KR102496852B1 - A locustacin 1 peptide isolated from Locusta migratoria and A use of the peptide - Google Patents

Abstract

The present invention relates to a peptide locustacin-1 having the amino acid sequence of KTHILSFFPSFLPLFLKK-NH ₂ derived from bell pepper and its use, since locustacin-1 has excellent antibacterial, antifungal or anti-inflammatory activity, so it can be used for antibacterial, antifungal or anti-inflammatory It can be used as a pharmaceutical composition for preparation, antibiotic, food composition, feed composition, food preservative, feed preservative, cosmetic preservative or pharmaceutical preservative.

Description

A locustacin 1 peptide isolated from Locusta migratoria and A use of the peptide}

The present invention is a bellows ( Locusta migratoria ) and uses thereof, and more preferably to antibacterial, antifungal or anti-inflammatory uses of locustacin-1 isolated from bell peppers.

Infection with pathogenic microorganisms is one of the most common and fatal causes of human disease. Unfortunately, due to the overuse of antibiotics, resistance of pathogenic microorganisms to antibiotics has been caused. In fact, the rate at which pathogenic microorganisms develop resistance to new antibiotics is much faster than the rate at which analogues of new antibiotics are developed. For example, Enterococcus faecalis , which can pose a threat to life, Mycobacterium tuberculosis , Pseudomonas aruginosa ( Pseudomonas ) aeruginosa ) have developed resistance to all known antibiotics.

Tolerance to antibiotics is a phenomenon distinct from resistance to antibiotics, which was first discovered in Pneumococcus sp. in the 1970s and provided important clues to the mechanism of action of penicillin. Resistant species stop growing in the presence of normal concentrations of antibiotics, but do not die as a result. This resistance occurs because when antibiotics inhibit cell wall synthase, the activity of bacterial autolytic enzymes such as autolysin does not occur. By activating, it kills bacteria, and bacteria also suppress their activity, resulting in survival even when treated with antibiotics.

It is very important clinically that pathogenic microorganisms have resistance to antibiotics, because if it is impossible to eradicate resistant microorganisms, the effectiveness of antibiotic treatment in clinical infections is reduced. In addition, development of resistance is considered a prerequisite for development of resistance to antibiotics, as it results in strains that survive antibiotic treatment. These strains acquire new genetic elements that are resistant to antibiotics and continue to grow in the presence of antibiotics. Since virtually all pathogenic microorganisms showing resistance are known to have resistance, the development of novel antibiotics capable of killing pathogenic microorganisms having such antibiotic resistance is urgent.

On the other hand, the inflammatory response is an innate immunity response that occurs first to defend the living body in the event of a wound or invasion of foreign substances, and can be divided into an acute inflammatory response and a chronic inflammatory response according to the action and duration of inflammation.

The acute inflammatory response is activated by the production of inflammatory mediators such as inflammatory cytokines, chemokines, nitric oxide (NO), and prostaglandin E2 (PGE2), which are produced when macrophages are activated by stimulation by external substances. After removing the foreign substance by the white blood cells and macrophages, it is terminated through tissue regeneration. Acute inflammatory response is an essential response to defend the body, but excessively secreted inflammatory mediators in this response can cause acute septic shock due to excessive vasodilation, and cause chronic inflammation or immunity by causing continued survival and activation of immune cells. Since it can cause hypersensitivity reactions and develop into chronic inflammatory diseases such as rheumatoid arthritis and autoimmune diseases, inflammatory response control is very important for maintaining life.

As described above, it is necessary to develop new drugs to prevent damage by pathogenic microorganisms that are resistant to antibiotics and to control inflammatory responses, and also have new antibiotic and anti-inflammatory effects that act independently of autolysin activity. It is necessary to develop drugs that show There is also a need to provide pharmaceutical compositions for effectively treating infections of pathogenic microorganisms with such new agents.

On the other hand, some of the substances that play an important role in the process for maintaining the homeostasis of living organisms are physiologically active substances derived from various living organisms. Until now, many studies have been conducted on a number of physiologically active substances, and among them, peptides isolated from various organisms are known to have antibacterial and/or anti-inflammatory effects on bacteria, fungi and viruses. Antimicrobial peptides are known to play important roles in host defense and the innate immune system. As for antibacterial related prior art, Korean Patent No. 10-0964136 discloses the antibacterial or antifungal activity of peptides isolated from Uldo Skylarvae, and Korean Patent No. 10-1953828 discloses the antibacterial activity of peptides derived from king crickets As prior art related to anti-inflammatory, Korean Patent No. 10-1730065 discloses the anti-inflammatory activity of an insect-derived natural resource extract, and Korean Patent No. 10-1700605 discloses the anti-inflammatory activity of a beetle extract. Initiates inflammatory activity, but studies on peptides derived from bell peppers and their new uses are lacking.

An object to be solved by the present invention is to provide a novel peptide locustacin-1 having antibacterial, antifungal or anti-inflammatory properties derived from bell pepper and its use.

The present invention relates to antibacterial, antifungal or anti-inflammatory peptides derived from Locusta migratoria and uses thereof.

