JP6952812B2 - Use of pharmaceutical compositions to improve central nervous system myelination prepared by helicin-elinaseus mycelium extract - Google Patents

Description

The present invention relates to the use of pharmaceutical compositions prepared with a helicin-elinaseus mycelium extract to improve central nervous system myelination.

The central nervous system of vertebrates contains two types of cells: neurons and glia. Glia is responsible for providing nutrients to neurons and maintaining a constant microenvironment, and is subdivided into microglia, astrocytes, and oligodendrocytes (OLGs). Oligodendrocytes (OLGs) first differentiate from oligodendrocyte precursor cells (OPCs) into premyelinated oligodendrocytes, and then continue to form. Mature OLG stretches its tentacles, wraps nerve cell axons, and forms a multi-layered myelin rich in lipid components, the protein component of which is primarily myelin basic protein (MBP). ) And myelin proteolipid protein (PLP). The insulating properties of the myelin sheath help transmit nerve signals and also protect neurons. When the structure of the myelin sheath is damaged or shed, nerve conduction is blocked, causing defects such as sensory, motor, and cognitive function.

With age, individuals may suffer from neurodegenerative diseases due to persistent axonal loss and neuronal cell death due to demyelination. In addition, renewal of OPC and decreased differentiation potential increase the difficulty of re-myelination. Diseases of the central nervous system include multiple sclerosis, leukodystrophy, chronic neurodegenerative disorders, etc., and are associated with the development of OLG demyelination or myelination. , Related to neurological inflammation.

Taking multiple sclerosis as an example, there are currently no drugs that can be treated. When acute multiple sclerosis develops, it is usually treated with corticosteroids, and general control and treatment is subcutaneous injection of interferon and Copaxone®. Therefore, a pharmaceutical composition for treating a disease of demyelination or myelination of the central nervous system, or a pharmaceutical composition for improving myelination of the central nervous system is developed, and a disease of the central nervous system is treated or ameliorated. There is an urgent need to help.

The applicant of the present application has finally come up with the present application, which considers the shortcomings of the prior art, and through careful experimentation and research, has a patient spirit and effectively overcomes the shortcomings of the prior art. A brief description of the present invention is as follows.

In order to develop a pharmaceutical composition for treating demyelination or myelination of the central nervous system and a pharmaceutical composition for improving myelination of the central nervous system, the present invention is based on Helicium erinaceus (Bull.). Pers) Obtain a natural and safe extract from myelin bodies. The extract contains active substances such as erinacine A, erinacine C, erinacine S and the like. Pharmaceutical compositions prepared from the extracts treat diseases of central nervous system demyelination or medullary sheath injury, improve central nervous system medullary sheathing, multiple sclerosis, white matter dystrophy, chronic neurodegeneration. Helps treat or ameliorate central nervous system disorders such as disorders, white matter damage and white matter inflammation. The helicin-elinaseus mycelium extract of the present invention also has an effect of improving inflammation of the central nervous system.

It is an object of the present invention to disclose the use of a pharmaceutical composition for improving central nervous system myelination prepared by a helicin-elinaseus mycelium extract, wherein the extracts are erinacin A, erinacin C, and the like. Includes one of the groups consisting of Elinacin S and combinations thereof.

In one embodiment, the extract is obtained by extracting the helicin-elinaseus myelin with a polar solution, which is an oligodendrocyte transcription factor 1 (oligodendrocyte transcription) of an individual oligodendrocyte (OLG). It is used to enhance the performance of factor 1; Olig1), oligodendrocyte transcription factor 2 (Olig2), myelin sheath basic protein (MBP) and myelin sheath proteolipid protein (PLP). In one embodiment, the polar solution is a methanol solution, an ethanol solution or water. In one embodiment, if the polar solution is a methanol solution, the extract is a methanol extract, and if the polar solution is an ethanol solution, the extract is an ethanol extract and the polar solution is. If it is water, the extract is a water extract. In one embodiment, the oligodendrocytes (OLGs) are differentiated from oligodendrocyte progenitor cells (OPCs) so that the oligodendrocyte progenitor cells (OPCs) secrete galactocerebroside (GC). used. In one embodiment, the extract is used to promote the differentiation of oligodendrocyte progenitor cells (OPCs) into oligodendrocytes (OLGs) and is damaged by the central nervous system (OLGs). Compensate for the number of.

