KR20210074869A - Composition for promoting myelination in nerve cell comprising daidzein and use thereof - Google Patents

Abstract

According to a method for treating diseases associated with demyelination of nerve cells, comprising daidzein, a pharmaceutically acceptable salt or solvate thereof, and a health functional food for preventing or alleviating diseases associated with demyelination of nerve cells, it is possible to promote demyelination by increasing the expression of SOX 10.

Description

Composition for promoting myelination of nerve cells, including daidzein, and their use {Composition for promoting myelination in nerve cell comprising daidzein and use thereof}

It relates to a composition comprising daidzein for promoting myelination of nerve cells, and to a use thereof.

Demyelinating disease refers to a disease of the nervous system in which the myelin sheath of neurons is damaged. This damage reduces signal transmission in the damaged nerve.

Myelin (myelin) is a multi-insulating layer that forms the axon surrounding the neuron, it promotes the transmission of shock, protects the nutrients around the axon, and serves to provide nutrition. It is also known to be produced by Schwann cells of the peripheral nervous system (PNS) and produced by oligodendrocytes of the central nervous system.

In turn, this decrease in transmission causes a decrease in sensation, movement, cognition, or other functions that depend on nerves. The disease may include those affecting the central nervous system and those affecting the peripheral nervous system. The peripheral nervous system demyelinating diseases include acute infectious polyneuritis (Guillain-Barre Syndrome) and Charcot Marie Tooth (CMT) and the like. The central nervous system demyelination disease includes multiple sclerosis.

The development of therapeutic agents for patients suffering from demyelination diseases as described above is being actively conducted. Currently, ascorbic acid is effective in animal models of peripheral neuropathy, but it is not effective in clinical trials, so there is a need for a fundamental treatment.

Therefore, in order to overcome the above problems, the present inventors have solved the above problems by developing a composition that is composed of natural compounds and minimizes side effects, while having an effective myelination effect.

One aspect provides a pharmaceutical composition for preventing or treating a disease associated with demyelination of nerve cells, comprising daidzein, a pharmaceutically acceptable salt or solvate thereof.

Another aspect provides a composition for promoting remyelination or inhibiting demyelination in a neuron comprising daidzein, a pharmaceutically acceptable salt or solvate thereof.

Another aspect provides a dietary supplement for preventing or ameliorating a disease associated with demyelination of nerve cells, comprising daidzein, a pharmaceutically acceptable salt or solvate thereof.

One aspect provides a pharmaceutical composition for preventing or treating a disease associated with demyelination of nerve cells, comprising daidzein, a pharmaceutically acceptable salt or solvate thereof.

Another aspect provides a composition for promoting remyelination or inhibiting demyelination in a neuron comprising daidzein, a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the daidzein may be in the form of a pharmaceutically acceptable salt thereof.

The daidzein and a pharmaceutically acceptable salt thereof may have the structure of Formula 1 below.

[Formula 1]

The daidzein may also be in the form of a solvate thereof. The term “solvate” refers to a complex or aggregate formed by one or more solute molecules, ie, a compound of Formula 1 or a pharmaceutically acceptable salt thereof, and one or more solvent molecules. The solvate may be, for example, a complex or aggregate formed with water, methanol, ethanol, isopropanol or acetic acid.

Said daidzein may also be in the form of its stereoisomers. The stereoisomer includes all stereoisomers such as enantiomers and diastereomers. The compound may be in a stereoisomerically pure form or a mixture of one or more stereoisomers, for example a racemic mixture. Separation of specific stereoisomers can be carried out by one of the conventional methods known in the art.

The daidzein may be chemically synthesized or purchased commercially.

In one embodiment, the composition may be a pharmaceutical composition. The composition may further include a pharmaceutically acceptable carrier. In the above compositions, "pharmaceutically acceptable carrier" refers to a substance used in combination with an active ingredient to aid in application of the active ingredient, usually an inert substance. The carrier includes conventional pharmaceutically acceptable excipients, additives or diluents. Such carriers may be, for example, fillers, binders, disintergrants, buffers, preservatives, antioxidants, glidants, flavoring agents, thickeners, coloring agents, emulsifying agents, suspending agents It may include one or more selected from a topical agent, a stabilizing agent, and an isotonic agent.

The pharmaceutical composition according to one embodiment may contain a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, an analgesic agent, Buffers, reducing agents, antioxidants and the like may be included as appropriate. Pharmaceutically acceptable carriers and agents suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995. The pharmaceutical composition according to one embodiment is formulated in a unit dosage form by using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. It can be prepared as or by putting it in a multi-dose container. Herein, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of a powder, granules, tablets or capsules.

The composition may include the daidzein or a pharmaceutically acceptable salt or solvate thereof in a “therapeutically effective amount”. In the composition, "therapeutically effective amount" means an amount sufficient to exhibit a therapeutic effect when administered to an individual in need of treatment.

The term “treatment” means treating a disease or medical condition, eg, a demyelination-associated disease, in a subject, eg, a mammal, including a human, including: (a) a disease or medical condition preventing the occurrence of symptoms, ie, prophylactic treatment of the patient; (b) alleviating the disease or medical condition, ie, causing the elimination or recovery of the disease or medical condition in the patient; (c) inhibiting the disease or medical condition, ie, slowing or arresting the progression of the disease or medical condition in a subject; or (d) alleviating the disease or medical condition in the subject.

