WO2022265195A1 - Novel antibacterial peptide and use thereof - Google Patents

Novel antibacterial peptide and use thereof Download PDF

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Publication number
WO2022265195A1
WO2022265195A1 PCT/KR2022/004429 KR2022004429W WO2022265195A1 WO 2022265195 A1 WO2022265195 A1 WO 2022265195A1 KR 2022004429 W KR2022004429 W KR 2022004429W WO 2022265195 A1 WO2022265195 A1 WO 2022265195A1
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peptide
composition
present
porphyromonas gingivalis
oral
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PCT/KR2022/004429
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French (fr)
Korean (ko)
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박준형
정호용
나타라잔사티시쿠마
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주식회사 쓰리빅스
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Publication of WO2022265195A1 publication Critical patent/WO2022265195A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present invention relates to novel antimicrobial peptides and uses thereof.
  • Antimicrobial peptides are mainly positively charged and have amphipathic properties due to hydrophobic amino acid residues. Due to this property, they bind to negatively charged microbial cell membrane lipids to change the potential of microbial cell membranes or to change the cell membrane potential. It destroys by puncturing or enters the cell membrane and inhibits cell function, thereby exhibiting antibacterial activity.
  • Antimicrobial peptides have proven effective against a wide range of pathogenic bacteria, fungi, parasites and viruses. Despite their excellent efficacy against pathogens, they have been rarely utilized until recently. Overuse or improper administration of classical antibiotics has caused serious problems in antimicrobial resistance. Thus, antimicrobial peptides can be considered as nature's alternative pharmacological resource against antibiotic-resistant microorganisms.
  • Periodontitis is an oral inflammatory disease caused by Gram-negative bacteria, particularly Porphyromonas gingivalis .
  • Treatment of periodontitis includes mechanical treatment such as scaling and antibiotics.
  • problems such as the appearance of antibiotic-resistant strains occur due to improper use of antibiotics. Therefore, there is a need for new therapeutic agents that can solve these problems and effectively treat oral diseases.
  • An object of the present invention is to provide a novel peptide having excellent antibacterial activity and an oral disease treatment composition using the same.
  • the present invention provides an antimicrobial peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
  • the present invention provides a polynucleotide encoding the antimicrobial peptide.
  • the present invention provides a pharmaceutical composition for preventing or treating oral diseases, a quasi-drug composition, and a health functional food composition comprising the antimicrobial peptide as an active ingredient.
  • the novel antibacterial peptide according to the present invention has excellent antibacterial activity against gram-negative bacteria or antibiotic-resistant bacteria related to oral diseases, and by using this, it can improve existing problems and more effectively prevent oral diseases such as tooth decay, periodontitis, and gingivitis, can be improved or cured.
  • MIC minimum inhibitory concentration
  • FIG. 2 is a graph showing the results of a cytotoxicity test according to an embodiment of the present invention.
  • the present inventors have synthesized a novel peptide, and the peptide is Porphyromonas gingivalis ( Porphyromonas gingivalis ), thereby completing the present invention.
  • the present invention provides an antimicrobial peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
  • peptide refers to a linear molecule formed by coupling amino acid residues to each other by a peptide bond.
  • the peptide is a synthetic peptide
  • the method for the synthesis may be a conventional chemical synthesis method of peptides in the art (W. H. Freeman and Co., Proteins; structures and molecular principles (1983)), specifically, a liquid phase solid phase method (Solution Phase Peptide Synthesis), Solid Phase Peptide Syntheses, Fragment Condensation Method, and F-moc or T-BOC Chemical Method, and more preferably, Liquid Phase Solid Phase Method (Merrifield, RB., J. Am. Chem Soc., 85.2149: 196), but is not limited thereto.
  • the peptide may have antibacterial activity against gram-negative bacteria, gram-positive bacteria, or antibiotic-resistant bacteria, preferably against gram-negative bacteria or antibiotic-resistant bacteria, and more preferably against gram-negative bacteria, but may have excellent antimicrobial activity, but is limited thereto It is not.
  • the gram-negative bacteria may be gram-negative bacteria of the genus Porphyromonas , and preferably, Porphyromonas gingivalis ( Porphyromonas gingivalis ), but is not limited thereto.
  • Porphyromonas gingivalis Porphyromonas gingivalis ; P. gingivalis )
  • P. gingivalis is one of several representative causative bacteria that cause oral diseases, and it has been reported that it occurs in the oral cavity where periodontal disease occurs, and is also found in the upper part of the gastrointestinal tract, respiratory tract and colon.
  • the gram-positive bacteria may be all gram-positive bacteria known in the art as gram-positive bacteria including Staphylococcus , Lactobacillus , and Bacillus , but are not limited thereto.
  • the antibiotic-resistant bacteria may be Porphyromonas gingivalis having antibiotic resistance, but is not limited thereto.
  • the antibiotics are aminoglycoside series (aminoglycoside, gentamicin, neomycin, etc.), penicillin series (ampicillin, etc.), sulfonamide series, beta-lactam series (beta-lactam, amoxicillin/clavulanic acid, etc.), chloramphenicol antibiotics of the antibiotic class, erythromycin class, florfenicol class, fosfomycin class, kanamycin class, lincomycin class, methicillin class, quinolone class, streptomycin class, tetracycline class, trimetsoprim class, and vancomycin class. It may be, but is not limited thereto.
  • the present invention provides a polynucleotide encoding the antimicrobial peptide.
  • the present invention provides a pharmaceutical composition for preventing or treating oral diseases comprising the antimicrobial peptide as an active ingredient.
  • the composition is Porphyromonas gingivalis ( Porphyromonas gingivalis ), it can be used as a pharmaceutical composition for the prevention or treatment of oral diseases caused by the strain.
  • the oral disease is various diseases occurring in the oral cavity, and may be one or more selected from the group consisting of tooth decay, periodontitis, and gingivitis, but is not limited thereto, and any disease occurring in the oral cavity may be included regardless of its condition.
  • the pharmaceutical composition according to the present invention can be prepared according to conventional methods in the pharmaceutical field.
  • the pharmaceutical composition may be formulated with an appropriate pharmaceutically acceptable carrier according to the formulation, and, if necessary, further include excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. It can be manufactured by The appropriate carrier and the like do not inhibit the activity and characteristics of the antimicrobial peptide according to the present invention, and may be selected differently depending on the dosage form and formulation.
  • the pharmaceutical composition may be applied in any dosage form, and more specifically, it may be formulated and used in parenteral dosage forms such as oral dosage forms, external preparations, suppositories and sterile injection solutions according to conventional methods.
