A novel antimicrobial peptide with excellent cytoplasmic elution effect on microorganisms

Abstract

The present invention relates to an antimicrobial peptide, a derivative or a fragment thereof, a polynucleotide encoding the antimicrobial peptide, the derivative or the fragment thereof, and a carrier for an external preparation for skin, a cosmetic composition, a quasi-drug composition and a pharmaceutical composition, each of which contains the antimicrobial peptide, the derivative or the fragment thereof.

Description

[Technical field]

[0001] The present invention relates to an antimicrobial peptide, a derivative or a fragment thereof, a polynucleotide encoding the antimicrobial peptide, the derivative or the fragment thereof, and a carrier for an external preparation for skin, a cosmetic composition, a quasi-drug composition and a pharmaceutical composition, each of which contains the antimicrobial peptide, the derivative or the fragment thereof. [Background technology]

[0002] In the current pandemic era, the demand for products with excellent antibacterial properties is increasing, and various attempts to develop antibacterial products are being actively carried out. As part of these efforts, research and development of natural antibiotics is being carried out.

[0003] Specifically, naturally derived antibiotics are known, such as an antibacterial composition containing a plant extract as an active ingredient (Patent Document 1). However, natural products such as extracts have relatively large particle sizes, making it difficult for a single natural product to exert a significant effect, and therefore there is an increasing need to develop new antibacterial substances. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Korean Patent Publication No. 10-2020-0017912 [Non-patent literature]

[0005] [Non-Patent Document 1] Sambrook et al., supra, 9.50-9.51, 11.7-11.8 [Non-Patent Document 2] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-Patent Document 3] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-Patent Document 4] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-Patent Document 5] Devereux, J., et al, Nucleic Acids Research 12: 387 (1984) [Non-Patent Document 6] Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990) [Non-Patent Document 7] Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994 [Non-Patent Document 8] [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073 [Non-Patent Document 9] Smith and Waterman, Adv. Appl. Math (1981) 2:482 [Non-Patent Document 10] Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) [Non-Patent Document 11] Gribskov et al(1986) Nucl. Acids Res. 14: 6745 [Non-Patent Document 12] J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 [Non-Patent Document 13] FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York [Non-Patent Document 14] E. Kaiser et al. Anal. Biochem., 1970, 34(2), 595~598. Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors have confirmed that the antimicrobial peptide of the present invention has a significantly superior antimicrobial effect compared to other peptides, and have thus completed the present invention. [Means for solving the problem]

[0007] The present invention aims to provide antimicrobial peptides, derivatives or fragments thereof.

[0008] Another object of the present invention is to provide a polynucleotide encoding the antimicrobial peptide, a derivative thereof, or a fragment thereof.

[0009] A further object of the present invention is to provide a carrier for an external preparation for skin, which contains the antimicrobial peptide, a derivative thereof, or a fragment thereof.

[0010] A further object of the present invention is to provide a cosmetic composition comprising the antimicrobial peptide, its derivative, or a fragment thereof.

[0011] A further object of the present invention is to provide a quasi-drug composition comprising the antimicrobial peptide, its derivative, or a fragment thereof.

[0012] A further object of the present invention is to provide a pharmaceutical composition comprising the antimicrobial peptide, a derivative thereof or a fragment thereof. Effect of the Invention

[0013] The peptide of the present invention has significantly improved antibacterial activity and reduced toxicity compared to other peptides, and is therefore expected to be industrially useful in the fields of preservatives, pharmaceuticals, cosmetics, and the like. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows the results of confirming the antibacterial activity and cytotoxicity of the peptide of SEQ ID NO: 1 in comparison with known peptides. [Diagram 2] FIG. 1 shows the results of confirming the antibacterial activity and cytotoxicity of the peptide of SEQ ID NO: 2 in comparison with the peptide of SEQ ID NO: 1. [Diagram 3] FIG. 1 shows the results of confirming the antibacterial activity and cytotoxicity of the peptides of SEQ ID NO:2 and SEQ ID NO:5 to SEQ ID NO:8. [Figure 4] FIG. 1 shows the results of confirming the antibacterial activity and cytotoxicity of peptides represented by SEQ ID NOs: 2 to 4. [Diagram 5] FIG. 1 shows the results of confirming the antibacterial effects of the antibacterial peptide of the present invention against Escherichia coli, Staphylococcus aureus, Pneumonia aeruginosa, and Candida albicans. [Figure 6] FIG. 2 shows the results of an analysis of the antibacterial effect of the antibacterial peptide of the present invention against Escherichia coli by SEM (scanning electron microscope). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will be described in detail below. Note that the description and embodiment of one aspect disclosed in the present invention also apply to the description and embodiment of other aspects with respect to common matters. In addition, all combinations of various elements disclosed in the present invention are included in the present invention. Furthermore, the present invention is not limited to the following specific description.

[0016] One aspect of the present invention provides an antimicrobial peptide consisting of an amino acid sequence of any one of SEQ ID NOs: 5 to 8, or a derivative or fragment thereof.

[0017] In the present invention, the "antimicrobial peptide" refers to a peptide having antimicrobial activity, and since the antimicrobial activity is not limited to the type of microorganism, the "antimicrobial" is used interchangeably with antibacterial, antifungal, and antibiotic. Specifically, the antimicrobial peptide of the present invention may include any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, and may include any of the derivatives of SEQ ID NO:5 to SEQ ID NO:8 or fragments thereof, so long as it has antimicrobial activity.

[0018] The antimicrobial peptides are substances expressed by the innate immune system (primary defense) of various species, such as plants, insects, amphibians, mammals, and humans, to protect individuals from microorganisms such as bacteria, and have the properties of superior antimicrobial activity and almost no resistance compared to conventional antibiotics, but are not limited to these as long as they function as antimicrobial peptides.

[0019] Furthermore, the antimicrobial peptide of the present invention may have any structure as long as it has antimicrobial activity, and the microorganism may be any microorganism that falls within the category of microorganisms, including bacteria, fungi, etc., and may be of any type or origin.

[0020] Specifically, an antimicrobial peptide consisting of any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof inhibits the growth of at least one organism selected from the group consisting of Escherichia coli, Staphylococcus aureus, Pneumonia aeruginosa, and Candida albicans, but is not limited to these.

[0021] In one embodiment of the present invention, it was confirmed that an antimicrobial peptide consisting of any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8 and its derivatives have antimicrobial activity against Escherichia coli, Staphylococcus aureus, Pneumonia aeruginosa and / or Candida albicans (Example 2).

[0022] The term "derivative" in the present invention refers to a derivative of an antimicrobial peptide consisting of any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, specifically, a peptide having an amino acid sequence in which a functional group has been added to the amino acid sequence of SEQ ID NO:5 to SEQ ID NO:8, or in which a portion of the amino acid sequence has been deleted, modified, substituted or added, but is not limited to these.

