KR20220050680A - Composition for preventing or treating of neuroinflammatory disease comprising Protaetiamycine-6 isolated from Protaetia brevitarsis seulensis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating neuroinflammatory diseases comprising the peptide protaetiamycin-6 having an amino acid sequence of PKARKLQKLSAYKTTLRN-NH_2. The protaetiamycin-6 according to the present invention has no side effects, excellent safety, and excellent anti-inflammatory efficacy, thereby being used as a preventive or therapeutic agent for neuroinflammatory diseases, a health food supplement, and a feed composition.

Description

TECHNICAL FIELD [0001] Composition for preventing or treating neuroinflammatory disease comprising Protaetiamycine-6 isolated from Protaetia brevitarsis seulensis

The present invention relates to a composition for preventing or treating neuroinflammatory diseases containing protetiamicin-6, and more particularly, to a neuroinflammatory disease comprising protetiamicin-6, a peptide derived from white spotted radish. It relates to a composition for prevention or treatment.

The central nervous system consists of neurons and glial cells. Glia cells account for about 90% of the total brain cells, and by volume account for about 50% of the total brain cells. Glia cells can be further classified into three types: astrocytes, microglia, and oligodendrocytes. Among them, microglia are a kind of differentiated macrophages, and are widely distributed in the brain. Microglia not only act as phagocytes that engulf tissue debris and dead cells, but also play a role in participating in the brain's biodefense activities. On the other hand, astrocytes are the most abundant cell type in the central nervous system and are activated in the presence of neuroinflammatory stimuli, and protein expression is changed.

Neuroinflammation is a type of immune response of the nervous system, and is closely related to many neurodegenerative diseases including Alzheimer's, Parkinson's, and multiple sclerosis, and is currently considered a typical characteristic of neurodegenerative diseases. Neuroinflammatory responses include activation of innate immune cells (microglia), release of inflammatory mediators such as nitric oxide (NO), cytokines and chemokines, and macrophage infiltration, leading to neuronal cell death. Inflammatory activation of microglia and astrocytes is thought to be a pathological marker and an important mechanism in the progression of neurodegenerative diseases. Tight regulation of microglia activity is essential for maintaining brain homeostasis and preventing infectious and inflammatory diseases.

Recently, interest in the pharmacological activity of insects is increasing, and many studies have been attempted to treat neuroinflammatory diseases. Korean Patent No. 10-1730065 discloses the anti-inflammatory activity of an insect-derived natural resource, grasshopper radish extract, and Korean Patent No. 10-1700605 discloses the anti-inflammatory activity of a beetle extract. No. 10-066500 discloses anti-inflammatory activity of a ladybug extract. However, there is a need for research on new therapeutic agents as no commercially available substances have been developed and their effects have been clearly verified so that they can be applied to a wide range of neuroinflammatory diseases.

White-spotted radish ( Protaetia ) brevitarsis seulensis ) is an insect belonging to the Coleoptera family, and it inhabits Korea, Japan, and Russia.

The present inventors completed the present invention by confirming that the peptides derived from white-spotted radish radish have excellent anti-inflammatory effects, especially anti-inflammatory effects on neuroinflammation, while studying various physiological activities using the white-spotted radish-derived peptides. did

The first problem to be solved by the present invention relates to a peptide protetiamycin-6 (Protaetiamycine-6) having the amino acid sequence of SEQ ID NO: 1 and a pharmaceutical composition for preventing or treating neuroinflammatory diseases including the same.

SEQ ID NO: 1: PKARKLQKLSAYKTTLRN-NH ₂

The second problem to be solved by the present invention relates to a food composition, feed composition or health functional food for the prevention or improvement of neuroinflammatory diseases comprising the peptide protethiamicin-6 having the amino acid sequence of SEQ ID NO: 1.

The present invention is white spotted radish ( Protaetia ) brevitarsis seulensis ) and its use.

In order to solve the above problems, the present invention provides a peptide represented by SEQ ID NO: 1 synthesized from a gene isolated from a transcript of Protaetia brevitarsis seulensis .

SEQ ID NO: 1: PKARKLQKLSAYKTTLRN-NH ₂

The method for providing the peptide according to the present invention is as follows. White Spotted Radish ( Protaetia brevitarsis) seulensis ) In order to confirm the derived peptide gene, first, the whole RNA is isolated by rapidly freezing the white-spotted radish in liquid nitrogen, and the genetic information about the white-spotted radish transcriptome is confirmed using the RNA seq analysis method. and a method of selecting a candidate through an anti-inflammatory activity test based on the sequence of amino acids translated from each transcript.

