KR102475368B1 - Composition for preventing or treating of neuroinflammatory disease comprising Teleogryllusin 1 - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases comprising the peptide tereogrileucine 1 having an amino acid sequence of VKWKRLNNNKVLQKIYFVKI-NH ₂ . Since tereogrileucine 1 according to the present invention has no side effects, excellent safety, and excellent anti-inflammatory efficacy, it can be used as a preventive or therapeutic agent for neuroinflammatory diseases, a health food supplement, and a feed composition.

Description

Composition for preventing or treating neuroinflammatory disease comprising Teleogryllusin 1

The present invention relates to a composition for preventing or treating neuroinflammatory diseases containing tereogrileucine 1, and more particularly, to a composition for preventing or treating neuroinflammatory diseases containing peptide tereogrileucine 1 derived from king cricket It is about.

The central nervous system consists of neurons and glial cells. Glial cells account for about 90% of all brain cells and about 50% of the brain in terms of volume. Glial cells can be further classified into three types: astrocytes, microglia, and oligodendrocytes. Among them, microglial cells are a type of specialized 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 biodefense activities in the brain. 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 their protein expression is changed.

Neuroinflammation is a kind of immune response of the nervous system, and is closely related to many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis, and is now considered a typical feature of neurodegenerative diseases. The neuroinflammatory response involves activation of innate immune cells (microglia), release of inflammatory mediators such as nitric oxide (NO), cytokines and chemokines, and macrophage infiltration, which leads 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 has increased, and many studies have been attempted to treat neuroinflammatory diseases. Korean Registered Patent No. 10-1730065 discloses anti-inflammatory activity of an insect-derived natural resource, extract of green radish radish, and Korean Registered Patent No. 10-1700605 discloses anti-inflammatory activity of a beetle extract. 10-066500 discloses the anti-inflammatory activity of ladybug extracts. However, since the effect has not been clearly verified and commercialized substances have not yet been developed so that it can be applied to a wide range of neuroinflammatory diseases, research on new therapeutic agents is needed.

The king cricket ( Teleogryllus emma ) is a kind of cricket that lives in East Asia and is an insect belonging to the order Grasshopper, Cricketidae, and King cricket.

The inventors of the present invention completed the present invention by confirming that the peptides derived from king crickets have excellent anti-inflammatory efficacy, especially for neuroinflammation, while studying various physiological activities using peptides derived from king crickets.

The first problem to be solved by the present invention relates to a pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases comprising the peptide tereogrillusin 1 having the amino acid sequence of SEQ ID NO: 1 below.

SEQ ID NO: 1: VKWKRLNNNKVLQKIYFVKI-NH ₂

The second problem to be solved by the present invention relates to a food composition, feed composition, or health functional food for preventing or improving neuroinflammatory diseases containing the peptide tereogrileucine 1 having the amino acid sequence of SEQ ID NO: 1.

The present invention relates to the protective effect of a peptide synthesized from a gene isolated from a transcript of Teleogryllus emma against neuroinflammation.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating neuroinflammatory diseases, including the peptide tereogrillusin 1 having the amino acid sequence of SEQ ID NO: 1 below.

SEQ ID NO: 1: VKWKRLNNNKVLQKIYFVKI-NH ₂

According to the present invention, the peptide tereogrillusin 1 (Teleogryllusin 1) may be derived from king cricket ( Teleogryllus emma ).

A method for providing the peptide according to the present invention is as follows. In order to identify the peptide gene derived from king cricket ( Teleogryllus emma ), first, the king cricket was rapidly frozen in liquid nitrogen to isolate total RNA, and RNA seq analysis was used to confirm genetic information about the king cricket transcriptome, and a method for selecting candidates through an anti-inflammatory activity test based on the sequence of amino acids translated from each of the transcripts.

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

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

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

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

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

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

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

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

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

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

The neuroinflammatory disease according to the present invention is 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 one or more selected ones.

The peptide tereogrileucine 1 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 inhibiting the expression of inducible nitric oxide synthase (iNOS), it may be to inhibit the production of nitric oxide.

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

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

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

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

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

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

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

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

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

According to the present invention, the health functional food treats neuroinflammatory diseases 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 dystrophy. Lateral sclerosis; may be for preventing or improving any one disease selected from the group consisting of.

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 0.01 to 95% by weight of tereogrileucine 1, preferably 1 to 80% by weight, based on the total weight of the composition.

