KR102492395B1 - Peptide, papiliocin-3 derived from papilio xuthus orientalis and uses thereof - Google Patents

Abstract

The present invention is a swallowtail butterfly ( Papilio xuthus ) papilyocin-3 peptide represented by the amino acid sequence of SEQ ID NO: 1 isolated from larvae and uses thereof.
The peptide represented by the amino acid sequence of SEQ ID NO: 1 according to the present invention has little cytotoxicity and has excellent antibacterial, antifungal and anti-inflammatory activity.

Description

Papiliocin-3, a peptide derived from swallowtail butterfly larvae and its uses {PEPTIDE, PAPILIOCIN-3 DERIVED FROM PAPILIO XUTHUS ORIENTALIS AND USES THEREOF}

The present invention relates to papiliosin-3, a peptide represented by the amino acid sequence of SEQ ID NO: 1, and uses thereof, and in detail, papiliosin-3 derived from swallowtail butterfly larvae and antibacterial, antifungal or anti-inflammatory including the same It is about a composition for

Insects have evolved in the direction of securing their own special physiological control mechanisms and materials to adapt to and survive in the living environment. Recently, research on the development of new drug candidates using insect physiologically active substances is being actively conducted [Insect Biochem . Mol. Biol. 41, 747-769]. Although swallowtail butterfly larvae are one of these insect candidates, little is known about the pharmacological mechanism or application method of the pharmaceutical composition using them.

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 numerous physiologically active substances, and among them, antibacterial peptides isolated from various living organisms are known to act in a variety of ways, ranging from bacteria, fungi and viruses. Antimicrobial peptides are also known to play an important role in host defense and the innate immune system.

Infection with pathogenic microorganisms is one of the most common and fatal causes of human disease. Unfortunately, the overuse of antibiotics has resulted in the resistance of pathogenic microorganisms to antibiotics. 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 the activity of bacterial autolytic enzymes, such as autolysin, does not occur when antibiotics inhibit cell wall synthase. 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.

Inflammation is one of the defense responses of biological tissues to certain stimuli, and refers to a complex lesion in which three things are combined: tissue deterioration, circulatory disorder and exudation, and tissue proliferation. Causes include mechanical injury, physical factors such as temperature and radiation, chemical factors such as poisons, and parasites such as bacterial infections, among which bacteria are the most common. In addition to these main causes, the occurrence is complicated by various secondary factors and the individual's predisposition or immunity. In addition, the inflammatory response can be said to be one of the most important responses among pathological mechanisms according to the host's defense mechanism against wounds, microbial infections, and the like. Macrophages are the most representative immune cells regulating such inflammatory responses, and activated macrophages secrete various inflammatory mediators such as NO (nitric oxide) and ROS (reactive oxygen species). On the other hand, overexpression of these inflammatory mediators induces rheumatoid arthritis, osteoporosis, sepsis, vascular disease, cancer, and the like (Lawrence, T. et al., 2012).

Accordingly, the present inventors were able to complete the present invention by confirming that papiliosin-3, a novel peptide, has antibacterial, antifungal or anti-inflammatory effects while studying various physiological activities using peptides derived from swallowtail butterfly larvae. .

Korean Patent Publication No. 10-2010-0132825 (2010.12.20.)

Biochemical and Biophysical Research Communications 408 (2011) 89-93.

The present invention provides a peptide represented by the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides an antibacterial, antifungal or anti-inflammatory pharmaceutical composition comprising the peptide represented by the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides an antibiotic comprising the peptide represented by the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a cosmetic preservative comprising the peptide represented by the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a pharmaceutical preservative containing the peptide represented by the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising the peptide represented by the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a food composition for preventing or improving inflammatory diseases comprising the peptide represented by the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a feed additive comprising the peptide represented by the amino acid sequence of SEQ ID NO: 1.

The present invention provides a peptide represented by the amino acid sequence of SEQ ID NO: 1.

The peptide is a swallowtail butterfly ( Papilio xuthus ) may be a novel peptide isolated from larvae. These peptides can be represented by the amino acid sequence of SEQ ID NO: 1 below.