[0023] Specifically, the derivative in the present invention may be at least one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4, and may function as an antimicrobial peptide similar to the antimicrobial peptide consisting of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 8. The antimicrobial peptide is as described above.

[0024] The antimicrobial peptides and derivatives thereof of the present invention consisting of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8 have the sequence of Chemical Formula 1 and have antimicrobial activity, but are not limited thereto.

[0025] [C1] [X1-X2- Leu -Phe-X3-X4-Ile-Leu-X5-Tyr-Leu-X6]

[0026] In Chemical Formula 1, X1 to X6 are selected from basic amino acids, and are selected from arginine, lysine, leucine, and phenylalanine. The antimicrobial peptide and its derivatives having any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8 preferably have the sequence of Chemical Formula 1, and the derivatives of the present invention preferably have the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:4, but are not limited thereto. SEQ ID NO: 1: H2N-[Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-Lys]-CO2H SEQ ID NO:2:H2N-[Arg-Arg-Leu-Phe-Arg-Arg-Ile-Leu-Arg-Tyr-Leu-Arg]-CO2H SEQ ID NO: 3: H2N-[Arg-Phe-Leu-Phe-Arg-Leu-Ile-Leu-Arg-Tyr-Leu-Arg]-CO2H SEQ ID NO: 4: H2N-[Arg-Arg-Leu-Phe-Arg-Leu-Ile-Leu-Arg-Tyr-Leu-Phe]-CO2H SEQ ID NO: 5: H2N-[Arg-Lys-Leu-Phe-Lys-Arg-Ile-Leu-Arg-Tyr-Leu-Lys]-CO2H SEQ ID NO: 6: H2N-[Lys-Arg-Leu-Phe-Arg-Lys-Ile-Leu-Lys-Tyr-Leu-Arg]-CO2H SEQ ID NO: 7: H2N-[Lys-Arg-Leu-Phe-Lys-Arg-Ile-Leu-Arg-Tyr-Leu-Lys]-CO2H SEQ ID NO: 8: H2N-[Arg-Lys-Leu-Phe-Arg-Lys-Ile-Leu-Lys-Tyr-Leu-Arg]-CO2H

[0027] Specifically, the derivative may be an antimicrobial peptide having an amino acid sequence of any one of SEQ ID NOs: 5 to 8, in which an amino acid corresponding to any one of the positions selected from the group consisting of the 1st, 2nd, 5th, 6th, 9th and 12th positions from the N-terminus is replaced with another basic amino acid.

[0028] Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues. Specifically, among amino acids having electrically charged side chains, positively charged (basic) amino acids include arginine (R), lysine (K), and histidine (H). Negatively charged (acidic) amino acids include glutamate (E) and aspartate (D). Among amino acids having uncharged side chains, nonpolar amino acids include glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), phenylalanine (F), tryptophan (W), and proline (P). Examples of polar or hydrophilic amino acids include serine (S), threonine (T), cysteine ​​(C), tyrosine (Y), asparagine (N) and glutamine (Q). Examples of aromatic amino acids among the nonpolar amino acids include phenylalanine, tryptophan and tyrosine.

[0029] In the present invention, the "substitution with another basic amino acid" may be any substitution with a basic amino acid different from the amino acid before substitution. That is, the amino acid before substitution at any position selected from the group consisting of the 1st, 2nd, 5th, 6th, 9th and 12th positions from the N-terminus of the antimicrobial peptide consisting of any of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 8 may be substituted with another basic amino acid, specifically, the amino acid at any position selected from the group consisting of the X1, X2, X3, X4, X5 and X6 positions may be substituted with another basic amino acid. More specifically, the basic amino acid may be selected from histidine, lysine and arginine, and may be lysine or arginine, but is not limited thereto.

[0030] For the purposes of the present invention, a derivative of an antimicrobial peptide consisting of any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8 is at least one selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:4, but is not limited thereto.

[0031] In one example of the present invention, it was confirmed that peptides represented by SEQ ID NO:5 to SEQ ID NO:8 and their derivatives, that is, peptides represented by SEQ ID NO:1 to 4, have the structure of Chemical Formula 1 and have antibacterial activity (Example 2).

[0032] Specifically, in one embodiment of the present invention, it was confirmed that the antimicrobial peptide or its derivatives consisting of the amino acid sequence of Chemical Formula 1 have excellent antimicrobial activity. More specifically, it was confirmed that SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 5 to SEQ ID NO: 8, more specifically, SEQ ID NO: 5 to SEQ ID NO: 8, and particularly specifically, SEQ ID NO: 6 have remarkably excellent antimicrobial activity (Examples 1 and 2).

[0033] In the present invention, a "fragment" refers to a partial sequence of a peptide having a specific sequence, and may be a partial sequence of an antimicrobial peptide or a derivative thereof consisting of any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, and may have antimicrobial activity similar to the antimicrobial peptide or a derivative thereof.

[0034] The antimicrobial peptide having an amino acid sequence of any one of SEQ ID NO:5 to SEQ ID NO:8, its derivative or its fragment is naturally derived, but is not limited thereto, and may be any sequence having the same activity as the antimicrobial peptide, its derivative or its fragment.

[0035] Although the antimicrobial peptide of the present invention is described as an antimicrobial peptide having an amino acid sequence of any one of SEQ ID NOs: 5 to 8, a derivative thereof, or a fragment thereof, this does not exclude meaningless addition of sequences before or after the amino acid sequence of the antimicrobial peptide having an amino acid sequence of any one of SEQ ID NOs: 5 to 8, a derivative thereof, or a fragment thereof, or silent mutations that maintain the same function as an antimicrobial peptide, and it will be clear to those skilled in the art that the antimicrobial peptide of the present invention includes any antimicrobial peptide having the same or corresponding activity as the antimicrobial peptide having an amino acid sequence of any one of SEQ ID NOs: 5 to 8, a derivative thereof, or a fragment thereof. As a specific example, the antimicrobial peptide of the present invention may be an antimicrobial peptide having an amino acid sequence of any one of SEQ ID NOs: 5 to 8, a derivative thereof, a fragment thereof, or a peptide having an amino acid sequence having 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto.

[0036] Even if the present invention describes "a peptide comprising an amino acid sequence represented by a specific SEQ ID NO," "a peptide consisting of an amino acid sequence represented by a specific SEQ ID NO," or "a peptide having an amino acid sequence represented by a specific SEQ ID NO," it goes without saying that a peptide having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added can be used in the present invention, so long as it has the same or corresponding activity as a peptide consisting of the amino acid sequence of the SEQ ID NO. For example, there can be mentioned an addition of a sequence that does not change the function of the peptide at the N-terminus and / or C-terminus of the amino acid sequence, a naturally occurring mutation, a silent mutation, or a conservative substitution thereof.

[0037] The term "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of the peptide.