In one embodiment, the helicin-elinaseus mycelium is obtained by liquid fermentation culture and the extract has an action concentration of 10 ng / mL to 100 μg / mL. In one embodiment, the helicin-elinaseus mycelium is cultured in medium, which medium comprises glucose, yeast extract, animal and plant derived proteins and hydrolysates thereof, magnesium sulfate and soybean powder. In one embodiment, the helicin-elinaseus mycelium is cultured and then dried, back-dissolved, and extracted to obtain the extract.

The present invention further discloses a method of treating a disease that is demyelination or myelin sheath injury of the central nervous system by using a helicin-elinaseus mycelium extract, an active substance or a prepared pharmaceutical composition. In the method, a pharmaceutically effective amount of extract, active substance or pharmaceutical composition is administered to the desired individual.

Therefore, a pharmaceutical composition for improving central nervous system myelination or treating a disease that is central nervous system demyelination or myelination injury according to the present invention is a medically effective amount of helicin. Includes an Elinaseus myelin extract, which contains active substances such as Elinacin A, Elinacin C, Elinacin S and the like. The pharmaceutical composition according to the present invention contains a pharmaceutically acceptable carrier, excipient, diluent or auxiliary agent. The pharmaceutical composition according to the present invention can be administered to a patient by oral administration, intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, sublingual administration and the like.

A fast liquid chromatogram of erinacin A. A fast liquid chromatogram of Elinacin C. A high-speed liquid chromatogram of Elinacin S. (A) Helicin-Erinaseus mycelium extract (HEM), (B) Erinacin A (HeA), (C) Erinacin C (HeC) and (D) Erinacin S (HeS) are examples of cell viability against OPC. It is a figure. Olig1 of OLG in which (A) helicin-elinaseus mycelium extract (HEM), (B) erinacin A (HeA), (C) erinacin C (HeC) and (D) erinacin S (HeS) were each differentiated from OPC. , Olig2, MBP and PLP1 mRNA expression levels. It is a schematic diagram of the differentiation from OPC treated with helicin-elinaseus mycelium extract (HEM) into (A) GC-positive OLG cells and (B) MBP-positive OLG cells. It is a schematic diagram of the differentiation from OPC treated with elinacin A (HeA) into (C) GC-positive OLG cells and (D) MBP-positive OLG cells. It is a schematic diagram of the differentiation from OPC treated with erinacin C (HeC) into (E) GC-positive OLG cells and (F) MBP-positive OLG cells. It is a schematic diagram of the differentiation from OPC treated with erinacin S (HeS) into (G) GC-positive OLG cells and (H) MBP-positive OLG cells.

The above objects and advantages of the present invention will become apparent to those skilled in the art more immediately after reference to the following detailed description and accompanying drawings.

The present invention mainly uses Helisium erinaseus, which is distributed in temperate regions at high latitudes. The polysaccharides in its offspring benefit the five organs, aid digestion, improve dyspepsia, improve physical weakness, and treat gastric ulcers, gastritis, stomach pain and bloating. Therefore, Helisium erinaseus is a mushroom for both food and medicine.

1. Experimental method:
1. 1. Liquid Fermentation Culture of Helisium Elinaseus Mycelium Helisium Elinaseus according to the embodiment of the present invention is a variety of No. BCRC35569 from the Food Industry Research and Development Institute (FIRDI) Bioresource Conservation Research Center (BCRC). However, the varieties of Helisium erinaseus suitable for the present invention are not limited to this.

First, helisium-elinaseus mycelium (BCRC number: 35669) was aseptically inoculated into a plate medium of potato dextrose agar (PDA) and cultured at 25 ° C. for 7 days. Next, aseptic techniques were used to scrape the helisium-elinaseus mycelium on the PDA plate medium with medium (2.0% by weight glucose, 0.1% by weight yeast extract, 0.1% by weight animal and plant derived protein). It was inoculated into a flask equipped with (containing the hydrolyzate) and shaken in a shaking incubator at 26 ° C., pH 5.0 and a rate of 120 rpm for 5 days. Then, the helisium-elinaseus mycelium in the flask is sterile inoculated into the fermentation tank containing the above medium, and stirred at 24 ° C. to 30 ° C., tank pressure 0.5 to 1.0 kg / cm2, pH 5.0, 10 to 150 rpm. When agitated at a rate or no agitation (air lift), air was introduced at an aeration rate of 0.5 to 1.0 VVM, and the mixture was cultured for 7 to 10 days to obtain a liquid culture fermentation broth of helisium-elinaseus mycelium. rice field. The supernatant is removed by centrifugation, and the remaining solid is the helisium-elinaseus mycelium. To facilitate cryopreservation, this solid was lyophilized to give Helisium-elinaseus mycelium powder.

