KR20150070576A - A preparation method of fractions from Phellinus igniarius extracts comprising enriched component for prevention and treatment of multiple sclerosis - Google Patents

Abstract

The present invention relates to a method for preparing a fraction by concentrating active ingredients with a multiple sclerosis alleviating activity from a willow bracket fungus (Phellinusigniarius) extract to significantly enhance the effectiveness thereof. The present invention also relates to a pharmaceutical composition including the fraction for preventing and treating autoimmune diseases as well as the multiple sclerosis.

Description

Technical Field [0001] The present invention relates to a method for preparing an extract fraction of horse mackerel mushroom which is enhanced in preventing and treating multiple sclerosis,

The present invention relates to a method for preparing a fraction capable of greatly enhancing the effect of concentrating an effective ingredient for improving multiple sclerosis from Phellinus igniarius extract, and a method for preventing and treating a multiple sclerosis and an autoimmune disease ≪ / RTI >

Multiple sclerosis is a typical autoimmune degenerative neurodegenerative disease. It occurs most frequently in Asian and African populations, but it is most common in European populations, especially in 30-year-old Caucasian women. In European white populations, 200 The incidence occurs with frequency.

Multiple sclerosis is accompanied by visual disturbances, myasthenia gravis and accompanying gait disorders, skin sensation disorders with pain or numbness, defecation and dysuria, language and hearing impairment, cognitive impairment, Often, it gets worse. These neurological disorders are caused by damage to the myelin sheath surrounding the axons of nerve cells due to inflammatory responses in the central nervous system. The lesion is diffuse in the white matter region of the brain and spinal cord. In the lesion, inflammatory reaction accompanied by typical infiltration of inflammatory cells around the vein and detachment of the nerve fibers in the vagina are observed. In addition, And proliferation of glial cells.

Although the pathogenesis and pathogenesis of multiple sclerosis has not yet been fully elucidated, it has been reported that it is an autoimmune response involving both cellular and humoral immune systems (Engelhardt, Clin. Exp. Neuroimmunol. 1: 79-93, 2010 ). The process of immunological development of multiple sclerosis is outlined as follows.

The autologous myelin-responsive self-reactive CD4 + T cells migrate from the blood to the substantia nigra of the central nervous system by various causes, such as encephalitis, and then the main histocompatibility complex class II molecules'. Reactivated CD4 + T cells proliferate by themselves and differentiate into several subset, leading to cellular immunity. These CD4 + T cells secrete other immune cells, macrophages and B cells, into the central nervous system, as well as secrete inflammatory substances.

When autologous reactive CD8 + T cells enter the central nervous system from the blood, they are reactivated by the 'main histocompatibility complex class II molecule' containing myelin and replicate themselves and cytotoxically attack and destroy antigenic determinant-containing cells.

The macrophage cooperates with the microglia present in the central nervous system to attract and activate CD4 + T cells and CD8 + T cells into the central nervous system, while activating B cells by secreting BAFF (B cell activating factor belonging to the TNF family) . In addition, macrophages secrete proinflammatory cytokines to destroy myelin and to eliminate myelin, which is labeled with antibodies and complement.

Antigen-specific B cells infiltrating the central nervous system act as antigen-presenting cells to T cells. When antigen - specific B cells interact with T cells, both B cells and T cells proliferate simultaneously. B cells, on the other hand, differentiate into plasma cells and secrete antibodies and complement to myelin.

In order for the immune cells to pass through the cerebral blood vessels, the cell adhesion molecules must be expressed in the cerebral vascular endothelial cells and immune cells. In the case of multiple sclerosis, the expression of vascular cell adhesion molecule-1 in the cerebral blood vessels and the expression of integrin-α4 in the immune cells increase, respectively.

Since multiple sclerosis is an immune disease involving both cellular and humoral immune systems as already mentioned, various immunotherapy methods have been tried: 1) sensitivity loss of autoreactive T cells using immuno-tolerant antigen; 2) Immunotherapy using a monoclonal antibody or interferon-γ for directed cytokines, B cell surface molecules or cell adhesion molecules, and 3) plasma separation and export methods. Although these treatments have positive results, their use is limited due to undesirable side effects (Steinman & Zamvil, Ann. Neurol. 60: 12-21, 2006). Therefore, it is necessary to develop safe drugs without side effects and toxicity for patients with multiple sclerosis.