In order to solve the above problems, the present invention is a locusta migratoria ) to provide a peptide represented by SEQ ID NO: 1 derived from.

SEQ ID NO: 1: KTHILSFFPSFLPLFLKK-NH ₂

A method for providing the peptide according to the present invention is as follows. In order to identify the antibacterial, antifungal or anti-inflammatory genes derived from Locusta migratoria , first, the bellows chives immunized with bellows chives were rapidly frozen in liquid nitrogen to isolate total RNA, and RNA seq analysis was used to analyze the transcriptome of bellows chives. ), and a method for selecting candidates through antibacterial, antifungal or anti-inflammatory activity tests based on amino acid sequences translated from each transcript.

In the present invention, "peptide" and "protein" are used interchangeably and include reference to polymers of amino acid residues. The terms apply to natural amino acid polymers as well as amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding natural amino acids. The terms also apply to those polymers that contain conservative amino acid substitutions such that the protein remains functional. In the present invention, the peptide may be synthesized using genetic recombination and a protein expression system, preferably synthesized in vitro through a peptide synthesizer or the like.

In the present invention, "biologically active" means a peptide fragment having a structural function similar to that of the polypeptide of SEQ ID NO: 1, and/or a similar regulatory function, and/or a similar biochemical function, as well as a peptide according to the present invention. do.

The present invention may include a variant of the sequence of SEQ ID NO: 1. Specifically, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% with the peptide sequence of SEQ ID NO: 1, Sequences that have at least 98%, or at least 99% sequence homology, that retain biological activity may be included within the scope of the present invention.

In the present invention, a "deletion" is defined as a change in a nucleotide or amino acid sequence that is missing one or more nucleotides or amino acid residues, respectively, compared to the wild-type polynucleotide or polypeptide.

In the present invention, an "insertion" or "addition" is a change in a nucleotide or amino acid sequence due to the addition of one or more nucleotides or amino acid residues, respectively, compared to a wild-type polynucleotide or polypeptide.

In the present invention, a "substitution" results from the replacement of one or more nucleotides or amino acids by a different nucleotide or amino acid, respectively, compared to the wild-type polynucleotide or polypeptide.

On the other hand, a preferred amino acid change at a substitution site may be one performed based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphiphilic properties of the residues. For example, but not limited to, negatively charged amino acids include aspartic acid and glutamic acid; Positively charged amino acids include lysine and arginine; And amino acids having uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine, threonine, phenylalanine and tyrosine; Such mutations may include conservative substitutions. A 'conservative substitution' is a substitution in which an amino acid is substituted with another amino acid having similar properties, and those skilled in the art can predict that the secondary structure and hydropathic nature (hydropathic nature, hydrophobic or hydrophilic nature) of the polypeptide are substantially unchanged. am. In general, the following groups of amino acids exhibit conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

In some cases, the variant is phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation and amylation It can also be modified as

In addition, the sequence according to the present invention may further include a cell penetrating peptide sequence for intracellular penetration. A cell-penetrating peptide is a kind of signal peptide, which is a peptide sequence intended to transmit substances into cells. It mainly consists of 7-30 amino acid peptide sequences, and any sequence may be included in the present invention as long as it has a signal peptide that can be delivered into cells.

For example, cell-penetrating peptides mainly contain basic amino acid residues such as lysine/arginine, and thus allow proteins fused thereto to permeate cell membranes and enter cells. The cell-penetrating peptide is HIV-1 Tat protein, Drosophila antennapedia homeodomain (Penetratin), HSV VP22 transcriptional regulatory protein, vFGF-derived MTS peptide, PTD-5, transportan or Pep-1 peptide Including sequences derived from, and the like, but are not limited thereto. As such, various CPPs (e.g., TAT48-60, penetratin (pAntp) 43-58, polyarginine, Pep-1, transportan, etc.) identified/synthesized from viruses or cationic peptides are biologically active substances. has an activity capable of internalizing cells to mediate the movement of drug carriers.

The amino acid sequence of the peptide is KTHILSFFPSFLPLFLKK-NH ₂ and is composed of 18 amino acids, which is referred to as locustacin-1.

According to the present invention, the peptide can exhibit excellent antibacterial or antifungal activity against gram-positive bacteria, gram-negative bacteria or fungi, so it is useful as a pharmaceutical composition for antibacterial or antifungal preparations. The gram-positive or gram-negative bacteria are, for example, Escherichia coli , Pseudomonas aeruginosa , Staphylococcus aureus , Klebsiella pneumoniae , MRSA ( Staphylococcus aureus subsp. aureus ), Bacillus subtilis ( Bacillus subtilis ), Vibrio parahaemolyticus , Propionibacterium acnes , Salmonella typhimurium , or Shigella flexneri , but is not limited thereto.

The fungus may be, for example, Candida albicans , but is not limited thereto.

