KR20240104493A - Peptides having activities for cell growth promotion and uses thereof - Google Patents

Abstract

The present invention relates to a peptide containing vespakinin, mastoparan, and eumenin mastoparan-AF as active ingredients. Since the peptide exhibits activity that promotes cell growth, it has the effect of inhibiting the production of beta-amyloid precursor protein (APP) or inhibiting neuroinflammatory responses.

Description

Peptides showing cell growth promoting activity and their uses {PEPTIDES HAVING ACTIVITIES FOR CELL GROWTH PROMOTION AND USES THEREOF}

It is about peptides that promote cell growth and their uses.

Alzheimer's disease (AD) is a disease that manifests symptoms of gradual memory decline and cognitive loss, and its incidence is significantly increasing as the average lifespan of humans increases. One of the pathological characteristics of Alzheimer's disease is senile plaques that accumulate on the outside of nerve cells, and the causative agent for this is beta-amyloid (Aβ).

Beta-amyloid (Aβ) is a protein fragment cleaved from amyloid precursor protein (APP), and the most important enzyme involved in beta-amyloid production is beta-site APP-cleaving enzyme. It is a beta-secretase named BACE1). APP cleaved by beta-secretase (BACE1) is divided into an N-terminal domain called sAPPβ of about 90 kDa and a cytoplasmic domain called CTFβ (C99). The sAPPβ produced in this way is It is secreted out of the cell, and C99 is again cleaved by gamma-secretase to produce 4 kDa beta-amyloid.

Due to abnormal metabolism of amyloid precursor protein, large amounts of beta-amyloid (Aβ) are produced, causing brain cell toxicity and causing Alzheimer's disease. Therefore, substances that inhibit the activity of amyloid precursor protein can be expected to prevent or treat Alzheimer's disease because they inhibit the production of beta-amyloid.

Most of the drugs currently on the market or in development are drugs that improve the symptoms of Alzheimer's patients and improve their quality of life, including tacrine and donet, which are acetylcholinesterase inhibitors developed based on the cholinergic nervous system hypothesis. Examples include donepezil, rivastigmine, galantamine, and memantine, which has an NMDA-glutamate receptor inhibitory function. However, most of these treatments only provide a temporary improvement in clinical symptoms in the early stages of the disease and also cause side effects, making treatment difficult (Korean Patent No. 10-2085358).

The present inventors studied to develop a therapeutic agent that can inhibit or decompose the production of beta-amyloid protein with the goal of fundamental treatment based on the cause of the invention of Alzheimer's disease, and developed a substance that inhibits the expression and/or activity of amyloid precursor protein. And based on this, the present invention was completed.

One aspect provides a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising at least one ingredient selected from vespakinin, mastoparan, and eumenin mastoparan-AF as an active ingredient.

Another aspect provides pharmaceutical use of the pharmaceutical composition for preventing or treating a degenerative brain disease, or a method of treating a degenerative brain disease comprising administering a therapeutically effective amount of the pharmaceutical composition to a subject. will be.

One aspect provides a pharmaceutical composition for preventing or treating degenerative brain diseases containing Vespakinin as an active ingredient.

Another aspect provides a pharmaceutical composition for preventing or treating degenerative brain diseases containing mastoparan as an active ingredient.

Another aspect provides a pharmaceutical composition for preventing or treating degenerative brain diseases comprising Eumenine Mastoparan-AF as an active ingredient.

As used herein, the term “prevention” refers to any action that inhibits or delays the onset of a disease by administering the composition.

As used herein, the term "treatment" refers to a treatment for an individual suffering from a disease or at risk of developing a disease, improving the condition of the individual (e.g., one or more symptoms), delaying the progression of the disease, delaying the onset of symptoms, or refers to any form of treatment that provides effects including slowing the progression of symptoms. Accordingly, the terms “treatment” and “prevention” are not intended to imply cure or complete elimination of symptoms.

The “subject” refers to a subject in need of treatment for a disease, and more specifically, refers to mammals such as human or non-human primates, mice, dogs, cats, horses, and cattle.