  • the solid dosage form is in the form of tablets, pills, powders, granules, capsules, etc., and includes at least one excipient such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc. may be prepared by mixing, and lubricants such as magnesium stearate and talc may be included in addition to simple excipients.
  • a liquid carrier such as fatty oil may be further included in addition to the above-mentioned materials.
  • liquid formulations include suspensions, solutions for internal use, emulsions, syrups, etc.
  • various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is.
  • the parenteral formulation may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized formulation, and a suppository.
  • Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.
  • As a base for the suppository witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogeratin, and the like may be used. It is not limited thereto, and all suitable agents known in the art may be used.
  • calcium or vitamin D 3 may be further added to the pharmaceutical composition according to the present invention to enhance therapeutic efficacy.
  • composition according to the present invention can be administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects.
  • the effective dosage level of the pharmaceutical composition depends on the purpose of use, the patient's age, sex, weight and health condition, disease type, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment Duration, combination, or factors including drugs used concurrently and other factors well known in the medical arts may be determined differently. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, may be administered once or several times a day. The dosage is not intended to limit the scope of the present invention in any way.
  • the pharmaceutical composition may be administered to any animal that may develop oral disease, and the animal may include, for example, humans and primates as well as livestock such as cattle, pigs, horses, and dogs.
  • the pharmaceutical composition may be administered by an appropriate administration route according to the formulation form, and may be administered through various oral or parenteral routes as long as it can reach the target tissue.
  • the method of administration is not particularly limited, and is administered by conventional methods, such as, for example, oral, rectal or intravenous, intramuscular, skin application, subcutaneous, intraventricular inhalation, intrauterine dural or intracerebroventricular injection. It can be.
  • the pharmaceutical composition may be used alone for the prevention or treatment of oral diseases, or may be used in combination with surgery or other drug treatment.
  • the present invention provides a quasi-drug composition for preventing or improving oral diseases comprising the antimicrobial peptide as an active ingredient.
  • the composition is Porphyromonas gingivalis ( Porphyromonas gingivalis ), it can be used as a quasi-drug composition for preventing or improving oral diseases caused by the strain.
  • the oral disease may be one or more selected from the group consisting of tooth decay, periodontitis and gingivitis, but is not limited thereto.
  • quasi-drugs means textiles, rubber products or similar products used for the purpose of treating, alleviating, treating or preventing human or animal diseases;
  • Non-machines and similar items items corresponding to one of preparations used for sterilization, insecticide, and similar purposes to prevent infection, and the quasi-drug composition according to the present invention may preferably include a quasi-drug for oral use .
  • the quasi-drug composition may further include ingredients commonly used in oral quasi-drug compositions in addition to the active ingredient, for example, abrasives, wetting agents, binders, foaming agents, sweeteners, preservatives, active pharmaceutical ingredients, flavoring agents, coloring agents, solvents , brighteners, solubilizers or pH adjusting agents.
  • abrasives for example, wetting agents, binders, foaming agents, sweeteners, preservatives, active pharmaceutical ingredients, flavoring agents, coloring agents, solvents , brighteners, solubilizers or pH adjusting agents.
  • the quasi-drug composition may be prepared in any dosage form conventionally prepared in the art, and for example, toothpaste, mouthwash, mouthwash, gum, candy, oral spray, oral ointment, oral varnish, mouthwash, and It may have a formulation such as gum massage cream, but is not limited thereto.
  • the quasi-drug composition may be used alone or overlapping, or overlapping with other oral quasi-drug compositions other than the present invention.
  • the present invention provides a health functional food composition for preventing or improving oral diseases comprising the antimicrobial peptide as an active ingredient.
  • the composition is Porphyromonas gingivalis ( Porphyromonas gingivalis ), it can be used as a health functional food composition for preventing or improving oral diseases caused by the strain.
  • the oral disease may be one or more selected from the group consisting of tooth decay, periodontitis and gingivitis, but is not limited thereto.
  • the health functional food may be prepared in powder, granule, tablet, capsule, syrup or beverage for the purpose of preventing or improving oral diseases, and the form that the food can take It is not limited and may include all foods in a conventional sense.
  • beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meat and their processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ), etc.
  • food used as feed for animals may also be included.
  • the health functional food composition may be prepared by further including food additives (food additives) commonly used in the art and appropriate other auxiliary ingredients.
  • food additives food additives commonly used in the art and appropriate other auxiliary ingredients.
  • the suitability as the food additive can be determined according to the standards and standards for the item in accordance with the general rules of the Food Additive Code and general test methods approved by the Ministry of Food and Drug Safety, unless otherwise specified.
  • Examples of the items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations; and the like.
  • the other auxiliary ingredients include, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, colorants, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents, etc. may additionally contain.
  • natural carbohydrate monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol may be used.
  • natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame may be used.
  • the effective dose of the antibacterial peptide contained in the health functional food according to the present invention may be appropriately adjusted according to the purpose of use, such as preventing or improving oral diseases.
  • the composition has the advantage of using food as a raw material and has no side effects that can occur when taking general medicines for a long time, and has excellent portability, so it can be taken as an adjuvant for preventing or improving oral diseases.
  • the peptide was synthesized by a solid phase method using Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group for the N ⁇ -amino group of an amino acid.
  • Rink Amide MBHA-Resin was used as a starting material for peptides with -NH 2 carboxyl terminals
  • Fmoc-amino acid-Wang Resin was used for peptides with carboxyl terminals -OH type as starting materials.
  • the elongation of the peptide chain by Fmoc-amino acid coupling was performed by the DCC (N-hydroxybenzotriazole (HOBt)-dicyclo-hexylcar-bodiimide) method.
  • DCC N-hydroxybenzotriazole (HOBt)-dicyclo-hexylcar-bodiimide
  • the Fmoc group was removed with a 20% piperidine/N-methylpyrrolidone (NMP) solution, washed several times with NMP and dichloromethane (DCM), and then nitrogen dried with gas
  • the molecular weight was calculated based on the sequence of the synthesized peptide, and the exact molecular weight was measured using a matrix assisted laser desorption ionization mass spectrometer (MALDI).
  • MALDI matrix assisted laser desorption ionization mass spectrometer
  • Example 2 Porphyromonas gingivalis ( Porphyromonas gingivalis ) Confirmation of antibacterial activity against
  • Example 2 After dissolving the peptide prepared in Example 1 in distilled water at a concentration of 1 mg/mL, a broth microdilution method assay was used to confirm the antibacterial activity.
