid selected from the group consisting of alanine, isoleucine, methionine, phenylalanine, proline, valine and leucine.

The term " amino acid " in the present invention naturally includes 22 standard amino acids as well as D-isomers and modified amino acids. Accordingly, in one aspect of the present invention, the peptide may be a peptide comprising a D-amino acid. In another aspect of the present invention, the peptide may include post-translationally modified non-standard amino acids and the like. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including, for example, acetylation, myristoylation and palmitoylation), alkylation ), Carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (such as, for example, beta-depleted deamidation , Deamidation) and structural changes (e.g., formation of a disulfide bridge). It also includes amino acid changes such as changes in amino acids, such as changes in amino groups, carboxy groups or side chains, caused by chemical reactions that take place during binding with crosslinkers to form peptide conjugates.

The antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the present invention may be at least one selected from the group consisting of gram-positive bacteria, gram-negative bacteria and drug- And has antibacterial activity against bacteria.

The Gram positive bacterium may be any one or more strains selected from Gram-positive bacteria including Bacillus subtilis and Staphylococcus aureus .

The Gram-negative bacteria are Escherichia coli Coli , and Pseudomonas aeruginosa . The strain may be a strain selected from the group consisting of Gram-negative bacteria including Pseudomonas aeruginosa and Pseudomonas aeruginosa .

The drug-resistant microorganism may be any one or more strains selected from drug resistant bacteria including Staphylococcus aureus and Pseudomonas aeruginosa .

The antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 according to the present invention exhibits a high antibacterial effect without losing activity even in the presence of physiological salt, It is not toxic to human cells and exhibits strong antibacterial activity against live bacteria in antibiotics as well as excellent bioavailability.

In the present invention, a pharmaceutical composition comprising an antibacterial activity peptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 as an active ingredient, Compositions or food additive compositions, which can be usefully employed as infectious agents, cosmetic additives, food preservatives or feed additives.

The pharmaceutical composition may further comprise an antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, can do.

The pharmaceutical composition of the present invention may be prepared by incorporating one or more pharmaceutically acceptable solvents in addition to the above-described effective ingredients for administration. The pharmaceutically acceptable solvent may be a mixture of saline, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components. In addition, other conventional additives such as antioxidants, buffers, And a dispersant, a diluent, and a surfactant may be formulated into injectable formulations, tablets, capsules, or granules, such as aqueous solutions, suspensions, emulsions, etc., by adding excipients, if necessary. In addition, the polymer can be made into a hydrogel or a film using a biomolecule.

The pharmaceutical composition of the present invention can be administered orally, intraperitoneally, intravenously and subcutaneously according to a desired method. The dosage may be appropriately selected according to the body weight, age, sex, health condition, diet, administration method, excretion rate, The range varies accordingly. The daily dose of the antimicrobial active peptide may be 1 to 10 mg / kg, preferably about 3 to 6 mg / kg, depending on the clinical result. .

The term " administering " as used herein is meant to provide any desired composition of the invention to an individual by any suitable method.

As used herein, the term " individual " means a mammal having a disease in which the growth of bacteria can be inhibited by administration of the composition of the present invention, a human, a monkey, a dog, Or all animals such as mice.

According to one embodiment of the present invention, a cosmetic additive composition comprising, as an active ingredient, an antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 May be prepared in the form of conventional emulsification and solubilization formulations. Cosmetics of emulsified form include nutrition lotion, cream, essence, etc., and cosmetics of solubilized form have softening longevity. In addition, the cosmetic additive composition of the present invention can be prepared in the form of an adjuvant that can be applied topically or systemically, which is conventionally used in the field of dermatological science by containing an acceptable medium or base in addition to the above-mentioned antimicrobial active peptide. In addition, as an excipient used in cosmetic additives, it may further contain arbutin, kojic acid, rucinol, vitamin C (vitamin C) and vitamin C derivatives and the like for the purpose of improving wrinkles And may further contain retinol and derivatives thereof, indole acetic acid and derivatives thereof, adenosine, tocoperol and derivatives thereof, and carnitine. In addition, the excipients include conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and fragrances, which are commonly used ingredients in cosmetic additives. Examples of suitable formulations of cosmetics include solutions, gels, solid or kneaded anhydrous products, antioxidants obtained by dispersing an oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic forms ), In the form of a non-ionic follicular dispersion, in the form of creams, skins, lotions, powders, ointments, sprays or conical sticks. It can also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. Also, it can be formed into a form such as encapsulation and grafting by using chitosan, dextran, and hyaluronic acid, which are biocompatible polymers, as a carrier.

The cosmetic additive composition of the present invention may further comprise an antimicrobial active peptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the present invention on the basis of the total weight of the cosmetic additive composition By weight to 0.01% by weight to 0.5% by weight. If the amount is less than 0.01% by weight, the antibacterial effect can not be exhibited. If the amount is more than 0.5% by weight, the difference in effect is insignificant.

According to one embodiment of the present invention, the food additive composition comprises an antimicrobial active peptide having an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, Together with one or more of the known food preservatives. Examples of the food preservative include sodium benzoate, potassium benzoate, calcium benzoate, dehydroacetic acid, potassium sorbate, calcium sorbate, methyl p-hydroxybenzoate, and p-hydroxybenzoic acid. Sodium propionate, calcium propionate, and the like, but are not limited thereto.

There is no particular limitation on the type of food using the food additive composition. Examples of the foods to which the above food additive composition can be added include dairy products including drinks, meat, sausage, bread, biscuits, rice cakes, candies, snacks, confectionery, pizza, ramen noodles, Soups, beverages, alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.

The food additive composition preferably contains 0.01 to 0.5% by weight, based on the total weight of the food additive composition, of the antimicrobial active peptide. If the amount is less than 0.01% by weight, the antibacterial effect can not be exhibited. If the amount is more than 0.5% by weight, the difference in effect is insignificant.