2. Preparation of Helisium-Erinaseus Mycelium Extract Helisium-Erinaceus Mycelium Powder is extracted with a polar solution for a few minutes (including but not limited to dipping, stirring, shaking or ultrasonic extraction), followed by vacuum concentration or freezing. It was dried by a drying method to obtain a mycelium extract of Helisium erinaseus. Polar solutions include, but are not limited to, methanol solutions, ethanol solutions or water. When the above solutions are used individually, a methanol extract, an ethanol extract or a water extract of the helisium-elinaseus mycelium can be obtained separately. The methanol solution is a mixture of methanol and water (eg, greater than or equal to 1% (v / v) and less than 100% (v / v)) or "pure" methanol. The ethanol solution can be a mixture of ethanol and water (eg, greater than or equal to 1% (v / v) and less than 100% (v / v)) or "pure" ethanol.

In this experiment, extraction is performed with a 95% (v / v) ethanol solution. Helisium-elinaseus mycelium powder was added to 25 times its weight of 95% (v / v) ethanol solution, the first ultrasonic shock extraction was performed for 1 hour, and the suspension was centrifuged to the first top. I got the Qing dynasty. Next, the supernatant was subjected to a second ultrasonic shock extraction with an 85% (v / v) ethanol solution for 1 hour, and the suspension was centrifuged to obtain a second supernatant. The second supernatant was concentrated under reduced pressure to obtain a paste-like Helisium-elinaseus mycelium extract (abbreviated as extract).

3. 3. Analysis of the active ingredient of the helisium-elinaseus mycelium extract The extract was extracted by liquid-liquid distribution of water and ethyl acetate (1: 1, v / v) to obtain an ethyl acetate layer. Furthermore, the ethyl acetate layer was analyzed by silica gel and LH-20 silica gel column chromatography. In high performance liquid chromatography (HPLC), on a Cosmosil R5C18-AR-II column, elute with 60% acetonitrile at 40 ° C, gradually increase to 65% acetonitrile in 20 minutes, flow rate 1 ml / min, wavelength It is 340 nm. As shown in Figure 1, erinacine A (symbol A, MW: 432.557 g / mol) appeared at 7.3 minutes. By HPLC, on a Cosmosil R5C18-AR-II column, it elutes at 40 ° C. with 60% acetonitrile and gradually increases to 65% acetonitrile in 20 minutes, with a flow rate of 1 ml / min and a wavelength of 210 nm. As shown in Figure 2, erinacine C (symbol C, MW: 434.573 g / mol) appeared at 10.4 minutes. By HPLC, on a Cosmosil R5C18-AR-II column, it elutes at 40 ° C. with 60% acetonitrile and gradually increases to 65% acetonitrile in 20 minutes, with a flow rate of 1 ml / min and a wavelength of 290 nm. As shown in Figure 3, erinacine S (symbol S, MW: 430.541 g / mol) appeared at 14.5 minutes. The chemical structural formulas of Elinacin A, Elinacin C and Elinacin S are shown below.

According to this, when the helicin-elinaseus mycelium cultured in a 20-ton fermenter is dried, about 120 kg of helicin-elinaseus mycelium powder is obtained. The extracted helicin-elinaseus mycelium extract contains active substances such as erinacin A, erinacin C and erinacin S. The helicin-elinaseus mycelium, powder and extract containing erinacin A, erinacin C and erinacin S can be prepared as required in various pharmaceutical compositions and dosage forms of health foods.