SEQ ID NO: 1: SMDYQFKLTILKAK-NH ₂

The peptide may exhibit antibacterial, antifungal and/or anti-inflammatory action effects.

In the present invention, the peptide of SEQ ID NO: 1 is SMDYQFKLTILKAK-NH ₂ , and is composed of 14 amino acids, which is named Papilocin-3.

In the present invention, swallowtail butterfly ( Papilio Xuthus ) is a lepidopteran insect and a member of the Swallowtail family. It is a medium-sized butterfly with a wingspan of 45-55 mm. There are black dots on a yellow background and beautiful stripes that develop along the grain. Its larvae feed on tangerine trees, Japanese pepper trees, tangerine trees, and hwangbyeok trees of the family Rutaceae. Larvae from 1st to 4th instar have a black body with a pattern that looks like a white band, and the larvae of the 5th instar, which is the species, are black. It turns green when it becomes green, and it is characterized by two black comb lines on both sides of the body.

In the present invention, antibacterial, antifungal, or anti-inflammatory means any action that inhibits the growth of bacteria and/or fungi in a subject, kills them, or reduces the activity of inflammation.

As used herein, the term "peptide" refers to a linear molecule formed by binding amino acid residues to each other by a peptide bond. The peptide may be prepared according to a chemical synthesis method known in the art, preferably according to a solid phase synthesis technique. In addition, the peptide is a swallowtail butterfly ( Papilio xuthus ) may be isolated from larvae.

The peptide according to the present invention may include an amino acid inserted or deleted in the amino acid sequence of SEQ ID NO: 1, as long as the antibacterial, antifungal and/or anti-inflammatory effect is not reduced. That is, this sequence is equivalent or similar to "biologically active", and 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 is also a peptide fragment according to the present invention. means peptide. 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, an "insertion" is a change in amino acid sequence compared to the wild-type peptide due to the addition of one or more amino acid residues.

In the present invention, a “deletion” is defined as a change in amino acid sequence that is missing one or more amino acid residues compared to the wild-type peptide.

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 characteristics, 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. 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. The cell-penetrating peptide is a kind of signal peptide, which is a peptide sequence that aims 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. It has an activity capable of cell internalization to mediate the movement of drug carriers.

In one embodiment of the present invention, a novel peptide having the amino acid sequence of SEQ ID NO: 1 was isolated from swallowtail butterfly larvae, named papiliocin-3, and the peptide was Escherichia coli, one of the Gram-negative bacteria. coli ) and Staphylococcus aureus , which is one of Gram-positive bacteria, antibacterial activity against, and antifungal activity against Candida albicans , which is one of the fungi, were confirmed. Anti-inflammatory activity was also confirmed to be excellent.

In the present invention, the term “gram-negative bacteria” refers to bacteria that are stained red when stained by the Gram staining method, and most of the cell walls of Gram-negative bacteria are thin (about 10 nm) and contain a large amount of lipopolysaccharide (LPS) on the outside. am. The gram-negative bacteria include, for example, Escherichia , Pseudomonas , Salmonella , Leptospira , Rickettsia , and the like, but are not limited thereto. In one embodiment of the present invention, Escherichia coli was used as a representative strain of Gram-negative bacteria.

In the present invention, the term "Gram-positive bacteria" is a bacterium whose cell wall is stained purple when stained by the Gram staining method, and the cell wall is composed of several layers of peptidoglycan. The Gram-positive bacteria include, for example, Staphylococcus , Listeria , Corynebacterium , Lactobacillus , Bacillus , and Propionibacterium . ), etc., but are not limited thereto. In one embodiment of the present invention, as representative strains of Gram-positive bacteria, Staphylococcus aureus and Propionbacterium acnes ( Propionibacterium acnes ) was used.

The gram-negative bacteria and gram-positive bacteria may be antibiotic-resistant bacteria, but are not limited thereto.

In the present invention, the term "fungi" refers to a group of microorganisms including molds, yeasts, and mushrooms, and belongs to eukaryotes having a nuclear membrane. In one embodiment of the present invention, Candida albicans was used as a representative strain of fungi.