In the present invention, the antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 is used to treat the surface of an organism or an organism, A composition capable of preventing or inhibiting a biofilm is provided.

The drug resistant strains are drug-resistant Staphylococcus aureus (Drug-resistant Staphylococcus aureus) and drug-resistant Pseudomonas rugi labor (Drug-resistant Pseudomonas in aeruginosa ), preferably Pseudomonas aeruginosa.

The composition for preventing or inhibiting biofilm of the present invention may contain, in addition to the above-mentioned effective ingredients, a buffering salt, a moisturizing agent, a softening agent, a wetting agent, a surfactant, a corrosion inhibitor, a solvent, a spraying agent, The above components may be mixed and used. The composition of the present invention may be provided in a concentrated form, a diluted form or a ready-to-use form. The concentrated composition of the present invention may be diluted at the time of use. The compositions of the present invention may also be further modified upon dilution by mixing the concentrated composition of the present invention with other concentrates, which may take the form of liquids, creams, foams or gels.

The surface of the living organism or the non-living matter means the surface of an organism or an organism which includes the surface of a tissue, an organ, or a medical article derived from an organism, and in which drug- resistant bacteria can form a biofilm. It includes surfaces of objects used in the home, industry, environment, medical field, as well as surfaces derived from living bodies.

As a specific example, the surface may be the surface of an organism-derived tissue or organ such as skin, teeth, and the like, and may be used for various medical facilities, equipment, instruments, temporary or permanent artificial insertion prostheses, lenses, prosthetic valves, pacemakers, An insertion catheter, a catheter, or the like, but is not limited thereto.

The prevention or inhibition of the biofilm can be achieved by bringing the composition comprising the antimicrobial active peptide of the present invention into contact with the drug-resistant microbes or by treating the surface of a tissue, an organ, or a medical article derived from an organism capable of proliferating or proliferating drug- . The contacting or treatment is carried out for a time sufficient for the composition of the present invention to inhibit the killing or proliferation of the drug-resistant microbes by being contacted, coated or sprayed on the biofilm formed by the drug-resistant bacteria or the drug-resistant bacteria.

In one specific embodiment, the composition may be applied to the surface of an organism or an organism and maintained for a period of time. The retention time can generally range from a few seconds to 30 minutes. The applied composition can then be rinsed with water to remove from the surface or to evaporate by heating the surface.

In one specific embodiment, the composition may be formulated as a disinfectant for the hands or the skin and applied to the skin or mucous membranes. For example, the composition can be applied as a hand sanitizer that can be applied to the skin in the form of a foam to sterilize the skin to prevent or inhibit the biofilm.

 In one specific embodiment, the compositions may be used as pre-operative preparations or scrubs and may be applied or sprayed onto the surface of a device or apparatus including a lumen surface.

As used herein, the terms " prevent " and " inhibition " refer to all actions of delaying or killing growth of pathogenic bacteria by the administration of the composition of the present invention and any action that delays the formation of biofilm or degrades the biofilm formed .

Thus, the antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 according to the present invention exhibits high antimicrobial activity against pathogenic bacteria and fungi, It is not toxic to cells and has strong antibacterial activity against drug resistant bacteria as well as excellent bioavailability. Accordingly, the antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the present invention and the antimicrobial active pharmaceutical composition, The food additive composition may be useful as an infectious agent, food preservative, feed additive or cosmetic additive. Further, it is possible to prevent or suppress the biofilm formed by the drug-resistant bacteria by contacting the composition containing the antimicrobial active peptide with the drug-resistant microbes or by treating the surface of a tissue, an organ, or a medical article derived from an organism capable of proliferating drug- Effect.

1 shows the structure of an antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 of the present invention, as http: //mobyle.rpbs.univ-aris -diderot.fr/cgi-bin/portal.py#forms.
FIG. 2 is a graph showing the distribution of the antimicrobial activity peptides comprising the amino acid sequences described in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 of the present invention on the Escherichia coli buffer solution 10 mM HEPES, 10 mM NaCl (pH 7.4) + 10% TSB, Buffer II: 10 mM HEPES, 150 mM NaCl (pH 7.4) + 10% TSB, Buffer III: 10 _{mM HEPES, 5 mM MgCl 2 (} pH 7.4) + 10% TSB, Buffer IV: 10 mM HEPES, 150 mM NaCl, 5 mM MgCl 2 (pH 7.4) + 10% TSB).
FIG. 3 is a graph showing the effect of the antimicrobial activity peptide of Staphylococcus aureus comprising the amino acid sequence of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, (10 mM HEPES, 10 mM NaCl (pH 7.4) + 10% TSB, Buffer II: 10 mM HEPES, 150 mM NaCl (pH 7.4) + 10% TSB, Buffer III: 10 mM HEPES, 5 mM MgCl 2 (pH 7.4) + 10% TSB, Buffer IV: 10 mM HEPES, 150 mM NaCl, 5 mM MgCl 2 (pH 7.4) + 10% TSB).
4 shows the hemolytic hemolysis (%) of the antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 of the present invention.
FIG. 5 is a graph showing the results of cytotoxicity tests on mouse skin fibroblasts (NIH3T3) of the antimicrobial active peptides comprising the amino acid sequences of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, .
FIG. 6 is a graph showing the relationship between the antimicrobial activity of Escherichia coli and Staphylococcus, comprising the amino acid sequence of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, The agar diffusion ability of Staphylococcus aureus is shown.
FIG. 7 is a graph showing the antimicrobial action of antimicrobial active peptides comprising the amino acid sequences of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 of the present invention. The artificial liposomes were treated with peptide to observe the degree of destruction by concentration. PE / PG is a lipid composition similar to the bacterial cell membrane and PC / Ch / SM is a lipid composition similar to that of animal cells.
FIG. 8 is a graph showing the effect of the antimicrobial activity peptide comprising the amino acid sequence of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 of the present invention on the drug susceptibility Pseudomonas aeruginosa ATCC 15692 and drug-resistant Pseudomonas This graph shows the inhibition of bacterial plankton growth (top two graphs) and biofilm removal (bottom two graphs) ability of strain 4007 of Luginosa.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples and Examples. The following examples and embodiments are illustrative of the present invention and are not intended to limit the scope of the present invention.