Mushrooms of the genus Phellinus have traditionally been used in the treatment of chronic diseases in Asia, and their medical efficacy has recently been revealed (Dai et al, Appl. Microbiol. Biotech. 87: 1587-1593, 2010 ). Β-glucan, a polysaccharide extracted from woody mud mushroom ( P. linteus ), has a powerful immunomodulatory effect and is effective in the treatment of cancer and immune diseases. In addition, horse mud mud mushrooms have been used as medicinal mushrooms in Asia and also have immunomodulatory effects.

Recently, the present inventors have found that the extract of P. igniarius (Linnearus: Fries Quelet 1886), a species of mud mushroom, contains a component that greatly improves experimental autoimmune meningitis, an experimental model of MS for multiple sclerosis, (Korean Patent Laid-Open Publication No. 2013-0078395). Although only the content of the ethanol precipitate fraction is mentioned in the specification of the patent, since the fraction contains a large amount of other ingredients than the active ingredient, it is possible to extract the active ingredient at a higher concentration to prevent or treat multiple sclerosis It is necessary to develop a technique that can further improve the effect of the present invention.

The inventors of the present invention conducted studies to improve the effect of extracting effective ingredients for preventing or treating multiple sclerosis from horse mackerel mushroom at a high concentration and found that the precipitate of the extract of horse mackerel mushroom was treated with ions such as HP- The present inventors have found that fractions obtained by fractionation under a specific condition exhibit excellent effects by using an exchange resin column.

Accordingly, it is an object of the present invention to provide a method for extracting and concentrating an effective ingredient for improving multiple sclerosis from horse mackerel mushroom to greatly improve its efficacy.

According to the present invention,

Adding the horse mud mushroom to an extraction solvent and heating to obtain an extract;

Mixing the extract with a C ₁ to C ₁₀ alcohol, cooling to -30 to 0 ° C to separate the precipitate formed;

Loading a mixed solution obtained by adding purified water to the separated precipitate into an ion exchange resin column and obtaining a fraction by using a mixed solvent in which C ₁ -C ₁₀ alcohol and water are selectively mixed,

Concentrating and lyophilizing the fraction;

And a method for producing a pharmaceutical composition for preventing and treating multiple sclerosis.

The present invention further provides a pharmaceutical composition for preventing and treating multiple sclerosis comprising the pharmaceutical composition prepared by the above-described method.

The pharmaceutical composition according to the present invention can be obtained by concentrating an effective ingredient for improving multiple sclerosis from an extract of a common horse mud mushroom by the production method of the present invention, thereby providing a pharmaceutical composition exhibiting excellent prophylactic or therapeutic effect of multiple sclerosis.

Figure 1 shows the improvement of ethanol extracts (Piwes) and ethanol precipitates (Piwep) of water extracts of horse mackerel mushroom mushroom on the clinical indices of experimental animals induced with experimental autoimmune meningitis using myelin oligodendrocyte glycoprotein (MOG) _35-55 . In the graph, MOG + Vehicle means MOG alone group, MOG + Piwes means MOG and Piwes 100 mg / kg combination group, MOG + Piwep: MOG and Piwep 100 mg / kg combination group.
FIG. 2 shows the concentration-dependent inhibitory effect of the respective fractions on the proliferation of lymphocytes isolated from the stool lymph node and spleen of the experimental animals to which MOG was administered.
FIG. 3 shows the concentration-dependent inhibitory effect of the Pi130 fraction on the clinical index of MOG-induced experimental autoimmune meningitis. MOG + Pi130 25 mg / kg, MOG + Pi130 12.5 mg / kg, MOG + Pi130 25 mg / kg, MOG + Pi130 25 mg / kg, MOG + Pi130 25 mg / mg / kg means the combined administration of MOG and Pi130 fraction 50 mg / kg.
FIG. 4 is a graph showing the effect of Pi130 on mRNA expression specific to CD4, CD8, CD11b, CD20, CXCR3, CCR5 and integrin-α4, the cell surface molecules of immune cells in experimental spinal cord of experimental autoimmune meningitis induced by MOG Inhibitory effect.
* Indicates statistical significance (p <0.05 or greater). N means normal group, V means MOG alone group, and P means MOG and Pi130 fraction 50 mg / kg combination group.

The present invention is characterized by a method for providing a pharmaceutical composition which is effective for prevention and treatment of multiple sclerosis by inhibiting infiltration of various immune cells into the central nervous system and inhibiting inflammation and detachment of aquatic plant from the central nervous system.