In one aspect of the present invention, the present invention provides an antibacterial, antifungal or anti-inflammatory pharmaceutical composition comprising papilyocin-3, a peptide represented by the amino acid sequence of SEQ ID NO: 1.

In another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating inflammatory diseases containing papilyocin-3, which is a peptide represented by the amino acid sequence of SEQ ID NO: 1.

In the present invention, the inflammatory disease refers to a disease in which inflammation is the main lesion, and is not limited thereto, but is not limited thereto, and allergic diseases (eg, allergic asthma, allergic rhinitis, allergic mucositis, urticaria and anaphylax), myopathy (including but not limited to systemic sclerosis, dermatomyositis and inclusion body myositis), inflammatory skin disorders (including but not limited to) , eg atopic dermatitis, psoriasis), arthritis, inflammatory bowel disease, inflammatory collagen vascular disease, glomerulonephritis, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, arthritis, tonsillitis, sore throat, bronchitis, pneumonia, pancreatitis, sepsis, cystitis , nephritis and neuritis, etc.

In the present specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.

In the present invention, the antibacterial or antifungal pharmaceutical composition may be a composition having an activity of inhibiting the growth of microorganisms such as bacteria and fungi. In addition, the anti-inflammatory pharmaceutical composition may be a pharmaceutical composition having an activity of suppressing or removing the occurrence of inflammation, and may specifically suppress the occurrence of inflammation by inhibiting the production of nitric oxide.

The peptides of the present invention may include all forms used in various fields requiring antibacterial, antifungal or anti-inflammatory effects. In addition, for example, it may be in the form of pharmaceuticals, quasi-drugs, food additives or feed additives. Specifically, for purposes such as antibiotics, antifouling agents, and therapeutic agents in medicine, for antiseptic or antibacterial purposes in food, for antibacterial, sterilization, and disinfection purposes in agriculture, and for suppressing dandruff and preventing athlete's foot in cosmetics and household goods It can be used for products directly related to microorganisms, such as anti-armpit collection, anti-acne, etc., or used for preservative, antibacterial or sterilization purposes, such as cleaning detergents or dishwashing detergents, but is not limited to these purposes.

In addition, the pharmaceutical composition of the present invention may contain at least one known pharmaceutically acceptable active ingredient that exhibits the same or similar efficacy as the peptide.

In the present invention, the phrase "pharmaceutically acceptable" refers to molecular substances and compositions that, where appropriate, do not cause side effects, allergic reactions or other undesirable reactions when administered to animals, such as humans. In light of this disclosure, one skilled in the art will recognize the preparation of pharmaceutical compositions comprising antibodies or additional active components. It will also be appreciated that for animal (eg, human) administration, preparations must meet sterility, pyrogenicity, general safety and purity standards, as required by the FDA Office of Biological Standards.

The pharmaceutical compositions for use in the present invention may be formulated by standard techniques using one or more physiologically acceptable carriers or excipients. Suitable pharmaceutical carriers are disclosed herein and in Remington: The Science and Practice of Pharmacy, 21st Ed., University of the Sciences in Philadelphia, Lippencott Williams & Wilkins (2005).

In addition, the peptides of the present invention may be formulated for administration by any suitable route, for example via inhalation, topically, nasally, orally, parenterally or rectally. Therefore, administration of the aforementioned pharmaceutical composition can be administered intradermal, subcutaneous, intravenous, intramuscular, intranasal, by inhalation, intracerebral, intratracheal, intraarterial, intraperitoneal, intravesical, intrapleural, intracoronary, subcutaneous or by intratumoral injection, using a syringe or other device. Transdermal administration is also contemplated, as is inhalation or aerosol administration. Tablets and capsules may be administered orally, rectally or intravaginally.

The antibacterial, antifungal or anti-inflammatory pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Preferably, it may contain peptides dissolved in an aqueous carrier, and the peptides may be provided together or separately.