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

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

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

The health functional food in the form of a pill can be prepared by molding a mixture of tereogrileucine 1, an excipient, a binder, a disintegrant, etc. in an appropriate method, and if necessary, a skin with sucrose or other suitable coating agent, or starch, talc or It can also be worn with a suitable material.

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

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

There is no particular limitation on the type of food. Examples of foods to which tereogrileucine 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 tereogrileucine 1 may be contained in 0.01 to 15% by weight based on the total weight of health functional food, and in the case of a health drink composition, 0.02 to 10 g, preferably 0.3 to 1, based on 100 g in total. can contain gram.

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

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

In addition, the present invention provides a feed composition for preventing or improving neuroinflammatory diseases containing tereogrileucine 1.

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

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

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

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

The peptide tereogrileucine 1 according to the present invention inhibits the expression of the inflammatory cytokines TNF-α and IL-6, and the proteins cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) Since it inhibits the expression of and has excellent anti-inflammatory efficacy by suppressing the production of nitric oxide, it can be used as a preventive or therapeutic agent for neuroinflammatory diseases, a health food supplement, and a feed composition.

1 is a result of assaying cytotoxicity of tereogrileucine 1 according to an embodiment of the present invention.
2 is a result of assaying the nitric oxide inhibitory effect of tereogrileucine 1 according to an embodiment of the present invention.
Figure 3 is a qRT-PCR gene expression suppression of inflammatory mediator cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) of tereogrileucin 1 according to an embodiment of the present invention This is the result of checking with
4 is a Western blot showing the protein expression inhibitory effect of the inflammatory mediators cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) of tereogrileucin 1 according to an embodiment of the present invention. This is the result of checking the method.
5 is a result confirming the effect of inhibiting inflammatory cytokine expression of tereogrileucine 1 according to an embodiment of the present invention.

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

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

gene selection

In order to identify peptide genes showing anti-inflammatory activity from king crickets, first, king crickets were rapidly frozen in liquid nitrogen to isolate total RNA, and RNA seq analysis was used to confirm genetic information on the king crickets transcriptome, Based on the amino acid sequence translated from each transcript, the entire Unigene was filtered based on the existing antibacterial peptide properties, and as a result, genes presumed to be anti-inflammatory peptides were selected. The selected amino acid sequence was VKWKRLNNNKVLQKIYFVKI-NH ₂ , which consisted of 20 amino acid sequences, and was named Teleogryllusin 1.

terogryleucine Synthesis of 1

In order to synthesize tereogrileucine 1 peptide, first, the peptide was prepared using Merrifield's liquid phase method (Merrifield, RB, J Am Chem Soc, 85, 2149, 1963) using Fmoc amino group protection container did The method of synthesizing the peptide was synthesized by a solid phase method using Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group for the Nα-amino group of an amino acid.

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

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

Here, 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 a purified reverse phase-HPLC column (Delta Pak, C18 300Å, 15 μm, 19.0 mm × 30 mm) with an acetonitrile gradient containing 0.1% TFA. , Waters).

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

As a result, it was confirmed that the antimicrobial peptide having the correct amino acid sequence was synthesized because the measured molecular weight and the calculated molecular weight matched.

< test example 1. Cell viability test>

cell culture

Mouse-derived microglia, BV-2 cells, were prepared in Dublecco's Modified Eagle Medium (DMEM) containing 50 μg/ml of Gentamicin (Gibco, NY, USA) and 5% fetal bovine serum (FBS; Gibco, MD, USA). , Gibco) was cultured in a 37° C., 5% CO2 incubator (Incubator; Thermo Scientific, IL, USA) using medium, and subcultures were performed at intervals of 3 days.

Cell viability measurement (MTS assay)

In order to measure cell viability, BV-2 cells, which are microglia, were dispensed at 2×10 ⁴ cells/well by 100 μl in a 96-well plate, and tereogrilyucine 1 was added by concentration (10, 20, 40, 80 μg/ml), and cultured for 24 to 48 hours in a 37° C., 5% CO2 incubator. Then, MTS reagent 10 μl CellTiter 96 ^ⓡ AQueous One Solution Reagent (Promega, USA) was added and reacted for 2 hours to confirm the color change. The cell viability was measured by measuring

As shown in FIG. 1, it was confirmed that all treatment groups (10, 20, 40, 80 μg/ml) treated with tereogrileucine 1 showed no cytotoxicity compared to the control group. The highest concentration of tereogrileucine 1 used in subsequent experiments was 80 μg/ml, which was not toxic to BV-2 cells.