The method for preparing a pharmaceutical composition for preventing and treating MS according to the present invention comprises the steps of: 1) extracting a horse mackerel mushroom in an extraction solvent and heating the extract; 2) mixing the extract with a C ₁ -C ₁₀ alcohol and cooling to -30 to 0 ° C to form a precipitate; 3) loading a mixed solution obtained by adding purified water to the separated precipitate into an ion exchange resin column, obtaining a fraction by using a mixed solvent in which a C ₁ -C ₁₀ alcohol and water are selectively mixed as a developing solvent, and 4) Concentrating the fraction and freeze-drying the fraction.

Hereinafter, each step of the method for preparing a pharmaceutical composition for the prevention and treatment of MS will be described in detail.

1. Preparation of crude extract

In the present invention, fruiting body powder of mulberry mulch is used to destroy the cell walls of thick and hard mushroom to obtain more effective ingredients efficiently. The horse mud mushroom fruiting body powder is added to an extraction solvent which is 10 to 20 times by weight and heated to sufficiently extract the active ingredient to obtain a crude extract. As the above-mentioned extraction solvent, it is preferable to use water to obtain a hot-water extract.

The heating temperature upon heating in the extraction solvent may vary depending on the extraction solvent, but is preferably 90 ° C to 120 ° C, more preferably 100 ° C to 120 ° C, . It is also preferable to use the extract obtained by removing the impurities by filtration of the crude extract obtained through the above extraction, and then concentrating the filtrate under reduced pressure within a temperature range of about 50 to 60 ° C.

2. Sediment Formation Step

The concentrated and concentrated extract is mixed with a C ₁ to C ₁₀ alcohol corresponding to 2 to 3 times its volume. At this time, ethanol is preferably used as the alcohol, and ethanol cooled to -30 to 0 占 폚 may be used, or ethanol may be added thereto, and the mixture may be cooled to -30 to 0 占 폚. After the ethanol is added, the solution is allowed to stand at -30 to 0 ° C for about 12 to 24 hours to form a precipitate in the solution. The mixture in which the precipitate is formed is centrifuged to separate the supernatant and the precipitate. Next, the separated precipitate is dissolved in distilled water and lyophilized.

3. Fractionation step by ion exchange resin column

The lyophilized precipitate is dissolved in distilled water corresponding to 10 to 15 times its weight to prepare a mixed solution, and the mixed solution is loaded into an ion exchange resin column. The ion exchange resin column is preferably an HP-20 column, but a column known in the art as having the function similar to the HP-20 column may also be used. When an HP-20 column treated with 100% ethanol as the ion exchange resin column is used, the mixed solution preferably loads an amount of about 1/15 to 1/10 volume of the column volume.

Next, fractions are obtained by using a mixed solvent of C ₁ -C ₁₀ alcohols, preferably ethanol and water, as a developing solvent. At this time, a mixed solvent in which the ethanol content increases sequentially to the HP-20 column is passed as a developing solvent to obtain a fraction, and the mixed solvent is added to the column at an amount corresponding to about 1 to 3 times the column volume . It is also preferable to pass the mixed solvent in which the ethanol content increases sequentially to the HP-20 column to obtain fractions.

4. Selection of fraction and freeze-drying

The fractions sequentially fractionated using the mixed solvent are concentrated under reduced pressure at about 30 to 50 ° C. and lyophilized to obtain a composition in which the final active ingredient is concentrated. At this time, it is preferable to use a fraction obtained by passing a solvent having a C ₁ to C ₁₀ alcohol content of not less than 50% by weight among a plurality of fractions having different ethanol concentrations, more preferably an alcohol content of 50 to 90 wt% %, Most preferably 50 to 80% by weight of alcohol.

The fraction obtained through the above process exhibits an excellent prophylactic and therapeutic effect of multiple sclerosis compared to the ethanol precipitate of the common horse mud mushroom extract.

Specifically, the composition significantly alleviates the clinical symptom index and incidence of experimental autoimmune meningitis.

In addition, the composition inhibits the infiltration of CD4 + T cells, CD8 + T cells, macrophages and B cells into non-vascular tissues in the central nervous system of experimental animals in which experimental autoimmune meningitis has been induced, Effect.

Thus, the composition is useful for the treatment of autoimmune diseases such as Type 1 diabetes mellitus, Hashimoto thyroiditis, celiac disease, giant cell arteritis, ringworm, autoimmune pancreatitis, autoimmune hepatitis or irritable bowel syndrome Prevention and treatment.

Meanwhile, the composition can be used as a pharmaceutical preparation for prevention and treatment of multiple sclerosis by being blended together with a carrier commonly accepted in the pharmaceutical field.