[0038] The peptide of the present invention can be prepared by, but is not limited to, a method of forming an amide bond by connecting at least one amino acid or a suitably protected amino acid to an amino acid backbone fixedly attached to a solid phase. In addition, the peptide can be inserted, substituted, or deleted with other amino acids as long as the stability is not significantly decreased, and these are also included in the present invention.

[0039] The peptide of the present invention may be prepared in a form in which a cell permeable peptide that promotes intracellular movement is bound to the C-terminus or N-terminus. For example, the cell permeable peptide may be TAT peptide (Arg-Lys-Lys-Arg-Arg-Tyr-Arg-Arg-Arg) and Tat-PTD peptide (Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg: Tat PTD), but the present invention is not limited thereto. Any cell permeable peptide known in the art may be used as long as it does not inhibit the activity of the peptide of the present invention.

[0040] The peptides and compounds of the present invention may be prepared in the form of metal complexes, the metal being selected from, but not limited to, copper, magnesium, calcium, iron, zinc, nickel, silver, germanium, and gallium, preferably, but not limited to, copper.

[0041] The peptide of the present invention may exist in the form of a salt. The salt form used in the present invention may be formed during the final isolation and purification of the compound, or by reacting an amino group with a suitable acid. For example, acid addition salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate. Examples of acids used to form acid addition salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc., and organic acids such as oxalic acid, maleic acid, succinic acid, citric acid, etc. The peptides of the present invention are preferably prepared in the form of trifluoroacetate or acetate salts.

[0042] The amino or carboxy groups of the amino acids used to prepare the peptides contained in the compositions of the invention may be protected by a suitable protecting group. The protected amino acid may be attached to a solid support and reacted in solution by adding the next amino acid under suitable conditions to form an amide bond. Alternatively, the protecting group may be completely removed before adding the amino acid protected by the suitable protecting group. Once all amino acids have been linked as desired, they are separated, either sequentially or simultaneously, from the remaining free protecting groups and the free solid support to obtain the final peptide of interest.

[0043] The most preferred synthetic method for preparing the peptide compounds of the present invention is solid-phase peptide synthesis using a solid-phase polymer support, and the α-amino group of the peptide prepared by the above method is protected by an acid- or base-sensitive functional group. Here, the protecting group of the amino acid must exhibit stable properties under peptide condensation reaction conditions and be easily removed without destroying the growing peptide chain or racemizing any chiral center contained therein. Thus, preferred protecting groups include 9-fluorenylmethoxycarbonyl (Fmoc), t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, (α,α)-dimethyl-3,5-dimethoxybenzyloxycarbonyl, O-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, etc., and other suitable protecting groups known in the art for the above purpose can also be used in the present invention.

[0044] As the protecting group for an amino acid in the peptide synthesis of the present invention, it is most preferable to use the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group.

[0045] In particular, the protecting groups of the amino acid residues used in the peptide synthesis of the present invention are t-butyl (t-Bu) for N-methylglutamic acid, t-butoxycarbonyl (Boc) for lysine, and t-butyl (t-C) for serine. t-Butyl (t-Bu) and preferably, for threonine and allothreonine, it is t-butyl (t-Bu), and for cysteine, it is trityl (Trt), although the present invention is not limited thereto.

[0046] In solid phase peptide synthesis, the C-terminal amino acid is attached to a suitable solid support or resin. Preferred solid supports useful in said synthesis are those materials which are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions and insoluble in the media used, such as 2-chlorotrityl chloride resin, rink amid or rink amid MBHA resin.

[0047] In particular, preferred solid supports for C-terminal peptides are 2-chlorotrityl chloride, rink amid or rink amide 4-methylbenzylhydrylamine resins available from Novabiochem Corporation.

[0048] The C-terminal amide can be prepared by cleaving the amide with 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BO) in a solvent such as dichloromethane, N-methylpyrrolidone (NMP) or DMF at a temperature of 10° C. to 50° C., preferably at 30° C., for 1 to 24 hours. Carboxylic acids can be activated with N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate] (HATU) or O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) in the presence or absence of PCI, and condensed (bound, coupled) to a resin or solid support by condensation.

[0049] When the solid support is Rink Amide 4-methylbenzylhydrylamine resin, the Fmoc functionality, which is the preferred protecting group, is cleaved prior to condensation with the C-terminal amino acid using an excess of a secondary amine solution, preferably 20% piperidine in DMF. As described above, preferred reagents for condensing the desired amino acid to the deprotected 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin include condensation reaction reagents such as N-methylmorpholine (NMM), 1-hydroxybenzotriazole (HOBt), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), N,N'-dicyclohexylcarbodiimide (DCC), and N,N'-diisopropylcarbodiimide (DIC) used in DMF solvent with an appropriately protected amino acid.

[0050] The condensation of successive amino acids in the present invention may be carried out using an automatic peptide synthesizer well known in the related technical field, or may be carried out directly manually. The preferred synthetic reaction conditions are to treat an α-amino acid protected with an Fmoc group with a secondary amine solution, preferably piperidine, to deprotect it, then wash it with a sufficient excess amount of a solvent, and then add each of the other protected amino acids to be condensed in a 3- to 7-fold molar excess, and carry out the reaction preferably in a DMF solvent.

[0051] In the final step of peptide synthesis using the solid-phase resin of the present invention, the peptide to be obtained may be removed from the resin in succession or in a single operation, and the protecting groups protecting each amino acid residue may be deprotected. Removal of the peptide from the resin and deprotection of the protecting groups present on the residues are generally carried out by treating with a cleavage reagent cocktail that cleaves the bond between the resin and the peptide, for example, a dichloromethane mixed cocktail solution consisting of trifluoroacetic acid (TFA), triisopropylsilane (TIS), thioanisole, water, ethanedithiol (EDT), etc. The mixed solution thus obtained is treated with an excess amount of diethyl ether solvent that has been kept refrigerated to produce a precipitate. The precipitate thus obtained is centrifuged to completely precipitate, and the excess amounts of trifluoroacetic acid, triisopropylsilane, thioanisole, water, ethanedithiol, etc. are primarily removed, and this procedure is repeated two or more times to obtain a solidified precipitate. Here, the completely deprotected peptide salt is separated and purified using a mixed solvent consisting of water and acetonitrile solvent and reverse-phase high performance liquid chromatography (HPLC). The separated and purified peptide solution is freeze-dried to completely concentrate and dry the peptide, thereby obtaining a solid peptide.

[0052] Another aspect of the present invention provides a polynucleotide encoding an antimicrobial peptide consisting of any of the amino acid sequences set forth in SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof.

[0053] The amino acid sequences, antimicrobial peptides, derivatives and fragments of any of SEQ ID NOs: 5 to 8 are as described above.