Example  One: Peptides  Preparation of derivatives

1, 2, 3 and 4, which shows an amphiphilic alpha-helix structure in which hydrophilic and hydrophobic are present in different planes, using lysine or arginine as the cationic amino acid and isoleucine or tryptophan as the hydrophobic amino acid, , 5, 6, 7, 8 and 9 were prepared.

An amphipathic alpha-helix structure in which lysine or arginine is used as a cationic amino acid, isoleucine or tryptophan is used as a hydrophobic amino acid, and glutamic acid, an anionic amino acid, is present on a surface having different hydrophilicity and hydrophobicity, 10 < / RTI >

10 (SEQ ID NO: 1), KWI-10 (SEQ ID NO: 2), WIK-10 (SEQ ID NO: 3), RIW- (SEQ ID NO: 5), WIR-10 (SEQ ID NO: 6), KIW-14 (SEQ ID NO: 7), KWI-14 (SEQ ID NO: 8), WIK-14 Respectively.

1 shows the structure of the peptide consisting of the amino acid sequence shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 in http://mobyle.rpbs.univ-paris-diderot.fr / cgi-bin / portal.py # forms.

Specifically, the peptides were synthesized using the following method. First, amidated peptides were synthesized using Rink Amide 4-methylbenzhydrylamine Resin (Novagiochem, 0.55 mmol / g). The imidized peptide was then precipitated with ether and cleaved from the resin to give a crude form of the peptide. The crude peptide was purified with a zorbax C18 column (21.2 x 250 mm, 300 Å, 7-μm) using 0-60% acetonitrile as a gradient in 0.1% trifluoroacetic acid Respectively. The purified peptide was measured for purity using a Shimadzu analytical HPLC system and the molecular weight of the peptide was determined using Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry. The results are shown in Table 2 below.

Peptides order Molecular Weight Net charge KIW-10 (SEQ ID NO: 1) KKIIKKIWKW 1370.7 +6 KWI-10 (SEQ ID NO: 2) KKIWKKWIKI 1370.7 +6 WIK-10 (SEQ ID NO: 3) WIKKIWKKIK 1370.7 +6 RIW-10 (SEQ ID NO: 4) RRIIRRIWRW 1510.8 +6 RWI-10 (SEQ ID NO: 5) RRIWRRWIRI 1510.8 +6 WIR-10 (SEQ ID NO: 6) WIRRIWRRIR 1510.8 +6 KIW-14 (SEQ ID NO: 7) KKIIKKIIKKIWKW 1853.4 +8 KWI-14 (SEQ ID NO: 8) KKIWKKWIKKIIKI 1853.4 +8 WIK-14 (SEQ ID NO: 9) WIKKIWKKIIKKIK 1853.4 +8 WIKE-14 (SEQ ID NO: 10) WIKKIWKKIIKEIK 1854.4 +6 Melittin (control) GIGAVLKVLTTGLPALISWIKRKRQQQ 2068.7 +5

An anti-inflammatory peptide having a purity of 95% or more similar to the amino acid sequence shown in Table 2 was synthesized. The measured molecular weights were in agreement with the molecular weights calculated from the amino acid sequences and confirmed that the antiinflammatory peptide consisting of the amino acid sequence set forth in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, I could confirm.

Test Example  1: Antimicrobial activity Of peptide  Minimal inhibitory concentration, MIC ) Measure

The minimum inhibitory concentrations of the antimicrobial active peptides (SEQ ID NOS: 1 to 10) prepared in Example 1 were measured using Gram-negative bacteria, Gram-positive bacteria and five drug-resistant bacteria.

In the case of bacteria, 10 ml of TSB (tripticase soy broth) was cultured at 37 ° C., and then transferred to a new medium for 6 hours. The microorganisms in the logarithmic growth phase were suspended in 10 mM sodium phosphate buffer (pH 7.4) Or a solution containing 10% by volume of TSB (Trycase soy broth) medium in physiologically active saline (PBS, NaCl 137 mM, KCl 2.7 mM, Na ₂ HPO ₄ 10 mM, KH ₂ PO ₄ 2 mM, pH 7.4) And diluted to a concentration of 1 x 106 CFU / ml. To each well of a polypropylene 96-well plate were mixed 90 μl of the microbial dilution and 10 μl of the peptide (10 μl) diluted twice in duplicate and then cultured at 37 ° C for 12-24 hours.

The absorbance of each well was then measured at 600 nm using a microplate reader to determine the lowest inhibitory concentration (MIC) as the lowest peptide concentration that completely inhibited the growth of the microorganism. The MIC values were determined as the mean value of three independent experiments, three at a time.

First, a gram-positive Bacillus sub-blocks bus (Bacillus subtilis) and Stability Philo nose kusu aureus (Staphylococcus aureus) and Listeria monocytogenes presence (Listeria As the gram-negative bacteria monocytogenes) Escherichia cyano coli (Escherichia coli) and rugi labor (Psedomonas Pseudomonas The antimicrobial active peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 was used as a control and Melittin peptide was used as a control. MIC values were measured and are shown in Table 3 below.