As the "pharmaceutically acceptable carrier", various aqueous carriers, such as buffered saline, can be used. These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium Lactate or the like may be contained. In addition, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. of additives can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component by using an appropriate method of sugar distribution or by using a method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA. The concentration of peptide in these formulations can vary widely and will be selected primarily on the basis of fluid volume, viscosity, body weight, etc., depending on the particular mode of administration selected and the needs of the patient.

The pharmaceutical composition of the present invention is suitable for parenteral administration, including intravenous administration or intracorporeal administration. The pharmaceutical composition of the present invention is suitable for parenteral administration, including intravenous administration or intracorporeal administration.

The peptides of the present invention may be formulated for parenteral administration by injection, eg bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are preferably prepared from fatty emulsions or suspensions. The composition may be sterilized and/or the composition may contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, dissolution promoters, salts for regulating osmotic pressure and/or buffers. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg sterile, pyrogen-free water, before use. In addition, the active ingredients may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods respectively, and they contain about 0.1 to 75%, preferably about 1 to 50% of the active ingredient.

For oral administration, the pharmaceutical composition or medicament may take the form of a tablet or capsule prepared, for example, by conventional means, together with pharmaceutically acceptable excipients. The active ingredient, i.e., the composition of the present invention, is mixed with (a) a diluent or filler such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose (eg ethyl cellulose, microcrystalline cellulose), glycine, pectin, polyacrylic salts and/or calcium hydrogen phosphate, calcium sulfate, (b) lubricants such as silica, talcum, stearic acid, its magnesium or calcium salts, metallic stearates, colloidal silicon dioxide, hydrogenated vegetable oils, corn starch, sodium with benzoate, sodium acetate and/or polyethylene glycol; for tablets also (c) binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone and/or hydroxypropyl methylcellulose; optionally (d) a disintegrant, for example starch (eg potato starch or sodium starch), glycolate, agar, alginic acid or its sodium salt, or effervescent mixtures; (e) wetting agents such as sodium lauryl sulfate, and/or (f) absorbents, colorants, flavors and sweeteners. Tablets and gelatin capsules are preferred.

The pharmaceutical composition of the present invention can be administered to a subject in a therapeutically effective amount to prevent, treat, or inhibit diseases caused by bacteria, fungi, or inflammation.

As used herein, "treatment" refers 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" refers 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 occurrence or recurrence of a disease or condition. refers to

In the present invention, "subject" refers to a human or non-human, such as a primate, mammal, or vertebrate. In certain embodiments, the subject is a human.

The pharmaceutical composition is administered to the patient in an amount sufficient to elicit an effective therapeutic or diagnostic response in the patient. An effective therapeutic or diagnostic response is one that at least partially arrests or slows the symptoms or complications of the disease or malignant condition. An amount adequate to accomplish this is defined as a “therapeutically effective amount”.

The dosage of the peptide to be administered varies depending on the species, body weight, age, individual condition of the mammal, the surface area of the area to be treated and the mode of administration. The size of the dose will also be determined by the presence, nature and extent of any side effects that accompany the administration of a particular compound in a particular patient.

Regarding the dosage of the peptide to be administered, a unit dose for administration to a mammal of about 50 to 80 kg, preferably a human, may contain an amount of about 1 mg/kg to 5 mg/kg of the peptide.

Typically, the dosage of the peptides of the invention is a dosage sufficient to achieve the desired effect. An optimal dosing schedule can be calculated from measurements of the peptide, accumulation in the patient's body. It may be given more than once per day, per week, per month or per year. Optimal dosages, administration methods and repetition rates can readily be determined by those skilled in the art. One skilled in the art will be able to determine the optimal dosage for administration of peptides to humans following established protocols known in the art and disclosed herein. However, it should be understood that the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the weight, age and sex of the patient, and therefore, the dosage is It does not limit the scope of the present invention in any way.

The present invention provides the use of the peptide represented by 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 a peptide having the amino acid sequence of SEQ ID NO: 1 to a subject in need thereof.

In one aspect, the present invention provides a food composition for preventing or improving inflammatory diseases comprising the peptide represented by the amino acid sequence of SEQ ID NO: 1.