[0054] Specifically, the antimicrobial peptide having an amino acid sequence of any one of SEQ ID NO:5 to SEQ ID NO:8 has a sequence of Chemical Formula 1, and the derivative has a sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:4, but is not limited to these.

[0055] Furthermore, the antimicrobial peptide, its derivative, or its fragment is encoded by any sequence selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 16, but is not limited thereto.

[0056] In the present invention, the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8 or derivatives thereof may be encoded by a polynucleotide containing the nucleic acid sequences of SEQ ID NO:9 to SEQ ID NO:16, for example.

[0057] In the present invention, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and refers to a DNA or RNA chain longer than a certain length. The polynucleotide in the present invention encodes an antimicrobial peptide having the amino acid sequence of SEQ ID NO: 5 to SEQ ID NO: 8, or a derivative or fragment thereof, but is not limited thereto.

[0058] The polynucleotide may be any polynucleotide that encodes a peptide having activity of the antimicrobial peptide, a derivative thereof, or a fragment thereof according to the present invention.

[0059] Specifically, the polynucleotide encoding the peptide exhibiting the activity of the antimicrobial peptide, its derivative, or its fragment may contain a base sequence encoding the amino acid represented by Chemical Formula 1, the antimicrobial peptide, its derivative, or its fragment. The polynucleotide may be modified in various ways in the coding region within the scope of not changing the amino acid sequence of the peptide, taking into consideration codon degeneracy or preferred codons in the organism in which the peptide is to be expressed. The polynucleotide may, for example, contain the base sequences of SEQ ID NO: 9 to SEQ ID NO: 16, or may be composed of a base sequence having 80% homology or identity thereto, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and even more specifically 99% or more, but is not limited thereto.

[0060] Furthermore, the polynucleotide of the present invention may be any sequence that encodes the amino acid sequence of SEQ ID NO:5 to SEQ ID NO:8, or a derivative or fragment thereof by hybridizing under stringent conditions with a probe, for example, a complementary sequence to all or a part of the base sequence. The "stringent conditions" refer to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (e.g., J. Sambrook et al.). For example, the conditions include conditions under which polynucleotides having high homology or identity, such as 40% or more, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and even more specifically 99% or more, hybridize with each other, while polynucleotides having lower homology or identity than that do not hybridize with each other, or conditions under which washing is performed once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to those used in normal Southern hybridization, specifically 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0061] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present invention may include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0062] Specifically, polynucleotides having homology or identity can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and may be appropriately adjusted by those skilled in the art depending on the purpose.

[0063] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, and variables are known in the art (see Non-Patent Document 1).

[0064] As used herein, "homology" or "identity" refers to the degree to which two given amino acid or nucleotide sequences are related, and is expressed as a percentage. Homology and identity are often used interchangeably.

[0065] Sequence homology or identity of conserved polynucleotides or peptides may be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize to at least about 50%, 60%, 70%, 80%, or 90% or more of the entire sequence or length under moderate or high stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides having common codons or codons that take into account codon degeneracy in the polynucleotide.

[0066] Whether any two polynucleotide or peptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters as in, for example, Non-Patent Document 2. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 4), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 3) (version 5.0.0 or later), which includes the GCG program package (Non-Patent Document 5), BLASTP, BLASTN, FASTA (Non-Patent Documents 6, 7 and 8). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0067] Homology, similarity or identity of polynucleotides or peptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 9, pp. 1111-1155, 2002. Briefly, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include (1) a binary comparison matrix (identity takes a value of 1 and non-identity takes a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 10, (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps.

[0068] Furthermore, whether any two polynucleotide or peptide sequences have homology, similarity or identity can be confirmed by comparing the sequences in a Southern hybridization experiment under defined stringent conditions, and appropriate hybridization conditions, as defined, are within the skill of the art and can be determined by methods well known to those skilled in the art (e.g., Non-Patent Documents 12 and 13).

[0069] Yet another aspect of the present invention provides a carrier for an external preparation for skin, comprising an antimicrobial peptide consisting of any of the amino acid sequences set forth in SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof.

[0070] The amino acid sequences, antimicrobial peptides, derivatives and fragments of any of SEQ ID NOs: 5 to 8 are as described above.

[0071] The term "carrier" in the present invention means a substance that carries any substance or component including a composition, and is used in combination with a carrier, an impregnating material, a medium, etc. In addition, the composition as an example of the carrier may be applied and delivered to the skin by an application means such as a hand, a puff, a tip, or a brush.

[0072] Furthermore, the carrier is included in cosmetic compositions, pharmaceutical compositions, quasi-drug compositions, etc., but is not limited thereto. For the purposes of the present invention, the carrier includes an antimicrobial peptide consisting of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof.

[0073] In the present invention, the term "external skin preparation" refers to a preparation form to be applied to the skin. In the present invention, the external preparation may be in any form as long as it is a preparation form to be applied to the skin, and the external preparation may be further classified into ointments, creams, lotions, gels, etc., but is not limited thereto.

[0074] Specifically, the ointment is a semi-solid external preparation in which the main ingredient to be applied to the skin is dissolved or dispersed in a base. The base is divided into oily ointments and water-soluble ointments depending on the ingredients. Ointments using an oily base have a skin softening effect, excellent sealing properties, and are suitable for application to wounds, but have the disadvantage of being difficult to remove. Since they have fat-soluble properties similar to those of the skin and a skin softening effect, they are an advantageous dosage form for locally permeating ingredients that are difficult to permeate through the skin.

[0075] The ointment is mainly formed of an oil phase such as oil, and has excellent moisturizing properties and is favorable for the permeation of fat-soluble components, but has the disadvantage of being sticky. The cream is a semi-solid external preparation emulsified into water-in-oil, and is a dosage form in which the viscosity of the ointment is improved by mixing the oil phase and the aqueous phase. In the emulsification process, the oil phase and the aqueous phase are mixed by using a surfactant or through a special process. In addition, the cream is a dosage form that is easier to apply and remove than the ointment, and the lotion is a dosage form that has properties more similar to water than the cream, and is an external preparation in which the main component is dissolved, emulsified, or finely dispersed in an aqueous liquid. The main component, additives, purified water, etc. are used to form a solution, suspension, or emulsion, and the whole is homogenized. The gel is a semi-solid external preparation made of organic molecules with a large molecular weight that have been permeated with a liquid. It may be one in which a gelling agent is dispersed in the aqueous phase, and gelling agents such as carbomers and synthetic polymers can be used. Various types of solvents are used depending on the active ingredient, purpose of use, method of use, etc., and the topical agent of the present invention is not limited to these.

[0076] Yet another aspect of the present invention provides a cosmetic composition comprising an antimicrobial peptide consisting of any of the amino acid sequences set forth in SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof.

[0077] The amino acid sequences, antimicrobial peptides, derivatives and fragments of any of SEQ ID NOs: 5 to 8 are as described above.