In the present invention, "peptide", and "protein" are used interchangeably and include reference to a polymer 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 acid. The terms also apply to those polymers containing conservative amino acid substitutions such that the protein remains functional. In the present invention, the peptide may be synthesized using a gene recombination and protein expression system, preferably synthesized in vitro through a peptide synthesizer or the like.

In the present invention, "biologically active" refers to 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, and also 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%, Sequences having at least 98%, or at least 99% sequence homology and retaining biological activity may be included within the scope of the present invention.

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

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

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

On the other hand, preferred amino acid mutation at the substitution position may be performed based on the similarity of residues in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphiphilic properties. 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 an uncharged polar head group having a similar hydrophilicity value 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' refers to a substitution in which an amino acid is substituted with another amino acid having similar properties so that those skilled in the art can predict that the secondary structure and hydropathic nature (hydropathic 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 (phosphorylation), sulfation (sulfation), acrylation (acrylation), glycosylation (glycosylation), methylation (methylation), farnesylation (farnesylation), acetylation (acetylation) and amylation (amidation) It can also be modified (modification) and the like.

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 and is a peptide sequence for the purpose of delivering a substance into a cell. It mainly consists of a peptide sequence of about 7-30 amino acids, and any sequence having a signal peptide that can be delivered into a cell may be included in the present invention.

For example, the cell-penetrating peptide mainly contains basic amino acid residues such as lysine/arginine, and thus serves to penetrate the fused proteins into the cell membrane and into the cell. The cell-penetrating peptide is HIV-1 Tat protein, Drosophila antennapedia homeodomain (phenetratin), HSV VP22 transcriptional regulatory protein, vFGF-derived MTS peptide, PTD-5, transpotan or Pep-1 peptide Sequences derived from, but are not limited thereto. As such, various CPPs identified/synthesized from viruses or cationic peptides (eg, TAT48-60, penetratin (pAntp) 43-58, polyarginine, Pep-1, transpotan, etc.) are biologically active substances. has an activity capable of internalization of cells to mediate the movement of drug carriers.

The amino acid sequence of the peptide consists of 18 amino acids as PKARKLQKLSAYKTTLRN-NH ₂ , and is named as Protaetiamycine-6.

According to the present invention, the peptide protects nerve cells from oxidative stress, can inhibit apoptosis of nerve cells, and exhibits anti-neuroinflammatory activity.

In addition, the present invention provides a pharmaceutical composition for preventing or treating neuroinflammatory diseases comprising the peptide protetiamycin-6 (Protaetiamycine-6) having the amino acid sequence of SEQ ID NO: 1.

The neuroinflammatory disease according to the present invention is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis, neuroblastoma, stroke, depression, schizophrenia, post-traumatic stress disorder and amyotrophic lateral sclerosis. It may be any one or more selected.

The peptide protetiamicin-6 according to the present invention inhibits the expression of neurotoxin-related inflammatory cytokines TNF-α and IL-6 generated in microglia, and the protein cyclooxygenase-2 (COX-2 ) and inducible nitric oxide synthase (iNOS), and may inhibit nitric oxide production.

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 the benefit of treatment of 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 onset or recurrence of a disease or condition.

In the present invention, “subject” and “patient” refer to a human or non-human, such as a primate, mammal, or vertebrate. 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 in connection with the medical treatment of such condition. This includes, but is not limited to, a decrease in the frequency or severity of signs or symptoms of a disease.

The protethiamicin-6 according to the present invention may be added to the pharmaceutical composition of the present invention in an amount of 0.001 to 95% by weight. The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., 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, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient, for example, starch, calcium carbonate to the peptide protethiamicin-6 of the present invention. , sucrose or lactose, gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can 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. Dosage determination based on these factors is within the level of one of ordinary skill 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 above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention may be administered to mammals such as mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection. The pharmaceutical composition of the present invention is suitable for parenteral administration, including intravenous or body cavity administration.

The present invention provides the use of the peptide protethiamicin-6 having the amino acid sequence of SEQ ID NO: 1 in the manufacture of a medicament for the treatment of a neuroinflammatory disease.

The present invention provides a method for treating a neuroinflammatory disease, comprising administering to a subject in need thereof a therapeutically effective amount of the peptide protethiamicin-6 having the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a food composition for preventing or improving neuroinflammatory diseases comprising the peptide protetiamicin-6 having 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 neuroinflammatory diseases comprising the peptide protethiamicin-6 having the amino acid sequence of SEQ ID NO: 1.