According to the present invention, there is provided a health functional food for preventing or improving inflammatory diseases comprising the peptide represented by the amino acid sequence of SEQ ID NO: 1.

According to the present invention, the health functional food may be for preventing or improving inflammatory diseases.

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 include the peptide represented by the amino acid sequence of SEQ ID NO: 1 in an amount of 0.01 to 95% by weight, 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 tablet form is obtained by granulating a mixture of the peptide represented by the amino acid sequence of SEQ ID NO: 1, an excipient, a binder, a disintegrant, and other additives in a conventional manner, and then adding a lubricant, etc. It can be compression molded by putting it, 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.

Hard capsules among health functional foods in the form of capsules can be prepared by filling ordinary hard capsules with a mixture of additives such as the peptide represented by the amino acid sequence of SEQ ID NO: 1 and excipients, or their granular material or coated granular material. Soft capsules can be prepared by filling a mixture of the peptide represented by the amino acid sequence of SEQ ID NO: 1 with 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 the peptide represented by the amino acid sequence of SEQ ID NO: 1, excipients, binders, disintegrants, etc. in an appropriate way, and if necessary, cover with sucrose or other suitable coating agent. , or it may be coated with starch, talc, or a suitable material.

The granular health functional food can be granulated by appropriately preparing a mixture of the peptide represented by the amino acid sequence of SEQ ID NO: 1, excipients, binders, disintegrants, etc., and, if necessary, flavoring agents, flavoring agents, etc. can do. 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 the peptide represented by the amino acid sequence of SEQ ID NO: 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 the peptide represented by the amino acid sequence of SEQ ID NO: 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 based on a total of 100 g, Preferably, it may contain 0.3 to 1 g.

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 restriction on the components added other than the peptide represented by the amino acid sequence of SEQ ID NO: 1, and various flavors or natural carbohydrates are added as in conventional beverages. It can be contained as a component. 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 extracts such as rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. .

In addition to the above, the food composition or health functional food containing the peptide represented by the amino acid sequence of SEQ ID NO: 1 is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and heavy substances (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. there is.

In another aspect, the present invention provides an antibiotic comprising the peptide.

The antibiotic refers to an agent that is provided to an individual in the form of a drug to kill bacteria, and may be used in combination with a disinfectant and an antibacterial agent.

In another aspect, the present invention provides a food preservative, cosmetic preservative or pharmaceutical preservative containing the peptide represented by the amino acid sequence of SEQ ID NO: 1.

The food preservative, cosmetic preservative, or pharmaceutical preservative may have antibacterial, antifungal, or anti-inflammatory effects.

At this time, food preservatives, cosmetic preservatives, and pharmaceutical preservatives are additives used to prevent deterioration, decay, discoloration, and chemical change of food or medicines, and may include bactericides and antioxidants, and may include microorganisms such as bacteria, fungi, and yeast. It also includes functional antibiotics such as inhibiting the growth of spoilage microorganisms or sterilizing action in foods and medicines by inhibiting proliferation. Ideal conditions for such food preservatives, cosmetics, and pharmaceutical preservatives should be non-toxic and effective even in small amounts.

Specifically, the food preservative of the present invention may be prepared by including one or more of known food preservatives. As food preservatives, dehydroacetic acid, potassium sorbate, calcium sorbate, sodium benzoate, potassium benzoate, calcium benzoate, methyl paraoxybenzoate, propyl paraoxybenzoate, sodium propionate, calcium propionate, etc. are used, but are not limited thereto.

The food preservative of the present invention preferably contains 0.01 to 1% by weight of the peptide based on the total weight of the food preservative, but is not limited thereto.

In addition, the cosmetic preservative or pharmaceutical preservative of the present invention means that it is used for preservation of cosmetics or pharmaceuticals.