The term “prevention” refers to inhibiting the development of a disease or medical condition, eg, a demyelination-associated disease, in a subject, eg, a mammal, including a human.

The terms “treatment” and “prevention” are not intended to mean cure or complete elimination of symptoms. They may refer to any form of treatment that provides an effect to a patient suffering from a disease, including ameliorating the patient's condition (eg, one or more symptoms), delaying the progression of the disease, and the like.

In addition, the composition comprises daidzein, a pharmaceutically acceptable salt thereof, or a solvate thereof in an "effective amount to promote remyelination or inhibit demyelination in neurons of a mammal" or "Schwann cells of a mammal" It may be included in an amount effective to increase the expression of SOX 10 in ".

The "effective amount" can be appropriately selected by those skilled in the art. The "effective amount" may be 0.01 mg to 10,000 mg, 0.1 mg to 1000 mg, 1 mg to 100 mg, 0.01 mg to 1000 mg, 0.01 mg to 100 mg, 0.01 mg to 10 mg, or 0.01 mg to 1 mg.

In one embodiment, the content or effective amount of the daidzein may be 2 to 30 uM. For example, 4-28 uM, 5-27 uM, 6-26 uM, 7-25 uM, 8-24 uM, 8-23 uM, 8.5-22 uM, 9-21 uM, 8-20 uM, 10 to 18 uM, 7.5 to 17 uM, 7 to 16 uM, 8 to 15 uM, 8 to 14 uM.

The composition may include daidzein, a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient. "Active ingredient" is intended to perform the function mentioned in the composition, and excludes those that do not perform the function because they are included in small amounts as impurities.

The dosage of the composition varies depending on various factors such as the patient's condition, administration route, judgment of the attending physician, and the like. The effective dosage can be estimated from the dose-response curves obtained from in vitro experiments or animal model tests, and the formulation method, administration method, age, weight, sex, morbidity, food, administration time, administration route, excretion rate, and It can be prescribed in various ways depending on factors such as response sensitivity, and those skilled in the art can appropriately adjust the dosage in consideration of these factors. The number of administration may be one time or two or more within the range of clinically acceptable side effects, and may be administered to one or two or more sites for the administration site. For animals other than humans, either use the same dose per kg as that of a human, or convert the above dose to, for example, the volume ratio (for example, average value) of an organ (heart, etc.) between the target animal and human. One dose can be administered.

The terms “administering,” “introducing” and “implanting” are used interchangeably and in one embodiment into a subject by a method or route that results in at least partial localization of the composition according to one embodiment to a desired site. It may mean the arrangement of the composition according to

The composition may be administered orally or parenterally, including intravenous, intraperitoneal, subcutaneous, rectal and topical administration. Accordingly, the composition may be formulated in various forms such as tablets, capsules, aqueous solutions or suspensions. In the case of oral tablets, excipients such as lactose and corn starch and lubricants such as magnesium stearate may be usually added. In the case of capsules for oral administration, lactose and/or dry corn starch may be used as diluents. If an aqueous suspension for oral use is required, the active ingredient may be combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring agents may be added. For intraneural, intramuscular, intraperitoneal, subcutaneous and intravenous administration, a sterile solution of the active ingredient is usually prepared, and the pH of the solution must be appropriately adjusted and buffered. For intravenous administration, the total concentration of solutes should be adjusted to render the formulation isotonic. The composition may be in the form of an aqueous solution containing a pharmaceutically acceptable carrier, such as saline having a pH of 7.4. The solution can be introduced into the patient's intramuscular or intraneural bloodstream by local bolus injection.

The daidzein, a pharmaceutically acceptable salt or solvate thereof may have an effect of inhibiting demyelination or restoring demyelinated neurons to myelinated neurons. The effect may be derived from increasing the expression level of the SOX 10 protein.

The composition may be combined with one or more other therapeutic agents to treat demyelination and associated diseases. In addition, the composition may include a daidzein compound or a pharmaceutically acceptable salt or solvate thereof without other active ingredients for treating demyelination and related diseases.

The composition may be for treating a disease associated with demyelination in neurons of a mammal. The disease associated with demyelination of a neuron (hereinafter, also referred to as “demyelinating disease”) refers to a disease of the nervous system in which the myelin sheath of neurons is damaged. The damage includes any damage that reduces signaling in neurons, including loss of myelin bound to neurons, a decrease in their amount, or a change in their structure. This damage reduces signal transmission in the damaged nerve. In turn, this decrease in transmission causes a decrease in sensation, movement, cognition, or other functions that depend on nerves.

The disease may include those affecting the central nervous system (hereinafter also referred to as “central nervous system demyelination disease”) and those affecting the peripheral nervous system (hereinafter also referred to as “peripheral nervous system demyelination disease”).

The central nervous system demyelinating diseases include multiple sclerosis, Devic' disease, inflammatory demyelinating diseases, CNS neuropathies such as caused by vitamin B12 deficiency, Tabes dorsalis and myelopathies, leukoencephalopathies such as progressive multifocal leukoencephalopathy, leukodystrophies, or a combination thereof.

The peripheral nervous system demyelination disease is Guillain-Barre syndrome and its chronic counterpart, chronic inflammatory demyelinating polyneuropathy, Dejerine-Sottas disease, Anti -MAG peripheral neuropathy, Charcot Marie Tooth (CMT), copper deficiency, progressive inflammatory neuropathy, or a combination thereof. The disease may be multiple sclerosis, acute infectious polyneuritis (Guillain-Barre Syndrome), Charcot Marie Tooth (CMT), or a combination thereof.