According to the present invention, the peptide is characterized in that it is non-toxic to cells. The cells may be, for example, mouse red blood cells, but are not limited thereto. In one embodiment of the present invention, it was confirmed that the peptide according to the present invention did not exhibit cytotoxicity as a result of treating mouse erythrocytes. Therefore, the peptide of the present invention can be usefully used as a safe antibacterial or antifungal agent.

The present invention provides an antibacterial or antifungal composition comprising locustacin-1, which is a peptide having the amino acid sequence of SEQ ID NO: 1 below.

SEQ ID NO: 1: KTHILSFFPSFLPLFLKK-NH ₂

In the present invention, the "antibacterial composition" or "antifungal composition" may mean the same as antibiotics, which is a generic term for antimicrobial agents, and may mean the same as antibacterial agents, antifungal agents, bactericides, preservatives, preservatives, or disinfectants. For example, bacteria that cause food poisoning or cause purulent infections, bacteria that can aggravate diseases due to infection when the body's resistance is lowered, such as surgery and burns, and bacteria that cause oral disease or vaginitis It means a substance capable of inhibiting, suppressing or killing the growth and life function of pathogenic microorganisms.

In addition, the present invention provides an antibiotic containing the peptide having the amino acid sequence of SEQ ID NO: 1, locustacin-1 (locustacin-1) as an active ingredient. Since the peptide locustacin-1 according to the present invention has antibacterial or antifungal activity against pathogenic microorganisms, particularly pathogenic microorganisms having antibiotic resistance, it can be usefully used as an antibiotic.

Use as the antibiotic includes use as a pharmaceutical composition for the purpose of treatment or prevention.

In addition, the present invention provides an anti-inflammatory composition comprising the peptide having the amino acid sequence of SEQ ID NO: 1, locustacin-1 as an active ingredient. The peptide locustacin-1 according to the present invention inhibits the expression of the inflammatory cytokines TNF-α and IL-6, and the proteins cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) Since it inhibits the expression of and can suppress the production of nitric oxide, it can be usefully used as an anti-inflammatory composition.

The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising the peptide having the amino acid sequence of SEQ ID NO: 1 and locustacin-1 as an active ingredient.

The inflammatory disease according to the present invention is atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatism , lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, tenosynovitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, and multiple sclerosis. there is.

The present invention also provides a pharmaceutical composition for preventing or treating sepsis comprising the peptide having the amino acid sequence of SEQ ID NO: 1 and locustacin-1 as an active ingredient.

As used herein, "treatment" and "treating" refer to the administration or application of a therapeutic agent to a subject or the performance of a procedure or modality in a subject for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. "Prevention", "preventing" and the like refer to an approach for preventing, inhibiting or reducing the likelihood of occurrence or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the development or recurrence of a disease or condition.

In the present invention, “subject” and “patient” refer to humans or non-humans, such as primates, mammals, and vertebrates. In certain embodiments, the subject is a human.

In the present invention, "therapeutic benefit" or "therapeutically effective" refers to anything that promotes or enhances the health of a subject with respect to medical treatment of such a condition. This includes, but is not limited to, a reduction in the frequency or severity of signs or symptoms of a disease.

The locustacin-1 according to the present invention may be added in an amount of 0.001 to 95% by weight in the pharmaceutical composition of the present invention. The pharmaceutical composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain the peptide locustacin-1 of the present invention and at least one excipient, for example, starch, calcium carbonate, water It is prepared by mixing cross, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions for oral 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. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. 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 suspensions. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. Determination of dosage based on these factors is within the level of those skilled in the art, and generally dosages range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, eg oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection. The pharmaceutical composition of the present invention is suitable for parenteral administration, including intravenous administration or intracorporeal administration.

The present invention provides the use of the peptide locustacin-1 having the amino acid sequence of SEQ ID NO: 1 in the preparation of a drug for the treatment of inflammatory diseases.

The present invention provides a method for treating an inflammatory disease comprising administering a therapeutically effective amount of locustacin-1 having the amino acid sequence of SEQ ID NO: 1 to a subject in need thereof.

In addition, the present invention provides a food composition for preventing or alleviating inflammatory diseases comprising the peptide locustacin-1 having the amino acid sequence of SEQ ID NO: 1.

According to the present invention, a health functional food for preventing or improving inflammatory diseases containing the peptide locustacin-1 having the amino acid sequence of SEQ ID NO: 1 is provided.

According to the present invention, the health functional food is an inflammatory disease such as atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, from the group consisting of gout, ankylosing spondylitis, rheumatism, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, tenosynovitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome and multiple sclerosis It may be for preventing or improving any one selected disease.

The food composition of the present invention may include conventional food additives, and the suitability as the "food additive" is determined according to the general rules of the food additive code approved by the Ministry of Food and Drug Safety and general test methods, etc., unless otherwise specified. It is judged according to the specifications and standards for the item.

The term “health functional food” refers to food manufactured and processed using raw materials or ingredients having useful functionalities for the human body in accordance with the Health Functional Food Act, and “functional” refers to food that is not related to the structure and function of the human body. It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological functions.

Items listed in the "Food Additive Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as dark pigment, licorice extract, crystalline cellulose, goyang pigment, guar gum, and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations.