The cosmetic preservative may be prepared in the form of a general emulsified formulation and a solubilized formulation. Emulsified cosmetics include nutrition lotion, cream, essence, etc., and solubilized cosmetics include softening lotion. In addition, the cosmetic preservative of the present invention can be prepared in the form of an adjuvant that can be applied topically or systemically, which is commonly used in the field of dermatology, by containing a dermatologically acceptable medium or base other than the peptide. In addition, suitable cosmetic formulations include, for example, solutions, gels, solid or kneaded anhydrous products, emulsions obtained by dispersing an oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic forms ( liposome), non-ionic vesicular dispersion, cream, toner, lotion, powder, ointment, spray or consil stick. It can also be prepared in the form of a foam (foam) or the form of an aerosol composition further containing a compressed propellant. In addition, as excipients used in cosmetic preservatives, arbutin, kojic acid, rucinol, vitamin C, and vitamin C derivatives may be additionally contained, and retinol may be used for the purpose of reducing wrinkles. (retinol) and its derivatives, indoleacetic acid and its derivatives, adenosine, tocopherol and its derivatives, cainitine, and the like may be further contained. In addition, the excipients may include components commonly used in cosmetic preservatives, and include, for example, stabilizers, solubilizers, vitamins, pigments, and flavors, and conventional adjuvants and carriers.

The cosmetic preservative of the present invention preferably contains 0.01 to 1% by weight of the peptide based on the total weight of the cosmetic preservative, but is not limited thereto.

In addition, in the present invention, the feed additive is appropriately mixed with the feed raw material and provided as a feed. As the feed raw material, grain oil, crude steel, vegetable oil meal, animal feed raw material, other feed raw materials, refined products, etc. are used, but are limited thereto. It doesn't work. The feed may further include conventional additives capable of increasing the preservability of the feed in addition to the peptide of the present invention.

In addition, other non-pathogenic microorganisms may be additionally added to the feed additive of the present invention. Microorganisms that can be added include Bacillus subtilis, which can produce proteolytic enzymes, lipolytic enzymes, and glycoconverting enzymes, such as Bacillus subtilis ; Strains ( Lactobacillus sp.), filamentous fungi such as Aspergillus oryzae and Saccharomyces cerevisiae , which show the effect of increasing the weight of livestock, increasing the milk yield of milk and increasing the rate of digestion and absorption of feed ) and may be selected from the group consisting of yeast.

In addition, the feed additive of the present invention may include binders, emulsifiers, preservatives, etc. added to prevent quality deterioration, and amino acids, vitamins, enzymes, probiotics, flavors, and non-protein forms added to feed to increase efficacy Nitrogen compounds, silicate agents, buffers, coloring agents, extractants, oligosaccharides, etc. may be included, and in addition, a feed mixture may be further included.

Matters mentioned in the use, composition, and treatment method of the present invention are equally applied unless contradictory to each other.

Since the peptide represented by the amino acid of SEQ ID NO: 1 of the present invention has little toxicity and excellent antibacterial, antifungal and anti-inflammatory activity, it can be used as a medicine, food, antibiotic, cosmetic preservative, pharmaceutical preservative, etc.

1 shows the results of analyzing the antibacterial and antifungal activity of the papiliocin-3 peptide according to the present invention.
Figure 2 shows the results of analyzing the cytotoxicity of the papiliocin-3 peptide according to the present invention.
Figure 3 shows the results of analyzing the cell viability of the papiliocin-3 peptide according to the present invention through the MTS Assay.
Figure 4 shows the results of analyzing the NO (nitric oxide) secretion inhibitory activity in order to confirm the anti-inflammatory activity effect of the papiliocin-3 peptide according to the present invention.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the content of the present invention is not limited by the examples.

In the present invention, the present invention was completed by confirming that papilyocin-3, a peptide represented by the amino acid sequence of SEQ ID NO: 1, has excellent antibacterial, antifungal or anti-inflammatory effects. Below, we look into this in detail.

< Example 1> Origin of swallowtail butterfly larvae peptide gene selection

In order to select peptides exhibiting antibacterial, antifungal or anti-inflammatory activity from swallowtail butterfly larvae, the following experiments were performed.