The formulation can be administered orally or parenterally. For oral use, the compositions are formulated in the form of tablets, capsules or liquids. In the case of parenteral administration, the composition is formulated in a liquid form by a method commonly known in the art to which the present invention belongs.

In addition, fractions that improve MS from the horse mud mushroom of the present invention may be used as health food or health functional food for the improvement of multiple sclerosis together with a pharmaceutically acceptable carrier

Hereinafter, the present invention will be described in detail with reference to examples. It should be understood, however, that the following examples are for the purpose of promoting understanding of the invention and are not to be construed as limiting the scope of the invention.

Example: Preparation of Extract from Horse Mud Mushroom

The fruiting body powder of horse mackerel mushroom was added to distilled water 10 times by weight and heated at 103 ° C for 3 hours to obtain hot water extract. Next, the hot-water extract was filtered to remove solid components, and the extract was concentrated under reduced pressure at 55 ° C.

The concentrated extract was mixed with twice the weight of cold ethanol (-20 占 폚), and allowed to stand at 4 占 폚 or lower for one day to form a precipitate.

The mixture in which the precipitate was formed was centrifuged to separate the supernatant and the precipitate. The separated supernatant was concentrated under reduced pressure at 48 ° C and then lyophilized (non-precipitated fraction, Piwes). The separated precipitate was dissolved in distilled water and lyophilized (precipitate fraction, Piwep).

The recoveries of Piwes and Piwep were 47.2% and 52.8%, respectively.

10 g of the lyophilized precipitate fraction (Piwep) was dissolved in 150 ml of distilled water to prepare a mixed solution. The mixed solution was applied to an HP-20 column (8 cm x 40 cm, about 2,000 ml) treated with 100 wt% Lt; / RTI &gt; Thereafter, 2,000 ml of each of distilled water, 25% by weight ethanol aqueous solution, 50% by weight ethanol aqueous solution and 100% by weight ethanol were sequentially eluted using the developing solvent to obtain respective fractions. Each fraction was concentrated under reduced pressure at 48 ° C and then lyophilized.

The recovery rates of the respective fractions by distilled water (Pi 110), 25 wt% ethanol aqueous solution (Pi 120), 50 wt% aqueous ethanol solution (Pi 130) and 100 wt% ethanol (Pi 140) were 42.2%, 49.0%, 5% and 1% appear.

Experimental Example 1: Induction of Experimental Autoimmune Meningitis

Experimental autoimmune encephalomyelitis (EAE) is used as a representative experimental animal model for studying multiple sclerosis, with clinical symptoms and pathologic findings being very similar to multiple sclerosis (Steinman & Zamvil, Ann. Neurol. 12-21, 2006). Therefore, in the present experiment, the efficacy of the extract of the above Example for the treatment of multiple sclerosis was confirmed using experimental autoimmune meningitis.

EAE was induced according to a conventional method as follows. 6-7 week old C57BL / 6 mouse females were adapted to a room temperature and humidity controlled environment. 200 μg of _myelin oligodendrocyte glycoprotein (MOG) _35-55 was dissolved in 100 μl of phosphate buffered saline (PBS) and mixed with an equal volume of complete Freund's adjuvant (containing 400 μg of Mycobacterium tuberculosis H37RaA) . The pertussis toxin was dissolved in PBS at a concentration of 400 ng / 200 μl and injected intraperitoneally into the experimental animals once at 0 and 2 days after the first injection of MOG. On the 7th day after injection of MOG, 200 μl of a mixture of MOG and complete Freund's adjuvant was inoculated.

After the first injection of MOG, the behavior of the experimental animals was observed daily and the degree of clinical symptoms of EAE was evaluated according to the following criteria and indexed.

Grade 0: Normal

Grade 1: Deflection of the tail

Grade 2: Sagging of the tail, debility of the hind legs

Grade 3: Sagging of the tail, partial paralysis of the hind leg, gait disturbance

Grade 4: Tail sagging, complete paralysis of both hind limbs

Grade 5: Permanent death or death

Experimental Example 2: Comparison between Piwes and Piwep

Piwes and Piwep obtained in the above example were each dissolved in physiological saline at a concentration of 20 mg / ml, sterilized using a microfilter, injected into the abdominal cavity of the experimental animal at a dose of 100 mg / kg every day from the first day of the first MOG injection Respectively.