The food composition of the present invention may contain 0.01 to 95% by weight of locustacin-1, preferably 1 to 80% by weight, based on the total weight of the composition.

In addition, the food composition of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like.

For example, the health functional food in the form of a tablet is obtained by granulating a mixture of locustacin-1, an excipient, a binder, a disintegrant, and other additives in a conventional manner, and then compression molding by adding a lubricant or the like, or The mixture can be directly compression molded. In addition, the health functional food in the form of a tablet may contain a corrigent, etc., if necessary, and may be coated with an appropriate coating agent, if necessary.

Among health functional foods in the form of capsules, hard capsules can be prepared by filling ordinary hard capsules with a mixture of locustacin-1 and additives such as excipients, granular materials thereof, or coated granular materials. It can be prepared by filling a mixture of Tacin-1 and additives such as excipients into a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

The health functional food in the form of a pill can be prepared by shaping a mixture of locustacin-1, excipients, binders, disintegrants, etc. in an appropriate way, and if necessary, it can be coated with sucrose or other suitable coating agent, or starch, talc, or a suitable coating agent. You can also wear clothes with materials.

The granular health functional food may be granularly prepared from a mixture of locustacin-1, an excipient, a binder, a disintegrant, and the like by an appropriate method, and may contain a flavoring agent, a flavoring agent, and the like, if necessary. For the health functional food in granular form, in the next particle size test using No. 12 (1680 μm), No. 14 (1410 μm) and No. 45 (350 μm) sieves, the total amount of the No. 12 sieve passed and the remaining amount on the No. 14 sieve 5.0% or less, and passing through a No. 45 sieve may be 15.0% or less of the total amount.

Definitions of terms for the excipients, binders, disintegrants, lubricants, corrigents, flavoring agents, etc. are described in documents known in the art, and include those having the same or similar functions (Korean Pharmacopoeia Explanatory Edition, Munseongsa, Korea Association of Colleges of Pharmacy, 5th Rev., p33-48, 1989).

There is no particular limitation on the type of food. Examples of foods to which locustacin-1 of the present invention can be added include beverages, chewing gum, vitamin complexes, and drinks, and include all health functional foods in the conventional sense.

The food composition may include a food supplement additive, and the food category includes, for example, beverages, gum, tea, vitamin complexes, and health supplements, such as capsules, tablets, powders, granules, liquids, pills, slices, and pastes. It can be used in the form of a phase, syrup, gel, beverage, jelly or wine.

The content of locustacin-1 may be contained in 0.01 to 15% by weight based on the total weight of the health functional food, and in the case of a health drink composition, 0.02 to 10 g, preferably 0.3 to 1 g, based on the total 100 g. can contain.

The supplementary food additives include conventional food additives in the art, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like. In addition, the health drink composition is an essential component in the indicated ratio, and there is no particular limitation on components added other than the locustacin-1, and various flavors or natural carbohydrates may be included as additional components as in conventional beverages. . Examples of the natural carbohydrate include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; polysaccharides such as dextrins, cyclodextrins; and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (eg rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. there is.

In addition to the above, the food composition or health functional food containing locustacin-1 is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.) , pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like.

In addition, the present invention provides a feed composition for preventing or improving inflammatory diseases containing locustacin-1.

In the present invention, the feed may be fish, bird, or mammal feed, and preferably, the wild habits defined in Article 2, Subparagraph 1 of the Livestock Act and each subparagraph of the Enforcement Rule of the same Act are purified and suitable for breeding. It can be feed for livestock or aquatic organisms that can contribute to farm household income increase. The livestock may be cattle, horses, mules, donkeys, goats, goats, sheep, deer, pigs, rabbits, poultry, etc., and poultry may be chickens, turkeys, ducks, ostriches, geese, quails, etc., preferably chickens, It is not limited thereto as long as it is suitable for breeding and obtaining livestock products. The above “livestock products” are meat, milk, eggs, honey and their processed products, hides (including raw fur), raw wool, and other livestock products produced from livestock, which are defined in Article 2, Subparagraph 3 of the Livestock Act, as determined by the Ordinance of the Ministry of Agriculture and Forestry. means that In addition, it may be a dog, cat, etc., including companion animals, but is not limited thereto.

The term "feed" in the present invention means any natural or artificial diet, meal, etc. or component of said meal, intended for or suitable for eating, ingestion and digestion by an animal. In one aspect, the feed composition containing locustacin-1 of the present invention may include concentrated feed, roughage and/or special feed.

Concentrated feed includes seed fruits including grains such as wheat, oats, and corn, bran including rice bran, wheat bran, and barley bran as by-products obtained after refining grains, soybeans, fluids, sesame seeds, linseed, and coco palm oil. Fish meal, which is a by-product obtained by extracting starch from sweet potatoes and potatoes, and residual starch, which is the main component of starch residues, fish meal, fish residue, and fish soluble, meat meal, which are concentrated fresh liquids obtained from fish. , animal feed such as blood meal, feather meal, skim milk powder, dried whey obtained by drying whey, which is the balance when cheese is produced from milk and casein is produced from skim milk, yeast, chlorella, and seaweed.