First, in the immunization of swallowtail butterfly larvae, E. coli , S. aureus , and C. albicans were first cultured in Tryptic Soy Broth (TSB) liquid medium at 37°C and 200 rpm in a shaking incubator for 18 hours, and then again under the same conditions. After secondary incubation for 2 hours and 30 minutes, wash with 10 mM sodium phosphate buffer (pH 7.4), and use a microinjector to take cells so that the number of cells is 5 × 10 ³ colony forming units (CFU) and injected into the body cavity, and incubated at 25° C. for 18 hours to induce immunization.

In order to maximize the induced expression of bioactive substances, RNA-seq was performed using total RNA of normal swallowtail butterfly larvae and immunized swallowtail butterfly larvae. First, normal swallowtail butterfly larvae and immunized swallowtail butterfly larvae were prepared, and in order to keep the tissues as fresh as possible, they were rapidly frozen in liquid nitrogen, prepared by grinding in a mortar, and then mixed with 100 mg of the sample and 1 ml of TRIZOL for 5 minutes. It was reacted at room temperature. 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 quality using an Agilent 2100 Bioanalyzer.

For RNA-seq analysis using the RNA obtained in this way, RNA-seq analysis was performed on unimmunized swallowtail butterfly larvae and immunized swallowtail butterfly larva samples using an Illumina HiSeq™ 2000 instrument, and as a result, genes presumed to be new physiologically active peptides was selected.

The selected amino acid sequence was SMDYQFKLTILKAK-NH ₂ , which consisted of 14 amino acids, and was named Papilocin-3 (Table 1).

peptide peptide amino acid sequence Papiliocin-3 SEQ ID NO: 1: SMDYQFKLTILKAK-NH ₂

< Example 2> Papiliocin -3 of peptides Antibacterial and antifungal activity assay

The antibacterial activity assay for the papiliocin-3 peptide selected in Example 1 was confirmed using a radial diffusion assay (RDA) test method. In the RDA method, bacteria cultured in a sterile underlay gel composed of citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH7.4), 1% (w/v) type I (low electroendosmosis) agarose, and 0.03% TSB (4 × 10 ⁶ colony forming units) were added, mixed, poured into a square plate, and when the underlay gel hardened, a hole with a diameter of 3 mm was made and 5 μl of peptides were loaded into the hole at each concentration. After incubation at 37°C for 3 hours to uniformly diffuse the peptide, 10 ml of overlay agar (6% TSB, 1% agarose) was poured and incubated again at 37°C to confirm the clear zone and measure the diameter. The strain is Escherichia coli, which is a representative strain of Gram-negative bacteria. Coli ) , Staphylococcus aureus , a representative strain of Gram-positive bacteria, and Candida albicans , a pathogenic fungus, were used as representatives. The results are shown in Figure 1.

As shown in Figure 1, as a result of confirming the antibacterial and antifungal activity against Escherichia coli, Staphylococcus aureus and Candida fungi against papiliocin-3 by RDA experiment, it was confirmed that there was antibacterial and antifungal activity against all of the test strains, which was confirmed at the concentration It was confirmed that it increased dependently.

< Example 3> Papiliocin -3 of peptides Cytotoxicity assay

A cytotoxicity assay was performed on rat red blood cells of the papiliocin-3 peptide selected in Example 1.

Specifically, in order to measure cytotoxicity to rat red blood cells, rat erythrocytes were washed three times with phosphate-sodium chloride buffer, pH=7.4 (PBS), diluted with phosphate-sodium chloride buffer, and then diluted to 5.0% A red blood cell solution of was prepared. Thereafter, 100 μl of the 5.0% red blood cell solution was dispensed into the tube, and papiliocin-3 was treated in a concentration-dependent manner. 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 0.1% 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 power = (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

The results of the cytotoxicity test on rat red blood cells of the papiliocin-3 peptide selected in Example 1 analyzed by the above method are shown in FIG. 2 .

As shown in FIG. 2, it was confirmed that papiliocin-3 had little ability to destroy red blood cells when treated at concentrations up to 200 μg/ml. On the other hand, it was found that melittin, a strong antibacterial peptide as a control, has high cytotoxicity even at low concentrations.

These results indicate that the papiliocin-3 peptide according to the present invention exhibits antibacterial and antifungal activity in the absence of cytotoxicity.