According to the present invention, the health functional food is a neuroinflammatory disease such as multiple sclerosis, neuroblastoma, stroke, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, schizophrenia and muscular atrophy It may be for preventing or improving any one disease selected from the group consisting of lateral sclerosis.

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

The term "health functional food" means a food manufactured and processed using raw materials or ingredients useful for the human body in accordance with the Health Functional Food Act, and "functionality" refers to the structure and function of the human body. It refers to ingestion for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological effects.

The items listed in the "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; Mixed preparations such as sodium L-glutamate preparation, noodle-added alkali agent, preservative agent, and tar color agent can be mentioned.

The food composition of the present invention may contain 0.01 to 95% by weight of protethiamicin-6, 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 manufactured 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 granulated with a mixture of protethiamicin-6, excipients, binders, disintegrants, and other additives in a conventional manner, and then compression-molded by adding a lubricant or the like. , the mixture can be directly compression molded. In addition, the health functional food in the form of tablets may contain a mating agent, etc., if necessary, and may be coated with a suitable skinning agent if necessary.

Among health functional foods in the form of capsules, hard capsules can be prepared by filling conventional hard capsules with a mixture of protethiamicin-6 and additives such as excipients, or their granules or coated granules. can be prepared by filling a mixture of protethiamicin-6 and additives such as excipients in a capsule base such as gelatin. The soft capsule formulation may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

A health functional food in the form of a ring can be prepared by molding a mixture of protethiamicin-6, excipients, binders, disintegrants, etc. in an appropriate way, and if necessary, the skin is coated with sucrose or other suitable skinning agent, or starch, talc Alternatively, the gown may be dressed with an appropriate material.

A health functional food in the form of granules can be prepared in a granular form by a suitable method of a mixture of protethiamicin-6, an excipient, a binder, a disintegrant, and the like, and may contain a flavoring agent, a flavoring agent, etc. if necessary. For health functional foods in granular form, use No. 12 (1680 μm), No. 14 (1410 μm), and No. 45 (350 μm) sieves to pass all the sieves in the next particle size test, and the remaining amount in No. 14 sieves 5.0% or less and passing through No. 45 may be less than 15.0% of the total amount.

The term definitions for the excipients, binders, disintegrants, lubricants, flavoring agents, flavoring agents, etc. are described in documents known in the art and include those having the same or similar functions (Explanation of the Korean Pharmacopoeia, Moonseongsa, Korea) Association of Colleges of Pharmacy, 5th ed., p33-48, 1989).

There is no particular limitation on the type of the food. Examples of foods to which protethiamicin-6 of the present invention can be added include beverages, gums, vitamin complexes, drinks, and the like, and include all health functional foods in a conventional sense.

The food composition may contain food supplement additives, and the food products include, for example, beverages, gum, tea, vitamin complexes, health supplements, etc., capsules, tablets, powders, granules, liquids, pills, slices, pastes. It can be used in the form of phases, syrups, gels, beverages, jellies or vines.

The content of protethiamicin-6 may be contained in an amount of 0.01 to 15% by weight based on the total weight of the health functional food, and in the case of a health beverage composition, 0.02 to 10 g, preferably 0.3 to 10 g, based on a total of 100 g It may contain 1 g.

The food supplement additives include food additives conventional 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 ingredient in the indicated ratio, and there is no particular limitation on the ingredients added other than the protethiamicin-6. can 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 described above, natural flavoring agents (taumatine, stevia extract (eg rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. there is.

In addition to the above, food compositions or health functional foods containing protethiamicin-6 are various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.

In addition, the present invention provides a feed composition for preventing or improving neuroinflammatory diseases comprising protethiamicin-6.

In the present invention, the feed may be a feed for fish, birds or mammals, and preferably, it is suitable for breeding as its wild habits are acclimatized, as defined in Article 2 No. 1 of the Livestock Act and Article 2 of the Enforcement Rule of the same Act. and may be feed for livestock or aquatic organisms that can contribute to the increase of farm household income. The livestock may include cattle, horses, mules, donkeys, goats, goats, sheep, deer, pigs, rabbits, poultry, etc., and the poultry includes chickens, turkeys, ducks, ostriches, geese, quails, etc., preferably chickens, If it is suitable for breeding and obtaining livestock products, it is not limited thereto. The above "livestock products" are meat, milk, eggs, honey and their processed products, raw hides (including raw fur), raw wool, and other livestock products, which are defined by Article 2, 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, a 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 a component of said meal, intended for or suitable for being eaten, consumed, and digested by an animal. In one aspect, the composition for feed containing protethiamicin-6 of the present invention may include a concentrated feed, roughage and/or special feed.