“Degenerative brain disease,” which is a disease to be prevented or treated by the pharmaceutical composition, may refer to a disease that causes various symptoms as degenerative changes occur in nerve cells of the nervous system due to gradual and steady death. The degenerative diseases include, for example, Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, mild cognitive impairment, cerebral amyloid angiopathy, amyloid stroke, Systemic amyloid disease, Dutch amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis, Spinocerebellar Atrophy, Tourette's Syndrome, Friedrich's Ataxia. (Friedrich`s Ataxia), Machado-Joseph`s disease, Lewy Body Dementia, Dystonia, Progressive Supranuclear Palsy and Frontotemporal Dementia Dementia), preferably Alzheimer's disease, but is not limited thereto.

As used herein, the term “Vespakinin” refers to the peptide of SEQ ID NO: 1, which is a toxic peptide targeting the bradykinin receptor. The vespakinin may refer to vespakinin-M or vespakinin-X.

As used herein, the term “Mastoparan subfamily peptide” refers to a peptide similar to mastoparan peptide, and may be a general term for tetratecapeptide amides contained in bee venom of the family Sparrow wasp.

In one embodiment, the mastoparan group peptide is mastoparan, eumenine mastoparan-AF (EMP-AF), eumenine mastoparan-OD (Eumenine mastoparan-OD) ), Eumenine mastoparan-EF, dominulin, and mastoparan-like peptide.

As used herein, the term “Mastoparan” refers to the peptide of SEQ ID NO: 2, which is a toxic peptide containing tetratecapeptide amide contained in bee venom. The mastoparan is mastoparan, mastoparan-1, mastoparan-2, mastoparan-3, mastoparan-X, mastoparan-J, mastoparan-X, mastoparan-M Or it may mean mastoparan-L.

As used herein, the term "Eumenine Mastoparan-AF (EMP-AF)" is a type of mastoparan group peptide and refers to the peptide of SEQ ID NO: 3.

The vespakinin, mastoparan, or EMP-AF can be obtained by extraction from bee venom, and synthetic or commercially available products can be used without limitation.

In one embodiment, the pharmaceutical composition comprises one selected from the group consisting of Vespakinin, Mastoparan, and Eumenine Mastoparan-AF (EMP-AF). It could be.

The vespakinin, mastoparan, or eumenin mastoparan-AF has chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., broad spectrum of biological activity), and reduced In order to acquire antigenicity, a protecting group may be attached to the N- or C-terminus of the active ingredient. In one embodiment, the N-terminus of the active ingredient is acetyl group, fluoreonylmethoxycarbonyl group, formyl group, palmitoyl group, myristyl group ( may be combined with any one protecting group selected from the group consisting of myristyl group, stearyl group, and polyethylene glycol (PEG); And/or the C-terminus of the active ingredient may be bonded to any one protecting group selected from the group consisting of amino group (-NH ₂ ) and azide (-NHNH ₂ ). In addition, the active ingredient may optionally additionally include a targeting sequence, a tag, a labeled residue, and an amino acid sequence prepared for the specific purpose of increasing half-life or stability.

As used herein, the term “stability” may mean not only in vivo stability, which protects the active ingredient from attack by protein cleavage enzymes in vivo, but also storage stability (e.g., room temperature storage stability).

In one embodiment, the content of vespakinin is 0.01 μM to 1 μM, 0.01 μM to 0.9 μM, 0.01 μM to 0.8 μM, 0.01 μM to 0.7 μM, 0.01 μM to 0.5 μM, 0.01 μM to 0.4 μM, 0.01 μM to 0.01 μM. It may be 0.3 μM, 0.01 μM to 0.2 μM, or 0.01 μM to 0.1 μM.

In one embodiment, the content of mastoparan is 0.01 μM to 100 μM, 0.01 μM to 90 μM, 0.01 μM to 80 μM, 0.01 μM to 70 μM, 0.01 μM to 60 μM, 0.01 μM to 50 μM, 0.01 μM to 40 μM, 0.01 μM to 40 μM. μM to 30 μM, 0.01 μM to 20 μM, 0.01 μM to 10 μM, 0.1 μM to 100 μM, 0.1 μM to 90 μM, 0.1 μM to 80 μM, 0.1 μM to 70 μM, 0.1 μM to 60 μM, 0.1 μM to 50 μM, 0.1 μM to 40μM, 0.1 It may be μM to 30 μM, 0.1 μM to 20 μM, or 0.1 μM to 10 μM.