In an embodiment, the Charcot Marie Tooth disease may be a CMT1A subtype, a CMT1E subtype, a CMT1 subtype, a CMT2 subtype, a CMT3 subtype, a CMT4 subtype, or a CMTX subtype. Preferably, it may be a CMT1A subtype or a CMT1E subtype, but is not limited thereto.

In the composition, the disease may be caused by low expression of SOX 10 protein. The SOX 10 protein is a protein encoded by the SOX 10 gene in humans.

Another aspect provides the use of daidzein, or a pharmaceutically acceptable salt or solvate thereof, as defined above, for the manufacture of a medicament for the treatment of a disease associated with demyelination.

In another aspect, by administering to a mammal an effective amount of daidzein, a pharmaceutically acceptable salt or solvate thereof, for preventing or treating a disease associated with demyelination of neurons, it is associated with demyelination of neurons. Provided is a method for preventing or treating a disease associated with demyelination of neurons in a mammal, comprising the step of preventing or treating the disease.

In another aspect, by administering to the mammal an amount of daidze, a pharmaceutically acceptable salt or solvate thereof, effective to promote remyelination or inhibit demyelination in a mammalian neuron, It provides a method for promoting remyelination or inhibiting demyelination in a mammalian neuron, including; promoting remyelination or inhibiting demyelination in a cell.

In another aspect, by administering to the mammal an effective amount of daidzein, a pharmaceutically acceptable salt or solvate thereof to increase the expression level of SOX 10 protein in the mammalian nerve cells, the mammalian nerve It provides a method of increasing the expression of SOX 10 protein in mammalian neurons, including; increasing the expression of SOX 10 protein in a cell.

In the method, the administration may be administered when the nerve cells are in the differentiation stage or myelination stage. In one embodiment, an effective amount may be administered in both the differentiation stage and the myelination stage. In the case of including the above steps, the myelination stage may be greater than when administered only in the differentiation stage or myelination stage.

In one embodiment, the nerve cells may include Schwann cells or may be Schwann cells. In one embodiment, by increasing the expression of SOX10 protein in Schwann cells of a mammal, it may have an effect of inhibiting myelination.

The "amount effective to prevent or treat a disease associated with demyelination of nerve cells" refers to a disease associated with demyelination of nerve cells when administered to an individual in need of prevention or treatment of a disease associated with demyelination of nerve cells. It means an amount sufficient to exhibit a prophylactic or therapeutic effect. "An amount effective to promote or inhibit demyelination in neurons of a mammal" is an amount that promotes or inhibits remyelination when administered to a subject in need of or in need of inhibiting demyelination. It means an amount sufficient to exhibit the effect of inhibiting , and the same term is construed as having the same meaning as previously mentioned, unless otherwise stated.

In the method, the dosage varies according to various factors such as the patient's condition, administration route, judgment of the attending physician, and the like, as described above. An effective dosage can be estimated from a dose-response curve obtained from in vitro experiments or animal model studies. The proportion and concentration of the compound of the present invention present in the composition to be administered may depend on the chemical nature, route of administration, therapeutic dosage, and the like. The dosage may be administered to an individual in an effective amount of about 1 μg/kg to about 1 g/kg per day, or about 0.1 mg/kg to about 500 mg/kg per day. The dose may be changed according to the age, weight, susceptibility, or symptoms of the individual.

In one embodiment, the content or effective amount of the daidzein may be 2 to 30 uM. For example, 4-28 uM, 5-27 uM, 6-26 uM, 7-25 uM, 8-24 uM, 8-23 uM, 8.5-22 uM, 9-21 uM, 8-20 uM, 10 to 18 uM, 7.5 to 17 uM, 7 to 16 uM, 8 to 15 uM, 8 to 14 uM.

The method may further comprise collecting information related to demyelination in neurons of the subject prior to said administration. Information related to demyelination may be a degree of demyelination, a rate of demyelination, a form of demyelination, and the like. The information related to the demyelination may be measured by visualizing the nerve cells, such as an optical or fluorescence microscope, or using a myelination-specific marker. Accordingly, the method may further comprise measuring demyelination in the neuron. The nerve cells may be derived from an individual having or suspected of having a disease associated with demyelination. In addition, the method may further include the step of determining whether demyelination has progressed or is in progress compared to a control group, based on the information collected by the step of collecting information related to demyelination. The control may be the degree of myelination of normal neurons derived from an individual without demyelination disease, or the degree of myelination of neurons of an average individual. In addition, the method includes the step of administering daidzein, a pharmaceutically acceptable salt thereof, or a solvate thereof to a mammal to an individual determined to be undergoing or undergoing demyelination compared to the control in the determining step. may include

In addition, in the method, the method may further include measuring the expression level of the SOX 10 protein in the nerve cells of the individual before the administration. The expression level may be mRNA or protein level expression. The expression level is measured by amplification using a primer nucleic acid specific for mRNA (eg, polymerase chain reaction (PCR)), Southern blotting, or sequencing, or by isolation of a PMP22-expressing protein, Western blotting, etc. can be The nerve cells may be derived from an individual having or suspected of having a disease associated with demyelination. In addition, the method may further include the step of determining whether the expression level measured by the measuring step is higher than that of the control. The control may be the degree of myelination of normal neurons derived from an individual without demyelination disease, or the degree of myelination of neurons of an average individual. In addition, the method includes administering daidzein, a pharmaceutically acceptable salt thereof, or a solvate thereof to a mammal for an individual whose expression of the SOX 10 protein is determined to be lower than that of the control in the determining step. may include

Another aspect is to contact a neuron in a mammal with a pharmaceutically acceptable salt or solvate thereof, which is a daid agent, in an amount effective to promote remyelination or inhibit demyelination, thereby causing remyelination in the neuron in the mammal. It provides a method for promoting remyelination or inhibiting demyelination in a mammalian neuron, including; promoting or inhibiting demyelination. The nerve cells may be isolated from the subject or may be present in vitro.

