KR102549333B1 - 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 the prevention or treatment of neuroinflammatory diseases containing the peptide proteiamycin-6 having an amino acid sequence of PKARKLQKLSAYKTTLRN-NH ₂ . Protethiamycin-6 according to the present invention has no side effects, excellent safety, and excellent anti-inflammatory efficacy, so 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 Protaetiamycin-6 isolated from Protaetia brevitarsis seulensis}

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

The central nervous system consists of neurons and glial cells. Glial cells account for about 90% of all brain cells and account for about 50% of the entire 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.

white-spotted bush ( Protaetia brevitarsis seulensis ) is an insect belonging to the coleoptera family Coleoptera and lives in Korea, Japan, and Russia.

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

The first problem to be solved by the present invention relates to a peptide proteiamycin-6 having the amino acid sequence of SEQ ID NO: 1 and a pharmaceutical composition for preventing or treating neuroinflammatory diseases comprising 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 preventing or improving neuroinflammatory diseases containing the peptide protethiamycin-6 having the amino acid sequence of SEQ ID NO: 1.

The present invention is a white-spotted mulberry ( 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

A method for providing the peptide according to the present invention is as follows. White-spotted bush ( Protaetia brevitarsis) seulensis ) In order to identify the derived peptide gene, first, the whole RNA was isolated by rapid freezing in liquid nitrogen, and the gene information on the transcriptome was confirmed using RNA seq analysis. and a method of selecting candidates through an anti-inflammatory activity test based on the amino acid sequence translated from each transcript.

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

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

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

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

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

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

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

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

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

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

The amino acid sequence of the peptide is PKARKLQKLSAYKTTLRN-NH ₂ and is composed of 18 amino acids, and is named Protaetiamycin-6.

According to the present invention, the peptide can protect neurons from oxidative stress, inhibit the death of neurons, and exhibit anti-neuroinflammatory activity.

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

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 proteiamycin-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 the expression of 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 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 protethiamycin-6 according to the present invention may be added in an amount of 0.001 to 95% by weight in the pharmaceutical composition of the present invention. The pharmaceutical composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient, for example, starch, calcium carbonate, in addition to the peptide proteiamycin-6 of the present invention. 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 proteiamycin-6 having the amino acid sequence of SEQ ID NO: 1 in the manufacture 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 proteiamycin-6 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 proteiamycin-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 containing the peptide proteiamycin-6 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 contain 0.01 to 95% by weight of protethiamycin-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 prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like.

For example, the health functional food in the form of a tablet is obtained by granulating a mixture of proteiamycin-6, excipients, binders, disintegrants, and other additives in a conventional manner, and then compression molding by adding a lubricant or the like. , the mixture can be directly compression molded. In addition, the health functional food in the form of a tablet may contain a corrigent, etc., if necessary, and may be coated with an appropriate coating agent, if necessary.

Among health functional foods in the form of capsules, hard capsules can be prepared by filling conventional hard capsules with a mixture of proteiamycin-6 and additives such as excipients, granular materials thereof, or coated granular materials. can be prepared by filling a mixture of proteiamycin-6 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 proteiamycin-6, an excipient, a binder, a disintegrant, etc. in an appropriate way, and if necessary, it can be coated with sucrose or other suitable coating agent, or starch or talc. Alternatively, it may be worn with a suitable material.

A health functional food in granular form can be granularly prepared from a mixture of proteiamycin-6, 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 proteiamycin-6 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 protethiamycin-6 may be contained in 0.01 to 15% by weight based on the total weight of the health functional food, and in the case of a health drink composition, 0.02 to 10 g, preferably 0.3 to 10 g, based on the total 100 g. It can contain 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 proteiamycin-6, and it may contain various flavoring agents or natural carbohydrates as additional components like conventional beverages. 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 mentioned above, natural flavoring agents (thaumatin, stevia extract (eg rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. there is.

In addition to the above, the food composition or health functional food containing proteiamycin-6 contains various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, and enhancers (cheese, chocolate) etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like.