4. Establishment and analysis of oligodendrocyte progenitor cell (OPC) to oligodendrocyte (OLG) differentiation A cerebral cortical layer was isolated from embryos of 13.5-14.5 day old Sprague-Dawley (SD) female rats. The cerebral cortex layer of the embryo is dispersed, passed through a filter membrane having a pore size of 40 μm, and the cortical layer is cultured in a cell culture dish not treated with poly-D-lysine (PDL) for 5 to 7 days to form a spherical shape. Neural stem cells were formed. Isolate nerve stem cells from cell culture dish with AccutaseR cell digestive enzyme, growth medium (2% B-27TM additive (Gibco), 1% N-2TM additive (Gibco), 10 ng / ml fibroblast growth factor 2) PDL-treated cells by resuspending cells with (FGF2), DMEM / F12) containing 10 ng / ml epithelial growth factor (EGF) and 10 ng / ml platelet-derived growth factor-AA ligand (PDGF-AA ligand) Planted in a culture dish. After 2 days, the growth medium was changed to the differentiation medium (4 mM L-glutamine), 1 mM sodium pyruvate (sodium pyruvate), 0.1% bovine serum albumin (BSA), 50 mg / ml apotransferrin. 5, 5 mg / ml insulin, 30 nM sodium pyruvate, 10 nM biotin, 10 nM hydrocortisone, 15 nM T3, 10 ng / ml pyriary neurotrophic factor (CNTF) and 5 mg / ml N-acetyl- The medium was changed to L-cysteine (DMEM medium containing N-acetyl-L-cysteine; NAC) and cultured for 2 days. It was treated with helicin-elinaseus mycelium extract (abbreviated as HEM), erinacin A (abbreviated as HeA), erinacin C (abbreviated as HeC) and erinacin S (abbreviated as HeS) according to experimental needs.

5. Cell viability of oligodendrocyte progenitor cells (OPC) differentiated into oligodendrocytes (OLG) This experiment evaluates whether HEM, HeA, HeC, and HeS affect the viability of OPC. First, 2 x 104 OPC cells were planted in 24-well culture plates, cultured for 2 days, and then treated with different concentrations of extract or 24 and 48 hours. 0.5 mg / ml 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide; MTT) was added, light was avoided for 4 hours, blue-purple crystalline formazan was dissolved in dimethyl sulfoxide (DMSO), and the absorbance at a wavelength of 595 nm was measured using a TECAN Sunrise ELISA Reader. , Cell viability was calculated.

6. OLG medullary ability This experiment uses a quantitative real-time polymerase chain reaction (Q-PCR) to measure specific gene expression in treated cells and OLG medullary sheath. The ability to transform was analyzed. First, OLG RNA (1 ug / sample) treated with helicin-elinaseus extract was transcribed into cDNA by M-MLV reverse transcriptase. cDNA, SYBR® Master Mixs, and rat oligodendrocyte (OLG) oligodendrocyte transcription factor 1 (Olig1), oligodendrocyte transcription factor 2 (Olig2), medullary sheath basic protein (MBP) and spinal cord Sheath proteolipid protein (PLP) primers were mixed (see Table 1), relative mRNA expression levels were measured, and the rat GAPDH gene was used as the control group gene. Experimental results were analyzed with StepOne software version 2.12 (Applied Biosystems).

7. Western blotting This experiment analyzed cell protein expression by conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. That is, after SDS-PAGE, the cell protein was transcribed on the nitrocellulose membrane, the primary antibody was added, and it acted at low temperature for 15 hours or more. After washing the nitrocellulose membrane with TBST buffer, a secondary antibody is added and the mixture acts at room temperature for 1 hour. The nitrocellulose film was washed with TBST buffer, cold-light colored with ECL, and developed into a negative film.

As used herein, the term "osteoporosis" refers to a disease caused by loss of bone mass and enlargement of holes in the honeycomb structure of bone, increasing the risk of fracture and increasing bone weakness.

8. Immunofluorescent stained cell slides were fixed with 4% paraformaldehyde for 15 minutes and then acted on with 0.1% Triton X100 PBS for 15 minutes. Washed 3 times with PBS, anti-MBP antibody (NB1018, Calbiochem) containing 1% horse serum or anti-galactosidase (GC) antibody (MAB342, Millipore) was added and acted overnight at 4 ° C. The cells were washed 3 times with PBS, a secondary antibody having biotin was added, and the mixture was allowed to act at room temperature for 1 hour. The cells were washed 3 times with PBS, a tertiary antibody containing avidin-Cy3 was added, and the mixture was allowed to act at room temperature for 45 minutes. After reacting with DAPI (1 μg / ml) for 1 minute, the slides were finally sealed with 90% glycerin and the results were observed under a microscope (FV1000, Japan).