Another aspect is to contact a mammalian neuron with an amount of daidze, a pharmaceutically acceptable salt or a solvate thereof, effective to reduce the expression of PMP22, thereby inhibiting the expression of SOX 10 protein in the mammalian neuron. It provides a method for increasing the expression of SOX 10 protein in mammalian neurons, comprising the step of increasing.

The nerve cells may be isolated from the subject or may be present in vitro.

In one embodiment, the nerve cells may include Schwann cells or may be Schwann cells.

In the method, the contacting may be performed in a liquid medium, for example, a culture medium of the neurons, or a buffer.

Another aspect provides a dietary supplement for preventing or ameliorating a disease associated with demyelination of nerve cells, comprising daidzein, a pharmaceutically acceptable salt or solvate thereof.

The term “improvement” refers to lessening the severity of a disease or medical condition, such as a demyelination-associated disease symptom, in a subject, for example, a mammal, including a human, and other terms are the same as described above.

A pharmaceutical composition for preventing or treating a disease associated with demyelination of nerve cells comprising daidzein, a pharmaceutically acceptable salt thereof, or a solvate thereof according to an aspect, and a health function for preventing or improving According to food, it is possible to promote myelination by increasing the expression of SOX 10.

Figure 1a is a schematic diagram showing the SOX10-MCS1C plasmid that can confirm the activity of the SOX10 promoter used in the experiment.
Figure 1b is a schematic diagram showing the SOX10-MCS4 plasmid that can confirm the SOX10 enhancer activity used in the experiment.
Figure 2a is a graph comparing the expression rates of SOX10 promoter of candidate compounds.
Figure 2b is a graph comparing the expression rate of SOX10 enhancer of the candidate compound.
Figure 3a is a graph showing the results of measuring the expression level of SOX 10 protein of two candidate compounds by Western blot.
Figure 3b is a graph quantitatively showing the results of measuring the expression level of SOX 10 protein of two candidate compounds by Western blot.
Figure 4a is a graph showing the degree of myelination of the experimental group treated with daidzein in the differentiation and myelination stage compared with the control group; MBP stands for myelin sheath, Tuj1 stands for neuron cell, and DAPI stands for cell nucleus.
FIG. 4B is a graph showing the degree of myelination shown in FIG. 4A by randomly designating three places and quantifying it with the imagej program.
Figure 5a is a graph showing the degree of myelination of the experimental group treated with daidzein in the differentiation stage compared with the control group; MBP stands for myelin sheath, Tuj1 stands for neuron cell, and DAPI stands for cell nucleus.
FIG. 5B is a graph showing the degree of myelination shown in FIG. 5A by randomly designating three places and quantifying it with the imagej program.

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

[Preparation example]

One. Preparation of animal models

Trembler J (Tr-j), a protein known to control the shape of myelin, is a mouse with a spontaneous mutation in the gene of PMP22. This mutation is named PMP22_L16P. The mouse is characterized in that it exhibits a phenotype in which the hind legs are trembling or withdrawn, unlike a normal mouse in which the hind legs are straightened when the tail is raised. This mouse is a strain frequently used when studying CMT1A and CMT1E among the Charco Marie tooth subtypes.

2. Preparation of Daidzein and Compounds

The FDA-approved Drug Library (L1300, 10mM in DMSO solution) purchased from Selleckchem was diluted 1/1000 in the medium and used at a concentration of 10uM.

Daidzein (Catalog No. S1849, 10 mM in DMSO) purchased from Selleckchem was diluted 1/1000 in the medium and used at a concentration of 10 uM.

Sulfasalazine (Catalog No. S0883-100G) purchased from Sigma was dissolved in DMSO to make a concentration of 10 mM, and then diluted at a ratio of 1/1000 in the medium to use at a concentration of 10 uM.

[Example]

One. Methods for screening compounds at the DNA level

1-1 Selection of candidate compounds

In order to select compounds capable of promoting myelination, the FDA-approved Drug Library (L1300) including a total of 1172 compounds was selected as a candidate group. The compounds were obtained from Selleckchem.

1-2 Primary screening using dual luciferase assay

1-2-1 Use of Schwannoma

For the primary screening (Screening), Schwannoma (rt-4d6p2t) was used. Schwannoma refers to cancer arising from Schwann cells, and the cells obtained here have immortality due to the nature of cancer. In order to obtain a sufficient number of Schwann cells obtained from the primary culture to be used for primary screening, a very large number of mice must be sacrificed, the division of Schwann cells itself is slow, and differences in individual status and experimental environment There are many difficulties due to the existence of Therefore, in the first screening, a cell line has already been established and an experiment was conducted on Schwannoma, which has similar properties to Schwann cells.