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

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 protethiamycin-6 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 proteiamycin-6 according to the present invention inhibits the expression of inflammatory cytokines TNF-α and IL-6, and inhibits the expression of proteins cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). ) and suppresses the production of nitric oxide to have excellent anti-inflammatory efficacy, so 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 the cytotoxicity of proteiamycin-6 according to an embodiment of the present invention.
2 is a result of examining the nitric oxide inhibitory effect of proteiamycin-6 according to an embodiment of the present invention.
Figure 3 shows the inhibition of gene expression of inducible nitric oxide synthase (iNOS) and inflammatory mediator cyclooxygenase-2 (COX-2) with proteiamycin-6 according to an embodiment of the present invention, respectively, qRT -This is the result confirmed by PCR (A: iNOS, B: COX-2).
Figure 4 shows the protein expression inhibitory effect of inflammatory mediators cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) of proteiamycin-6 according to an embodiment of the present invention, Western This is the result confirmed by the blot method.
Figure 5 is a result confirming the effect of inhibiting inflammatory cytokine expression of proteiamycin-6 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 Protaetiamycin -6 (Protaetiamycine-6)>

experimental insect

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

gene selection

In order to identify the peptide gene showing anti-inflammatory activity from the white-spotted radish, first, the white-spotted radish was rapidly frozen in liquid nitrogen to isolate the total RNA, and RNA seq analysis was used to convert the white-spotted radish to the transcriptome. Gene information was confirmed, 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 were selected. did The selected amino acid sequence was PKARKLQKLSAYKTTLRN-NH ₂ , consisting of 18 amino acid sequences, and was named Protaetiamycin-6.

proteiamycin Synthesis of -6

To synthesize the proteiamycin-6 peptide, first, the peptide was prepared using Merrifield's liquid-solid phase method (Merrifield, RB, J Am Chem Soc, 85, 2149, 1963) using an Fmoc amino group protection container. manufactured. 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 cultured in Dublecco's Modified Eagle Medium (DMEM, Gibco) containing 5% fetal bovine serum (FBS) and 50 μg/ml gentamicin at 37°C and 5% CO. ₂ incubator (Incubator; Thermo Scientific, IL, USA).

Cell viability measurement (MTS assay)

In order to confirm the cytotoxicity of proteiamycin-6 on BV-2 cells, which are microglia, BV-2 cells were dispensed in a 96-well plate at 2×10 ⁴ cells/well for 24 hours. cultured. Next, proteiamycin-6 was treated at different concentrations (0, 5, 10, 20, 50, 80 μg/ml), further cultured for 24 hours, and then MTS ((3-(4,5-dimethyl Thiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) reagent was used to measure cell viability. Absorbance was measured by a multi detector (Beckman, DTX8800, CA, USA) at 490 nm CTR: control (non-treated sample).

As shown in Figure 1, it was confirmed that all treatment groups (5, 10, 20, 50, 80 μg/ml) treated with proteiamycin-6 showed no cytotoxicity compared to the control group. Then, in the neuroinflammation inhibition efficacy test, it was used at a concentration of 80 μg/ml or less, which does not exhibit cytotoxicity.

< test example 2. Neuroinflammation inhibition activity assay>

nitric oxide inhibitory ability black

Nitric oxide (NO), a type of reactive oxygen species, is produced by LPS, interferon-gamma (IFN-γ), β-amyloid, etc., and reacts easily with biological substances due to its unstable and high oxidizing power. Therefore, when nitric oxide is produced in the human body, oxidative stress is caused. This oxidative stress oxidizes cell membranes and DNA to induce aging and transformation of cells, resulting in oxidative damage, which promotes damage to the blood-brain barrier and causes various degenerative diseases such as cancer, Alzheimer's disease, and Parkinson's disease.

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

Specifically, after dividing BV-2 cells into 96-well at 4 × 10 ⁴ cells/well and culturing for 24 hours, proteiamycin-6 was added at concentrations (0, 5, 10, 20, 50, 80 μg/ml) for 1 hour, and then treated with 100 ng/ml of Lipopolysaccharide (LPS) and incubated for 24 hours. After taking 100 μl of the supernatant of the cultured cells and reacting with Griess reagent, absorbance was measured at 540 nm with a multi detector (Beckman, DTX 8800, CA, USA) to measure NO production. The concentration of NO produced was calculated based on the standard curve of sodium nitrate (NaNO ₂ ) solution, which is a standard substance. CTR: control (non-treated sample).

As shown in FIG. 2, in the experimental group (0 μg/ml) in which proteiamycin-6 was not added to the BV-2 cells and only LPS was treated, the amount of NO production increased about 50-fold compared to the control group, but the proteiamycin-6 was not added. It was confirmed that treatment with thiamycin-6 decreased NO secretion in a concentration-dependent manner. These results show that proteiamycin-6 according to the present invention can effectively inhibit neuroinflammation.