[0078] In the present invention, the cosmetic composition may be prepared in a dosage form selected from the group consisting of a solution, an ointment for external use, a cream, a foam, a nutritious lotion, a softening lotion, a perfume, a pack, a softening lotion, a milky lotion, a makeup base, an essence, a soap, a liquid cleanser, a bath additive, a sunscreen cream, a sun oil, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a surfactant-containing cleanser, an oil, a powder foundation, an emulsion foundation, a wax foundation, a patch, and a spray, but is not limited thereto.

[0079] The cosmetic composition of the present invention may further contain one or more cosmetically acceptable carriers that are generally incorporated into skin cosmetics, and may appropriately contain, as typical ingredients, for example, oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, colorants, preservatives, fragrances, etc., but is not limited to these.

[0080] The cosmetically acceptable carrier contained in the cosmetic composition of the present invention varies depending on the formulation of the cosmetic composition.

[0081] When the dosage form of the present invention is an ointment, paste, cream or gel, the carrier component may be, but is not limited to, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. These may be used alone or in combination of two or more.

[0082] When the dosage form of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. are used as carrier components, and particularly when the dosage form is a spray, propellants such as hydrochlorofluorocarbons, propane / butane, or dimethyl ether are further included, but are not limited to these. These may be used alone or in combination of two or more.

[0083] When the dosage form of the present invention is a solution or emulsion, a solvent, a solubilizer, an emulsifier, etc. are used as a carrier component, for example, water, glycerin, ethanol, isopropanol, diethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, etc. are used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerin aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan are used, but are not limited thereto. These may be used alone or in combination of two or more.

[0084] When the dosage form of the present invention is a suspension, the carrier component may be, but is not limited to, a liquid diluent such as water, glycerin, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester, or microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tragacanth. These may be used alone or in combination of two or more.

[0085] When the dosage form of the present invention is a soap, the carrier component may be, but is not limited to, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolyzate, an isethionate salt, a lanolin derivative, an aliphatic alcohol, a vegetable oil, glycerin, a sugar, etc. These may be used alone or in combination of two or more kinds.

[0086] Yet another aspect of the present invention provides a quasi-drug composition comprising an antimicrobial peptide consisting of any of the amino acid sequences set forth in SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof.

[0087] The amino acid sequences, antimicrobial peptides, derivatives and fragments of any of SEQ ID NOs: 5 to 8 are as described above.

[0088] In the present invention, the term "quasi-drug" refers to an article used for the purpose of diagnosing, curing, improving, mitigating, treating or preventing a disease in humans or animals, and which has a milder effect than a drug. For example, according to the Pharmaceutical Affairs Law, quasi-drugs refer to articles other than those used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases in humans and animals, and products that have a mild effect on the human body or that do not have a direct effect.

[0089] The quasi-drug composition of the present invention may be prepared in a dosage form selected from the group consisting of body cleansers, foams, soaps, masks, ointments, creams, lotions, essences, and sprays, but is not limited thereto.

[0090] In the cosmetic composition or quasi-drug composition of the present invention, the antimicrobial peptide consisting of any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, its derivative or its fragment may be contained in an amount of 0.01% by weight to 100.0% by weight, or 0.1% by weight to 10% by weight, based on the total composition.

[0091] Yet another aspect of the present invention provides a pharmaceutical composition for external application to the skin, comprising an antimicrobial peptide consisting of any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof.

[0092] The amino acid sequence of any one of SEQ ID NO:5 to SEQ ID NO:8, the antimicrobial peptide, the derivative, the fragment, and the topical skin preparation are as described above.

[0093] The pharmaceutical composition of the present invention may further contain a pharma- ceutical acceptable carrier in addition to containing an antimicrobial peptide consisting of an amino acid sequence of any of SEQ ID NOs: 5 to 8, a derivative thereof, or a fragment thereof.

[0094] The term "pharmaceutical acceptable" in the present invention means a substance that does not stimulate an organism when administered, does not impair the biological activity and properties of the administered compound, and is commonly used in the pharmaceutical field. The pharmaceutical composition of the present invention is formulated with the carrier and is used as a food, drug, feed additive, drinking water additive, etc.

[0095] The type of the carrier is not particularly limited, and any carrier commonly used in the art can be used. Examples of the carrier include, but are not limited to, saline, sterilized water, Ringer's solution, buffered saline, albumin, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerin, and ethanol. These may be used alone or in combination of two or more, but are not limited to these.

[0096] In addition, the pharmaceutical composition of the present invention may be used by adding other pharma- ceutically acceptable additives, such as excipients, diluents, antioxidants, buffers, bacteriostatic agents, etc., if necessary, and may further include, but is not limited to, fillers, extenders, wetting agents, disintegrants, dispersants, surfactants, binders, lubricants, etc.

[0097] The pharmaceutical composition of the present invention can be formulated into various dosage forms suitable for use as an external agent for the skin.

[0098] Examples of the topical skin preparation include, but are not limited to, aerosols, sprays, cleansers, ointments, powders for application, oils, creams, etc., and any substance that functions as a topical skin preparation may be used.

[0099] In order to formulate the pharmaceutical composition of the present invention for use as an external preparation for skin, a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, an external preparation, etc. may be used. Examples of the non-aqueous solvent and suspension include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and esters such as ethyl oleate.

[0100] More specifically, when the pharmaceutical composition of the present invention is formulated, the pharmaceutical composition of the present invention is mixed with a stabilizer or a buffer in water to form a solution or suspension, which is then formulated into a unit such as an ampoule. Furthermore, when the pharmaceutical composition of the present invention is formulated into an aerosol, a propellant for spraying a water-dispersed concentrate or a wet powder is mixed with an additive, but the additives are not limited thereto.

[0101] Furthermore, when the pharmaceutical composition of the present invention is formulated into an ointment, cream or the like, it is formulated using animal oils, vegetable oils, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. as a carrier, but is not limited to these.

[0102] It is more preferable that the pharmaceutical composition of the present invention is formulated for topical application to the skin and has a dosage form selected from the group consisting of gel, patch, spray, ointment, plaster, lotion, liniment, paste and cataplasm, but is not limited thereto.

[0103] The pharmacologic effective amount of the pharmaceutical composition of the present invention, and the effective dosage for topical preparations, vary depending on the formulation method, administration method, administration time and / or administration route of the pharmaceutical composition, and vary depending on various factors including the type and degree of reaction to be achieved by administration of the pharmaceutical composition, the type, age, weight, general health condition, symptoms and degree of disease, sex, diet, excretion, drugs used simultaneously or at different times in the individual, other components of the composition, and similar factors well known in the medical field, and a person having ordinary knowledge in the art can easily determine and prescribe an effective dosage for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or in several divided doses. Therefore, the dosage does not limit the present invention in any aspect.