1-2-2 Method of Plasmid Transfection

Dual luciferase assay was used to determine whether DNA expression was increased. Luciferase is a luminescent protein that can be used to measure changes in DNA expression level.

We transfected Schwannoma with a plasmid before treating the compound with Schwannoma to make it express luciferase.

More specifically, three plasmids were used. at first. MCS-1C with firefly luciferase, MCS4 with sox10 enhancer activity, firefly luciferase, and finally pRL-TK with renilla luciferase as a control for firefly luciferase luminescence.

As shown in FIGS. 1A and 1B , MCS-1C and MCS4 contain the luciferase gene, and when used for transfection, the gene can be expressed in schwannoma, a host cell.

The transfection was performed with MCS-1C and pRL-TK, or two combinations of MCS4 and pRL-TK.

1-2-3 Dual luciferase assay method

After the transfection was completed, 2*10^3 cells/well of Schwannoma was administered to each well of a 96-well plate, the candidate compound was treated, and a dual luciferase assay was performed after a certain time elapsed.

If the compound increased the expression level by activating the Sox10 promoter, the level of luminescence of firefly luciferase in the MCS-1C-transfected cells would increase. was expected to increase. Also, at this time, to check whether the Firefly value simply changed due to a large number of cells or a small number of cells, the Renilla value was also measured.

1-2-4 Selection of the top 4 compounds

The results of the dual luciferase assay were confirmed by the fold-change method in which the final result value was determined by comparing firefly and renilla, and the result value of the control group was set to 1. In addition, as compared to the control group treated with DMSO, the solvent of the compound, compounds having both high MCS1C and MCS4 results were selected.

Table 1 below is a table showing the results of the Dual luciferase assay of MCS1C plasmid-transfected cells.

Table 2 below is a table showing the results of Dual luciferase assay of MCS4 plasmid-transfected cells.

As a result, as shown in Tables 1 and 2, the results of Nos. 62, 391, 470, and 625 of the top four candidate compounds were confirmed.

2. Method for secondary screening of compounds at the genetic level

The transcriptional activity of the SOX10 gene was investigated using MCS1C and MCS4 among the four primary selected 62, 391, 470, and 625 using a dual luciferase assay.

As a result, as shown in FIGS. 2A and 2B , it was possible to confirm the transcriptional activity results of SOX10 at Nos. 62, 391, 470, and 625 of the top four candidate compounds.

As a result, as shown in FIG. 2 , it was confirmed that the sox 10 gene transcription activity was increased by the compounds 62 and 391 rather than the compound 625.

3. Confirmation of increased expression of SOX 10 protein in Schwann cells of daidzein

The present inventors conducted an experiment to confirm that not only the expression at the DNA level is increased, but also the expression of the sox10 protein in Schwannoma is increased by the candidate compound.

More specifically, Schwannoma was seeded with 5x10^3 cells/1 well (based on 12 wells) and prepared by diluting compounds (391, 62) to a concentration of 10uM in medium (DMEM-10% FBS, 1% antibiotic) the next day and 24 hours. processed. After each compound was treated at a concentration of 10 uM for 24 hours, the sox10 protein expressed by Schwanooma was confirmed using western blot.

As a result, as shown in FIG. 3, it was confirmed that the expression of sox 10 was more effectively increased in the group treated with compound No. 391 when the compound No. 62 and No. 391 were compared. Therefore, 391 compound, daidzein, was selected.

Table 3 below is data obtained by quantifying FIG. 3B. More specifically, it was quantified how much sox10 was increased by the compound compared to the experimental group by the fold-change method in which the control group (DMSO, negative control) was set to 1.

experimental group sox10 actin sox10/actin normalization DMSO 24605.51 11698.05 2.103386506 One 62 19817.05 8593.974 2.305923313 1.096290818 391 (DZ) 29035.66 7053.296 4.116608746 1.957133762

4. Demonstrating the myelination effect of daidzein in the in vitro myelination system

By applying the compound with the highest grades to the in vitro myelination system, an experiment was conducted to see how the compound actually affects myelination.

DRG explant culture was performed using an animal model for the top four candidate substances as a result of the primary selection. DRG explant culture, which is a primary culture of DRG (Dorsal Root Ganglia) itself, can be said to be an in vitro myelination system in the peripheral nervous system.

The in vitro myelination system was sequentially divided into three stages: a cell growth stage, a differentiation stage, and a myelination stage.

Table 4 below shows the composition of the medium in each step.

Step media name Span condition cell growth Neuron Growth media
(NG media) 7 days Neurobasal (P/S 1%)
D-Glucose 4mg/ml
B27 Supplement 1X
NGF 50ng/ml
L-glutamine 2mM Differentiation Differentiation media 7-10 days DMEM/F-12 (P/S 1%)
NGF 50ng/ml
N2 Supplement 1X
L-glutamine 2mM Myelination Myelination media Min.
21 days MEM (Horse Serum 5%, P/S 1%)
D-Glucose 4mg/ml
Bovine pituitary extract 50ug/ml
Forskolin 0.5 uM
NGF 50ng/ml
N2 Supplement 1X
L-Ascorbic acid 50ug/ml
L-glutamine 2mM Name Cat. NO. Note Neurobasal Gibco, 21103-049 DMEM/F12 Welgene, LM 002-04 with 15mM HEPES, with L-glutamine,
with sodium bicarbonate MEM Gibco, 11095-080 D-Glucose Sigma, G7528 Stock solution = cell culture grade water B27 supplement Thermo, 17504-044 NGF Alomone labs, N-100 Stock solution = cell culture grade water N2 supplement Thermo, 17502-048 Bovine pituitary extract Thermo, 13028-014 Forskolin Cayman, 11018 Stock solution = DMSO L-Ascorbic Sigma, A5960 Stock solution = cell culture grade water L-glutamine Gibco, 25030-081 200mM stock

Using the in vitro myelination system, Sulfasalazine, a positive control, and daidzein, a natural compound, were treated at the stage of differentiation and myelination of Schwann cells to demonstrate the myelination effect. At this time, the concentration of the compound was 10uM for both Sulfasalazine and daidzein, and the compound was treated again every time the medium was changed 3 times a week.