< Example 4> Papiliocin -3 of peptides Cell viability measurement

MTS assay was performed to confirm the cell viability of the papiliocin-3 peptide selected in Example 1.

Specifically, the experiment was conducted using Raw264.7 cells, which are mouse macrophages mainly used for anti-inflammatory-related evaluation. 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.

After dispensing 2 × 10 ⁴ cell/well cells in 100 μl each in a 96-well culture vessel, the papilyocin-3 peptide 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). And the results are shown in Figure 3.

As shown in FIG. 3, it was confirmed that the cell viability of the papiliocin-3 peptide of the present invention was 95% or higher regardless of the concentration. Accordingly, it was confirmed that the papiliocin-3 peptide was not toxic to Raw264.7 cells up to the measured maximum concentration of 200 μg/ml.

< Example 5> Papiliocin -3 of peptides Anti-inflammatory activity assay (Nitric oxide inhibitory ability measurement)

In order to measure the anti-inflammatory activity of the papiliosin-3 peptide according to Example 1, the amount of NO secretion was confirmed in Raw264.7 cells. Since Raw264.7 cells are known to produce NO and secrete it extracellularly when inflammation is induced, the amount of extracellular secretion of NO was confirmed through experiments.

Specifically, each 8 × 10 ⁴ cell / ml cell number was prepared in 96-well, and the peptide was treated for 1 hour at each concentration. Thereafter, 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 using the NO detection kit (intron), how much the amount of NO produced by LPS is reduced by the peptide was measured by using a multi detector (Beckman DTX8800) to measure the absorbance at 550 nm wavelength. It was confirmed through measurement. The results are shown in FIG. 4 .

As can be seen in Figure 4, it was confirmed that the amount of NO secretion decreased when the papilyocin-3 peptide according to Example 1 was treated.

From the above results, it can be seen that the papiliosin-3 peptide according to the present invention exhibits excellent antibacterial, antifungal and anti-inflammatory activities in the absence of cytotoxicity. It is suggested that it can be used as a red composition, antibiotic, food preservative, cosmetic preservative, medicine, etc.

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are merely preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION <120> PEPTIDE, PAPILIOCIN-3 DERIVED FROM PAPILIO XUTHUS ORIENTALIS AND USES THEREOF <130> 1131 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 14 <212> PRT <213> artificial sequence <220> <223> PAPILIOCIN-3 <400> 1 Ser Met Asp Tyr Gln Phe Lys Leu Thr Ile Leu Lys Ala Lys 1 5 10

Claims (13)

Peptide represented by the amino acid sequence of SEQ ID NO: 1:
SEQ ID NO: 1: SMDYQFKLTILKAK-NH _2. The method of claim 1, wherein the peptide has at least one activity selected from the group consisting of antibacterial and anti-inflammatory, and the antibacterial activity is an activity against Escherichia Coli or Staphylococcus aureus. , peptide. According to claim 1,
The peptide is a swallowtail butterfly ( Papilio xuthus ) Peptide, derived from larvae. Claims 1 to 3 of the antibacterial or anti-inflammatory pharmaceutical composition comprising any one of the peptides. An antibiotic comprising the peptide of any one of claims 1 to 3. A food preservative comprising the peptide of any one of claims 1 to 3. A cosmetic preservative comprising the peptide of any one of claims 1 to 3. A pharmaceutical preservative comprising the peptide of any one of claims 1 to 3. A pharmaceutical composition for preventing or treating inflammatory diseases comprising the peptide of any one of claims 1 to 3, wherein the inflammatory diseases include dermatitis, arthritis, inflammatory bowel disease, inflammatory collagen vascular disease, glomerulonephritis, sarcoidosis, and retinitis. , Gastritis, hepatitis, enteritis, tonsillitis, sore throat, bronchitis, pneumonia, pancreatitis, sepsis, cystitis, nephritis and neuritis, which is selected from the group consisting of, a pharmaceutical composition. delete A health functional food composition for anti-inflammatory comprising the peptide of any one of claims 1 to 3. delete A feed additive comprising the peptide of any one of claims 1 to 3.

Images