Forage, raw grass feed such as wild grass, grass, and green cutting, turnip for feed, beet for feed, root vegetables such as Lutherbearer, a type of turnip, raw grass, green cut crops, grain, etc. are put into silos. There are silage (silage), which is a stored feed filled and fermented with lactic acid, grass, hay cut and dried, straw from breeding crops, and leaves of leguminous plants. Special feeds include mineral feeds such as oyster shells and rock salt, urea feeds such as urea or its derivative, diureide isobutane, and supplements for ingredients that tend to be insufficient when only natural feed ingredients are mixed, or formulated feeds to improve the storage life of feeds. There are feed additives, which are substances added in small amounts.

In addition, the present invention provides an antibacterial, antifungal or anti-inflammatory skin external preparation comprising locustacin-1, which is a peptide having the amino acid sequence of SEQ ID NO: 1. Since the peptide locustacin-1 according to the present invention has antimicrobial activity against gram-positive or gram-negative bacteria and antifungal activity against fungi, it can be usefully used as an external skin preparation for antibacterial or antifungal use, and the inflammatory cytokine TNF-α And anti-inflammatory because it can inhibit the expression of IL-6, inhibit the expression of the protein cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), and inhibit nitric oxide production It can be usefully used as an external skin preparation.

In addition, the present invention provides food preservatives, feed preservatives, cosmetic preservatives, and pharmaceutical preservatives containing the peptide locustacin-1. At this time, food preservatives, feed preservatives, cosmetic preservatives, and pharmaceutical preservatives are additives used to prevent deterioration, decay, discoloration, and chemical changes in food, feed, medicines, and cosmetics, and include disinfectants and antioxidants, and include bacteria, fungi, Also included are functional antibiotics, such as suppressing the growth of microorganisms such as yeast, inhibiting the growth of spoilage microorganisms in food or medicine, or sterilizing action. Ideal conditions for such food preservatives, cosmetics or pharmaceutical preservatives should be non-toxic and effective even in small amounts.

In particular, since the novel peptide locustacin-1 of the present invention exhibits antibacterial or antifungal activity and has minimal toxicity, it can be usefully used as a preservative for food or feed, or as a preservative for cosmetics or pharmaceuticals.

The use of the above food or feed as a preservative is added to general food or feed materials, and there is no limit to the type of food or feed.

For use as a cosmetic or pharmaceutical preservative as above, a substance selected arbitrarily may be added according to the purpose of use. For example, purified water, oil, a surfactant, a moisturizer, a higher alcohol, a thickener, a chelating agent, a pigment, a fatty acid, a wax, a pH adjuster, and a flavoring agent may be added.

In addition, the peptide according to the present invention can be used for various purposes and uses requiring antibacterial, bactericidal or antifungal activity. Specifically, it can be used as a skin cleaner, hair cleaner, vaginal cleaner, mouthwash, bathroom cleaner, kitchen cleaner, food additive, feed additive, disinfectant or humidifier sterilizer material.

For use as above-mentioned food and/or feed preservatives, cosmetics or pharmaceutical preservatives, skin cleaners, hair cleaners, vaginal cleaners, mouthwashes, bathroom cleaners, kitchen cleaners, food additives, feed additives, disinfectants, or materials for humidifier disinfectants, etc. In the case, the peptide may be included in an amount of 0.00001 to 50% by weight based on the total weight of the additive, and may be included in a concentration of 1 μg / ml to 10000 μg / ml. In addition, other known antibacterial substances having additional antibacterial activity or substances capable of imparting other functionalities may be further included.

Since the peptide locustacin-1 derived from bell peppers according to the present invention has little cytotoxicity and exhibits excellent antibacterial, antifungal or anti-inflammatory activity, it is used as a pharmaceutical composition for antibacterial, antifungal or anti-inflammatory, antibiotics, food or feed preservatives, cosmetics or It can be usefully applied as a pharmaceutical preservative.

1 is a result of confirming the antibacterial and antifungal activity of locustacin-1 according to the present invention.
Figure 2 is a result of assaying the cytotoxicity of locustacin-1 according to an embodiment of the present invention.
Figure 3 is a result of assaying the cell viability of locustacin-1 according to an embodiment of the present invention.
Figure 4 is a result of assaying the nitric oxide inhibitory effect of locustacin-1 according to an embodiment of the present invention.
Figure 5 is the result of confirming the effect of inhibiting the expression of cytokines IL-6 and IL-1β related to the induction of locustacin-1 according to an embodiment of the present invention by ELISA.
Figure 6 is the result of confirming the expression suppression of the inflammatory mediator inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) of locustacin-1 according to an embodiment of the present invention.

Hereinafter, preferred embodiments are presented to aid understanding of the present invention, but the following examples are merely illustrative of the present invention, and various changes and modifications are possible within the scope and spirit of the present invention. It is obvious to those skilled in the art, It goes without saying that these variations and modifications fall within the scope of the appended claims.