 Name MIC  ( μM ) Escherichia  coli Pseudomonas  aeruginosa Staphylococcus aureus Bacillus subtilis Listeria  monocytogenes LIS HIS LIS HIS LIS HIS LIS HIS LIS HIS KIW-10 8 64 16 64 16 128 4 32 4 128 KWI-10 8 64 16 64 16 128 2 32 2 64 WIK-10 8 32 8 32 8 64 2 8 2 16 RIW-10 8 16 16 32 8 16 2 4 2 8 RWI-10 2 4 16 32 8 16 One 4 2 8 WIR-10 2 4 4 8 4 8 One One 2 2 KIW-14 4 4 2 2 4 8 One 2 0.5 One KWI-14 4 4 2 2 4 8 One 2 0.5 One WIK-14 4 4 2 2 2 2 One One 0.5 One WIKE-14 4 64 8 32 16 128 2 8 4 8 Melittin 2 2 2 16 2 2 2 2 One 2

LIS: Low Ionic Strength buffer, 10 mM Sodium Phosphate buffer, HIS: High Ionic Strength, PBS, NaCl 137 mM, KCl 2.7 mM, Na ₂ HPO ₄ 10 mM , KH ₂ PO ₄ 2 mM)

As shown in Table 3 above, Melittin, a control group in a solution with low ionic strength, showed MICs of 1-2 μM in five bacteria, while 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- KWI-14 (SEQ ID NO: 8), WIK-14 (SEQ ID NO: 9) and WIKE-14 (SEQ ID NO: 10) peptides showed MICs of 0.5 to 16 μM in five bacteria. In particular, KIW-14 (SEQ ID NO: 7), KWI-14 (SEQ ID NO: 8) and WIK-14 (SEQ ID NO: 9) peptides showed MICs of 0.5-4 μM, Value was maintained as it was.

Test Example  2: Antimicrobial activity Of peptide  Antimicrobial activity measurement

The antibacterial activity of the antimicrobial active peptides (SEQ. ID. Nos. 1 to 10) prepared in Example 1 was measured in order to verify the practical use of the antimicrobial active peptides of the present invention as a therapeutic agent for infectious microorganism infection.

Drug-resistant Pseudomonas aeruginosa (Drug-resistant Pseudomonas spp. aeruginosa , PA) and drug-resistant Staphylococcus aureus (Sa). In Test Example 2, the same method as that of Test Example 1 was performed except that the test was performed only in 10 mM sodium phosphate buffer.

The MIC values for the drug resistant Pseudomonas aeruginosa are shown in Table 4, and the MIC values for the drug resistant Staphylococcus aureus are shown in Table 5 below. On the other hand, oxacillin, tobramycin and erythromycin were used as positive control groups for comparison.

MIC (μM) Pa
(Antibiotic susceptibility bacteria) Pa-01
(Drug resistant bacteria) Pa-02
(Drug resistant bacteria) Pa-03
(Drug resistant bacteria) Pa-04
(Drug resistant bacteria) Pa-05
(Drug resistant bacteria) KIW-10 16 16 16 8 16 16 KWI-10 16 16 16 8 8 8 WIK-10 8 8 8 8 8 8 RIW-10 16 16 16 8 16 16 RWI-10 16 8 8 8 8 8 WIR-10 4 4 4 4 4 4 KIW-14 2 2 2 2 2 2 KWI-14 2 2 2 2 2 2 WIK-14 2 2 2 One One One WIKE-14 8 16 8 8 8 8 Melittin 2 2 4 4 4 2 Oxacillin 4 32 8 > 128 > 128 > 128 Erythromycin One One 32 > 128 > 128 > 128

MIC (μM) Sa
(Antibiotic susceptibility bacteria) Sa-01
(Drug resistant bacteria) Sa-02
(Drug resistant bacteria) Sa-03
(Drug resistant bacteria) Sa-04
(Drug resistant bacteria) Sa-05
(Drug resistant bacteria) KIW-10 16 16 16 16 16 16 KWI-10 16 16 16 8 16 8 WIK-10 8 8 4 4 8 4 RIW-10 8 8 8 8 8 8 RWI-10 8 8 8 8 4 4 WIR-10 4 4 2 2 4 2 KIW-14 4 4 2 2 2 4 KWI-14 4 2 2 4 2 2 WIK-14 2 2 2 2 2 2 WIKE-14 16 8 16 8 16 16 Melittin 2 2 2 4 2 2 Tobramycin 2 > 128 2 > 128 64 2 Erythromycin 2 > 128 64 > 128 > 128 16

As shown in Tables 4 and 5, the 10 kinds of antimicrobial active peptides (SEQ ID NOS: 1 to 10) of the present invention are drug-resistant Pseudomonas aeruginosa and 5 drugs resistant Staphylococcus aureus And it was confirmed that it showed strong antibacterial activity against.

Test Example  3: Antimicrobial activity Of peptide  Measurement of antimicrobial activity at physiologically active salt concentration

10 mM HEPES, 10 mM NaCl (pH 7.4) + 10% TSB, and Buffer II: 10 in order to confirm the antimicrobial activity of the antimicrobial active peptides (SEQ ID NOS: 1 to 10) 10 mM HEPES, 150 mM NaCl pH 7.4 + 10% TSB, Buffer III: 10 mM HEPES, 5 mM MgCl ₂ (pH 7.4) + 10% TSB, Buffer IV: 10 mM HEPES, 150 mM NaCl, 5 mM MgCl ₂ (pH 7.4) + 10% Antibacterial test was carried out by the method of Test Example 1 in TSB.