In one embodiment, the content of eumenin mastoparan-AF is 0.01 μM to 10 μM, 0.01 μM to 9 μM, 0.01 μM to 8 μM, 0.01 μM to 6 μM, 0.01 μM to 5 μM, 0.01 μM to 4 μM, 0.01 μM to 0.01 μM. 3 μM, 0.01 μM to 2 μM, 0.01 μM to 1 μM, 0.01 μM to 0.9 μM, 0.01 μM to 0.8 μM, 0.01 μM to 0.7 μM, 0.01 μM to 0.5 μM, 0.01 μM to 0.4 μM, 0.01 μM to 0.3 μM, .01μM It may be from 0.2 μM or from 0.01 μM to 0.1 μM.

If the content of vespakinin, mastoparan or eumenin mastoparan-AF is less than or exceeds the above value, the formation of amyloid precursors may increase and the treatment effect for degenerative brain disease may be reduced.

In one embodiment, the degenerative brain disease may be Alzheimer's disease.

The Alzheimer's disease may be caused by abnormal metabolism of amyloid precursor protein, which produces a large amount of beta-amyloid, causing brain cell toxicity. Protase may be involved in the decomposition of the beta-amyloid.

The beta-amyloid (Aβ) is a protein fragment cut from amyloid precursor protein (APP).

In one embodiment, the pharmaceutical composition may inhibit beta-amyloid (β-amyloid) production.

In one embodiment, the pharmaceutical composition may reduce the expression of beta-amyloid precursor protein (APP).

In one embodiment, the pharmaceutical composition may increase the activity or expression of a protease that decomposes beta-amyloid precursor protein, amyloid plaques, or fragments thereof.

In one embodiment, the protease may be Neprilysin or insulin degradation enzyme (IDE).

In one embodiment, the pharmaceutical composition may increase the expression of Brain Derived Neurotropic Factor or promote the growth of neurites.

According to one embodiment, the pharmaceutical composition inhibits the expression of amyloid precursor protein, regenerates nerves, and increases the expression of Neprilysin or insulin degradation enzyme (IDE), thereby regenerating nerves. By reducing the inflammatory response, it may be effective in treating or preventing degenerative brain diseases.

In one embodiment, the pharmaceutical composition contains two or more selected from the group consisting of Vespakinin, Mastoparan, and Eumenine Mastoparan-AF (EMP-AF). It may include For example, it may contain two or three peptides.

In one embodiment, the pharmaceutical composition may include the following combination of ingredients as active ingredients. (a) vespakinin and mastoparan; (b) mastoparan and eumenin mastoparan-AF; (c) vespakinin and eumenin mastoparan-AF; or (d) vespakinin, mastoparan, and eumenin mastoparan-AF.

In one embodiment, the pharmaceutical composition may contain vespakinin and mastoparan as active ingredients, and the pharmaceutical composition may contain mastoparan and eumenin mastoparan-AF as active ingredients. The pharmaceutical composition may include vespakinin and eumenin mastoparan-AF as active ingredients.

In one embodiment, the pharmaceutical composition may include vespakinin, mastoparan, and eumenin mastoparan-AF as active ingredients.

The content of each of the components is 0.001 to 10 uM, 0.001 to 1 uM, and 0.001 to 0.1 uM. It may be 0.001 to 0.01 uM, 0.01 to 10 uM, 0.01 to 1 uM, 0.01 to 0.1 uM, 0.1 to 10 uM, 0.1 to 1 uM, but is not limited thereto, and the weight ratio between each component is also 1:1 to 1: It may be 1000, but it can be changed as needed within the range of effective efficacy.

In one embodiment, the pharmaceutical composition may be formulated with a preparation selected from the group consisting of tablets, soft or hard capsules, pills, powders, suspensions, syrups, injections, and granules.