In the enriched feed, seed fruits containing grains such as wheat, oats, and corn, bran containing rice bran, bran, barley bran, etc. Fish-soluble, meat powder, which is a concentrated product of fish meal, fish waste, and fresh liquids obtained from fish meal, fish waste, and residual starch, which is the main component of starch residue, which is the remainder of starch residue obtained by subtracting starch from sweet potatoes and potatoes. , animal feed such as dried whey, yeast, chlorella, seaweed, etc., which are dried whey, which is the remainder of the production of casein from milk, cheese, and casein from skim milk.

For roughage, raw grass feed such as wild grasses, grasses, and green cuttings, turnips for feed, beets for feed, root vegetables such as ruther beargers, a type of turnip, raw herbs, green crops, grains, etc. are placed in a silo. There are silage, which is stored feed fermented with lactic acid, wild grass, hay dried after cutting grass, straw for breeding crops, and leaves from legumes. Special feed includes mineral feed such as oyster shells and rock salt, urea feed such as urea or its derivative diureide isobutane, and supplementation of ingredients that are likely to be lacking when only natural feed ingredients are mixed, or added to formulated feed to increase feed storage. There are feed additives, which are substances added in trace amounts.

The peptide protethiamicin-6 according to the present invention suppresses the expression of inflammatory cytokines TNF-α and IL-6, and the proteins cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) ), and has excellent anti-inflammatory efficacy by inhibiting nitric oxide production, so it can be used as a preventive or therapeutic agent for neuroinflammatory diseases, health food supplements and feed compositions.

1 is a result of assaying the cytotoxicity of protetiamicin-6 according to an embodiment of the present invention.
2 is a result of assaying the nitric oxide inhibitory efficacy of protethiamicin-6 according to an embodiment of the present invention.
3 is qRT of gene expression inhibition of inducible nitric oxide synthase (iNOS) and inflammatory mediator cyclooxygenase-2 (COX-2) of protethiamicin-6 according to an embodiment of the present invention, respectively; -This is the result confirmed by PCR (A: iNOS, B: COX-2).
Figure 4 is a western protein expression inhibitory effect of protetiamycin-6, an inflammatory mediator cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), according to an embodiment of the present invention; This is the result confirmed by the blot method.
5 is a result confirming the inhibitory effect on inflammatory cytokine expression of protetiamycin-6 according to an embodiment of the present invention.

Hereinafter, preferred examples are presented to help the understanding of the present invention, but the following examples are merely illustrative of the present invention, and it will be apparent to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention, It goes without saying that such variations and modifications fall within the scope of the appended claims.

< Example 1. Peptide having the amino acid sequence of SEQ ID NO: 1 Preparation of Protaetiamycine -6>

experimental insect

White-spotted flower radish ( Protaetia ) brevitarsis seulensis ) was bred in the insect breeding building of the National Academy of Agricultural Sciences, using fermented sawdust as a food source, and was bred in a photoperiod of 16L:8D under conditions of 28℃ and 70% relative humidity.

gene selection

In order to identify the peptide gene exhibiting anti-inflammatory activity from the white-spotted radish, the total RNA was isolated by rapidly freezing the white-spotted radish with liquid nitrogen, and the transcriptome of the white-spotted radish was prepared using RNA seq analysis. Genetic information was checked, and the entire Unigene was filtered based on the existing antibacterial peptide properties based on the amino acid sequence translated from each transcript, and as a result, genes presumed to be anti-inflammatory peptides did The selected amino acid sequence is PKARKLQKLSAYKTTLRN-NH ₂ , and consists of an 18 amino acid sequence, and was named as Protaetiamycine-6.

protetiamicin -6 synthesis

To synthesize the protethiamicin-6 peptide, first, the peptide was prepared using the liquid-solid-phase method of Merrifield (Merrifield, RB, J Am Chem Soc, 85, 2149, 1963) using an Fmoc amino group protective container. prepared. The peptide synthesis method was a solid phase method using Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group of the Nα-amino group of an amino acid.