< Example 1. Preparation of the peptide locustacin-1 having the amino acid sequence of SEQ ID NO: 1 >

Collection and immunization of insect samples

Pulmuchi ( Locusta migratoria ) was immunized with Escherichia coli ( E. coli ), Staphylococcus aureus ( S. aureus ) and Candida albicans ( C. albicans ) in Tryptic Soy Broth (TSB) liquid medium at 37 ° C. in a shaking incubator at 200 rpm for 18 days. After the primary culture for 2 hours and 30 minutes under the same conditions, the secondary culture was performed, washed with 10 mM sodium phosphate buffer (pH 7.4), and the cell count was 5 × 10 ³ colony forming units (CFU). It was taken so as to be injected into the body cavity using a microinjector and raised for 18 hours at 25 ° C. to induce immunization.

transcriptome RNA isolation

In order to maximize the induced expression of physiologically active substances, RNA-seq was performed using total RNA of normal bell peppers and immunized bell peppers. First, after preparing the normal bell peppers and immunized bell peppers, rapidly freeze them in liquid nitrogen to keep the tissues as fresh as possible, prepare samples by grinding them in a mortar, mix 100 mg of the sample with 1 ml of TRIZOL, and incubate at room temperature for 5 minutes. reacted After adding and mixing 200 μl chloroform, the mixture was reacted at room temperature for 3 minutes. Next, after centrifugation at 12,000 × g at 4 ° C. for 15 minutes, the supernatant was taken, 500 μl isopropanol was added, reacted at room temperature for 10 minutes, and then centrifuged at 4 ° C. at 12,000 × g for 10 minutes. The precipitated total RNA was washed with 1 ml of 75% ethanol and centrifuged at 7,500×g for 5 minutes at 4°C. After centrifugation, ethanol was removed, dried, and RNA was prepared by dissolving it in sterile water. The prepared total RNA was checked for purity and quantity using an Agilent 2100 Bioanalyzer.

gene selection

For transcriptome sequencing, RNA-seq analysis was performed on non-immunized and immunized bell pepper samples using the Illumina HiSeq™ 2000 device, and as a result, genes presumed to be antibacterial, antifungal, or anti-inflammatory peptides were selected. . The selected amino acid sequence was KTHILSFFPSFLPLFLKK-NH ₂ , consisting of 18 amino acid sequences, and was named locustacin-1.

< test example 1. Evaluation of antibacterial/antifungal activity against pathogenic microorganisms>

Radial diffusion assay (RDA) was performed to evaluate the antibacterial activity of the peptide locustacin-1 against pathogenic microorganisms. In the present invention, the antimicrobial peptide was stored at -20 ° C and used after being dissolved in sterilized tertiary distilled water.

<Antibacterial activity assay>

For the antibacterial activity assay, first, a citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4) and 1% (w/v) type I (low electroendosmosis) agarose, consisting of 0.03% TSB Cultured bacteria (4×10 ⁶ colony forming units/ml) are added to the sterilized underlay gel, mixed, poured into a square plate, and when the underlay gel hardens, a hole with a diameter of 3 mm is made by making a hole for each concentration. 5 μl of the peptide (locustacin-1) at (0, 12.5, 25, 50, 100 and 200 μg/ml) was put into the hole. As for the use of strains, pathogenic bacteria Staphylococcus aureus ( Staphylococcus aureus ) and Escherichia coli ( Escherichia coli ) were used.

The results of confirming the antibacterial activity of locustacin-1 against Escherichia coli and Staphylococcus aureus by RDA test are shown in FIG. 1 . It was confirmed that all of the tested strains had a concentration-dependent effect and exhibited a wide range of antibacterial activities as a whole.

Through this, it was confirmed that the peptide locustacin-1 according to the present invention has high availability as a therapeutic agent for known harmful strains and excellent antibacterial activity.

< Antifungal activity assay >

For the measurement of the antifungal activity of the peptide, citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH7.4) and 1% (w / v) type I (low electroendosmosis) agarose, 0.03% Cultured bacteria (4×10 ⁶ colony forming units/ml) are added to the sterilized underlay gel composed of TSB, mixed, poured into a square plate, and when the underlay gel hardens, a hole with a diameter of 3 mm is made. 5 μl of the peptide (locustacin-1) at each concentration (0, 12.5, 25, 50, 100 and 200 μg/ml) was put into the hole. As for the use of the strain, Candida albicans , a pathogenic fungus, was used.

The results of confirming the antifungal activity of locustacin-1 against Candida fungi by RDA experiment are shown in FIG. 1 . It was confirmed that it had a concentration-dependent effect and exhibited antifungal activity.

Through the above antibacterial and antifungal activity assay, it was confirmed that the peptide locustacin-1 according to the present invention has excellent antibacterial and antifungal activity against a wide range of strains.