9. Establishment of animal experiment model The cerebral brain of a 7-day-old SD rat offspring was cut into 300 μm-thick sections using a vibrating biological tissue slicer (Microslicer ™ DTK-1000), and an insert cell culture medium (0.4 μm Millicell®) was used. ) Cell culture insert, placed in Millipore®), Cerbellar slice culture medium; minimum essential medium (MEM) containing 50% Earle's salt, 35% Earl Equilibrium salt solution, 15% heat-inactivated horse serum and 1% GlutaMAX ™ aid) were cultured for 3 days. Sections were treated with various concentrations of helicin erinaseus extract or erinacin for 11 days, fixed on day 14 of co-culture with 4% paraformaldehyde for 2 hours and acted on 1% Triton-X100 PBS for 2 days. bottom. Anti-myelin basic protein mouse (anti-MBP) mAb, NE1018, Calbiochem by immunofluorescence and antibody against neurofilament H (anti-neurofilament H antibody, NF200, AB5539, Millipore) ) Was double-fluorescent, and the results were observed under a microscope (FV1000, Japan).

10. Statistical analysis Experimental data is expressed as "mean ± standard error", differences between groups are analyzed by t-test, and p <0.05 is considered to be statistically significant.

Second, experimental results:
1. 1. Cell viability of HEM, HeA, HeC and HeS against OPC See (A), (B), (C) and (D), (A) Helicin-Erinaseus mycelium extract (HEM), (B). ) Erinacin A (HeA), (C) Erinacin C (HeC) and (D) Erinacin S (HeS) are schematic diagrams of cell viability against OPC. The control group of FIGS. 4 (A) to 4 (D) is a test in which an extract or an active substance is not added. In FIGS. 4 (A) to 4 (D), treatment of OPC cells with 10 ng / ml to 100 μg / ml HEM for 24 to 48 hours does not cause toxicity to OPC cells (FIG. 4 (A)). When HeA, HeC, and HeS are 0.001 ng / ml to 10 ng / ml, no toxicity occurs to OPC cells even after treatment for 24 to 48 hours (FIGS. 4 (B), (C) and (D). )). Therefore, at appropriate concentrations, helicin-elinaseus mycelium extract (HEM) and active substances (HeA, HeC and HeS) do not affect the cell viability of OPC and are safe and non-toxic to OPC. Can be proved.

2. Effects of HEM, HeA, HeC and HeS on OPC to OLG differentiation and gene expression Refer to FIGS. 5 (A), (B), (C) and (D), and (A) helicin-elinaseus mycelium extract. (HEM), (B) helicin A (HeA), (C) helicin C (HeC) and (D) helicin S (HeS) for Olig1, Olig2, MBP and PLP1 mRNA expression levels of OLG differentiated from OPC, respectively. It is a schematic diagram. The control group of FIGS. 5 (A) to 5 (D) is a test in which the treatment is performed without adding an extract or an active substance. In FIG. 5 (A), 100 ng / ml to 1 μg / ml HEM increased Olig2 mRNA expression in OLG cells (see (A) -2), while also increasing MBP and PLP1 mRNA expression in mature OLG cells. (See (A) -3 and (A) -4). In addition, OPC cells treated with 100 ng / ml and 1 μg / ml HEM had more differentiated OLG cells than the control group (results not shown).

In FIG. 5 (B), 0.001 ng / ml to 10 μg / ml HeA also increased the expression level of Olig2 mRNA in OLG cells (see (B) -2), and HeA also increased the expression level of MBG and PLP1 mRNA in OLG cells. Promoted an increase in (see (B) -3 and (B) -4).

In FIG. 5 (C), different concentrations of HeC increase Olig1, Olig2, MBP, and PLP1 mRNA expression levels in OLG cells, eg, when HeC is 0.01 ng / ml-10 ng / ml, OLG cells. Olig1 mRNA expression level is increased (see (C) -1), and when HeC is 0.1 ng / ml to 10 ng / ml, Olig2 mRNA expression level in OLG cells is increased (see (C) -2). , When HeC is 0.01 ng / ml, the MBP mRNA expression level in OLG cells is significantly increased (see (C) -3), and HeC is not significantly different from the PLP1 mRNA expression level in OLG cells. ((C) -4).