As shown in FIG. 2 and FIG. 3, all of the 10 peptides inhibited the bacterial growth of 95% or more at 32 μM or less in the buffer I. KIW-10 (SEQ ID NO: 1), KWI-10 (SEQ ID NO: 2) and WIK-10 (SEQ ID NO: 3) peptides having an amino acid number of 10 and hydrophilic lysine in buffers I, RIW-10 (SEQ ID NO: 4), RWI-10 (SEQ ID NO: 5) and WIR-10 (SEQ ID NO: 6) peptides having an amino acid number of 10 and hydrophilic arginine, 14 (SEQ ID NO: 7), KWI-14 (SEQ ID NO: 8), WIK-14 (SEQ ID NO: 9) and WIKE-14 (SEQ ID NO: 10) in which the number of amino acids was 14, Peptide showed antibacterial activity almost similar to buffer I. 10) (SEQ ID NO: 1), KWI-10 (SEQ ID NO: 2), WIK-10 (SEQ ID NO: 3), RIW- (SEQ ID NO: 6), KWI-14 (SEQ ID NO: 8), WIK-14 (SEQ ID NO: 9), WIKE-14 (SEQ ID NO: 10) peptides were judged to be applicable to both internal and external preparations since the activity of the antifungals was maintained in the in vivo salt concentration.

Test Example  4: Antimicrobial activity Of peptide  Measurement of erythrocyte hemolytic activity

To determine whether the antimicrobial active peptides of the present invention exhibited cytotoxicity, the erythrocyte-disrupting activity of the antimicrobial conjugated peptides was examined as follows.

Human red blood cells were diluted with a phosphate buffer solution (PBS, pH 7.0) to a concentration of 8%, and each of the peptides prepared in Example 1 was diluted successively with 1/2 concentration and reacted at 37 ° C for 1 hour After centrifugation at 1,000 g, the amount of hemoglobin contained in the supernatant was measured by measuring the 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. The cell destruction ability of 0.1% Triton X-100 was set to 100% And calculated according to the following equation (1).

In this formula,

Absorbance A is the absorbance of the group treated with the peptide solution at a wavelength of 414 nm,

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

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

FIG. 4 is a graph comparing antimicrobial activity peptides (SEQ. ID. Nos. 1 to 10) of the present invention with the results of measurement of erythropoietic activity of melittin. As shown in FIG. 4, the KIW-14 (SEQ ID NO: 7) peptide showed a hemolytic effect at 200 μM, the KWI-14 (SEQ ID NO: 8) peptide showed a hemolytic effect at 200 μM at 6.84%, the WIK- (SEQ ID NO: 1), KWI-10 (SEQ ID NO: 2), WIK-10 (SEQ ID NO: 3) and RIW-10 (SEQ ID NO: 9) showed a hemolysis phenomenon of 16.17% 4), RWI-10 (SEQ ID NO: 5), WIR-10 (SEQ ID NO: 6) and WIKE-14 (SEQ ID NO: 10) peptides showed almost no hemolysis at 200 μM. From these results, it was confirmed that the peptide consisting of the amino acid sequence of SEQ ID NOS: 1 to 10 of the present invention is a stable peptide that hardly causes erythrocyte hemolysis.

Test Example  5: Antimicrobial activity Of peptide  Cytotoxicity measurement

In order to examine the toxicity of the antimicrobial active peptides (SEQ ID NOS: 1 to 10) of the present invention to normal cells, MTT assay was performed after treating the mouse fibroblast cell line (NIH3T3) with peptides. NIH3T3 cells (10,000 cells / well) were cultured in a 96-well plate in DMEM (Dulbecco's modified eagle medium) containing 10% FBS in the presence of 5% CO ₂ for 16 hours. (SEQ ID NOS: 1 to 10), respectively, and further cultured for 36 hours. Then, it was developed by MTT assay and absorbance was measured at 570 nm to confirm cell viability. The cell viability [Viability (%)] was determined by the following equation (2).

In this formula,

Absorbance A is the absorbance of the peptide solution at the 570 nm wavelength,

Absorbance B is the absorbance of the group not treated with the peptide at a wavelength of 570 nm,

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

5 is a graph showing cytotoxicity of the antimicrobial active peptides (SEQ ID NOS: 1 to 10) of the present invention to mouse fibroblast cell line (NIH3T3) in comparison with melitin. As shown in FIG. 5, the survival rate of NIH3T3 cells was 91.42% at 100 μM, the survival rate of NIH3T3 cells at 92.59% at 100 μM, and the survival rate of NIH3T3 cells of KWI-14 (SEQ ID NO: 10 (SEQ ID NO: 1), KWI-10 (SEQ ID NO: 2), WIK-10 (SEQ ID NO: 3) and RIW The peptides of -10 (SEQ ID NO: 4), RWI-10 (SEQ ID NO: 5), WIR-10 (SEQ ID NO: 6) and WIKE-14 (SEQ ID NO: 9) showed 100% survival rates of NIH3T3 cells at 100 μM . The peptide of the present invention showed a survival rate of 85% or more at a concentration of 100 [mu] M, confirming that there was almost no cytotoxicity.

From the above results, it can be seen that the antimicrobial active peptide comprising the amino acid sequence of SEQ ID NOS: 1 to 10 of the present invention exhibits excellent antimicrobial effect and is not cytotoxic, so that it can be effectively used as a safe antimicrobial agent in the human body.

Test Example  6: Identification of antimicrobial activity mechanism

In order to confirm the action mechanism of the antimicrobial activity of the antimicrobial active peptides of the present invention, the antimicrobial active peptides (SEQ ID NOS: 1 to 10) prepared in Example 1 were treated with an artificial liposome to measure physical phenomena over time .

1: 1, w / w) and animal cells (PC / CH / SM, 1: 1: 1, w / w), and then the mixed lipid membrane was firstly removed with argon gas. Then, in order to completely remove the chloroform, a freeze dryer was used for 3 hours in a vacuum state Followed by freeze-drying. After that, the mixture was mixed with the phospholipid using a buffer, vortexed and mixed thoroughly, followed by sonication at room temperature for 30 minutes. Liposomes of a certain size are then separated using a mini-extruder. A polycarbonate membrane with a size of 0.1 μm was used for a small unilamellar vesicle and a polycarbonate membrane with a size of 0.2 μm was used for a large unilamellar vesicle.