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

It has been reported that iNOS is a biosynthetic pathway of NO, COX is involved in the synthesis of pepsinogen (PG), and the expression of both iNOS and COX-2 is increased by infection with microorganisms or external stimuli. In addition, iNOS induces harmful effects to the living body such as pathological vasodilation, cytotoxicity, and tissue damage. Therefore, effectively inhibiting the expression of iNOS and COX-2 in the inflammatory response state of microglia is recognized as a general approach for the treatment and prevention of neuroinflammation and neurodegenerative diseases along with inhibition of inflammatory cytokine production. Therefore, in order to confirm the effect of proteiamycin-6 on the expression of iNOS and COX-2 in BV-2 cells excessively activated by LPS, the present applicant evaluated expression levels using real-time PCR and Western blot analysis confirmed through

1. Real time RT- PCR

BV-2 microglial cells were dispensed in a 6-well plate at a cell count of 4 × 10 ⁵ cells/well and cultured for 24 hours, and proteiamycin-6 was added at each concentration (10, 20, 40, 80 μg/well). ml) for 1 hour, and then treated with lipopolysaccharide (LPS) 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), and total RNA was extracted with TRIZOL reagent (Invitrogen, Carlsbad, CA), and High Capacity cDNA Reverse Transcription Kit from the same amount of RNA (2 μg) (Applied Biosystems, Foster City, CA) was used to synthesize cDNA. Expression of inflammation-related genes was measured using AMPIGENE® qPCR Green Mix Lo-ROX (Enzo Life Sciences, Farmingdale, NY) with the respective primers shown in Table 1, using an ABI 7500 Real Time PCR System (PE Applied Biosystems, Foster City, CA, USA), which is shown in Figure 3.

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

2. western blot black

BV-2 cells were divided into 6-well plates at 4 × 10 ⁵ cells/well and cultured for 24 hours, and then proteiamycin-6 was added at concentrations (10, 20, 40, 80 μg/ml). ) for 1 hour, and in order to induce inflammation, LPS was treated at a concentration of 100 ng/ml 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 by 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 then transferred to a PVDF membrane. The membrane was reacted with 5% skim milk for 1 hour to block the reactivity to non-specific proteins, and then reacted with anti-iNOS and anti-COX-2 (Cell signaling Technology, Denver, MA, USA) antibodies, respectively, followed by horseradish perox-idase (HRP) was reacted for 1 hour with a conjugated secondary antibody. Between each reaction, it was washed three times for 10 minutes with 0.05% TBST. Thereafter, the protein bands corresponding to the antibody were identified using an ECL kit (Amersham Phar-macia Biotech, UK) using FluorChem (Alpha Innotech Corporation, San Leandro, CA, USA) image analysis equipment.

As shown in FIG. 4 , it was confirmed that the amount of protein expression of iNOS and COX-2 significantly decreased in a concentration-dependent manner after treatment with proteiamycin-6. When an inflammatory response was induced in BV-2 cells with LPS, the expression of iNOS and COX-2 increased about 14-fold and 3-fold compared to the control group, however, treatment with proteiamycin-6 showed a concentration-dependent effect on iNOS. and inhibiting the expression of COX-2.

That is, it was confirmed that the protetiamicin-6 of the present invention not only exhibits no toxicity to cells, but also significantly reduces inflammatory mediators generated in activated microglial cells, which indicates that the protetiamicin-6 of the present invention -6 can be used as a functional substance for the treatment of neuroinflammation caused by microglia and various degenerative brain diseases based on the inflammatory response.