[0104] Furthermore, administration in the present invention is as an external preparation, and the dosage of the pharmaceutical composition of the present invention is preferably 1 mg / kg to 1,000 mg / kg per day.

[0105] The administration route and administration method of the pharmaceutical composition of the present invention may be independent of each other, and the method is not particularly limited, and any administration route and administration method can be used as long as the pharmaceutical composition can be delivered to the target site. The pharmaceutical composition may be administered as a skin topical agent. Specifically, the composition is applied or sprayed to the diseased site, but is not limited thereto.

[0106] The pharmaceutical composition of the present invention is preferably administered by coating or spraying, specifically by topical application to areas in need of improving skin condition or areas in need of preventing, improving or treating skin diseases.

[0107] Yet another aspect of the present invention provides the use of the antimicrobial peptide of the present invention, a derivative thereof or a fragment thereof for antimicrobial purposes.

[0108] The term "antibacterial" refers to antimicrobial activity, and is not limited to the type of microorganism, so that the term "antibacterial" is used interchangeably with "antibacterial," "antifungal," and "antibiotic."

[0109] The antimicrobial peptides, derivatives and fragments are as described above.

[0110] Yet another aspect of the present invention provides an antimicrobial method comprising the step of treating a subject with an antimicrobial peptide, derivative or fragment thereof of the present invention.

[0111] The target may be any target requiring antibacterial treatment and treated with an antibacterial peptide, a derivative thereof, or a fragment thereof, and the treatment may be any treatment, such as spraying, coating, etc., that requires antibacterial treatment and that brings the antibacterial peptide, a derivative thereof, or a fragment thereof into contact with the target. The antibacterial peptide, derivative, and fragment are as described above. EXAMPLES

[0112] The present invention will be described in more detail below with reference to examples and experimental examples. However, these examples and experimental examples are merely illustrative of the present invention, and the present invention is not limited to these examples and experimental examples.

[0113] Production Example 1: Preparation of antibacterial peptides In order to confirm the function of the antimicrobial peptides, SEQ ID NO:5 to SEQ ID NO:8 and their derivative peptides were prepared.

[0114] 1-1: Synthesis of peptide of SEQ ID NO:1 The abbreviations and names of the amino acids used in the present invention are as follows: Phe: phenylalanine / Ile: isoleucine / Lys: lysine / Leu: leucine / Arg: arginine / Tyr: tyrosine

[0115] 71.4 mg (0.10 mmol) of 2-chlorotrityl chloride resin (resin loaded at 1.4 mmol / g) purchased from Novabiochem Corporation was weighed out, and the resin was reacted with 7.5 ml of methyl chloride solvent for 5 minutes, after which the methyl chloride was removed.

[0116] Then, Fmoc-Lys(Boc)-OH (281.1 mg, 0.60 mmol) was completely dissolved in 7.5 ml of MC solvent, and then N,N'-diisopropylethylamine (DIPEA) (0.21 ml, 1.2 mmol) was added to the resin. The reaction solution was shaken at room temperature for 12 hours, and then washed four times with 10 ml of DMF solvent.

[0117] Next, 7.5 ml of 20% (w / v) piperidine DMF solution was added and shaken for 15 minutes to completely remove the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group from the substance added to the resin, and the resin was washed four times with 10 ml of DMF solvent (four times with 10 ml each). In this step, the Kaiser test [Non-Patent Document 14] was performed to confirm the presence or absence of deprotection reaction of the Fmoc protecting group.

[0118] Meanwhile, Fmoc-Leu-OH (212.0 mg, 0.60 mmol), hexafluorophosphate benzotriazole tetramethyluronium (HBTU) (227.6 mg, 0.60 mmol), and hydroxybenzotriazole (HOBt) (81.0 mg, 0.6 mmol) were completely dissolved in 7.5 ml of DMF solvent, and then DIPEA (0.21 ml, 1.2 mmol) was added to the resin. The reaction solution was shaken at room temperature for 3 hours, and then washed four times with 10 ml of DMF solvent. At this step, the Kaiser test was performed to confirm the completion of the reaction.

[0119] The peptides were then successively condensed (coupled) in subsequent synthetic cycles. (1) Wash four times with DMF solvent (10 ml) (2) Deprotection using 20% ​​(w / v) piperidine in DMF (7.5 ml) for 15 min (3) Wash four times with DMF solvent (10 ml) (4) Addition of Fmoc-amino acids, HBTU, HOBt, and DIPEA (5) Add HBTU, HOBt, and DIPEA condensation reagents, activate amino acids, and condense for 3 hours. (6) Wash four times with DMF solvent (10 ml)

[0120] (1) to (6) were continuously repeated. Here, the Fmoc-protected amino acids (0.60 mmol) after Fmoc-Leu-OH were added to the resin reaction vessel in the following order for condensation. (i) Fmoc-Tyr(tBu)-OH (ii) Fmoc-Lys(Boc)-OH (iii) Fmoc-Leu-OH (iv) Fmoc-Ile-OH (v) Fmoc-Lys(Boc)-OH (vi) Fmoc-Lys(Boc)-OH (vii) Fmoc-Phe-OH (viii) Fmoc-Leu-OH (ix) Fmoc-Lys(Boc)-OH (x) Fmoc-Lys(Boc)-OH

[0121] After Fmoc-Lys(Boc)-OH condensation (6), the product was finally treated with 20% piperidine in DMF (7.5 ml), and then washed with 10 ml portions of DMF solvent three times, MC solvent three times, and diethyl ether solvent three times.

[0122] Immediately after the completion of these syntheses, the peptide was cleaved from the resin with a mixture (10 ml) of trifluoroacetic acid / triisopropylsilane / water (95:2.5:2.5) over a period of 3 hours. The mixed solution thus obtained was treated with 100 ml of diethyl ether solvent stored in a refrigerator to produce a precipitate. The precipitate thus obtained was centrifuged to completely precipitate, and the first removal was performed with trifluoroacetic acid. The above procedure (the step of adding 100 ml of diethyl ether solvent to wash the precipitate and centrifuging - the work of removing the first removal attempt of trifluoroacetic acid) was repeated twice to obtain a solidified precipitate.