  • Porphyromonas gingivalis ( Strain number: KCTC5352 ) was cultured for 24 hours, and then a certain amount of the culture solution was added to a new BHI (Brain Heart Infusion) liquid medium and cotton red blood cells. After diluting to 0.1 at OD600 with a spectrophotometer in a medium supplemented with 5%, the peptide and positive control, oxytetracycline, were treated at a concentration of 50 ⁇ g/mL to 100 ⁇ g/mL and then treated for 24 hours. cultured. After culturing, absorbance was measured at OD600.
  • BHI Brain Heart Infusion
  • the human keratin cell line HaCat cell line is a human epidermal cell, and was identified by culturing in an environment of 37° C., 5% CO 2 using DMEM medium containing 1% penicillin-streptomycin and 10% FBS. .
  • HaCaT was seeded at 3 ⁇ 10 5 /well using DMEM medium supplemented with 10% FBS in a 6-well cell culture plate and cultured for one day. Thereafter, the cells were washed with a serum-free medium, treated with the peptide at each concentration in the serum-free medium, and then cultured for 24 hours, and cell death was confirmed.

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Abstract

The present invention relates to a novel antibacterial peptide and a use thereof, and, more specifically, provides an antibacterial peptide made of an amino acid sequence represented by SEQ ID NO: 1, and a pharmaceutical composition, a quasi-drug composition, and a health functional food composition for preventing or treating oral diseases, comprising the antibacterial peptide as an active ingredient. The antibacterial peptide has excellent antibacterial activity against gram-negative bacteria or antibiotic-resistant bacteria related to oral diseases, and can thus be utilized to prevent, alleviate, or treat oral diseases more effectively.

Description

신규한 항균 펩타이드 및 이의 용도Novel antimicrobial peptides and uses thereof
본 발명은 신규한 항균 펩타이드 및 이의 용도에 관한 것이다.The present invention relates to novel antimicrobial peptides and uses thereof.
인간 및 생명체는 외부와 내부에서 다양한 미생물에 노출되어 있어 스스로를 보호할 수 있는 선천성 면역체계를 가지고 있으며 이 중 하나가 항균 펩타이드로 약 2~50개의 아미노산으로 구성된 작은 크기의 펩타이드로서 생물체가 병원균에 감염되었을 때 1차 방어물질로 작용한다. 항균 펩타이드는 주로 양전하(cationic)를 띠며 소수성(hydrophobic) 아미노산 잔기에 의해 양친매성(amphipathic) 특성을 가지는데 이러한 특성으로 음전하를 띠는 미생물 세포막 지질과 결합하여 미생물의 세포막의 전위를 변화시키거나 세포막에 구멍을 내어 파괴하거나 세포막내로 들어가 세포 기능을 저해함으로 항균 활성을 나타내게 된다. 항균 펩타이드는 광범위한 병원성 박테리아, 곰팡이, 기생충 및 바이러스에 대해 효과적인 것으로 입증되었다. 병원균에 대해 탁월한 효능에도 불구하고, 이들은 최근까지 거의 활용되지 않았다. 고전적 항생제의 남용 또는 부적절한 투여는 항균 저항성에 심각한 문제를 일으켰다. 따라서, 항균 펩타이드는 항생제 내성 미생물에 맞선 자연의 대체 약리학적 자원으로 간주될 수 있다.Humans and living organisms have an innate immune system that can protect themselves from exposure to various microorganisms both externally and internally. One of these is an antibacterial peptide, a small-sized peptide composed of about 2 to 50 amino acids, which protects organisms from pathogens. It acts as the first line of defense when infected. Antimicrobial peptides are mainly positively charged and have amphipathic properties due to hydrophobic amino acid residues. Due to this property, they bind to negatively charged microbial cell membrane lipids to change the potential of microbial cell membranes or to change the cell membrane potential. It destroys by puncturing or enters the cell membrane and inhibits cell function, thereby exhibiting antibacterial activity. Antimicrobial peptides have proven effective against a wide range of pathogenic bacteria, fungi, parasites and viruses. Despite their excellent efficacy against pathogens, they have been rarely utilized until recently. Overuse or improper administration of classical antibiotics has caused serious problems in antimicrobial resistance. Thus, antimicrobial peptides can be considered as nature's alternative pharmacological resource against antibiotic-resistant microorganisms.
구강 점막(Oral mucosa)은 다양한 미생물의 틈새로, 미생물 군의 변화는 충치(dental caries), 치주염(periodontitis) 및 치은염(gingivitis)과 같은 질병으로 이어진다. 치주염은 그람-음성 박테리아, 특히 포르피로모나스 진지발리스(Porphyromonas gingivalis)에 의한 구강 염증 질환이다. 치주염의 치료는 스켈링 및 항생제와 같은 기계적 치료가 포함된다. 그러나 항생제의 부적절한 사용으로 항생제 내성 균주가 출현하는 등의 문제가 발생하는 바, 이러한 문제점을 해결하고 구강 질환을 효과적으로 치료할 수 있는 새로운 치료제가 필요한 실정이다.Oral mucosa is a niche for various microorganisms, and changes in the microbial community lead to diseases such as dental caries, periodontitis and gingivitis. Periodontitis is an oral inflammatory disease caused by Gram-negative bacteria, particularly Porphyromonas gingivalis . Treatment of periodontitis includes mechanical treatment such as scaling and antibiotics. However, problems such as the appearance of antibiotic-resistant strains occur due to improper use of antibiotics. Therefore, there is a need for new therapeutic agents that can solve these problems and effectively treat oral diseases.
본 발명의 목적은 항균 활성이 우수한 신규 펩타이드 및 이를 이용한 구강 질환 치료 조성물을 제공하는 데에 있다.An object of the present invention is to provide a novel peptide having excellent antibacterial activity and an oral disease treatment composition using the same.
상기의 목적을 달성하기 위하여, 본 발명은 서열번호 1로 표시되는 아미노산 서열로 이루어진 항균 펩타이드를 제공한다.In order to achieve the above object, the present invention provides an antimicrobial peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
본 발명은 상기 항균 펩타이드를 코딩하는 폴리뉴클레오티드를 제공한다.The present invention provides a polynucleotide encoding the antimicrobial peptide.
또한, 본 발명은 상기 항균 펩타이드를 유효성분으로 포함하는 구강 질환 예방 또는 치료용 약학 조성물, 의약외품 조성물, 및 건강기능식품 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating oral diseases, a quasi-drug composition, and a health functional food composition comprising the antimicrobial peptide as an active ingredient.
본 발명에 따른 신규한 항균 펩타이드는 구강 질환과 관련된 그람 음성균이나 항생제 내성균에 대해 우수한 항균 활성을 가지는 바, 이를 활용하여 기존의 문제점을 개선하고 보다 효과적으로 충치, 치주염, 치은염 등의 구강 질환을 예방, 개선 또는 치료할 수 있다.The novel antibacterial peptide according to the present invention has excellent antibacterial activity against gram-negative bacteria or antibiotic-resistant bacteria related to oral diseases, and by using this, it can improve existing problems and more effectively prevent oral diseases such as tooth decay, periodontitis, and gingivitis, can be improved or cured.