Figure 1 shows the effect of Piwes and Piwep on EAE clinical indices. The maximum clinical symptom index was 3.00 ± 0.84 in the MOG + vehicle group treated with MOG alone. On the other hand, the maximum clinical index in the MOG + Piwes group combined with MOG and Piwes was 1.87 ± 0.50, and the maximum clinical index in the MOG + Piwep group combined with MOG and Piwep was 0.30 ± 0.20.

Therefore, it was confirmed through the above test that the ingredient improving the clinical symptoms of EAE was mainly contained in Piwep as compared with Piwes.

Experimental Example 3: Comparison of effect of each fraction by HP-20 column

The activity of each fraction by the HP-20 column was compared by measuring the inhibitory effect on MGF-induced lymphocyte proliferation. The animals were injected with 100 μg of MOG intraperitoneally. Three days later, the lymph nodes and spleen were removed and lymphoid cells were collected. The lymphocytes were distributed at 3.6 × 10 ⁵ per well according to a conventional method, and the Pi110, Pi120, Pi130 and Pi140 fractions were administered to the wells at concentrations of 10, 50, 100 and 200 μg / ml, respectively, ^3- thymidine uptake was measured.

Figure 2 shows the effect of each fraction on lymphocyte proliferation. As shown in FIG. 2, Pi110 fraction promoted lymphocyte proliferation rather than PI120 fraction, and did not affect lymphocyte proliferation.

In contrast, the Pi130 and Pi140 fractions inhibited lymphocyte proliferation in a concentration-dependent manner, indicating that the Pi140 fraction had a greater inhibitory effect than the Pi130 fraction.

Although the inhibitory effect of the Pi140 fraction on the lymphocyte proliferation was greater than the efficacy of the Pi130 fraction, the recovery of the Pi140 fraction was very low. Therefore, it is considered preferable to apply the Pi130 fraction having a relatively high recovery rate In the following, the concentration-dependent inhibitory effect of the Pi130 fraction was specifically confirmed.

Experimental Example 4: Confirmation of effect of Pi130 fraction

The Pi130 fraction was dissolved in physiological saline at a concentration of 2.5 mg / ml, 5 mg / ml and 10 mg / ml, sterilized using a microfilter, and then 12.5 mg / kg, 25 mg / mg / kg, and 50 mg / kg, respectively.

Figure 3 shows the concentration-dependent inhibitory effect of the Pi130 fraction on EAE. As shown in the left figure, when the Pi130 fraction was administered at 12.5 mg / kg, 25 mg / kg and 50 mg / kg, Were respectively inhibited to 1.22 ± 0.20, 0.30 ± 0.21 and 0.06 ± 0.06 in a concentration dependent manner.

On the other hand, the photograph A in FIG. 3 shows the normal experimental animal, and the photograph B shows the experimental animal showing the paralysis symptom of the clinical index 4 induced by EAE. In addition, Photo C stained with Luxol Fast Blue (LFB) and Periodic Acid Schiff (PAS) according to the usual method in the spinal cord of a laboratory animal showing paralysis of clinical index 4, indicating that myelin was severely damaged in the spinal cord I could confirm.

In addition, the inhibitory efficacy of Piwep and Pi130 fractions against EAE were compared to confirm that the component inhibiting the clinical index of EAE was enriched in the Pi130 fraction.

Table 1 below shows the inhibitory effects of Piwep and Pi130 fractions on the clinical indices and incidence of EAE.

sample Vehicle Piwep Pi130 Dose - 100 mg / kg 50 mg / kg 25 mg / kg 12.5 mg / kg Maximum clinical index 3.11 ± 0.28 0.30 0.15 0.06 0.06 * 0.30 0.21 1.22 0.20 Maximum incidence rate 90% 20% 10% 20% 60%

* : Significantly lower ( p <0.01, t test) than Piwep (100 mg / kg).

As shown in Table 1, the maximum clinical index and the maximum incidence of EAE were 0.30 ± 0.15 and 20% when 100 mg / kg of Piwep was administered, respectively. These results were significantly higher than those of 50 mg / kg of Pi130 (0.06 ± 0.06, 10%), but the Pi130 fraction was similar to that of 25 mg / kg (0.30 ± 0.21, 20%) .

Therefore, since the Pi130 fraction suppresses the clinical index of EAE by about 4 times as much as that of Piwep, the active ingredient for improving EAE is efficiently and efficiently administered to the Pi130 fraction of Example 1 as compared with the ethanol precipitate of general water extract It was confirmed that it was concentrated.