In one embodiment, the pharmaceutical composition may be for oral or parenteral administration.

The pharmaceutical composition includes conventional fillers, extenders, binders, disintegrants, anti-aggregants, lubricants, wetting agents, pH adjusters, nutrients, vitamins, electrolytes, alginic acid and its salts, pectic acid and its salts, protective chloride, glycerin, It may contain fragrances, emulsifiers, or preservatives.

The pharmaceutical composition may include a pharmaceutically acceptable carrier, examples of such carriers include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin. , calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, propylhydroxybenzoate, One or more selected from the group consisting of talc, magnesium stearate, mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, and physiological saline may be mentioned, but are not limited thereto, and any conventional pharmaceutically acceptable carrier can be used. do.

The formulation of the pharmaceutical composition may vary depending on the method of use, and may be formulated using methods well known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal. It can be.

Preparations for oral administration include tablets, soft or hard capsules, pills, powders, suspensions, syrups, injections and granules, and these preparations may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose. It can be manufactured by mixing gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Preparations for parenteral administration may be creams, lotions, ointments, warning agents, solutions, aerosols, fluid extracts, elixirs, needles, sachets, patches, or injections.

Another aspect provides a method of treating, preventing, or ameliorating a degenerative brain disease by administering the pharmaceutical composition to a subject.

Among the terms or elements mentioned in the description of the active ingredient, those that are the same as those already mentioned are as described above.

The method is applicable to any animal, and animals may include humans and primates, as well as domestic animals such as cattle, pigs, sheep, horses, dogs, and cats.

The term “improvement” refers to any action that results in at least a reduction in the severity of the parameters associated with the condition being treated, such as symptoms.

The dosage of the composition for treatment, prevention, or improvement can be determined considering the method of administration, the age and gender of the recipient, the severity and condition of the patient, the absorption of the active ingredient in the body, the inactivation rate, and the concomitant drug, Based on the daily active ingredient, 0.1 mg/kg (body weight) to 500 mg/kg (body weight), 0.1 mg/kg (body weight) to 400 mg/kg (body weight), or 1 mg/kg (body weight) to 300. It can be administered in mg/kg (body weight) and can be administered once or in several divided doses, but is not limited to this.

Another aspect provides a health functional food composition for preventing or improving degenerative brain disease, comprising at least one ingredient selected from vespakinin, mastoparan, and eumenin mastoparan-AF as an active ingredient.

Among the terms or elements mentioned in the description of the active ingredient, those that are the same as those already mentioned are as described above.

The above-mentioned “health functional food” refers to a food manufactured or processed using specific ingredients as raw materials or specific ingredients contained in food ingredients by methods such as extraction, concentration, purification, mixing, etc., for the purpose of health supplementation. It refers to food designed and processed to fully exert bioregulatory functions on the living body, such as defense, regulation of biological rhythm, prevention and recovery of disease, etc.

The health functional food may include food supplements that are foodologically acceptable, and may include an appropriate carrier commonly used in the manufacture of health functional foods.

The health functional food composition is a compound of astragalin, tiliroside, kaempferol, or 5-O-caffeoylshikimic acid isolated from the fern extract according to the present invention, a pharmaceutically acceptable salt thereof, or a combination thereof. It may contain 0.001 to 99% by weight of the total food weight.

The pharmaceutical composition according to one aspect includes at least one ingredient selected from vespakinin, mastoparan, and eumenin mastoparan-AF as an active ingredient, and the pharmaceutical composition contains beta-amyloid precursor protein (APP). It can inhibit the production of or suppress neuroinflammatory responses. Therefore, the pharmaceutical composition can be used as a preventive or therapeutic agent for degenerative brain diseases, including Alzheimer's dementia.

Figure 1 shows cells overexpressing the beta-amyloid precursor gene treated with Vespakinin, Mastoparan, Eumenine Mastoparan-AF (EMP-AF), or a combination thereof. This is a graph showing the expression level of Brain Derived Neurotropic Factor (BDNF).
Figure 2 shows the results confirming the effect of promoting neurite growth when mastoparan was added to a human nerve cell line.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[Preparation example]

1. Cell culture method and experimental group settings

SH-SY5Y human neuroblastoma cell line cells were cultured in DMEM (Dulbecco's modified) containing 5% fetal bovine serum and penicillin/streptomycin at 37°C and 5% CO ₂ conditions. Subculture was carried out in Eagle's medium) medium.