< test example 2. Anti-inflammatory activity assay>

nitric oxide inhibitory ability black

In order to measure the inhibitory effect of tereogrileucine 1 on microglia activity, BV-2 cells were treated with the extract, and then the amount of nitric oxide (NO) produced by LPS was measured. Cells were prepared in 96-well at 4 × 10 ⁴ cell/well, cultured for 24 hours for cell stabilization, and then prepared tereogrileucine 1 was added by concentration (10, 20, 40, 80 μg/ml) was pretreated for 1 hour. Then, to induce inflammation, Lipopolysaccharide (LPS) was treated at a concentration of 100 ng/ml and cultured for 24 hours. NO was measured using a NO detection kit (Intron, Korea) using 100 μl of the supernatant of the cultured cells.

As shown in FIG. 2, it can be confirmed that the amount of NO secretion is decreased in a concentration-dependent manner of tereogrileucine 1.

quantitative reverse transcription polymerase chain reaction, qRT - PCR

The gene expression levels of COX-2 and iNOS, which are inflammatory mediators of BV-2 cells by tereogrileucine 1, were verified by real-time PCR. Specifically, after preparing 4 × 10 ⁵ cell/well cells for each BV-2 microglia in a 6-well plate, tereogrileucine 1 was added by concentration (10, 20, 40, 80 μg/ml) was pretreated for 1 hour. Thereafter, lipopolysaccharide (LPS) was treated at a concentration of 100 ng/ml to induce neuroinflammation and cultured for 24 hours. Then, the supernatant was removed, and the cells were harvested after washing with PBS. The harvested cells were mixed with 1 ml of TRIZOL solution and reacted at room temperature for 5 minutes. After adding and mixing 200 μl of chloroform, centrifuged at 4°C at 12,000×g for 15 minutes, took the supernatant, added 500 μl of isopropanol, reacted at room temperature for 10 minutes, and centrifuged at 4°C at 12,000×g for 10 minutes. do. The precipitated RNA was washed three times with 75% ethanol, dried, and dissolved in sterile water before use. For cDNA synthesis, cDNA was synthesized with a cDNA synthesis kit using 1 μg of total RNA, and qRT-PCR experiments were performed using the primers shown in Table 1 below.

division Sequence sequence number 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'-GAGGATACCACTCCCAACAGACC-3' SEQ ID NO: 8 Reverse 5'-AAGTGCATCATCGTTGTTCATACA-3' SEQ ID NO: 9 GAPDG Forward 5'-AAGGTCATCCCAGAGCTGAA-3' SEQ ID NO: 10 Reverse 5'-CTGCTTCACCACCTTCTTGA-3' SEQ ID NO: 11

As shown in FIG. 3 below, it was confirmed that the expression levels of iNOS and COX-2 significantly decreased depending on the treatment concentration of tereogrileucine 1. The expression level of iNOS was reduced by about 30% and 80%, respectively, compared to the positive control when tereogrileucine 1 was treated at a concentration of 20 μg/ml or 40 μg/ml, and the expression level of COX-2 was When leucine 1 was treated at a concentration of 40 μg/ml or 80 μg/ml, it was confirmed that the concentration decreased by about 30% and 60%, respectively, compared to the positive control group. This shows that tereogrileucine 1 has an excellent effect on neuroinflammatory diseases.

Western blot black

BV-2 cells were prepared in 6-well at 2 × 10 ⁵ cells/well, and then treated with tereogrileucine 1 at each concentration for 1 hour. Thereafter, in order to induce inflammation, LPS was treated at a concentration of 100 ng/ml and cultured for 24 hours. Then, the supernatant was removed, washed with PBS, and cells were harvested using lysis buffer. The supernatant was removed, washed with PBS, and proteins were extracted using RIPA lysis buffer. Protein was quantified using BCA kit (Thermo Fisher), and developed using SDS-PAGE gel. The developed proteins were transferred to a PVDF membrane, blocked with 5% skim milk for 1 hour, diluted with primary antibody (1:1000), overnighted at 4˚C, and washed three times with TBST at 10 minute intervals. Then, each secondary antibody was diluted 1:10,000 and reacted at room temperature for 1 hour at room temperature. After washing with TBST three times for 10 minutes, it was reacted with ECL solution, and signal confirmation was measured using Image analyzer FluorChem (Alpha Innotech, USA).