도 1은 본 발명의 일 실시예에 따른 포르피로모나스 진지발리스(Porphyromonas gingivalis)에 대한 최소억제농도(MICminimum inhibitory concentration, MIC)를 측정한 결과 그래프이다.1 is a graph of the results of measuring the minimum inhibitory concentration (MIC) for Porphyromonas gingivalis according to an embodiment of the present invention.
도 2는 본 발명의 일 실시예에 따른 세포 독성 실험 결과 그래프이다.2 is a graph showing the results of a cytotoxicity test according to an embodiment of the present invention.
이하, 본 발명을 상세하게 설명하기로 한다.Hereinafter, the present invention will be described in detail.
본 발명자는 신규한 펩타이드를 합성하였고, 상기 펩타이드가 포르피로모나스 진지발리스(Porphyromonas gingivalis)에 대해 항균 활성을 나타냄을 확인함으로써, 본 발명을 완성하였다.The present inventors have synthesized a novel peptide, and the peptide is Porphyromonas gingivalis ( Porphyromonas gingivalis ), thereby completing the present invention.
이에, 본 발명은 서열번호 1로 표시되는 아미노산 서열로 이루어진 항균 펩타이드를 제공한다.Thus, the present invention provides an antimicrobial peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
- 서열번호 1 : RVLTHVFKCKLKLR- SEQ ID NO: 1: RVLTHVFKCKLKLR
본 명세서에 있어서, "펩타이드"는 펩타이드 결합에 의해 아미노산 잔기들이 서로 결합되어 형성된 선형의 분자를 의미한다.In the present specification, "peptide" refers to a linear molecule formed by coupling amino acid residues to each other by a peptide bond.
상기 펩타이드는 합성 펩타이드로서, 상기 합성을 위한 방법은 당업계의 통상적인 펩타이드의 화학적 합성 방법(W. H. Freeman and Co., Proteins; structures and molecular principles (1983))일 수 있으며, 구체적으로는 액상 고상법(Solution Phase Peptide synthesis), 고상 고상법(Solidphase peptide syntheses), 단편 응축법 및 F-moc 또는 T-BOC 화학법일 수 있으며, 보다 바람직하게는 액상 고상법(Merrifield, RB., J. Am. Chem. Soc., 85.2149: 196)으로 합성할 수 있으나, 이에 제한되는 것은 아니다.The peptide is a synthetic peptide, and the method for the synthesis may be a conventional chemical synthesis method of peptides in the art (W. H. Freeman and Co., Proteins; structures and molecular principles (1983)), specifically, a liquid phase solid phase method (Solution Phase Peptide Synthesis), Solid Phase Peptide Syntheses, Fragment Condensation Method, and F-moc or T-BOC Chemical Method, and more preferably, Liquid Phase Solid Phase Method (Merrifield, RB., J. Am. Chem Soc., 85.2149: 196), but is not limited thereto.
상기 펩타이드는 그람 음성균, 그람 양성균 또는 항생제 내성균에 대해 항균 활성을 가질 수 있으며, 바람직하게는 그람 음성균 또는 항생제 내성균에 대해, 보다 바람직하게는 그람 음성균에 대해 우수한 항균 활성을 가질 수 있으나, 이에 제한되는 것은 아니다.The peptide may have antibacterial activity against gram-negative bacteria, gram-positive bacteria, or antibiotic-resistant bacteria, preferably against gram-negative bacteria or antibiotic-resistant bacteria, and more preferably against gram-negative bacteria, but may have excellent antimicrobial activity, but is limited thereto It is not.
상기 그람 음성균은 포르피로모나스 속(Porphyromonas) 그람 음성균일 수 있으며, 바람직하게는, 포르피로모나스 진지발리스(Porphyromonas gingivalis)일 수 있으나, 이에 제한되는 것은 아니다.The gram-negative bacteria may be gram-negative bacteria of the genus Porphyromonas , and preferably, Porphyromonas gingivalis ( Porphyromonas gingivalis ), but is not limited thereto.
본 명세서에서, "포르피로모나스 진지발리스(Porphyromonas gingivalis; P.gingivalis)"는 구강질환을 일으키는 몇 가지 대표적인 원인균 중 하나로, 치주질환이 발생한 구강 내에서 발생되며, 그 외에도 위장관 상부, 호흡기 및 결장에서도 발견되는 것으로 보고되었다.In the present specification, "Porphyromonas gingivalis ( Porphyromonas gingivalis ; P. gingivalis )" is one of several representative causative bacteria that cause oral diseases, and it has been reported that it occurs in the oral cavity where periodontal disease occurs, and is also found in the upper part of the gastrointestinal tract, respiratory tract and colon.
상기 그람 양성균은 스타필로코커스 속(Staphylococcus), 락토바실러스 속(Lactobacillus) 및 바실러스 속(Bacillus)을 포함하는 그람 양성균으로 당업계에 공지된 모든 그람 양성균일 수 있으나, 이에 제한되는 것은 아니다.The gram-positive bacteria may be all gram-positive bacteria known in the art as gram-positive bacteria including Staphylococcus , Lactobacillus , and Bacillus , but are not limited thereto.
상기 항생제 내성균은 항생제 내성을 갖는 포르피로모나스 진지발리스(Porphyromonas gingivalis)일 수 있으나, 이에 제한되는 것은 아니다.The antibiotic-resistant bacteria may be Porphyromonas gingivalis having antibiotic resistance, but is not limited thereto.
상기 항생제는 아미노글리코사이드 계열 (아미노글리코사이드, 겐타마이신, 네오마이신 등), 페니실린 계열 (앰피실린 등), 술폰아미드 계열, 베타-락탐 계열 (베타-락탐, 아목시실린/클라불란산 등), 클로람페니콜 계열, 에리트로마이신 계열, 플로르페니콜 계열, 포스포마이신 계열, 카나마이신 계열, 린코마이신 계열, 메티실린 계열, 퀴놀론 계열, 스트렙토마이신 계열, 테트라사이클린 계열, 트리메소프림 계열 및 반코마이신 계열의 항생제를 포함할 수 있으나, 이에 제한되는 것은 아니다.The antibiotics are aminoglycoside series (aminoglycoside, gentamicin, neomycin, etc.), penicillin series (ampicillin, etc.), sulfonamide series, beta-lactam series (beta-lactam, amoxicillin/clavulanic acid, etc.), chloramphenicol antibiotics of the antibiotic class, erythromycin class, florfenicol class, fosfomycin class, kanamycin class, lincomycin class, methicillin class, quinolone class, streptomycin class, tetracycline class, trimetsoprim class, and vancomycin class. It may be, but is not limited thereto.