Next, cDNA of the beta-amyloid precursor (APP) gene was transformed into the SH-SY5Y cell line to overexpress APP. Thereafter, the overexpressed cells are treated with Vespakinin, Mastoparan, Eumenine Mastoparan-AF (EMP-AF), Bradykinin, or a combination thereof. One experimental group was cultured.

Table 1 shows the peptide sequences of vespakinin, mastoparan, and Eumenine Mastoparan-AF (EMP-AF) used in the experiment.

sequence number denomination Sequence information (N→C) One Vespakinin GRPPGFSPFR 2 mastoparan INLKALAALAKKIL 3 Eumenin Mastopalan-AF INLLKIAKGIIKSL

Specifically, the treatment groups for each combination of active ingredients include a group treated with vespakinin alone, a group treated with mastoparan alone, a group treated with EMP-AF alone, a group treated with vespakinin and mastoparan, a group treated with mastoparan and EMP-AF, The experimental groups were set as vespakinin and EMP-AF treatment group, and Vespakinin, Mastoparan, and EMP-AF treatment group. In the case of the single treatment group, the concentration of each active ingredient was set to 0.01mM.

In addition, as treatment groups by active ingredient dose, for the group treated with Vespakinin alone, 0μM, 0.01μM, 0.1μM, 1μM, and 10μM were treated to the cells, and for the group treated with Mastoparan only, 0μM , 0.01μM, 0.1μM, 1μM and 10μM, 50μM and 100M were treated to the cells. For the EMP-AF treatment group alone, 0μM, 0.01μM, 0.1μM, 1μM, 10μM and 50μM were treated to the cells.

2. Performance of RT-PCR

To isolate mRNA from cells treated with Vespakinin, Mastoparan, Eumenine Mastoparan-AF (EMP-AF), Bradykinin, or a combination thereof. , Trizol reagent was used in the cells of each experimental group. Afterwards, these quantitative changes were confirmed through RT-PCR using agarose gel. Additionally, to perform real-time quantitative PCR (qPCR), Biorad's Real-time PCR CFX96 system was used using Bioneer's Accupower Greenstar qPCR premix. was used.

3. Performance of Western Blot

20 ug of protein in each experimental group treated with Vespakinin, Mastoparan, Eumenine Mastoparan-AF (EMP-AF), Bradykinin, or a combination thereof was extracted. The extracted proteins were separated using 10% SDS-PAGE and then transferred to a nitrocellulose (NC) membrane. Next, the cells were blocked in 5% skim milk for 1 hour and reacted with the primary antibody, APP/β-Amyloid antibody, for more than 16 hours. Afterwards, it was reacted with HRP-conjugated secondary antibody for more than 2 hours. Finally, the results of Western blot were shown by reacting with ECL (enhanced chemiluminescence, GE) reagent.

[Example]

Example 1. Confirmation of expression level of APP gene

1.1 Confirmation of expression level of each combination of active ingredients

To cells overexpressing the beta-amyloid precursor gene, Vespakinin, Mastoparan, Eumenine Mastoparan-AF (EMP-AF), or a combination thereof (1 uM each) In order to observe the expression level of the beta-amyloid precursor gene upon treatment, RT-PCR and Western blot were performed on the cells of the treatment group for each combination of active ingredients in Preparation Example 1 above.

As a result, compared to the negative control untreated group (APP), the expression of beta-amyloid precursor genes and proteins decreased in the experimental group treated with Vespakinin, Mastoparan, EMP-AF, or a combination thereof. could be confirmed. These results mean that Vespakinin, Mastoparan, EMP-AF, or a combination thereof has a significant effect in preventing or treating degenerative brain diseases by reducing beta-amyloid precursors. do.