In addition, according to the cell cycle of Schwann cells, it is more effective to treat the compound at any time by dividing it into an experimental group treated with the compound only in the differentiation stage (Fig. 5), and an experimental group treated with the compound in both differentiation and myelination stages (Fig. 4). Induction of myelination was also confirmed.

5. Comparison of myelination effects of Sulfasalazine and Daidzein

As shown in Example 4, the degree of myelination was confirmed by using the ICC staining method on the 14th day after the in vitro myelination system was treated with the compound and myelination was induced.

More specifically, the myelination induction period was set as 14 days, which is the minimum period required for myelination to take place, and to check the difference in the degree of myelination between each experimental group, Imagej (a Java-based image processing program developed by NIH) was used to check the difference between compounds. The myelinated points were quantified and determined. Specifically, by using the above program, the resulting photos were randomly assigned to three places, and the degree of myelination was quantified and presented as a graph.

Table 5 below shows the measurement of the degree of myelination of the experimental group treated with the compound in both differentiation and myelination stages.

Differentiation-myelination stage DMSO SSZ DZ One 2.926 8.125 5.97 2 1.647 4.046 7.696 3 2.361 3.401 5.627 average 2.311333 5.190667 6.431 normalized One 2.245746 2.782377

Table 6 below is a measurement of the degree of myelination of the experimental group treated with the compound only in the differentiation stage.

differentiation stage DMSO SSZ DZ One 0.642 3.899 6.48 2 0.964 2.261 4.606 3 1.222 3.778 6.443 average 0.942667 3.312667 5.843 normalized One 3.514144 6.198373

As a result, as shown in Figures 4a and 5a, it was confirmed that the myelination effect of the natural extract daidzein was higher than that of the synthetic compound Sulfasalazine. MBP stands for myelin sheath, Tuj1 stands for neuron cell, and DAPI stands for cell nucleus.

In addition, as a result of quantifying the images of FIGS. 4A and 5A using the imegej program, as shown in FIGS. 4B and 5B , it was confirmed that daidzein, a natural extract, had a higher myelination effect than Sulfasalazine.

In addition, it was confirmed that the case of FIGS. 4a and b treated with the compound in both the differentiation and myelination stages was superior to the myelination effect of FIGS. 5a and b, which is the experimental group treated with the compound only in the differentiation stage. This suggests that treatment with the compound at both the differentiation and myelination phases of the cell cycle is more suitable for inducing myelination.