본 발명은 상기 항균 펩타이드를 코딩하는 폴리뉴클레오티드를 제공한다.The present invention provides a polynucleotide encoding the antimicrobial peptide.
본 발명은 상기 항균 펩타이드를 유효성분으로 포함하는 구강 질환 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating oral diseases comprising the antimicrobial peptide as an active ingredient.
상기 조성물은 포르피로모나스 진지발리스(Porphyromonas gingivalis)에 대해 우수한 항균 활성을 가지는 바, 상기 균주에 의한 구강 질환의 예방 또는 치료를 위한 약학 조성물로 활용할 수 있다.The composition is Porphyromonas gingivalis ( Porphyromonas gingivalis ), it can be used as a pharmaceutical composition for the prevention or treatment of oral diseases caused by the strain.
상기 구강 질환은 구강영역에서 발생하는 여러 가지 질환으로, 충치, 치주염 및 치은염으로 이루어진 군에서 선택되는 하나 이상일 수 있으나 이에 제한되지 않고, 구강에 발생하는 질환이라면 그 병증에 관계없이 모두 포함될 수 있다.The oral disease is various diseases occurring in the oral cavity, and may be one or more selected from the group consisting of tooth decay, periodontitis, and gingivitis, but is not limited thereto, and any disease occurring in the oral cavity may be included regardless of its condition.
본 발명에 따른 약학 조성물은 약학적 분야의 통상적인 방법에 따라 제조될 수 있다. 상기 약학 조성물은 상기 유효성분 이외에 제형에 따라 약학적으로 허용가능한 적절한 담체와 배합될 수 있고, 필요에 따라, 부형제, 희석제, 분산제, 유화제, 완충제, 안정제, 결합제, 붕해제, 용제 등을 더 포함하여 제조될 수 있다. 상기 적절한 담체 등은 본 발명에 따른 항균 펩타이드의 활성 및 특성을 저해하지 않는 것으로, 투여 형태 및 제형에 따라 달리 선택될 수 있다.The pharmaceutical composition according to the present invention can be prepared according to conventional methods in the pharmaceutical field. In addition to the active ingredient, the pharmaceutical composition may be formulated with an appropriate pharmaceutically acceptable carrier according to the formulation, and, if necessary, further include excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. It can be manufactured by The appropriate carrier and the like do not inhibit the activity and characteristics of the antimicrobial peptide according to the present invention, and may be selected differently depending on the dosage form and formulation.
상기 약학 조성물은 어떠한 제형으로도 적용될 수 있고, 보다 상세하게는 통상의 방법에 따라 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 비경구형 제형으로 제형화하여 사용될 수 있다.The pharmaceutical composition may be applied in any dosage form, and more specifically, it may be formulated and used in parenteral dosage forms such as oral dosage forms, external preparations, suppositories and sterile injection solutions according to conventional methods.
상기 경구형 제형 중 고형 제형은 정제, 환제, 산제, 과립제, 캡슐제 등의 형태로, 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로스, 락토오스, 솔비톨, 만니톨, 셀룰로오스, 젤라틴 등을 섞어 조제할 수 있고, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 포함될 수 있다. 또한, 캡술제형의 경우 상기 언급한 물질 외에도 지방유와 같은 액체 담체를 더 포함할 수 있다.Among the oral dosage forms, the solid dosage form is in the form of tablets, pills, powders, granules, capsules, etc., and includes at least one excipient such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc. may be prepared by mixing, and lubricants such as magnesium stearate and talc may be included in addition to simple excipients. In addition, in the case of a capsule formulation, a liquid carrier such as fatty oil may be further included in addition to the above-mentioned materials.
상기 경구형 제형 중 액상 제형은 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Among the oral formulations, liquid formulations include suspensions, solutions for internal use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is.
상기 비경구 제형은 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함될 수 있다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다. 이에 제한되지 않고, 당해 기술 분야에 알려진 적합한 제제를 모두 사용 가능하다.The parenteral formulation may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized formulation, and a suppository. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogeratin, and the like may be used. It is not limited thereto, and all suitable agents known in the art may be used.
또한, 본 발명에 따른 약학 조성물은 치료 효능의 증진을 위해 칼슘이나 비타민 D3 등을 더 첨가할 수 있다.In addition, calcium or vitamin D 3 may be further added to the pharmaceutical composition according to the present invention to enhance therapeutic efficacy.
본 발명에 따른 약학 조성물은 약학적으로 유효한 양으로 투여될 수 있다. The pharmaceutical composition according to the present invention can be administered in a pharmaceutically effective amount.
본 명세서에서, "약학적으로 유효한 양"이란, 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미한다.In the present specification, "pharmaceutically effective amount" means an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects.
상기 약학 조성물의 유효 용량 수준은 사용 목적, 환자의 연령, 성별, 체중 및 건강 상태, 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 달리 결정될 수 있다. 예를 들어, 일정하지는 않지만 일반적으로 0.001 내지 100mg/kg으로, 바람직하게는 0.01 내지 10mg/kg을 일일 1회 내지 수회 투여될 수 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The effective dosage level of the pharmaceutical composition depends on the purpose of use, the patient's age, sex, weight and health condition, disease type, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment Duration, combination, or factors including drugs used concurrently and other factors well known in the medical arts may be determined differently. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, may be administered once or several times a day. The dosage is not intended to limit the scope of the present invention in any way.
상기 약학 조성물은 구강 질환이 발생할 수 있는 임의의 동물에 투여할 수 있고, 상기 동물은 예를 들어, 인간 및 영장류뿐만 아니라 소, 돼지, 말, 개 등의 가축 등을 포함할 수 있다.The pharmaceutical composition may be administered to any animal that may develop oral disease, and the animal may include, for example, humans and primates as well as livestock such as cattle, pigs, horses, and dogs.
상기 약학 조성물은 제제 형태에 따른 적당한 투여 경로로 투여될 수 있고, 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있다. 투여 방법은 특히 한정할 필요 없이, 예를 들면, 경구, 직장 또는 정맥, 근육, 피부 도포, 피하, 호흡기내 흡입, 자궁내 경막 또는 뇌혈관내(intracere-broventricular) 주사 등의 통상적인 방법으로 투여될 수 있다.The pharmaceutical composition may be administered by an appropriate administration route according to the formulation form, and may be administered through various oral or parenteral routes as long as it can reach the target tissue. The method of administration is not particularly limited, and is administered by conventional methods, such as, for example, oral, rectal or intravenous, intramuscular, skin application, subcutaneous, intraventricular inhalation, intrauterine dural or intracerebroventricular injection. It can be.