1.2 Confirmation of expression level of mastoparan by content

In order to observe the expression level of the beta-amyloid precursor gene when cells overexpressing the beta-amyloid precursor gene were treated with Mastoparan by content, the cells in the treatment group according to the dosage of the active ingredient in Preparation Example 1 above were subjected to treatment. RT-PCR was performed.

As a result, it was confirmed that the expression level of the beta-amyloid precursor gene was relatively decreased in the group treated with 0.01 μM to 50 μM mastoparan compared to the negative control untreated group (0 μM). In addition, in the group treated with 100 μM mastoparan, the expression of the beta-amyloid precursor gene was confirmed to be similar to that of the untreated group. These results mean that a composition containing a certain amount of mastoparan has a significant effect in preventing or treating degenerative brain diseases by reducing beta-amyloid precursors.

1.3 Confirmation of expression level of vespakinin by content

In order to observe the expression level of the beta-amyloid precursor gene when cells overexpressing the beta-amyloid precursor gene were treated with Vespakinin by content, the cells in the treatment group according to the dosage of the active ingredient in Preparation Example 1 above were tested. RT-PCR was performed.

As a result, it was confirmed that the expression level of the beta-amyloid precursor gene was relatively decreased in the group treated with 0.01 μM to 1 μM of vespakinin compared to the negative control untreated group (0 μM). In addition, in the group treated with 10 μM vespakinin, the expression of the beta-amyloid precursor gene was confirmed to be higher than that in the untreated group. These results mean that a composition containing a certain amount of vespakinin has a significant effect in preventing or treating degenerative brain diseases by reducing beta-amyloid precursors.

1.4 Confirmation of expression level of EMP-AF by content

In order to observe the expression level of the beta-amyloid precursor gene when cells overexpressing the beta-amyloid precursor gene are treated with Eumenine Mastoparan-AF (EMP-AF) by content, the above preparation example RT-PCR was performed on cells from treatment groups for each combination of active ingredients in 1.

As a result, it was confirmed that the expression level of the beta-amyloid precursor gene was relatively decreased in the group treated with 0.01 μM to 0.1 μM EMP-AF compared to the negative control untreated group (0 μM). In addition, in the group treated with 10 μM EMP-AF, the expression of beta-amyloid precursor gene was confirmed to be higher than that of the untreated group. These results mean that a composition containing a certain amount of EMP-AF has a significant effect in preventing or treating degenerative brain diseases by reducing beta-amyloid precursors.

Example 2. Confirmation of expression level of IL-10 cytokine

Anti-inflammatory effect when cells overexpressing the beta-amyloid precursor gene are treated with Vespakinin, Mastoparan, Eumenine Mastoparan-AF (EMP-AF), or a combination thereof In order to observe the expression level of the cytokine IL-50, RT-PCR was performed on cells from treatment groups for each combination of active ingredients in Preparation Example 1.

As a result, it was confirmed that the expression level of IL-10 was relatively increased when treated with Vespakinin, Mastoparan, EMP-AF, or a combination thereof. In particular, when EMP-AF and Vespakinin or Mastoparan were combined, the expression level was higher than the group treated with EMP-AF alone. These results show that the composition containing Vespakinin, Mastoparan, EMP-AF, or a combination thereof increases the expression of IL-10, an anti-inflammatory cytokine, thereby suppressing neuroinflammation. This means that it is significantly effective in preventing or treating degenerative brain diseases.

Example 3. Confirmation of expression level of neprilysin

When cells overexpressing the beta-amyloid precursor gene are treated with Vespakinin, Mastoparan, Eumenine Mastoparan-AF (EMP-AF), or a combination thereof, amyloid precursors Alternatively, in order to observe the expression level of Neprilysin, a protease that decomposes amyloid, RT-PCR was performed on cells from treatment groups for each combination of active ingredients in Preparation Example 1.

As a result, it was confirmed that the expression level of neprilysin was relatively increased when treated with Vespakinin, Mastoparan, EMP-AF, or a combination thereof. In particular, when EMP-AF, Vespakinin, and Mastoparan were combined, the expression level was higher than the group treated with Mastoparan alone. These results show that, in the case of a composition containing Vespakinin, Mastoparan, EMP-AF, or a combination thereof, the expression of amyloid precursors or proteases that decompose amyloid is increased, thereby reducing amyloid precursors. This means that it has a significant effect in preventing or treating degenerative brain diseases.