< test example 2. Cytotoxicity test>

A hemolysis assay was performed to investigate the cytotoxicity of the locustacin-1 peptide. After washing mouse erythrocytes three times with phosphate-sodium chloride buffer, pH=7.4 (PBS), the cells were diluted with phosphate-sodium chloride buffer to prepare a 5.00% red blood cell solution. Next, 100 μl of the 5.0% red blood cell solution was dispensed into each tube, and then treated with locustacin-1 at concentrations (0, 12.5, 25, 50, 100 and 200 μg/ml) for 24 hours. Phosphate-sodium chloride buffer, pH = 7.4 (PBS) was added to all tubes so that the total amount was 200 μl, and incubated for 30 minutes in a 37° C. incubator. After incubation, the supernatant was centrifuged for 10 minutes at a rotational speed of 1000 rpm in a centrifuge, and the supernatant was transferred to a 96-well culture vessel, and then confirmed by measuring absorbance at a wavelength of 550 nm using a multi detector (Beckman DTX8800). As a control, the value when treated with 1% Triton X-100 was calculated as 100% disruptive capacity, and the value when only red blood cells were added was calculated as 0% disruptive capacity. At this time, the destructive power of the peptide was calculated by the following equation.

[Equation 1]

% destructive activity = (absorbance of solution treated with peptide 550 nm - absorbance of phosphate-sodium chloride buffer 550 nm) / (absorbance of solution treated with Triton X-100 550 nm - absorbance of phosphate-sodium chloride buffer 550 nm) × 100

As shown in Figure 2, the peptide locustacin-1 showed insignificant ability to destroy red blood cells even when treated at high concentrations up to 200 μg/ml. On the other hand, melittin, a strong antibacterial peptide used as a control, was evaluated to have high cytotoxicity even at low concentrations.

Through this, it was confirmed that the peptide locustacin-1 according to the present invention has minimal cytotoxicity, so it can be used as a safe antibacterial, antifungal and antibiotic agent, as well as usefully applied as a food or feed preservative, and a cosmetic or pharmaceutical preservative.

<Test Example 3. Cell viability test>

① Cell culture

In order to verify the anti-inflammatory effect, experiments were conducted using mouse macrophages, Raw264.7 cells. The cells were cultured in a DMEM medium containing antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin) and 10% fetal bovine serum (FBS) in a 37°C, 5% CO ₂ incubator and used for experiments.

② MTS assay

After dispensing 2 × 10 ⁴ cell/well cells by 100 μl in a 96-well culture vessel, locustacin-1 was treated for 24 hours in a concentration-dependent manner. Then, MTS reagent 10 μl CellTiter 96 ^® AQueous One Solution Reagent (Promega, USA) was added and reacted for 3 to 4 hours to confirm the color change, which was confirmed by measuring absorbance at 590 nm wavelength using a multi detector (Beckman DTX8800). did

As shown in Figure 3, it was confirmed that the cell viability of locustacin-1 was 95% or higher regardless of the concentration. Therefore, it was confirmed that locustacin-1 was not toxic to Raw264.7 cells up to the measured maximum concentration of 200 μg/ml.

<Test Example 4. Anti-inflammatory activity test>

① Nitric oxide (NO) assay

In order to measure the anti-inflammatory activity of locustacin-1, the amount of NO secretion was confirmed in Raw264.7 cells. Cells were prepared in 6 wells at 1 × 10 ⁶ cells/ml, and then treated with peptides for 1 hour at each concentration. After that, lipopolysaccharide (LPS) was treated at 100 ng/ml to induce inflammation and cultured for 24 hours. 100 μl of the supernatant of the cultured cells was used for the experiment, and the absorbance at 550 nm wavelength was measured using a multi detector (Beckman DTX8800) to determine how much the amount of NO secreted by LPS was reduced by the peptide using the NO detection kit (intron). It was confirmed through measurement.

As shown in Figure 4, it was confirmed that the amount of NO secretion decreased dependently from 50 ug/ml concentration when treated with locustacin-1.

② Quantitative RT-PCR (qRT-PCR)

Cells were prepared in 6 wells at 1 × 10 ⁶ cells/ml, and then treated with peptides for 1 hour at each concentration. Thereafter, 100 ng/ml of LPS was treated to induce inflammation and cultured for 24 hours. Then, the supernatant was removed, and the cells were harvested after washing with PBS. The harvested cells were mixed with 1 ml of TRIZOL solution and reacted at room temperature for 5 minutes. After adding and mixing 200 μl chloroform, centrifuged at 4 ℃ at 12,000 × g for 15 minutes, took the supernatant, added 500 μl isopropanol, reacted at room temperature for 10 minutes, and centrifuged at 4 ℃ at 12,000 × g for 10 minutes did Precipitated RNA was washed with 1 ml 75% ethanol and centrifuged at 7,500×g for 5 minutes at 4°C. After centrifugation, ethanol was removed, dried, and RNA was prepared by dissolving it in sterile water. After that, cDNA was prepared using cDNA synthesis kit using 1 μg of total RNA, and qRT-PCR experiments were performed. The primers used are as follows.

[Table 1]

As shown in Figure 5, it was confirmed that the expression levels of all the cytokines used were decreased in a concentration-dependent manner, indicating that locustacin-1 has an excellent effect on inflammatory diseases.