As shown in FIG. 4 , it was confirmed that the protein expression levels of iNOS and COX-2 were significantly decreased in a concentration-dependent manner with tereogrilyucine 1 treatment. That is, it was confirmed that the tereogrileucine 1 of the present invention not only exhibits no toxicity to cells, but also significantly reduces inflammatory mediators generated in activated microglia. It shows that it effectively suppresses the neuroinflammation caused by these microglia.

Verification of inflammatory cytokine reduction

When inflammation is induced in cells, the expression level of cytokines, which are inflammation-inducing factors, is increased. Therefore, in this experiment, the expression levels of IL-6 and TNF-α, which are representative substances among cytokines, were investigated using ELISA and real-time PCR test methods.

As shown in Figure 5, it was confirmed that the expression level of all cytokines used in the experiment decreased in a concentration-dependent manner, which shows that tereogrileucine 1 has an excellent effect on neuroinflammatory diseases.

Through the above results, it was confirmed that tereogrileucine monovalent according to the present invention suppresses the expression of inflammation inducing factors and inflammatory mediators, and reduces the secretion of nitric oxide, thereby having excellent effects in preventing, improving, and treating neuroinflammatory diseases. did

<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Composition for preventing or treating neuroinflammatory disease comprising Teleogryllusin 1 <130> nsh1.133 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> PRT <213> artificial sequence <220> <223> teleogryllus emma <400> 1 Val Lys Trp Lys Arg Leu Asn Asn Asn Lys Val Leu Gln Lys Ile Tyr 1 5 10 15 Phe Val Lys Ile 20 <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 (12)

of at least one neuroinflammatory disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis and stroke, including the peptide teleogryllusin 1 having the amino acid sequence of SEQ ID NO: 1 A pharmaceutical composition for prevention or treatment.
SEQ ID NO: 1: VKWKRLNNNKVLQKIYFVKI-NH ₂ delete According to claim 1,
The tereogrileucine 1 inhibits the expression of neurotoxin-related neuroinflammatory cytokines TNF-α and IL-6 generated in microglia, and cyclooxygenase-2 (COX-2), which is a protein, and induces oxidation Inhibiting the expression of nitrogen synthase (iNOS) and inhibiting the production of nitric oxide, a pharmaceutical composition. of at least one neuroinflammatory disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis and stroke, including the peptide teleogryllusin 1 having the amino acid sequence of SEQ ID NO: 1 A food composition for prevention or improvement.
SEQ ID NO: 1: VKWKRLNNNKVLQKIYFVKI-NH ₂ delete According to claim 4,
The tereogrileucine 1 inhibits the expression of neurotoxin-related neuroinflammatory cytokines TNF-α and IL-6 generated in microglia, and cyclooxygenase-2 (COX-2), which is a protein, and induces oxidation A food composition that inhibits the expression of nitrogen synthase (iNOS) and inhibits the production of nitric oxide. of at least one neuroinflammatory disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis and stroke, including the peptide teleogryllusin 1 having the amino acid sequence of SEQ ID NO: 1 Feed composition for prevention or improvement.
SEQ ID NO: 1: VKWKRLNNNKVLQKIYFVKI-NH ₂ delete According to claim 7,
The tereogrileucine 1 inhibits the expression of neurotoxin-related neuroinflammatory cytokines TNF-α and IL-6 generated in microglia, and cyclooxygenase-2 (COX-2), which is a protein, and induces oxidation To inhibit the expression of nitrogen synthase (iNOS) and inhibit the production of nitric oxide, a feed composition. of at least one neuroinflammatory disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis and stroke, including the peptide teleogryllusin 1 having the amino acid sequence of SEQ ID NO: 1 Health functional food for prevention or improvement.
SEQ ID NO: 1: VKWKRLNNNKVLQKIYFVKI-NH ₂ delete According to claim 10,
The tereogrileucine 1 inhibits the expression of neurotoxin-related neuroinflammatory cytokines TNF-α and IL-6 generated in microglia, and cyclooxygenase-2 (COX-2), which is a protein, and induces oxidation A health functional food that inhibits the expression of nitrogen synthase (iNOS) and inhibits the production of nitric oxide.

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