Specifically, Rink Amide MBHA-Resin was used as a starting material for a peptide having a carboxyl terminus of -NH ₂ , and Fmoc-amino acid-Wang Resin was used as a starting material for a peptide having a carboxyl terminus of -OH. The elongation of the peptide chain by Fmoc-amino acid coupling was performed by DCC (N-hydroxybenzo-triazole (HOBt)-dicyclohexylcar-bodiimide) method.

After coupling the Fmoc-amino acid at the amino terminus of each peptide, the Fmoc group was removed with 20% piperidine/N-methylpyrrolidone (NMP) solution and washed several times with NMP and dichloromethane (DCM), followed by nitrogen dried with gas

To this, a mixed solution of TFA (trifluoroacetic acid):phenol:thioanisole:H ₂ O:triisopropylsilane (85:5:5:2.5:2.5, v:v:v:v:v) was added and 3 After reacting for a period of time to remove the protecting group and separate the peptide from the resin, the peptide was precipitated with diethyl ether. The crude peptide thus obtained was subjected to an acetonitrile gradient containing 0.1% TFA and purified by reverse phase-HPLC column (Delta Pak, C18 300 Å, 15 μm, 19.0 mm × 30 mm). , Waters) was used for purification.

After hydrolysis of the synthetic peptide with 6N HCl at 110° C. for 24 hours, the obtained residue was concentrated under reduced pressure, dissolved in 0.02N HCl, and the amino acid composition was measured with an amino acid analyzer (Hitachi 8500 A). In addition, the molecular weight was calculated based on the sequence of the synthesized peptide, and the exact molecular weight was measured using the MALDI mass spectrometer (matrix-assisted laser desorption ionization mass spectrometer).

As a result, since the measured molecular weight and the calculated molecular weight were identical, it was confirmed that an antibacterial peptide having an accurate amino acid sequence was synthesized.

< test example 1. Cell viability test>

cell culture

BV-2 cells, which are mouse-derived microglia, were prepared using Dublecco's Modified Eagle Medium (DMEM, Gibco) medium containing 5% fetal bovine serum (FBS) and 50 μg/ml gentamicin at 37°C, 5% CO2. ₂ incubator (Incubator; Thermo Scientific, IL, USA).

Cell viability measurement (MTS assay)

In order to confirm the cytotoxicity of protethiamicin-6 to BV-2 cells, which are microglai, BV-2 cells were aliquoted in a 96-well plate at 2×10 ⁴ cells/well for 24 hours. cultured. Next, protethiamicin-6 was treated at different concentrations (0, 5, 10, 20, 50, 80 μg/ml), and after further incubation for 24 hours, MTS ((3-(4,5-dimethyl Cell viability was measured using thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reagent. DTX8800, CA, USA) was measured at 490 nm CTR: control (non-treated sample).

As shown in FIG. 1 , it was confirmed that all treatment groups (5, 10, 20, 50, 80 μg/ml) treated with protethiamicin-6 did not exhibit cytotoxicity compared to the control group. Then, in the neuroinflammation inhibitory efficacy experiment, it was used at a concentration of 80 μg/ml or less, which does not show cytotoxicity.

< test example 2. Neuroinflammation Inhibitory Activity Assay>

nitric oxide deterrent black

Nitric oxide (NO), a kind of reactive oxygen species, is produced by LPS, interferon-gamma (IFN-γ), beta-amyloid, etc. Therefore, when nitric oxide is generated in the human body, oxidative stress occurs. Such oxidative stress oxidizes cell membranes and DNA to induce cell aging and transformation, thereby causing oxidative damage to promote cerebrovascular barrier damage, and induce various degenerative diseases such as cancer, Alzheimer's disease, and Parkinson's disease.

Accordingly, BV-2 cells are treated with lipopolysaccharide (LPS) to induce a neuroinflammatory response, and nitric oxide (NO), a representative indicator of the inflammatory response according to the concentration of protethiamicin-6 treatment, is The inhibitory effect on neuroinflammation was investigated by measuring the production amount of Griess reagent (iNtRON, Korea).

Specifically, BV-2 cells were seeded into 96-wells each at the number of 4 × 10 ⁴ cells/well and cultured for 24 hours. 50, 80 μg/ml) for 1 hour, then treated with Lipopolysaccharide (LPS) 100 ng/ml and cultured for 24 hours. After taking 100 μl of the supernatant of the cultured cells and reacting with the Griess reagent, the amount of NO production was measured by measuring the absorbance at 540 nm with a multi detector (Beckman, DTX 8800, CA, USA). The concentration of NO generated was calculated based on the standard curve of the standard sodium nitrate (NaNO ₂ ) solution. CTR: control (non-treated sample).