Experimental Example 5: Expression of CD4, CD8, CD11b, CD20, CXCR3, CCR5 and integrin-α4 specific mRNA in spinal cord

In order to examine the effect of Pi130 fraction on the spinal cord infiltration of immune cells in EAE, the amount of each receptor-specific mRNA expressed on the surface of immune cells in the spinal cord was measured by reverse transcription-polymerase chain reaction (RT-PCR ) Were measured as follows.

Three weeks after the first injection of MOG, the animals were anesthetized, perfused through the heart with PBS, and spinal cord was collected. Total RNA was extracted from the spinal cord according to conventional methods and the expression of specific mRNAs for CD4, CD8, CD11b, CD20, CXCR3, CCR5 and integrin-4 was measured. A cDNA synthesis kit was used to synthesize a first cDNA strain from 1 ug RNA. Oligonucleotide primers are as follows:

CD4: forward 5'-TGT GCC GAG CCA TCT CTC TTA GG-3 ', reverse 5'-GCA CTG AGA GTG TCA TGC CGA AC-3';

CD8: forward 5'-ATG CAG CCA TGG CTC TGG C-3 ', reverse 5'-GCA TGT CAG GCC CTT CTG GGT-3';

CD11b: forward 5'-GGG CAC GGT GGC AGG TGA-3 ', reverse 5'-GCT GGC TGT GGG AGG CAC TG-3';

CD20: forward 5'-AAA ACC TCC AGG AAG AGT TTG GTC-3 ', reverse 5'-CGA TCT CAT TTT CCA CTG GCA AG-3';

CXCR3: forward 5'-GAA CGT CAA GTG CTA GAT GCC TCG-3 ', reverse 5'-GTA CAC GCA GAG CAG TGC G-3';

CCR5: forward 5'-GTA TGT CAG CAC CCT GCC AA-3 ', reverse 5'-AAG ATG AGC CTC ACA GCC CTG-3';

integrin-α: forward 5'-CCA CTA CGA TCG CTC CGC CTG T-3 ', reverse 5'-CCG GTT GGC ACC CCG AAG TA-3';

β-actin: forward 5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3 ', reverse 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3';

The RT-PCR reaction was separated on agarose gel, stained with ethidium bromide, and the amount of RNA was determined using Image J.

FIG. 4 shows the results of measurement of specific mRNA expression for CD4, CD8, CD11b, CD20, CXCR3, CCR5 and integrin-α4 in the spinal cord of an experimental autoimmune meningitis-induced animal. Expression of specific mRNAs for CD4, CD8, CD11b, CD20, CXCR3, CCR5, and integrin-α4 in the EAE-induced spinal cord was greatly elevated and the Pi130 fraction showed significant increase in mRNA expression specific to these receptors Respectively.

From the above experimental results, it is considered that the Pi130 fraction of the present invention inhibits infiltration of various immune cells into the central nervous system tissues accompanied by EAE.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. . Accordingly, the true scope of the present invention is to be determined by the following claims.

Claims (6)

Adding the horse mud mushroom to an extraction solvent and heating to obtain an extract;
Mixing the extract with a C ₁ to C ₁₀ alcohol, cooling to -30 to 0 ° C to separate the precipitate formed;
Loading a mixed solution obtained by adding purified water to the separated precipitate into an ion exchange resin column and obtaining a fraction by using a mixed solvent in which C ₁ -C ₁₀ alcohol and water are selectively mixed,
Concentrating and lyophilizing the fraction;
Or a pharmaceutically acceptable salt thereof, and a method for the preparation of a pharmaceutical composition for the prevention and treatment of multiple sclerosis.
The method according to claim 1,
Wherein the step of obtaining the fraction is a step of passing the mixed solvent in which the content of the C ₁ to C ₁₀ alcohol is gradually increased to the ion exchange resin column as a developing solvent to prepare a pharmaceutical composition for preventing and treating multiple sclerosis Gt;
3. The method of claim 2,
Wherein the freeze-dried fraction is obtained by passing a mixed solvent having a C ₁ -C ₁₀ alcohol content of 50 wt% or more.
4. The method according to any one of claims 1 to 3,
Wherein the C ₁ -C ₁₀ alcohol is ethanol. The method of claim _1, wherein the C ₁ -C ₁₀ alcohol is ethanol.
4. The method according to any one of claims 1 to 3,
Wherein the ion exchange resin column is an HP-20 column. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
A pharmaceutical preparation for the prevention and treatment of multiple sclerosis, which comprises a composition obtained by the method of any one of claims 1 to 3.

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