<110> Daegu Gyeongbuk Institute of Science and Technology <120> Composition for promoting myelination in nerve cell comprising daidzein and use thereof <130> PN130040 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 5567 <212> DNA <213> Artificial Sequence <220> <223> pE1B-C-F/Sox10-MCS1C, pE1B-C-F/Sox10-MCS4 (Identification of neural crest and glial enhancers at the mouse Sox10 locus through transgenesis in zebrafish) <400> 1 ttgtacaaag tggtgatggg ctcgagatct cagggatata aagctgggtt ggtgttgctt 60 tgaatagttc atcttaagct tggcattccg gtactgttgg taaagccacc atggaagacg 120 ccaaaaacat aaagaaaggc ccggcgccat tctatccgct ggaagatgga accgctggag 180 agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt gcttttacag 240 atgcacatat cgaggtggac atcacttacg ctgagtactt cgaaatgtcc gttcggttgg 300 cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta tgcagtgaaa 360 actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt gcagttgcgc 420 ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgggcatt tcgcagccta 480 ccgtggtgtt cgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa aaaaagctcc 540 caatcatcca aaaaattatt atcatggatt ctaaaacgga ttaccaggga tttcagtcga 600 tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat tttgtgccag 660 agtccttcga tagggacaag acaattgcac tgatcatgaa ctcctctgga tctactggtc 720 tgcctaaagg tgtcgctctg cctcatagaa ctgcctgcgt gagattctcg catgccagag 780 atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt gttccattcc 840 atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt cgagtcgtct 900 taatgtatag atttgaagaa gagctgtttc tgaggagcct tcaggattac aagattcaaa 960 gtgcgctgct ggtgccaacc ctattctcct tcttcgccaa aagcactctg attgacaaat 1020 acgatttatc taatttacac gaaattgctt ctggtggcgc tcccctctct aaggaagtcg 1080 gggaagcggt tgccaagagg ttccatctgc caggtatcag gcaaggatat gggctcactg 1140 agactacatc agctattctg attacacccg agggggatga taaaccgggc gcggtcggta 1200 aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa acgctgggcg 1260 ttaatcaaag aggcgaactg tgtgtgagag gtcctatgat tatgtccggt tatgtaaaca 1320 atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct ggagacatag 1380 cttactggga cgaagacgaa cacttcttca tcgttgaccg cctgaagtct ctgattaagt 1440 acaaaggcta tcaggtggct cccgctgaat tggaatccat cttgctccaa caccccaaca 1500 tcttcgacgc aggtgtcgca ggtcttcccg acgatgacgc cggtgaactt cccgccgccg 1560 ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat tacgtcgcca 1620 gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac gaagtaccga 1680 aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata aaggccaaga 1740 agggcggaaa gatcgccgtg taattctaga gtcggggcgg ccggccgctt cgagcagaca 1800 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct 1860 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac 1920 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg 1980 ttttttaaag caagtaaaac ctctacaaat gtggtaaaat cgataaggat ccgtcgaccg 2040 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc 2100 gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg 2160 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 2220 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 2280 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 2340 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 2400 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 2460 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 2520 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 2580 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 2640 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 2700 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 2760 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 2820 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 2880 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 2940 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 3000 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 3060 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 3120 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 3180 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 3240 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 3300 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 3360 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 3420 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 3480 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 3540 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 3600 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 3660 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 3720 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 3780 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 3840 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 3900 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 3960 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 4020 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 4080 aaaagtgcca cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 4140 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 4200 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 4260 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 4320 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 4380 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta 4440 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat 4500 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt gccattcgcc 4560 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca 4620 gcccaagcta ccatgataag taagtaatat taaggtacgg gaggtacttg gagcggccgc 4680 aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta 4740 ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc 4800 cccagtgcaa gtgcaggtgc cagaacattt ctctatcgat aggtaccgag ctcttacgcg 4860 tgctagccca tcacaagttt gtacaaaaaa gcaggcttgc ggtccagctc ggggctgggg 4920 agtgacgctg atggactggg agggaggggc tggggggccc tgagcctcag gccactgcct 4980 tgtctggacg gagcctgaat cctcaccacc aaacaccctg gctggacagc cgagactgac 5040 tgagccgctg cagacggctg gggaaggaag gagggggagg ggagggggaa aggagggagg 5100 gaagggcaga aggtggagcc tccagcgctg ccttcatccc caacacccag gacgcctcca 5160 ctctgcccag cccccccccc cccaacccca gccctgtgac ccgcactgct gcctaggccc 5220 gtctccctgg actcagcttg ggtttggcat ctggtgtgag catggggata ttttaggtct 5280 gggaagggag agatacaggt ggcacagtca gaacctgttg cctgggaccc taaaccattg 5340 ccacaggtgg tggcatggac agttgacaaa ggactggaga gaggtgtgag gagagtggat 5400 aacttgaaag ggtcgtggcc tgtgttgctt catttgagaa atcccactga gtcccactgt 5460 gtgctaggca ccgcagcctg cagtaggtat cagaccctcg ccttctcctg tcctctccaa 5520 cacagctgac ccaagccaag ccccccagga ccgcagaccc agctttc 5567 <210> 2 <211> 5729 <212> DNA <213> Artificial Sequence <220> <223> pE1B-C-F/Sox10-MCS1C, pE1B-C-F/Sox10-MCS4 (Identification of neural crest and glial enhancers at the mouse Sox10 locus through transgenesis in zebrafish) <400> 2 ttgtacaaag tggtgatggg ctcgagatct cagggatata aagctgggtt ggtgttgctt 60 tgaatagttc atcttaagct tggcattccg gtactgttgg taaagccacc atggaagacg 120 ccaaaaacat aaagaaaggc ccggcgccat tctatccgct ggaagatgga accgctggag 180 agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt gcttttacag 240 atgcacatat cgaggtggac atcacttacg ctgagtactt cgaaatgtcc gttcggttgg 300 cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta tgcagtgaaa 360 actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt gcagttgcgc 420 ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgggcatt tcgcagccta 480 ccgtggtgtt cgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa aaaaagctcc 540 caatcatcca aaaaattatt atcatggatt ctaaaacgga ttaccaggga tttcagtcga 600 tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat tttgtgccag 660 agtccttcga tagggacaag acaattgcac tgatcatgaa ctcctctgga tctactggtc 720 tgcctaaagg tgtcgctctg cctcatagaa ctgcctgcgt gagattctcg catgccagag 780 atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt gttccattcc 840 atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt cgagtcgtct 900 taatgtatag atttgaagaa gagctgtttc tgaggagcct tcaggattac aagattcaaa 960 gtgcgctgct ggtgccaacc ctattctcct tcttcgccaa aagcactctg attgacaaat 1020 acgatttatc taatttacac gaaattgctt ctggtggcgc tcccctctct aaggaagtcg 1080 gggaagcggt tgccaagagg ttccatctgc caggtatcag gcaaggatat gggctcactg 1140 agactacatc agctattctg attacacccg agggggatga taaaccgggc gcggtcggta 1200 aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa acgctgggcg 1260 ttaatcaaag aggcgaactg tgtgtgagag gtcctatgat tatgtccggt tatgtaaaca 1320 atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct ggagacatag 1380 cttactggga cgaagacgaa cacttcttca tcgttgaccg cctgaagtct ctgattaagt 1440 acaaaggcta tcaggtggct cccgctgaat tggaatccat cttgctccaa caccccaaca 1500 tcttcgacgc aggtgtcgca ggtcttcccg acgatgacgc cggtgaactt cccgccgccg 1560 ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat tacgtcgcca 1620 gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac gaagtaccga 1680 aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata aaggccaaga 1740 agggcggaaa gatcgccgtg taattctaga gtcggggcgg ccggccgctt cgagcagaca 1800 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct 1860 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac 1920 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg 1980 ttttttaaag caagtaaaac ctctacaaat gtggtaaaat cgataaggat ccgtcgaccg 2040 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc 2100 gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg 2160 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 2220 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 2280 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 2340 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 2400 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 2460 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 2520 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 2580 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 2640 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 2700 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 2760 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 2820 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 2880 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 2940 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 3000 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 3060 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 3120 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 3180 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 3240 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 3300 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 3360 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 3420 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 3480 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 3540 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 3600 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 3660 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 3720 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 3780 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 3840 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 3900 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 3960 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 4020 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 4080 aaaagtgcca cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 4140 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 4200 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 4260 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 4320 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 4380 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta 4440 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat 4500 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt gccattcgcc 4560 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca 4620 gcccaagcta ccatgataag taagtaatat taaggtacgg gaggtacttg gagcggccgc 4680 aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta 4740 ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc 4800 cccagtgcaa gtgcaggtgc cagaacattt ctctatcgat aggtaccgag ctcttacgcg 4860 tgctagccca tcacaagttt gtacaaaaaa gcaggctggg gtcactaaga agacctcctc 4920 caacaagact gtcacttggg aaaggatgaa gcctggccca aaggcatctt ggagccccacg 4980 cctccctgta ttatactctg ccaccagccg gccccaggat gctccaggaa gtgctgtgca 5040 tgacacccgc ccccaggcgc cacctcttag agaagggacc attgtctccg aagggttaac 5100 gagggatgca aggaaggggc cctttagcca tgatgccctc tgacctttca taaatcagag 5160 aggggctggg ggcgaggggc tgcttggcag gacttggtgg ggtggggact tagaagcagc 5220 tgcgcgcgac gttgacattg ttcccaccat tctgagtgca acaaatccct ctattgtgtt 5280 cctggtttat ctggttcctc ttgtttatga gcagggctcc tttggcagtg gttccagccc 5340 tgggcgggtg gaaagagtgc tggcacgcac tgaggggggga ggggcgggag ggggcgctgt 5400 caatgcccgc attgtccccc cgcgcttttt tgtttctact gtaatgacat gttgagagaa 5460 agagagagag agagagagag agagagagag agagagagag aaagtgagtt ggctggggag 5520 agaaaagcac agcaaagaaa gccagccgag caagagctct ggtgtggatg tgtggattga 5580 tatctgggcc tgggcggatg tgctggcagc tgggcggcag ggctgcatgg agccagggaa 5640 gggtgcctgg ggggcatggg gcttgagggg tcagccagac gccccccccc agacacccta 5700 gcctctgctg cctggcacac ccagctttc 5729