As shown in FIG. 2, the experimental group (0 μg/ml) treated only with LPS without adding protethiamicin-6 to the BV-2 cells increased the amount of NO production by about 50 times compared to the control group, but It was confirmed that the treatment of thiamycin-6 decreased the amount of NO secretion in a concentration-dependent manner. These results show that protetiamicin-6 according to the present invention can effectively inhibit neuroinflammation.

with iNOS Assessing the effect on the expression of COX-2

iNOS is a biosynthetic pathway of NO, COX is involved in the synthesis of pepsinogen (PG), and it has been reported that both iNOS and COX-2 expression are increased by infection or external stimuli by microorganisms. In addition, iNOS induces harmful effects in the living body, such as pathological vasodilation, cytotoxicity, and tissue damage. Therefore, effectively suppressing the expression of iNOS and COX-2 in the inflammatory state of microglia is recognized as a general approach for the treatment and prevention of neuroinflammation and neurodegenerative diseases along with suppression of the production of inflammatory cytokines. Accordingly, the present applicant wanted to determine the effect of protethiamicin-6 on the expression of iNOS and COX-2 in BV-2 cells overly activated by LPS, expression level evaluation using real-time PCR and Western blot analysis was confirmed through

1. Real time RT- PCR

BV-2 microglia were seeded in a 6-well plate at each 4 × 10 ⁵ cell/well cell number and cultured for 24 hours. ml) for 1 hour, lipopolysaccharide (LPS) was treated at a concentration of 100 ng/ml to induce neuroinflammation, and cultured for 5 hours. The cultured BV-2 cells were washed twice with phosphate buffered saline (PBS), total RNA was extracted with TRIZOL reagent (Invitrogen, Carlsbad, CA), and the High Capacity cDNA Reverse Transcription Kit was used from the same amount of RNA (2 μg). (Applied Biosystems, Foster city, CA) was used to synthesize cDNA. Expression of inflammation-related genes using AMPIGENE® qPCR Green Mix Lo-ROX (Enzo Life Sciences, Farmingdale, NY) together with each primer shown in Table 1 ABI 7500 Real Time PCR System (PE Applied Biosystems, Foster City, CA, USA), which is shown in FIG. 3 .

division Sequence SEQ ID NO: iNOS Forward 5'-CAGCACAGGAAATGTTTCAGC-3' SEQ ID NO: 2 Reverse 5'-TAGCCAGCGTACCGGATGA-3' SEQ ID NO: 3 COX-2 Forward 5'-CAGACAACATAAACTGCGCCTT-3' SEQ ID NO: 4 Reverse 5'-GATACACCTCTCCACCAATGACC-3' SEQ ID NO: 5 TNF-α Forward 5'-ATGAGAAGTTCCCAAATGGC-3' SEQ ID NO: 6 Reverse 5'-CTCCACTTGGTGGTTTGCTA-3' SEQ ID NO: 7 IL-6 Forward 5'-GAGGATAACCACTCCCAACAGACC-3' SEQ ID NO: 8 Reverse 5'-AAGTGCATCATCGTTGTTCATACA-3' SEQ ID NO: 9 GAPDH Forward 5'-AAGGTCATCCCAGAGCTGAA-3' SEQ ID NO: 10 Reverse 5'-CTGCTTCACCACCTTCTTGA-3' SEQ ID NO: 11

Figure 3A is the result of confirming the iNOS expression level according to the treatment concentration of protetiamicin-6, Figure 3B is the result of confirming the expression level of COX-2 according to the treatment concentration of protetiamicin-6. It was confirmed that the expression levels of iNOS and COX-2 were significantly reduced depending on the treatment concentration of protethiamicin-6. When the inflammatory response was induced in BV-2 cells with LPS, the expression of iNOS and COX-2 were increased by about 4 times and by about 2 times compared to the control group. And it was confirmed to reduce the expression level of COX-2. In particular, it was confirmed that the treatment of 40 ~ 80 ㎍ / ㎖ of protethiamicin-6 to lower the expression of iNOS and COX-2 to the control level. This shows that protethiamicin-6 has an excellent effect on NO production inhibition and neuroinflammatory diseases.