③ Enzyme-linked immunosorbent assay (ELISA)

Raw264.7 cells were prepared in 6 wells at 1 × 10 ⁶ cells/ml, and then treated with peptides for 1 hour at each concentration. Thereafter, 100 ng/ml of LPS was treated to induce inflammation and cultured for 24 hours. The supernatant of the cultured cells was collected, and the absorbance was measured and quantified with a multi detector (Beckman DTX8800) using an ELISA kit to secrete IL-6 and IL-1β.

As shown in Figure 6, it was confirmed that the expression of iNOS and COX-2, which are inflammatory mediators, were reduced in a concentration-dependent manner of locustacin-1, which shows that locustacin-1 has an excellent effect on inflammatory diseases.

<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> A locustacin 1 peptide isolated from Locusta migratoria larva and A use of the peptide <130> NSH1-156 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> PRT <213> artificial sequence <220> 223 <223> <400> 1 Lys Thr His Ile Leu Ser Phe Phe Pro Ser Phe Leu Pro Leu Phe Leu 1 5 10 15 Lys Lys <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> TNF-alpha Forward <400> 2 atgagaagtt cccaaatggc 20 <210> 3 <211> 20 <212> DNA <213> artificial sequence <220> <223> TNF-alpha Reverse <400> 3 ctccacttgg tggtttgcta 20 <210> 4 <211> 23 <212> DNA <213> artificial sequence <220> <223> IL-6 Forward <400> 4 gaggatacca ctcccaacag acc 23 <210> 5 <211> 24 <212> DNA <213> artificial sequence <220> <223> IL-6 Reverse <400> 5 aagtgcatca tcgttgttca taca 24 <210> 6 <211> 21 <212> DNA <213> artificial sequence <220> <223> IL-1beta Forward <400> 6 ccttccagga tgaggacatg a 21 <210> 7 <211> 21 <212> DNA <213> artificial sequence <220> <223> IL-1beta Reverse <400> 7 tgagtcacag aggatgggct c 21 <210> 8 <211> 21 <212> DNA <213> artificial sequence <220> <223> iNOS Forward <400> 8 cagcacagga aatgtttcag c 21 <210> 9 <211> 19 <212> DNA <213> artificial sequence <220> <223> iNOS Reverse <400> 9 tagccagcgt accggatga 19 <210> 10 <211> 22 <212> DNA <213> artificial sequence <220> <223> COX-2 Forward <400> 10 cagacaacat aaactgcgcc tt 22 <210> 11 <211> 23 <212> DNA <213> artificial sequence <220> <223> COX-2 Reverse <400> 11 gatacacctc tccaccaatg acc 23 <210> 12 <211> 20 <212> DNA <213> artificial sequence <220> <223> GAPDH Forward <400> 12 aaggtcatcc cagagctgaa 20 <210> 13 <211> 20 <212> DNA <213> artificial sequence <220> <223> GAPDH Reverse <400> 13 ctgcttcacc accttcttga 20

Claims (17)

Peptide represented by SEQ ID NO: 1:
SEQ ID NO: 1: KTHILSFFPSFLPLFLKK-NH ₂ According to claim 1,
The peptide is a peptide that exhibits antibacterial, antifungal or anti-inflammatory activity. According to claim 2,
The peptide is Escherichia coli , Pseudomonas aeruginosa , Staphylococcus aureus , Klebsiella pneumoniae , Bacillus subtilis , Vibrio parahaemolyticus , Propionibacter Showing antibacterial or antifungal activity against any one or more selected from the group consisting of Propionibacterium acnes , Salmonella typhimurium , Shigella flexneri and Candida albicans phosphorus, peptides. According to claim 2,
The peptide is a peptide that exhibits anti-inflammatory activity. An antibacterial or antifungal composition comprising the peptide locustacin-1 consisting of the amino acid sequence of SEQ ID NO: 1. According to claim 5,
The peptide is Escherichia coli , Pseudomonas aeruginosa , Staphylococcus aureus , Klebsiella pneumoniae , Bacillus subtilis , Vibrio parahaemolyticus , Propionibacter Leeum acnes ( Propionibacterium acnes ), rat titus bacteria ( Salmonella typhimurium ) and Shigella flexneri ( Shigella flexneri ) Which exhibits antibacterial activity against at least one selected from the group consisting of, antibacterial or antifungal composition. According to claim 5,
The peptide exhibits antifungal activity against Candida albicans , an antibacterial or antifungal composition. delete delete delete An antibiotic comprising the peptide of any one of claims 1 to 4 as an active ingredient. An anti-inflammatory food composition comprising the peptide of any one of claims 1 to 4. An anti-inflammatory health functional food comprising the peptide of any one of claims 1 to 4. An anti-inflammatory feed composition comprising the peptide of any one of claims 1 to 4. An antibacterial, antifungal or anti-inflammatory external skin preparation comprising the peptide of any one of claims 1 to 4. A food or feed preservative comprising the peptide of any one of claims 1 to 4. A cosmetic or pharmaceutical preservative comprising the peptide of any one of claims 1 to 4.

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