The fluorescence emission spectrum of the tryptophan residues of the peptides was measured on the buffer solution and on the liposomes to determine the ability of the peptides to bind to the cell membrane. The lipid mixture was incubated with 10 mM HEPES buffer solution and sonicated to produce a small membrane sac (SUV). Peptides were prepared by adding liposomes and peptides at a ratio of liposomes from 0 to 0.6 mM, and reacted at 20 ° C for 10 minutes. The excitation wavelength of the spectrofluorimeter was scanned at 280 nm and the emission wavelength was set at 300 to 400 nm. The blue shift value of the peptide was determined by calculating the difference in wavelength representing the maximum value of the emission spectrum of the peptide. The results of the measurement are shown in Table 6 below.

Peptides Solution without liposome PE / PG liposomes (7: 3, w / w) PG / CL liposome (1: 1, w / w) PC / CH / SM liposome (1: 1: 1, w / w / w) Buffer I Buffer IV Buffer I Buffer IV Buffer I Buffer IV Buffer IV Buffer IV KIW-10 352 356 343 346 340 341 355 351 KWI-10 354 354 338 345 340 341 354 352 WIK-10 354 354 334 339 331 332 354 354 RIW-10 352 356 339 341 339 339 356 353 RWI-10 353 355 338 338 337 338 355 353 WIR-10 352 355 337 337 337 338 354 354 KIW-14 353 357 340 340 339 340 349 343 KWI-14 353 356 332 332 331 332 349 352 WIK-14 356 356 318 318 321 321 342 350 WIKE-14 353 356 332 337 322 328 353 352

Buffer I: 10 mM HEPES (pH 7.4) containing 10 mM NaCl, buffer IV: 10 mM HEPES (pH 7.4) with 150 mM NaCl and 5 mM MgCl ₂ ,

As shown in Table 6, all of the antimicrobial active peptides (SEQ ID NOS: 1 to 10) exhibited the maximum emission intensity at 352 to 357 nm in buffer I and IV in the liposome-free solution, and PE / PG composition liposomes showed the maximum emission intensity in buffer I at 318 to 343 nm. Therefore, it was found that the antimicrobial active peptides (SEQ ID NOS: 1 to 10) bind strongly to the bacterial cell membrane in the buffer I. Also, the maximum emission intensity of 318 to 346 nm in buffer IV means that the binding force with the bacterial cell membrane is weak, which is consistent with the results of FIGS. 2 and 3.

The PG / CL liposomes in Table 6 are the same as the composition of the inner membrane of negative bacteria, with results similar to those of PE / PG liposomes. The PC / CH / SM liposome, which is the cell membrane composition of normal cells, is composed of the amino acid sequence as shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8 and 10 except for WIK-14 (SEQ ID NO: The peptides were determined to have a maximum emission intensity shift of 1-4 nm relative to the liposome-free solution, consistent with the results of the erythrocyte hemolytic activity and cytotoxicity of FIGS. 4 and 5 without binding to PC / CH / SM liposomes.

On the other hand, the 14 nm shift of WIK-14 (SEQ ID NO: 9) peptide in buffer I shows a strong binding, but a shift of 6 nm in buffer IV shows weak binding. This is consistent with the results of the cytotoxicity test and is unlikely to indicate serious cytotoxicity.

Test Example  7: Antimicrobial activity Of peptide  Cell membrane Destructiveness  Measure

In order to confirm the cell membrane-destructive ability of the antimicrobial active peptides of the present invention, PE / PG and PC / Ch / SM liposome carrying 80 mM calcein fluorescent staining solution were prepared and the antimicrobial active peptides ( SEQ ID NOS: 1 to 10).

The lipid weight of the desired mole ratio was measured and placed in a 10 mL round bottle flask, wrapped with foil to block the light, and filled with nitrogen in a round bottom flask. To make liposomes with uniform lipid distribution, lipids were completely dissolved in 2 mL of volatile organic solvent anhydrous chloroform in a round bottom flask. The lipids were thoroughly vortexed for 5 minutes to allow uniform mixing of the lipids and the organic solvent was evaporated with nitrogen gas. At this time, the lipid film was finely adhered to the surface of the round bottom flask while being careful not to bloom. After that, it was left in a vacuum dryer for about 3 hours to completely remove the remaining organic solvent. After making a 80 mM calcein solution, the calcein solution was added to a round-bottomed flask containing lipid completely removed from the organic solvent. Multilamellar vesicles (MLV) were made by vortexing for 10 min to allow the lipid and calcine solutions to mix well. A round bottom flask with a mixture of lipid and calcine solution was placed in an ultrasonic disperser (Sonicator) for 20 seconds. At this time, since some heat may be generated and damage to the lipid may occur, the process of completely cooling down the ice water is repeated. The round bottom flask was then immersed in liquid nitrogen to completely freeze the liposomes. Freeze-thawing was repeated ten times with rapid freezing and slow melting at room temperature while shaking the round bottom flask in a circular motion to uniformly freeze the liposome. In order to obtain a uniform liposome of 200 nm or less, an Avanti mini-extruder (USA) equipped with 400 nm and 200 nm polycarbonate membranes (Nuclepore corporation, Pleasanton CA, USA) And filtered. To remove the remaining fluorescent material without being trapped in the liposome, the liposome filtered by an extruder was loaded onto a gel permeation chromatography (GPC), which was packed with Sephadex G-50 (pharmacia, Sweden) as a filler ). Liposomes that are relatively large in size compared to fluorescent materials can easily escape from a small hole in the GPC. When liposomes are first separated due to the size difference between the fluorescent material and the liposome, they are received in a micro tube, Respectively.