Evaluation of effects on inflammatory cytokine expression

When inflammation is induced in cells, the expression level of cytokines, which are inflammation-inducing factors, is increased. TNF-α is produced in many cells, including monocytes, macrophages, mast cells, and lymphocytes, and is one of the mediators involved in infection or inflammatory responses, and can interfere with normal cell functions. When TNF-α is 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 blood clot formation, and promotes lipogenetic It is known to inhibit metabolism. In addition, among inflammatory cytokines, IL-6 is known to be involved in the differentiation of B lymphocytes and T lymphocytes, act as a signal to inform tissue damage and infection, exhibit anticancer effects, and activate B lymphocytes to increase antibody production. It has been reported that IL-6 is always increased when this occurs. Accordingly, in order to confirm that proteiamycin-6 can inhibit the expression and reaction products of inflammatory cytokines by inducing an inflammatory response through LPS stimulation, the present applicant examined IL-6 and TNF-6, 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 in a 6-well plate at 4 × 10 ⁵ cells/well and cultured for 24 hours, and then proteiamycin-6 was added at concentrations (10, 20, 40, 80 μg/ml). ) for 1 hour, and LPS was treated at a concentration of 100 ng/ml and cultured for 24 hours. Thereafter, 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 alone treatment group rapidly increased the expression levels of IL-6 and TNF-α compared to the control group, and the treatment with proteiamycin-6 concentration-dependently increased the expression of IL-6 and TNF-α. levels were observed to decrease. This shows that proteiamycin-6 has an excellent effect on neuroinflammatory diseases.

The above results show that the protetiamicin-6 according to the present invention inhibits the expression of inflammatory inducing factors and inflammatory mediators and reduces the secretion of nitric oxide, thereby having excellent effects in preventing, improving, and treating neuroinflammatory diseases. Confirmed.

<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Composition for preventing or treating 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)

Peptide, consisting of the amino acid sequence of SEQ ID NO: 1:
SEQ ID NO: 1: PKARKLQKLSAYKTTLRN-NH ₂ According to claim 1,
The peptide is a peptide that exhibits anti-neuroinflammatory activity. According to claim 1,
The peptide is a peptide that protects neurons from oxidative stress and inhibits the death of neurons. A pharmaceutical composition for preventing or treating neuroinflammatory diseases comprising the peptide Protaetiamycin-6 consisting of the amino acid sequence of SEQ ID NO: 1,
The neuroinflammatory disease is any one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis and stroke, a pharmaceutical composition for preventing or treating neuroinflammatory diseases. delete According to claim 4,
The proteiamycin-6 inhibits the expression of neurotoxin-related neuroinflammatory cytokines TNF-α and IL-6 generated in microglia, and the protein cyclooxygenase-2 (COX-2) and inducible A pharmaceutical composition for preventing or treating neuroinflammatory diseases, which inhibits the expression of nitric oxide synthase (iNOS) and inhibits the production of nitric oxide. A food composition for preventing or improving neuroinflammatory diseases comprising the peptide Protaetiamycin-6 consisting of the amino acid sequence of SEQ ID NO: 1,
The neuroinflammatory disease is any one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis and stroke, a food composition for preventing or improving neuroinflammatory diseases. delete According to claim 7,
The proteiamycin-6 inhibits the expression of neurotoxin-related neuroinflammatory cytokines TNF-α and IL-6 generated in microglia, and the protein cyclooxygenase-2 (COX-2) and inducible A food composition for preventing or improving neuroinflammatory diseases, which inhibits the expression of nitric oxide synthase (iNOS) and inhibits the production of nitric oxide. A feed composition for preventing or improving neuroinflammatory diseases comprising the peptide Protaetiamycin-6 consisting of the amino acid sequence of SEQ ID NO: 1,
The neuroinflammatory disease is any one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis and stroke, a feed composition for preventing or improving neuroinflammatory diseases. delete According to claim 10,
The proteiamycin-6 inhibits the expression of neurotoxin-related neuroinflammatory cytokines TNF-α and IL-6 generated in microglia, and the protein cyclooxygenase-2 (COX-2) and inducible Inhibiting the expression of nitric oxide synthase (iNOS) and inhibiting the production of nitric oxide, a feed composition for preventing or improving neuroinflammatory diseases. As a health functional food for preventing or improving neuroinflammatory diseases containing the peptide Protaetiamycin-6 (Protaetiamycine-6) consisting of the amino acid sequence of SEQ ID NO: 1,
The neuroinflammatory disease is any one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis and stroke, a health functional food for preventing or improving neuroinflammatory diseases. delete According to claim 13,
The proteiamycin-6 inhibits the expression of neurotoxin-related neuroinflammatory cytokines TNF-α and IL-6 generated in microglia, and the protein cyclooxygenase-2 (COX-2) and inducible A health functional food for preventing or improving neuroinflammatory diseases, which inhibits the expression of nitric oxide synthase (iNOS) and inhibits the production of nitric oxide.

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