[0123] The precipitate (peptide) was purified by HPLC using a 5%-100% acetonitrile / water gradient solvent system containing 0.001% trifluoroacetic acid over 50 minutes on a C-18 column. The purified fraction was lyophilized to obtain 155 mg of the antimicrobial peptide of SEQ ID NO: 1 as a white powder of trifluoroacetate. SEQ ID NO: 1: H2N-[Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-Lys]-CO2H MS(ESI)m / e,[M+H]+=1550.17;(155mg)

[0124] 1-2: Synthesis of SEQ ID NO:2 to SEQ ID NO:8 The same production process as in Production Example 1-1 was used, but the order of the Fmoc-protected amino acids was changed to prepare the following peptides represented by SEQ ID NO: 2 to SEQ ID NO: 8. SEQ ID NO:2:H2N-[Arg-Arg-Leu-Phe-Arg-Arg-Ile-Leu-Arg-Tyr-Leu-Arg]-CO2H MS(ESI)m / e,[M+H]+=1718.23;(171mg) SEQ ID NO: 3: H2N-[Arg-Phe-Leu-Phe-Arg-Leu-Ile-Leu-Arg-Tyr-Leu-Arg]-CO2H MS(ESI)m / e,[M+H]+=1666.18;(166mg) SEQ ID NO: 4: H2N-[Arg-Arg-Leu-Phe-Arg-Leu-Ile-Leu-Arg-Tyr-Leu-Phe]-CO2H MS(ESI)m / e,[M+H]+=1666.18;(166mg) SEQ ID NO: 5: H2N-[Arg-Lys-Leu-Phe-Lys-Arg-Ile-Leu-Arg-Tyr-Leu-Lys]-CO2H MS(ESI)m / e,[M+H]+=1634.2;(153mg) SEQ ID NO: 6: H2N-[Lys-Arg-Leu-Phe-Arg-Lys-Ile-Leu-Lys-Tyr-Leu-Arg]-CO2H MS(ESI)m / e,[M+H]+=1634.2;(143mg) SEQ ID NO: 7: H2N-[Lys-Arg-Leu-Phe-Lys-Arg-Ile-Leu-Arg-Tyr-Leu-Lys]-CO2H MS(ESI)m / e,[M+H]+=1634.2;(132mg) SEQ ID NO:8:H2N-[Arg-Lys-Leu-Phe-Arg-Lys-Ile-Leu-Lys-Tyr-Leu-Arg]-CO2H MS(ESI)m / e,[M+H]+=1634.2;(172mg) EXAMPLES

[0125] Antibacterial and cytotoxicity evaluation of SEQ ID NO:5 to SEQ ID NO:8 and their derivatives In order to confirm the function of the antimicrobial peptide of the present invention, the antimicrobial activity and cytotoxicity were evaluated. The antimicrobial peptides of the present invention consisting of any one of the amino acid sequences selected from SEQ ID NO:5 to SEQ ID NO:8 and their derivatives, SEQ ID NO:1 to SEQ ID NO:4, were evaluated for their antimicrobial activity and cytotoxicity.

[0126] Specifically, to evaluate the antibacterial activity, the antibacterial activity against Escherichia coli was measured, and the survival rate of the cells was measured after treating the E. coli with the antibacterial peptide, and then the results were compared.

[0127] In addition, in order for an antimicrobial peptide to function, it must only inhibit the survival of microorganisms and minimize cytotoxicity. Therefore, we also evaluated the cytotoxicity of the peptide to human keratinocytes by treating the cells with the antimicrobial peptide and measuring the cell viability.

[0128] Methods for evaluating antibacterial activity The E. coli used in this experiment was provided by the Korea Technical College of Bioscience and Biotechnology (KTCC) and cultured in LB medium. Human keratinocytes (HaCaT) were purchased from the American Type Culture Collection (ATCC, USA) and cultured in a 5% CO2 incubator at 37°C using Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% antibiotic-antimycotic (Gibco, USA).

[0129] To test the antibacterial activity of antibacterial peptides, E. coli was cultured in LB medium for about 8 hours, and then the cultured bacteria was dispensed into a 96-well plate. Antibacterial peptide samples of various concentrations were placed in each well and cultured at 37°C for 24 hours. After that, the absorbance was measured at 600 nm using a microplate reader, and the results were expressed as a percentage (%) compared to a group not treated with the sample.

[0130] Cytotoxicity assessment against human keratinocytes To examine the viability of HaCaT cells against antimicrobial peptides, an MTT assay was performed. HaCaT cells were cultured in a 96-well plate at 2 × 10 5 The cells were cultured at 1000x1000 cells / ml for 24 hours, then treated with samples at various concentrations and cultured for another 24 hours. The culture medium was removed, and the cells were treated with MTT solution (0.5mg / ml in PBS). After 4 hours, the MTT solution was removed, DMSO was added to each well, and formazan was dissolved at 37℃ for 30 minutes. The absorbance was then measured at 570nm using a microplate reader (Molecular Devices Spectra MAX, Sunnyvale, CA, USA). For all experiments, the average of three wells for each concentration was calculated and statistically analyzed, and the relative survival rate (%) was expressed compared to the untreated group.

[0131] 1-1: Antibacterial activity and cytotoxicity evaluation of the antibacterial peptide of sequence number 1 Among the peptides of SEQ ID NO: 1 to SEQ ID NO: 8, the antibacterial activity and cytotoxicity of the antibacterial peptide of SEQ ID NO: 1 were measured preferentially, and the antibacterial activity and cytotoxicity of SEQ ID NO: 1 were compared with those of known antibacterial peptides. The sequences of the antibacterial peptides used in this example are as follows. · Known peptide sequence (BP-100): KKLFK KILKY L

[0132] As a result of the evaluation of antibacterial activity against E. coli, it was confirmed that the peptide of SEQ ID NO: 1 had significantly superior antibacterial activity compared to known peptides (Figure 1A). Moreover, it was confirmed that the peptide of SEQ ID NO: 1 maintained a toxicity level against human keratinocytes at the same level as known peptides (Figure 1B).

[0133] These results confirmed that SEQ ID NO:1 has a significant effect as an antimicrobial peptide compared to known peptides, even though it is a known peptide sequence with one additional amino acid.

[0134] 1-2: Antibacterial activity and cytotoxicity evaluation of the antibacterial peptide of sequence number 2 The antibacterial activity and cytotoxicity of SEQ ID NO:1 and SEQ ID NO:2 were comparatively analyzed.

[0135] Antibacterial activity against E. coli: SEQ ID NO:1 Compared to peptides , SEQ ID NO:2 The peptide was found to have a remarkably excellent antibacterial activity (Fig. 2A). Furthermore, its toxicity against human keratinocytes was SEQ ID NO:2 The peptide SEQ ID NO:1 It was confirmed that the level was maintained at a level equivalent to that of the control (Figure 2B).

[0136] These results confirmed that SEQ ID NO:2 has a significant effect as an antimicrobial peptide.

[0137] 1-3: Antibacterial activity and cytotoxicity evaluation of antibacterial peptides of SEQ ID NO:5 to SEQ ID NO:8 The antibacterial activity and cytotoxicity of SEQ ID NO:5 to SEQ ID NO:8 were comparatively analyzed.

[0138] For the evaluation of antibacterial activity against E. coli, Example 1-1 and Example 1-2 In addition to the experiments performed in the above, MIC (Minimum inhibitory concentration) and IC 50 Measurements were carried out.