Leakage assay shows almost no fluorescence due to self-quenching of each other when the fluorescent substance is captured in the liposome. When the fluorescent substance in the liposome is leaked by the peptide, the fluorescent substance captured by the peptide concentration This is an experiment that examines the degree of release by the change of fluorescence. Fluorescence was observed using a Perkin Elmer LS-55 fluorescence analyzer and the wavelength range was set differently depending on the fluorescent material captured in the liposome. For calcein, one of the fluorophores, fluorescence was measured at 490 nm and emission at 520 nm. Herein, 100% calcein leakage was calculated according to the following equation (3) using a 0.1% Triton X-100 solution as a positive control.

In this formula,

The absorbance F ₀ is the fluorescence value measured when only the liposomes are in the cuvette before adding the antimicrobial active peptide

The absorbance F _(t) is a measure of the increase in fluorescence over time after treatment of the liposomes with the antimicrobial active peptide

The absorbance F _x represents the fluorescence of a liposome treated with a Triton X-100 solution.

The measurement results are shown in Fig. The results shown in FIG. 6 indicate the extent of the efflux of the calcein by treating the peptide. The higher the efflux rate, the more severe the cell membrane destruction, which means that there is strong antibacterial activity and cytotoxicity. As shown in FIG. 6, the peptides KIW-10 (SEQ ID NO: 1), KWI-10 (SEQ ID NO: 2) and WIK-10 (SEQ ID NO: 3) produced almost no leakage of calcein in the bacterial cell membrane composition PE / PG liposomes , And the RIW-10 (SEQ ID NO: 4), RWI-10 (SEQ ID NO: 5) and WIR-10 (SEQ ID NO: 6) peptides eluted some of the calcein. The 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 were found to severely leak calcein in a concentration dependent manner. This shows that the peptide of KIW-10 (SEQ ID NO: 1), KWI-10 (SEQ ID NO: 2) and WIK-10 (SEQ ID NO: 3) infiltrated into cells without almost destroying the cell membrane, 4), RWI-10 (SEQ ID NO: 10) and WIR-10 (SEQ ID NO: 6) peptides can be described as having an antimicrobial mechanism that induces a slight cell membrane breakdown upon passage through the cell membrane, , 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 directly destroy bacterial membranes, Can be described as having an antimicrobial mechanism to kill bacteria by allowing them to leak.

Test Example  8: Antibacterial activity Of peptide  Agar Permeability  Measure

In order to confirm the antimicrobial activity of the antimicrobial active peptides (SEQ ID NOS: 1 to 10) prepared in Example 1 of the present invention, the agar diffusion assay method was used. In order to measure the ability of the antimicrobial active peptides to penetrate the agar, Escherichia coli was selected as Gram-negative bacteria and staphylococcus aureus was selected as Gram-positive bacteria.

A single colony of bacteria was inoculated into freshly prepared TSB medium and reacted overnight at 37 ° C. A portion of the culture was transferred to fresh TSB medium and allowed to react for another 5 hours at 37 ° C (OD600 = 0.4). Petri dishes were prepared by adding 6 ml of agar dissolved in LB medium, and 100 μl of the bacterial suspension was inoculated (OD600 = 0.4). The peptide solution was dispensed into a 5 mm diameter hole and reacted overnight at 37 ° C to observe a clearing zone showing antimicrobial activity. The above observation result is shown in Fig.

As shown in FIG. 7, KIW-14 (SEQ ID NO: 7) and KWI-14 (SEQ ID NO: 7) exhibited stronger skin penetration and antibacterial activity as the size of the clearing zone of the medium inoculated with Escherichia coli and Staphylococcus aureus was larger. (SEQ ID NO: 8) and WIK-14 (SEQ ID NO: 9) peptides, respectively, compared to peptides consisting of the amino acid sequences of SEQ ID NOS: 1 to 6 and 10 and melanin as a control.

Test Example  9: Antimicrobial activity Of peptide Biofilm  Inhibition and elimination effect

The effect of inhibiting and eliminating the biofilm formed by Pseudomonas aeruginosa in the antimicrobial active peptides (SEQ ID NOS: 1 to 10) prepared in Example 1 of the present invention is that the growing cells are cultured in a polypropylene 96-well microtiter The ability to attach to the surface of the plate was determined by evaluating it according to a fixed biofilm assay.

Pseudomonas aeruginosa strains were inoculated into 5 ml MHB liquid medium and incubated overnight at 37 ° C. The bacteria were then inoculated at 1 x 10 7 cfu / mL in 96-well tissue culture plates containing 100 μl / well LB liquid medium and incubated at 37 ° C for 48 hours. The medium was removed, carefully washed once with PBS, and the desired concentration of the peptide solution was dispensed into the biofilm formation wells. The wells were left at 37 ° C for 24 hours, and the wells were washed three times with 200 μL / well dH20 to remove any remaining planktonic cells, taking care not to disturb any biofilm formed. A 1% solution of crystal violet was added at 125 μl / well to stain adherent cells (biofilm) and incubated at room temperature for 15 minutes. The plates were then thoroughly washed with dH2O to remove excess bacteria.

Crystal Violet bacteria as a means for measuring the degree of the biofilm formed were recovered with 200 μl / well of 95% ethanol. The absorbance of 125 μl ethanol / crystal violet was measured at 590 nm and the results are shown in FIG.

As shown in Fig. 8, the peptide consisting of the amino acid sequence of SEQ ID NOS: 1 to 10 strongly inhibited the growth of the drug-susceptible Pseudomonas aeruginosa ATCC15692 and the drug-resistant Pseudomonas aeruginosa 4007 as a result of growth inhibition And the existing drugs piperacillin and oxacillin did not substantially inhibit the growth of the drug resistant Pseudomonas eruginosa 4007. Further, in the biofilm reduction results, it was confirmed that the peptide consisting of the amino acid sequence shown in SEQ ID NOS: 1 to 10 had a strong elimination ability. As a result, it was confirmed that all of the peptides consisting of the amino acid sequences of SEQ ID NOS: 1 to 10 had excellent biofilm inhibition and elimination ability.