In FIG. 5 (D), 0.01 ng / ml HeS can significantly increase the expression levels of Olig1, MBP and PLP mRNA in OLG cells ((D) -1, (D) -3 and (D). ) -4), 0.001 ng / ml to 0.1 ng / ml HeS can increase the expression level of Olig2 mRNA in OLG cells (see (D) -2). 0.01 ng / ml HeS promotes MBP and PLP protein expression in OLG cells (Western blotting results not shown).

With reference to FIGS. 6 (A)-(H), the results of the immunofluorescent staining experiment show that the extract (HEM) and the active substances (HeA, HeC and HeS) are GC-positive from OPC (FIG. 6 (A)). ), (C), (E) and (G)) and OLG with MBP positivity (FIGS. 6 (B), (D), (F) and (H)) can be promoted. Quantitative results show that the ability of HeS to promote the differentiation of OPC into GC-positive OLG and MBP-positive OLG (FIGS. 6 (G) and (H)) is HeA (FIG. 6 (C) and (D)). )) Or HeC (FIGS. 6 (E) and (F)) is also shown to be superior.

In summary, the helisium-elinaseus myelin extracts of the present invention and their active substances (HeA, HeC and HeS) are safe and non-toxic to oligodendrocyte precursor cells (OPC), and from OPC to oligodendrocytes (HeA, HeC and HeS). Oligodendrocyte transcription factor 1 (Olig1), oligodendrocyte transcription factor 2 (Olig2), myelin sheath basic protein (MBP) in the myelination process in which OLG wraps axons, which can promote differentiation into OLG) ) And myelin sheath proteolipid protein (PLP) mRNA expression level and protein expression level are promoted, and the OLG becomes an OLG having galactocerebroside (GC) positive and MBP positive. Therefore, the helisium-elinaseus myelin extract according to the present invention and the elinacin A, elinacin C and elinacin S contained therein improve the myelination of the central nervous system of an individual, or demyelinate or myelinate the central nervous system. It can be used in pharmaceutical compositions for treating diseases that are sheath injuries.

Although the present invention has been described above using the embodiments, the technical scope of the present invention is not limited to the scope described in the above embodiments. It will be apparent to those skilled in the art that various changes or improvements can be made to the above embodiments. It is clear from the description of the claims that such modified or improved forms may also be included in the technical scope of the present invention.

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Claims (10)

A pharmaceutical composition for improving myelination of the central nervous system, which contains erinacin S as an active ingredient. The erinacin S is contained in the extract of helicin-elinaseus mycelium,
The extract was obtained by extracting the helicin-elinaseus mycelium with a polar solution.
The extracts are oligodendrocyte transcription factor 1 (Olig1), oligodendrocyte transcription factor 2 (Olig2), myelin sheath basic protein (MBP) and myelin sheath proteolipid protein (PLP) of individual oligodendrocytes (OLG). The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is used to enhance the performance of). The pharmaceutical composition according to claim 2, wherein the polar solution is a methanol solution, an ethanol solution, or water. If the polar solution is a methanol solution, the extract is a methanol extract.
If the polar solution is an ethanol solution, the extract is an ethanol extract.
The pharmaceutical composition according to claim 3, wherein when the polar solution is water, the extract is a water extract. The oligodendrocyte (OLG) is differentiated from oligodendrocyte precursor cell (OPC) and is differentiated.
The pharmaceutical composition according to claim 2, wherein the oligodendrocyte progenitor cell (OPC) is used to secrete galactocerebroside (GC). The extract is used to promote the differentiation of oligodendrocyte progenitor cells (OPCs) into oligodendrocytes (OLGs) to compensate for the number of oligodendrocytes (OLGs) damaged by the central nervous system. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition comprises. The helicin-elinaseus mycelium was obtained by liquid fermentation culture.
The pharmaceutical composition according to claim 2 , wherein the extract has an action concentration of 10 ng / mL to 100 μg / mL. It said extract or pharmaceutical composition, the pharmaceutical composition according to claim 2, characterized in that it is used to treat the demyelinating or disease is a myelin damage in the central nervous system. The helicin-elinaseus mycelium was cultured in a medium and
The pharmaceutical composition according to claim 2 , wherein the medium contains glucose, yeast extract, a protein derived from animals and plants and a hydrolyzate thereof, magnesium sulfate and soybean powder. The pharmaceutical composition according to claim 2 , wherein the helicin-elinaseus mycelium is cultured and then dried, back-dissolved, and extracted to obtain the extract.

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