[0139] Specifically, the experimental method used was an antibacterial activity effect experiment, and the MIC and IC 50 The values ​​were calculated by evaluating the minimum inhibitory concentration and 50% inhibitory concentration, respectively, using absorbance OD values.

[0140] As a result, it was confirmed that the antibacterial activity of the peptides of SEQ ID NO:5 to SEQ ID NO:8 was superior to that of the peptide of SEQ ID NO:2. In particular, it was confirmed that the antibacterial activity of the peptides of SEQ ID NO:6 and SEQ ID NO:8 was the most superior, followed by the antibacterial activity of the peptides of SEQ ID NO:5 and SEQ ID NO:7 (FIG. 3A and Table 1).

[0141] It was also confirmed that the toxicity levels of the peptides of SEQ ID NO:5 to SEQ ID NO:8 against human keratinocytes were not only significantly lower than known peptides, but also lower than the peptide of SEQ ID NO:2 (FIG. 3B).

[0142] These results demonstrate that peptides of sequence numbers 5 to 8, particularly sequence numbers 6 and 8, have significantly improved antibacterial activity and significantly reduced cytotoxicity compared to peptide of sequence number 1, and are therefore significantly effective as antibacterial peptides.

[0143] [Table 1]

[0144] Comparative Example: Antibacterial and Cytotoxicity Evaluation of Peptides of SEQ ID NO:3 and SEQ ID NO:4 To assess whether peptides of similar size and sequence all have similar antibacterial activity, the antibacterial activity and cytotoxicity of SEQ ID NO:3 and SEQ ID NO:4 were analyzed in comparison to SEQ ID NO:2.

[0145] As a result of the evaluation of antibacterial activity against E. coli, it was confirmed that the peptide of SEQ ID NO:2 had significantly superior antibacterial activity compared to the peptides of SEQ ID NO:3 and SEQ ID NO:4 (FIG. 4A).In addition, it was confirmed that the toxicity level of the peptide of SEQ ID NO:2 against human keratinocytes was significantly lower compared to the peptides of SEQ ID NO:3 and SEQ ID NO:4 (FIG. 4B).

[0146] From these results, although SEQ ID NO:3 and SEQ ID NO:4 are similar to the peptide sequence of SEQ ID NO:2, the antibacterial activity of SEQ ID NO:2 is significantly improved and the cytotoxicity is reduced compared to SEQ ID NO:3 and SEQ ID NO:4, confirming that SEQ ID NO:2 is much more effective as an antibacterial peptide than the peptides of SEQ ID NO:3 and SEQ ID NO:4, and that not all peptides having a size and sequence similar to that of the present invention have excellent antibacterial activity. EXAMPLES

[0147] Antibacterial activity and cytotoxicity evaluation of antibacterial peptides against other bacteria In order to evaluate whether the antimicrobial peptide of the present invention has antimicrobial activity against bacteria other than E. coli, the antimicrobial effect against Escherichia coli, Staphylococcus aureus, Pneumonia aeruginosa, and Candida albicans, which are strains designated by the Ministry of Food and Drug Safety, was confirmed.

[0148] Specifically, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were cultured in LB medium, and Candida was cultured in YM medium, and the cultured bacteria were then dispensed into a 96-well plate. Each well was placed in a sample of antimicrobial peptide at various concentrations and cultured at 37°C for 24 hours. The absorbance was then measured at 600 nm using a microplate reader, and the results were expressed as a percentage (%) compared to a group not treated with the sample.

[0149] As a result, IC50 peptides of SEQ ID NO:1 to SEQ ID NO:8 showed antibacterial activity against four types of bacteria. 50 The values ​​are as follows, and all of the sequence numbers except sequence numbers 3 and 4 showed strong antibacterial activity against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida (Table 2).

[0150] [Table 2]

[0151] In addition, the following experiment was carried out to perform the disk diffusion method on the four types of bacteria.

[0152] Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans were cultured in each medium, and then the unsolidified solid medium was treated with a specified amount of the cultured bacteria to solidify it. The surface of the solidified solid medium was treated with 10 ul of antimicrobial peptide sample, and a dried 3M paper disk was placed on it. After that, it was cultured at 37°C for 16 hours, and the size of the inhibition zone was observed.

[0153] As a result, when the culture medium for each bacteria for the disk diffusion method (Kirby-Bauer) was treated with the peptide of sequence number 6, it was confirmed that a clear inhibition zone with a clear boundary appeared for all bacteria, including E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida (Figure 5).

[0154] Therefore, it was confirmed that the antimicrobial peptides of SEQ ID NO:5 to SEQ ID NO:8 of the present invention and their derivatives exhibit significant antimicrobial activity not only against Escherichia coli, but also against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida. EXAMPLES

[0155] Antibacterial activity of antibacterial peptides by disrupting microbial cell membranes To confirm in detail the effect of the antimicrobial peptide of the present invention on microorganisms, E. coli was treated with the peptide of SEQ ID NO: 6 for 30 minutes, and then the antimicrobial effect was analyzed by SEM (scanning electron microscope).

[0156] Specifically, for SEM analysis, E. coli was cultured in LB medium, and then a predetermined amount was taken and treated with peptide of SEQ ID NO:6 for 30 minutes, washed twice with PBS, and then fixed for 2 hours with a primary fixative of 2% paraformaldehyde in PBS. After fixation, the cells were washed twice with PBS, and then dehydrated with 30, 50, 70, 80, 95, and 100 (%) ethanol for 10 minutes at each step (the final 100% treatment was repeated three times), and the pellets were freeze-dried. Platinum coating was performed using a coating machine, and then analyzed by SEM.

[0157] As a result, when treated with the peptide of SEQ ID NO:6, the bacterial cell membrane of E. coli was totally destroyed, large pores were formed, and the contents inside the cells were observed to be eluted (FIG. 6).

[0158] These results demonstrate that the antimicrobial peptide of the present invention, its derivatives or fragments thereof exhibit excellent antimicrobial activity and reduced cytotoxicity, and therefore have remarkable effectiveness as antimicrobial peptides.

[0159] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be interpreted as including all modifications and alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. An antimicrobial peptide consisting of any of the amino acid sequences set forth in SEQ ID NO:5 to SEQ ID NO:

8.

2. The antimicrobial peptide of claim 1, which inhibits the growth of at least one selected from the group consisting of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans.

3. A polynucleotide encoding the antibacterial peptide described in claim 1 or 2.

4. The polynucleotide according to claim 3, wherein the antimicrobial peptide is encoded by any sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO:

16.

5. A carrier for a topical skin preparation comprising the antibacterial peptide described in claim 1 or 2.

6. A cosmetic composition comprising the antibacterial peptide described in claim 1 or 2.

7. A quasi-drug composition comprising the antibacterial peptide described in claim 1 or 2.

8. A pharmaceutical composition for topical skin application comprising the antibacterial peptide described in claim 1 or 2.

Images