상기 약학 조성물은 구강 질환 예방 또는 치료를 위하여 단독으로 사용될 수 있고, 수술 또는 다른 약물 치료 등과 병용하여 사용될 수 있다.The pharmaceutical composition may be used alone for the prevention or treatment of oral diseases, or may be used in combination with surgery or other drug treatment.
본 발명은 상기 항균 펩타이드를 유효성분으로 포함하는 구강 질환 예방 또는 개선용 의약외품 조성물을 제공한다.The present invention provides a quasi-drug composition for preventing or improving oral diseases comprising the antimicrobial peptide as an active ingredient.
상기 조성물은 포르피로모나스 진지발리스(Porphyromonas gingivalis)에 대해 우수한 항균 활성을 가지는 바, 상기 균주에 의한 구강 질환의 예방 또는 개선을 위한 의약외품 조성물로 활용할 수 있다.The composition is Porphyromonas gingivalis ( Porphyromonas gingivalis ), it can be used as a quasi-drug composition for preventing or improving oral diseases caused by the strain.
상기 구강 질환은 충치, 치주염 및 치은염으로 이루어진 군에서 선택되는 하나 이상일 수 있으나, 이에 제한되는 것은 아니다.The oral disease may be one or more selected from the group consisting of tooth decay, periodontitis and gingivitis, but is not limited thereto.
본 명세서에서, "의약외품"이란 사람이나 동물의 질병을 치료, 경감, 처치 또는 예방할 목적으로 사용되는 섬유, 고무제품 또는 이와 유사한 것, 인체에 대한 작용이 약하거나 인체에 직접 작용하지 아니하며, 기구 또는 기계가 아닌 것과 이와 유사한 것, 감염형 예방을 위하여 살균, 살충 및 이와 유사한 용도로 사용되는 제제 중 하나에 해당하는 물품으로서, 본 발명에 따른 의약외품 조성물은 바람직하게는 구강용 의약외품을 포함할 수 있다.In this specification, “quasi-drugs” means textiles, rubber products or similar products used for the purpose of treating, alleviating, treating or preventing human or animal diseases; Non-machines and similar items, items corresponding to one of preparations used for sterilization, insecticide, and similar purposes to prevent infection, and the quasi-drug composition according to the present invention may preferably include a quasi-drug for oral use .
상기 의약외품 조성물은 상기 유효성분 이외에 구강용 의약외품 조성물에 통상적으로 이용되는 성분들을 더 포함할 수 있고, 예를 들어, 연마제, 습윤제, 결합제, 기포제, 감미제, 방부제, 약효성분, 향미제, 색소, 용제, 증백제, 가용화제 또는 pH 조정제를 포함할 수 있다.The quasi-drug composition may further include ingredients commonly used in oral quasi-drug compositions in addition to the active ingredient, for example, abrasives, wetting agents, binders, foaming agents, sweeteners, preservatives, active pharmaceutical ingredients, flavoring agents, coloring agents, solvents , brighteners, solubilizers or pH adjusting agents.
상기 의약외품 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 치약, 구강세정제, 구강청정제, 껌, 캔디류, 구강스프레이, 구강용 연고제, 구강용 바니쉬, 구강양치액 및 잇몸 마사지 크림 등의 제형을 가질 수 있으나, 이에 제한되는 것은 아니다.The quasi-drug composition may be prepared in any dosage form conventionally prepared in the art, and for example, toothpaste, mouthwash, mouthwash, gum, candy, oral spray, oral ointment, oral varnish, mouthwash, and It may have a formulation such as gum massage cream, but is not limited thereto.
상기 의약외품 조성물은 단독 또는 중복하여 사용하거나, 본 발명 이외의 다른 구강용 의약외품 조성물과 중복하여 사용할 수 있다.The quasi-drug composition may be used alone or overlapping, or overlapping with other oral quasi-drug compositions other than the present invention.
또한, 본 발명은 상기 항균 펩타이드를 유효성분으로 포함하는 구강 질환 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving oral diseases comprising the antimicrobial peptide as an active ingredient.
상기 조성물은 포르피로모나스 진지발리스(Porphyromonas gingivalis)에 대해 우수한 항균 활성을 가지는 바, 상기 균주에 의한 구강 질환의 예방 또는 개선을 위한 건강기능식품 조성물로 활용할 수 있다.The composition is Porphyromonas gingivalis ( Porphyromonas gingivalis ), it can be used as a health functional food composition for preventing or improving oral diseases caused by the strain.
상기 구강 질환은 충치, 치주염 및 치은염으로 이루어진 군에서 선택되는 하나 이상일 수 있으나, 이에 제한되는 것은 아니다.The oral disease may be one or more selected from the group consisting of tooth decay, periodontitis and gingivitis, but is not limited thereto.
본 발명에 따른 건강기능식품 조성물에 있어서, 상기 건강기능식품은 구강 질환 예방 또는 개선의 목적으로 분말, 과립, 정제, 캡슐, 시럽 또는 음료 등으로 제조될 수 있고, 상기 식품이 취할 수 있는 형태에는 제한이 없으며, 통상적인 의미의 식품을 모두 포함할 수 있다. 예를 들어, 음료 및 각종 드링크, 과실 및 그의 가공식품(과일통조림, 잼 등), 어류, 육류 및 그 가공식품(햄, 베이컨 등), 빵류 및 면류, 쿠키 및 스낵류, 유제품(버터, 치즈 등) 등이 가능하며, 통상적인 의미에서의 기능성 식품을 모두 포함할 수 있다. 또한, 동물을 위한 사료로 이용되는 식품도 포함할 수 있다.In the health functional food composition according to the present invention, the health functional food may be prepared in powder, granule, tablet, capsule, syrup or beverage for the purpose of preventing or improving oral diseases, and the form that the food can take It is not limited and may include all foods in a conventional sense. For example, beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meat and their processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ), etc., and may include all functional foods in a conventional sense. In addition, food used as feed for animals may also be included.
상기 건강기능식품 조성물은 당업계에서 통상적으로 사용되는 식품학적으로 허용 가능한 식품 첨가제(식품 첨가물) 및 적절한 기타 보조 성분을 더 포함하여 제조될 수 있다. The health functional food composition may be prepared by further including food additives (food additives) commonly used in the art and appropriate other auxiliary ingredients.