Formulation example  1. Preparation of pharmaceutical compositions

1-1. Liquid  Produce

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

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to a conventional liquid preparation method, the lemon flavor was added in an appropriate amount, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 mL, And sterilized to prepare a liquid preparation.

1-2. Sanje  Produce

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

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

1-3. Injection preparation

K 10 mg of the antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ .2H ₂ O 26 mg

(2 mL) per 1 ampoule in accordance with the usual injection preparation method.

1-4. Manufacture of tablets

K 10 mg of the antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

1-5. Preparation of capsules

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

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  2. Cosmetics  Of the additive composition Manufacturing example

2-1. Manufacture of softening longevity (skin lotion)

0.25% by weight of an antibacterial activity peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,

Beta-1,3-glucan 1.0 wt%

Butylene glycol 2.0 wt%

Propylene glycol 2.0 wt%

Carboxyvinyl polymer 0.1 wt%

0.2% by weight of phage-12 nonylphenyl ether

Polysorbate 80 0.4 wt%

Ethanol 10.0 wt%

0.1% by weight triethanolamine

Preservative, pigment, perfume

The amount to make the purified water 100% by weight

And each of them is manufactured in accordance with a conventional method for producing softened water.

2-2. Nutrition lotion ( Milk lotion )

0.5% by weight of an antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,

Beta-1,3-glucan 1.0 wt%

4.0 wt%

Polysorbate 60 1.5 wt%

1.5% by weight of sorbitan sesquioleate

0.5% by weight liquid paraffin

Caprylic / capric triglyceride 5.0 wt%

Glycerin 3.0 wt%

3.0% by weight of butylene glycol

3.0% by weight of propylene glycol

Carboxyvinyl polymer 0.1 wt%

0.2% by weight triethanolamine

Preservative, pigment, perfume

The amount to make the purified water 100% by weight

It is manufactured in accordance with the usual nutrition lotion production method.

2-3. Manufacture of nutrition cream

0.5% by weight of an antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,

Beta-1,3-glucan 5.0 wt%

Wax 10.0 wt%

Polysorbate 60 1.5 wt%

≪ tb > < tb > < tb >

0.5% by weight of sorbitan sesquioleate

Liquid paraffin 10.0 wt%

Squalane 5.0 wt%

Caprylic / capric triglyceride 5.0 wt%

Glycerin 5.0 wt%

3.0% by weight of butylene glycol

3.0% by weight of propylene glycol

0.2% by weight triethanolamine

Preservative, pigment, perfume

The amount to make the purified water 100% by weight

They are prepared in accordance with the usual nutritional cream manufacturing method.

Formulation example  3. Food preservatives Manufacturing example

0.5% by weight of an antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,

0.1% by weight of dehydroacetic acid

0.1% by weight potassium sorbate

0.2% by weight calcium sorbate

0.5% by weight sodium benzoate

0.1% by weight potassium benzoate

Calcium benzoate 0.5 wt%

0.1% by weight of methyl paraoxybenzoate

0.1% by weight of p-hydroxybenzoic acid propylate

0.1% by weight sodium propionate

0.1% by weight calcium propionate

They are prepared in accordance with a conventional food preservative manufacturing method.

Formulation example  4. Drug resistant bacteria Biofilm  Composition for preventing or inhibiting the formation of Manufacturing example

20% by weight of the antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,

5% by weight of surfactant

10% by weight of ethanol

Sterile water balance

Each of which is prepared according to a conventional disinfectant preparation method.

Thus, the antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 according to the present invention exhibits a high antibacterial effect And is not toxic to human cells, exhibits strong antibacterial activity against live bacteria in antibiotics, and has excellent bioavailability. Accordingly, the antimicrobial pharmaceutical composition, the cosmetic composition and the food additive composition comprising the antimicrobial active peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the present invention Can be usefully used as an infectious agent, a food preservative, a feed additive or a cosmetic additive, and can be effectively used as a composition for preventing or inhibiting the biofilm formed by drug resistant bacteria.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION OF SUNCHON NATIONAL UNIVERSITY <120> Alpha-helical peptide having antimicrobial activity against          drug-resistant bacteria and biofilm and antimicrobial composition          comprising the same <130> PA-17-0129 <160> 10 <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

Claims (8)

1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 in the amino acid sequence shown in SEQ ID NO: The method according to claim 1,
Wherein the antimicrobial active peptide is substituted with any one selected from the group consisting of isoleucine, alanine, methionine, phenylalanine, tryptophan, proline, valine and leucine in the amino acid sequence. The method according to claim 1,
Wherein the antimicrobial active peptide is substituted with any one selected from the group consisting of tryptophan, alanine, methionine, phenylalanine, proline, valine and leucine in the amino acid sequence. The method according to claim 1,
Wherein the antimicrobial active peptide has an antimicrobial activity against at least one bacterium selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and drug-resistant bacteria. An antimicrobial pharmaceutical composition comprising the antimicrobial active peptide according to any one of claims 1 to 4 as an active ingredient. An antimicrobial cosmetic additive composition comprising the antibacterial activity peptide of any one of claims 1 to 4 as an active ingredient. An antimicrobial food additive composition comprising the antibacterial activity peptide of any one of claims 1 to 4 as an active ingredient. A composition for preventing or inhibiting drug-resistant microbial biofilm formation comprising the antimicrobial active peptide according to any one of claims 1 to 4 as an active ingredient.

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