2. western blot black

BV-2 cells were dispensed into a 6-well plate at each 4 × 10 ⁵ cell/well cell number and cultured for 24 hours. ) was pretreated for 1 hour, and LPS was treated at a concentration of 100 ng/ml to induce inflammation and cultured for 24 hours. Next, the culture medium was removed, the cells were lysed using M-PER™ Mammalian Protein Extraction Reagent (Thermo scientific, MA, USA), and the supernatant was collected by centrifugation (12,000 rpm, 15 min). The amount of protein was quantified with Pierce™ BCA protein assay kit (Rockford, IL, USA), and the same amount of protein was separated by sodium dodecyl sulfate-polyacrlyamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. The membrane was reacted with 5% skim milk for 1 hour to block reactivity to non-specific proteins, and after reacting with anti-iNOS and anti-COX-2 (Cell signaling Technology, Denver, MA, USA) antibodies, respectively, horseradish perox-idase (HRP) was reacted with the bound secondary antibody for 1 hour. Between each reaction, it was washed 3 times with 0.05% TBST for 10 minutes each. Thereafter, the corresponding protein band for the antibody was confirmed with an image analysis device of FluorChem (Alpha Innotech Corporation, San Leandro, CA, USA) using an ECL kit (Amersham Phar-macia Biotech, UK).

As shown in FIG. 4 , it was confirmed that the protein expression levels of iNOS and COX-2 also significantly decreased in a concentration-dependent manner with protetiamycin-6 treatment. When the inflammatory response was induced in BV-2 cells with LPS, the expression of iNOS was increased by about 14-fold and the expression of COX-2 by about 3-fold compared to the control group, but the treatment with protetiamicin-6 was concentration-dependently iNOS. and COX-2 expression.

That is, it was confirmed that protetiamicin-6 of the present invention not only does not show toxicity to cells, but also significantly reduces inflammatory mediators generated in activated microglia, which is the protetiamicin of the present invention. -6 can be used as a functional material for the treatment of various degenerative brain diseases based on neuroinflammation caused by microglia and further inflammatory response.

Assessment of Effect on Inflammatory Cytokine Expression

When inflammation is induced in cells, the expression level of cytokines, which is an inflammation-inducing factor, is increased. TNF-α is produced in many cells, including monocytes, macrophages, mast cells, and lymphocytes, and is one of the mediators involved in the infection or inflammatory response, and can interfere with normal cell function. When excessively activated by LPS from immune cells and a large amount of TNF-α is secreted, it plays a very important role in the development of inflammatory diseases, induces insulin resistance in metabolic diseases such as cancer and obesity, promotes thrombus formation, and has a lipogenetic effect. It is known to inhibit metabolism. In addition, among the inflammatory cytokines, IL-6 is involved in the differentiation of B lymphocytes and T lymphocytes, acts as a signal to inform tissue damage and infection, and exhibits anticancer effects, and is known to increase antibody production by activating B lymphocytes. It has been reported that IL-6 is always increased when this occurs. Accordingly, the present applicant inducing an inflammatory response through LPS stimulation to determine whether protetiamicin-6 can inhibit the expression and reaction products of inflammatory cytokines, IL-6 and TNF-, which are representative substances among cytokines The expression level of α was evaluated using ELISA and Real-time PCR test methods.

BV-2 cells were dispensed into a 6-well plate at each 4 × 10 ⁵ cell/well cell number and cultured for 24 hours. ) was pretreated for 1 hour, and LPS was treated at a concentration of 100 ng/ml and cultured for 24 hours. After that, the culture medium was recovered and IL-6 and TNF-α released in the culture medium were measured using an ELISA kit (ThermoFisher, Waltham, MA).

As shown in FIG. 5 , the LPS-treated group rapidly increased the expression levels of IL-6 and TNF-α compared to the control group, and the treatment with protetiamicin-6 was concentration-dependent on the expression of IL-6 and TNF-α. was observed to lower the level. This shows that protetiamicin-6 has an excellent effect on neuroinflammatory diseases.

According to the above results, protetiamicin-6 according to the present invention suppresses the expression of inflammatory factors and inflammatory mediators, and reduces the amount of nitric oxide secretion, thereby showing that there is an excellent effect in the prevention, improvement and treatment of neuroinflammatory diseases. Confirmed.