상기 식품 첨가물로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전처에 승인된 식품첨가물공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정할 수 있다. 상기 '식품첨가물공전'에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼슘, 니코틴산, 계피산 등의 화학적 합성물; 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물; L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료 제제, 타르색소제제 등의 혼합 제제류 등을 들 수 있다. The suitability as the food additive can be determined according to the standards and standards for the item in accordance with the general rules of the Food Additive Code and general test methods approved by the Ministry of Food and Drug Safety, unless otherwise specified. Examples of the items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations; and the like.
상기 기타 보조 성분은 예를 들어, 향미제, 천연 탄수화물, 감미제, 비타민, 전해질, 착색제, 펙트산, 알긴산, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산화제 등을 추가로 함유할 수 있다. 특히, 상기 천연 탄수화물로는 포도당, 과당과 같은 모노사카라이드, 말토스, 수크로오스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜을 사용할 수 있으며, 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다.The other auxiliary ingredients include, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, colorants, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents, etc. may additionally contain. In particular, as the natural carbohydrate, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol may be used. As the sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame may be used.
본 발명에 따른 건강기능식품에 함유된 항균 펩타이드의 유효용량은 구강 질환 예방 또는 개선 등 그 사용 목적에 따라 적절하게 조절될 수 있다. 상기 조성물은 식품을 원료로 하여 일반 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나, 구강 질환 예방 또는 개선을 위한 보조제로 섭취될 수 있다.The effective dose of the antibacterial peptide contained in the health functional food according to the present invention may be appropriately adjusted according to the purpose of use, such as preventing or improving oral diseases. The composition has the advantage of using food as a raw material and has no side effects that can occur when taking general medicines for a long time, and has excellent portability, so it can be taken as an adjuvant for preventing or improving oral diseases.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
<실시예 1> 신규한 펩타이드의 제조<Example 1> Preparation of novel peptides
신규한 펩타이드를 합성하기 위하여, 본 발명자들은 Fmoc 아미노기 보호용 기를 이용한 메리필드(Merrifield)의 액상 고상법 (Merrifield, RB., J. Am. Chem. Soc., 85, 2149, 1963)을 사용하여 펩타이드를 제조하였다.In order to synthesize a novel peptide, the present inventors used Merrifield's liquid phase solid phase method (Merrifield, RB., J. Am. Chem. Soc., 85, 2149, 1963) using the Fmoc amino group protecting group to synthesize peptides. was manufactured.
상기 펩타이드는 Fmoc (9-fluorenylmethoxycarbonyl)를 아미노산의 Nα-아미노 그룹(amino group)의 보호기(protecting group)로 사용하는 고상법(solid phase method)으로 합성하였다. The peptide was synthesized by a solid phase method using Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group for the Nα-amino group of an amino acid.
구체적으로, 카르복실 말단이 -NH2 형태인 펩타이드는 Rink Amide MBHA-Resin을 출발물질로 사용하였으며, 카르복실 말단이 -OH 형태의 펩타이드는 Fmoc-아미노산-Wang Resin을 출발물질로 사용하였다.Specifically, Rink Amide MBHA-Resin was used as a starting material for peptides with -NH 2 carboxyl terminals, and Fmoc-amino acid-Wang Resin was used for peptides with carboxyl terminals -OH type as starting materials.
Fmoc-아미노산의 커플링(coupling)에 의한 펩타이드 사슬의 연장 (elongation)은 DCC (N-hydroxybenzotriazole(HOBt)-dicyclo-hexylcar-bodiimide)법에 의하였다.The elongation of the peptide chain by Fmoc-amino acid coupling was performed by the DCC (N-hydroxybenzotriazole (HOBt)-dicyclo-hexylcar-bodiimide) method.
각 펩타이드의 아미노 말단의 Fmoc-아미노산을 커플링 시킨 후, 20% 피페리딘/N-메틸피롤리돈(NMP)용액으로 Fmoc 기를 제거하고 NMP 및 디클로로메탄(DCM)으로 여러 번 씻어준 다음 질소 가스로 말렸다.After coupling the Fmoc-amino acid at the amino terminal of each peptide, the Fmoc group was removed with a 20% piperidine/N-methylpyrrolidone (NMP) solution, washed several times with NMP and dichloromethane (DCM), and then nitrogen dried with gas
여기에 TFA (trifluoroacetic acid)-phenol-thioanisole-H2O-triisop- ropylsilane (85 : 5 : 5 : 2.5 : 2.5, vol./vol.) 용액을 가하고 3시간 동안 반응시켜 보호기의 제거 및 레진으로부터 펩타이드를 분리시킨 다음, 디에틸에테르로 펩타이드를 침전시켰다. 이렇게 하여 얻은 조(crude) 펩타이드는 0.1% TFA가 포함된 아세토니트릴 농도구배(acetonitrile gradient)로 하여 정제형 역상-HPLC(reverse phase-HPLC) column (Delta Pak, C18 300Å, 15μm, 19.0mm x 30mm, Waters)을 이용하여 정제하였다.A solution of TFA (trifluoroacetic acid)-phenol-thioanisole-H 2 O-triisop-ropylsilane (85 : 5 : 5 : 2.5 : 2.5, vol./vol.) was added and reacted for 3 hours to remove the protecting group and remove the resin from the resin. After the peptide was isolated, the peptide was precipitated with diethyl ether. The crude peptide thus obtained was subjected to an acetonitrile gradient containing 0.1% TFA on a purified reverse phase-HPLC (Delta Pak, C18 300Å, 15 μm, 19.0 mm x 30 mm) column. , Waters).
합성 펩타이드를 6N-HCl로 110℃에서 24시간 동안 가수분해한 후, 얻어진 잔사를 감압농축 한 뒤, 0.02N-HCl에 녹여서 아미노산 분석기 (Hitachi 8500 A)로 아미노산 조성을 측정하였다.After hydrolyzing the synthesized peptide with 6N-HCl at 110° C. for 24 hours, the obtained residue was concentrated under reduced pressure, dissolved in 0.02N-HCl, and the amino acid composition was measured using an amino acid analyzer (Hitachi 8500 A).
또한, 합성된 펩타이드의 시퀀스(sequence)를 바탕으로 분자량을 계산하였고, MALDI 질량 분석법 (matrixassisted laser desorption ionization mass spectrometer)을 이용하여 정확한 분자량을 측정하였다.In addition, the molecular weight was calculated based on the sequence of the synthesized peptide, and the exact molecular weight was measured using a matrix assisted laser desorption ionization mass spectrometer (MALDI).
그 결과, 측정된 분자량과 계산된 분자량이 일치하여 하기와 같이 정확한 아미노산 서열을 가지는 항균 펩타이드가 합성되었음을 확인하였다.As a result, it was confirmed that the measured molecular weight and the calculated molecular weight matched, and antimicrobial peptides having the correct amino acid sequence were synthesized as follows.