Claims (13)

A pharmaceutical composition for preventing or treating a disease associated with demyelination of nerve cells, comprising daidzein, a pharmaceutically acceptable salt or solvate thereof. The pharmaceutical composition of claim 1, wherein the disease is a disease associated with low expression of SOX 10. The method according to claim 1, wherein the disease is the demyelinating disease is multiple sclerosis, Guillain-Barre syndrome (Guillain-Barre Syndrome), Dejerine-Sottas disease (Dejerine-Sottas disease) and at least one composition selected from the group consisting of Charcot Marie Tooth (CMT). The composition of claim 3, wherein the Charcot Marie Tooth disease is CMT1A subtype, CMT1E subtype, CMT1 subtype, CMT2 subtype, CMT3 subtype, CMT4 subtype or CMTX subtype. The composition according to claim 1, wherein the content of the daidzein, a pharmaceutically acceptable salt thereof, or a solvate thereof is 2 to 30 uM. By administering to the mammal an amount of daidzein, a pharmaceutically acceptable salt thereof or a solvate thereof effective to promote remyelination or inhibit demyelination in the mammalian nerve cell, the mammalian neuronal cell A method for preventing or treating a disease associated with demyelination of neurons in a mammal, comprising the step of promoting remyelination in or inhibiting demyelination. The method according to claim 6, wherein the neuron is demyelinated or is in a demyelinated state by low expression of SOX 10. The method according to claim 6, wherein the administering is administered when the nerve cells are in the differentiation stage or myelination stage. The method according to claim 1, wherein the content of the daidzein, a pharmaceutically acceptable salt thereof, or a solvate thereof is 2 to 30 uM. The method according to claim 6, The demyelination disease is multiple sclerosis (multiple sclerosis), Guillain-Barre syndrome (Guillain-Barre Syndrome) Dejerine-Sottas disease (Dejerine-Sottas disease) and Charcot Marie Tooth (CMT). The method of claim 10 , wherein the Charcot Marie Tooth disease is CMT1A subtype, CMT1E subtype, CMT1 subtype, CMT2 subtype, CMT3 subtype, CMT4 subtype or CMTX subtype. Daidzein (Daidzein), a health functional food for preventing or improving a disease associated with demyelination of nerve cells comprising a pharmaceutically acceptable salt or solvate thereof. The method according to claim 12, wherein the demyelinating disease is multiple sclerosis, Guillain-Barre Syndrome, Dejerine-Sottas disease. And Charcot Marie Tooth disease (Charcot Marie Tooth: CMT) is at least one health functional food selected from the group consisting of.

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