Example 4. Confirmation of expression level of insulin decomposing enzyme

When cells overexpressing the beta-amyloid precursor gene are treated with Vespakinin, Mastoparan, Eumenine Mastoparan-AF (EMP-AF), or a combination thereof, amyloid precursors Alternatively, to observe the expression level of insulin degradation enzyme (IDE), a protease that decomposes amyloid, RT-PCR was performed on cells treated with each combination of active ingredients in Preparation Example 1.

As a result, it was confirmed that the expression level of IDE was relatively increased when treated with Vespakinin, Mastoparan, EMP-AF, or a combination thereof. In particular, when two or more were treated in combination, the expression level was higher than the group treated with vespakinin alone. These results show that compositions containing Vespakinin, Mastoparan, EMP-AF, or a combination thereof increase the expression of amyloid precursors or proteases that decompose amyloid, thereby reducing amyloid precursors. By reducing it, it means that there is a significant effect in preventing or treating degenerative brain diseases.

Example 5. Confirmation of expression level of brain-derived neurotrophic factor

When cells overexpressing the beta-amyloid precursor gene are treated with Vespakinin, Mastoparan, Eumenine Mastoparan-AF (EMP-AF), or a combination thereof, neuronal cells In order to observe the expression level of Brain Derived Neurotropic Factor (BDNF), which regenerates, RT-PCR was performed on cells from treatment groups for each combination of active ingredients in Preparation Example 1.

Figure 1 shows brain-derived neurotrophic factor (BDNF) when cells overexpressing the beta-amyloid precursor gene are treated with Vespakinin, Mastoparan, EMP-AF, or a combination thereof. This is a graph showing the expression level of .

As a result, as shown in Figure 1, it was confirmed that the expression level of BDNF was relatively increased when treated with Mastoparan, EMP-AF, or a combination thereof. In particular, when two or more were treated in combination, the expression level was higher than the group treated with each active ingredient alone. These results show that the composition containing Vespakinin, Mastoparan, EMP-AF, or a combination thereof increases the expression of BDNF, which regenerates nerve cells, thereby repairing nerve damage and preventing degenerative disease. This means that it has a significant effect in preventing or treating brain diseases.

Example 6. Confirmation of neurite growth promotion effect

In this example, in order to confirm the regeneration of nerve cells by treatment with mastoparan, the growth of neurites was examined. Specifically, mastoparan was added to SH-SY5Y, a human neural cell line, at a concentration of 0.001, 0.01, 0.1, or 1 mM, and cultured for 24 hours. Afterwards, the grown neurites were confirmed through a microscope, and their length was measured and evaluated.

As a result, as shown in Figure 2, the length of neurites was increased by the addition of mastoparan according to one embodiment.

Through the above results, it was found that the peptide according to one embodiment can contribute to suppressing the occurrence and progression of degenerative brain diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis.

Claims (4)

A peptide that exhibits cell growth-promoting activity and contains Vespakinin, Mastoparan, and Eumenine Mastoparan-AF as active ingredients. In claim 1,
The peptide exhibits cell growth promoting activity, having the following characteristics:
(a) Inhibition of amyloid precursor protein (APP) expression;
(b) increased expression of anti-inflammatory cytokines;
(c) increased expression of Neprilysin and IDE (insulin degrading enzyme) proteins;
(d) Increased expression of Brain Derived Neurotropic Factor; or
(e) Promoting neurite growth. In claim 1,
The N-terminus of the component is acetyl group, fluoreonylmethoxycarbonyl group, formyl group, palmitoyl group, myristyl group, and stearyl group. A peptide exhibiting cell growth promoting activity, characterized in that it is combined with any one protecting group selected from the group consisting of (stearyl group) and polyethylene glycol (PEG). In claim 1,
The C-terminus of the component exhibits cell growth promoting activity, characterized in that it is combined with any one protecting group selected from the group consisting of amino group (-NH ₂ ) and azide (-NHNH ₂ ). Peptide.