<110> REPUBLIC OF KOREA (MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Composition for preventing or treating of neuroinflammatory disease comprising Protaetiamycine-6 isolated from Protaetia brevitarsis seulensis <130> NSH1-159 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Protaetia brevitarsis seulensis <400> 1 Pro Lys Ala Arg Lys Leu Gln Lys Leu Ser Ala Tyr Lys Thr Thr Leu 1 5 10 15 Arg Asn <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> iNOS Forward <400> 2 cagcacagga aatgtttcag c 21 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> iNOS Reverse <400> 3 tagccagcgt accggatga 19 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> COX-2 Forward <400> 4 cagacaacat aaactgcgcc tt 22 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> COX-2 Reverse <400> 5 gatacacctc tccaccaatg acc 23 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF alpha Forward <400> 6 atgagaagtt cccaaatggc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF alpha Reverse <400> 7 ctccacttgg tggtttgcta 20 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IL-6 Forward <400> 8 gaggatacca ctcccaacag acc 23 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IL-6 Reverse <400> 9 aagtgcatca tcgttgttca taca 24 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Forward <400> 10 aaggtcatcc cagagctgaa 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Reverse <400> 11 ctgcttcacc accttcttga 20

Claims (15)

A peptide represented by SEQ ID NO: 1 below:
SEQ ID NO: 1: PKARKLQKLSAYKTTLRN-NH ₂ According to claim 1,
Wherein the peptide exhibits anti-neuroinflammatory activity. According to claim 1,
The peptide protects nerve cells from oxidative stress and inhibits the death of nerve cells, a peptide. A pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases comprising the peptide protetiamycin-6 (Protaetiamycine-6) having the amino acid sequence of SEQ ID NO: 1. 5. The method of claim 4,
The neuroinflammatory disease is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis, neuroblastoma, stroke, depression, schizophrenia, post-traumatic stress disorder and amyotrophic lateral sclerosis The above, a pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases. 5. The method of claim 4,
The protethiamicin-6 inhibits the expression of TNF-α and IL-6, which are neurotoxin-related neuroinflammatory cytokines generated in microglia, and cyclooxygenase-2 (COX-2) and inducibility proteins Inhibiting the expression of nitric oxide synthase (iNOS) and inhibiting the production of nitric oxide, a pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases. A food composition for preventing or improving neuroinflammatory diseases comprising a peptide protetiamycin-6 (Protaetiamycine-6) having the amino acid sequence of SEQ ID NO: 1. 8. The method of claim 7,
The neuroinflammatory disease is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis, neuroblastoma, stroke, depression, schizophrenia, post-traumatic stress disorder and amyotrophic lateral sclerosis The above, a food composition for the prevention or improvement of neuroinflammatory diseases. 8. The method of claim 7,
The protethiamicin-6 inhibits the expression of TNF-α and IL-6, which are neurotoxin-related neuroinflammatory cytokines generated in microglia, and cyclooxygenase-2 (COX-2) and inducibility proteins Inhibiting the expression of nitric oxide synthase (iNOS) and inhibiting the production of nitric oxide, a food composition for the prevention or improvement of neuroinflammatory diseases. A feed composition for preventing or improving neuroinflammatory diseases comprising a peptide protetiamycin-6 (Protaetiamycine-6) having the amino acid sequence of SEQ ID NO: 1. 11. The method of claim 10,
The neuroinflammatory disease is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis, neuroblastoma, stroke, depression, schizophrenia, post-traumatic stress disorder and amyotrophic lateral sclerosis The above, a feed composition for the prevention or improvement of neuroinflammatory diseases. 11. The method of claim 10,
The protethiamicin-6 inhibits the expression of TNF-α and IL-6, which are neurotoxin-related neuroinflammatory cytokines generated in microglia, and cyclooxygenase-2 (COX-2) and inducibility proteins Inhibiting the expression of nitric oxide synthase (iNOS) and inhibiting the production of nitric oxide, a feed composition for preventing or improving neuroinflammatory diseases. A health functional food for the prevention or improvement of neuroinflammatory diseases comprising the peptide protetiamycin-6 (Protaetiamycine-6) having the amino acid sequence of SEQ ID NO: 1. 14. The method of claim 13,
Any one selected from the group consisting of multiple sclerosis, neuroblastoma, stroke, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, schizophrenia and amyotrophic lateral sclerosis; One or more, health functional food for the prevention or improvement of neuroinflammatory diseases. 14. The method of claim 13,
The protethiamicin-6 inhibits the expression of TNF-α and IL-6, which are neurotoxin-related neuroinflammatory cytokines generated in microglia, and cyclooxygenase-2 (COX-2) and inducibility proteins A health functional food for preventing or improving neuroinflammatory diseases, which suppresses the expression of nitric oxide synthase (iNOS) and suppresses the production of nitric oxide.

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