- RVLTHVFKCKLKLR (서열번호 1)- RVLTHVFKCKLKLR (SEQ ID NO: 1)
<< 실시예Example 2> 2> 포르피로모나스Porphyromonas 진지발리스gingivalis (( PorphyromonasPorphyromonas gingivalisgingivalis )에 대한 항균 활성 확인) Confirmation of antibacterial activity against
상기 실시예 1에서 제조된 펩타이드를 증류수에 1mg/mL 농도로 녹인 후, 항균 활성을 확인하기 위해 액체배지 미량희석법(broth microdilution method assay)을 사용하였다.After dissolving the peptide prepared in Example 1 in distilled water at a concentration of 1 mg/mL, a broth microdilution method assay was used to confirm the antibacterial activity.
제조된 펩타이드의 항균 활성을 측정하기 위해 포르피로모나스 진지발리스(Porphyromonas gingivalis, Strain number: KCTC5352)를 24시간 동안 배양한 후 배양 균액의 일정액을 새 BHI(Brain Heart Infusion) 액체배지와 면양적혈구가 5% 첨가된 배지에서 분광광도계로 OD600에서 0.1이 되도록 희석한 후, 펩타이드 및 양성 대조군(positive control)인 옥시테트라사이클린(Oxytetracycline)은 50μg/mL 부터 100μg/mL의 농도로 처리한 후 24시간 동안 배양하였다. 배양한 후 OD600에서 흡광도를 측정하였다.In order to measure the antibacterial activity of the prepared peptide, Porphyromonas gingivalis ( Strain number: KCTC5352 ) was cultured for 24 hours, and then a certain amount of the culture solution was added to a new BHI (Brain Heart Infusion) liquid medium and cotton red blood cells. After diluting to 0.1 at OD600 with a spectrophotometer in a medium supplemented with 5%, the peptide and positive control, oxytetracycline, were treated at a concentration of 50 μg/mL to 100 μg/mL and then treated for 24 hours. cultured. After culturing, absorbance was measured at OD600.
그 결과, 하기 표 1 및 도 1에 나타난 바와 같이, 상기 펩타이드에 의해 처리한 경우 양성 대조군보다 더 낮은 최소 억제 농도(minimum inhibitory concentration, MIC)로 포르피로모나스 진지발리스를 억제함을 확인하였다.As a result, as shown in Table 1 and FIG. 1 below, it was confirmed that treatment with the peptide inhibited Porphyromonas gingivalis with a lower minimum inhibitory concentration (MIC) than that of the positive control group.
minimum inhibitory concentration (μg/mL)minimum inhibitory concentration (μg/mL)
옥시테트라사이클린oxytetracycline 서열번호 1의 펩타이드Peptide of SEQ ID NO: 1
PorphyromonasPorphyromonas gingivalis gingivalis
50
50
25
25
<< 실시예Example 3> 세포 독성 확인 3> Confirmation of cytotoxicity
인간 각질 세포주 HaCat cell line은 사람의 표피 세포로, 1% 페니실린-스트렙토마이신(penicillin-streptomycin) 및 10% FBS를 포함하는 DMEM 배지를 사용하여 37℃, 5% CO2의 환경에서 배양하여 확인하였다.The human keratin cell line HaCat cell line is a human epidermal cell, and was identified by culturing in an environment of 37° C., 5% CO 2 using DMEM medium containing 1% penicillin-streptomycin and 10% FBS. .
먼저, 6-well 세포 배양 플레이트에 10% FBS가 첨가된 DMEM 배지를 사용하여 HaCaT를 3×105/well로 시딩하여 하루 배양하였다. 그 후 무혈청(serum-free) 배지로 세척하고 무혈청 배지에 상기 펩타이드를 농도별로 처리한 다음 24시간 동안 배양한 후 세포 사멸을 확인하였다.First, HaCaT was seeded at 3×10 5 /well using DMEM medium supplemented with 10% FBS in a 6-well cell culture plate and cultured for one day. Thereafter, the cells were washed with a serum-free medium, treated with the peptide at each concentration in the serum-free medium, and then cultured for 24 hours, and cell death was confirmed.
그 결과, 도 2에 나타난 바와 같이, 세포 독성이 거의 확인되지 않았다.As a result, as shown in FIG. 2, cytotoxicity was hardly confirmed.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 즉, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다.Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. Do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

  1. 서열번호 1로 표시되는 아미노산 서열로 이루어진 항균 펩타이드.An antibacterial peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
  2. 제 1 항에 있어서,According to claim 1,
    상기 펩타이드는,The peptide,
    포르피로모나스 진지발리스(Porphyromonas gingivalis)에 대해 항균 활성을 가지는 것을 특징으로 하는, 항균 펩타이드.Porphyromonas gingivalis ( Porphyromonas gingivalis ), characterized in that it has antibacterial activity against, antibacterial peptide.
  3. 제 1 항에 따른 항균 펩타이드를 코딩하는 폴리뉴클레오티드.A polynucleotide encoding the antimicrobial peptide according to claim 1.
  4. 제 1 항에 따른 항균 펩타이드를 유효성분으로 포함하는 구강 질환 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating oral diseases comprising the antimicrobial peptide according to claim 1 as an active ingredient.
  5. 제 4 항에 있어서,According to claim 4,
    상기 조성물은,The composition,
    포르피로모나스 진지발리스(Porphyromonas gingivalis)에 대해 항균 활성을 가지는 것을 특징으로 하는, 약학 조성물.Porphyromonas gingivalis ( Porphyromonas gingivalis ), characterized in that it has an antibacterial activity against, a pharmaceutical composition.
  6. 제 4 항에 있어서,According to claim 4,
    상기 구강 질환은, The oral disease,
    충치, 치주염 및 치은염으로 이루어진 군에서 선택되는 하나 이상인 것을 특징으로 하는, 약학 조성물.Characterized in that at least one selected from the group consisting of caries, periodontitis and gingivitis, a pharmaceutical composition.
  7. 제 1 항에 따른 항균 펩타이드를 유효성분으로 포함하는 구강 질환 예방 또는 개선용 의약외품 조성물.A quasi-drug composition for preventing or improving oral diseases comprising the antimicrobial peptide according to claim 1 as an active ingredient.
  8. 제 1 항에 따른 항균 펩타이드를 유효성분으로 포함하는 구강 질환 예방 또는 개선용 건강기능식품 조성물.A health functional food composition for preventing or improving oral diseases comprising the antimicrobial peptide according to claim 1 as an active ingredient.
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