WO2013100717A2

WO2013100717A2 - Composition for preventing and treating multiple sclerosis and autoimmune diseases, containing phellinus igniarius extract

Info

Publication number: WO2013100717A2
Application number: PCT/KR2012/011776
Authority: WO
Inventors: 박형진
Original assignee: Park Hyoung Jin
Priority date: 2011-12-30
Filing date: 2012-12-28
Publication date: 2013-07-04
Also published as: KR101713166B1; KR20130078395A; WO2013100717A3

Abstract

The present invention relates to a composition containing a Phellinus igniarius extract. The composition containing a Phellinus igniarius extract 1) improves clinical symptoms by inhibiting inflammation and demyelinating plaque of the central nervous system, (2) inhibits the infiltration of immunocytes such as T cells, macrophages, B cells and the like into the central nervous system, (3) inhibits the expression of vascular cell adhesion molecule (VCAM)-1 in endothelial cells of the central nervous system, and (4) inhibits the expression of integrin-α4 in lymphocytes, in experimental animals in which experimental autoimmune meningitis is induced. Therefore, the composition of the present invention can be useful for preventing and treating multiple sclerosis and various immune diseases. In addition, the present invention can provide a Phellinus linteus or Phellinus baumii extract in the form of a composition useful for preventing and treating multiple sclerosis and various immune diseases.

Description

Composition for the prevention and treatment of multiple sclerosis and autoimmune diseases, including horse mud extract

The present invention provides a composition effective for the prevention and treatment of multiple sclerosis and autoimmune diseases comprising the extract of Phellinus igniarius . The present invention also provides a method for extracting the extract.

Multiple sclerosis is a representative disease of autoimmune inflammatory neurodegenerative disease, which is rare in Asians and Africans, but most frequently in Europeans, especially Caucasian women in their 30s. In Caucasian whites, the frequency affects 200 people per 100,000.

Multiple sclerosis involves visual impairment, dysfunction of the limbs and resulting gait disturbances, skin sensation impairment with pain or numbness, defecation and urination disorders, speech and hearing disorders, cognitive impairment, etc. Often it gets worse. This neurological disorder is due to damage to myelin sheaths surrounding the axons of nerve cells due to inflammatory responses in the central nervous system. The lesions are scattered in the white matter areas of the brain and spinal cord. In the lesions, inflammatory reactions involving perivenous inflammatory cell infiltration and bullous myelin sheaths of nerve fibers are observed. In addition, loss and appearance of oligodendrocytes Proliferation of glial cells.

The cause and pathological progress of multiple sclerosis have not yet been fully understood, but it is known to be an autoimmune reaction involving both cellular and humoral immune systems (Engelhardt, Clin. Exp. Neuroimmunol. 1: 79-93, 2010) ). The immunological course of multiple sclerosis is outlined below.

Autoreactive CD4 + T cells, which respond to self myelin, migrate from the blood to the parenchymal tissues of the central nervous system for various causes, such as encephalitis, and are known as' major histocompatibility complexes, including myelin provided by antigen-supplying cells present there. class II molecules'. Reactivated CD4 + T cells multiply themselves and differentiate into several subsets to induce cellular immunity. These CD4 + T cells secrete substances that attract other immune cells, such as macrophages and B cells, into the central nervous system, while also secreting proinflammatory substances.

When autoreactive CD8 + T cells enter the central nervous system in the blood, they are reactivated by 'major histocompatibility complex class II molecules' containing myelin, which replicate and proliferate themselves and destroy cytotoxic agents by cytotoxicly attacking them.

Macrophages, in cooperation with microglia in the central nervous system, attract and activate CD4 + T and CD8 + T cells into the central nervous system, while activating B cells by releasing B cell activating factor belonging to the TNF family (BAFF). . In addition, macrophages secrete proinflammatory cytokine, destroying myelin, and feeding and removing myelin labeled with antibodies and complement.

Antigen-specific B cells infiltrating the central nervous system act as antigen supplying cells to T cells. In addition, when antigen-specific B cells and T cells interact, B cells and T cells proliferate simultaneously. B cells, on the other hand, differentiate into plasma cells to secrete antibodies and complement to myelin.

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

Multiple sclerosis is an immunological disease involving both cellular and humoral immune systems, as mentioned earlier, and various immunotherapies have been attempted: 1) desensitization of autoreactive T cells using immuno-tolerant antigens, and 2) T cells. Immunization using monoclonal antibodies or interferon-γ against directional cytokine, B cell surface molecules or cell adhesion molecules, 3) plasma separation and the like have been attempted. These therapies have positive results, but their use is limited due to undesirable side effects (Steinman & Zamvil, Ann. Neurol. 60: 12-21, 2006). Therefore, the development of a safe drug without side effects and toxicity for patients with multiple sclerosis is urgently needed.

Mushrooms of 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 Phellinus linteus , has potent immunomodulatory effects and is effective in treating cancer and immune diseases. In addition, dung mud mushrooms have been used as medicinal mushrooms in Asia and also have immunomodulatory effects.

The present invention mud mud mushroom An object of the present invention is to provide a novel composition which is effective in the prevention and treatment of multiple sclerosis comprising an extract, and which has low side effects and low toxicity. In addition, the present invention is another species belonging to the genus Mud mushrooms wood mud mushrooms and Baumi mud mushrooms (Phellinus baumii) The present invention also seeks to provide a new composition having the same effect in the prevention and treatment of multiple sclerosis.

The present invention is one or more mushrooms selected from the group consisting of dung mud mushrooms, wood mud mushrooms and Baumi mud mushrooms It provides a composition for preventing and treating multiple sclerosis comprising the extract.

Preferably, the extract

i) hydrothermal extraction of one or more mushrooms selected from the group consisting of dung mud mushrooms, wood mud mushrooms and Baumi mud mushrooms;

ii) mixing the hot water extract with cold ethanol to obtain a precipitate, and

iii) obtained by dissolving the precipitate in distilled water and freeze drying.

Experimental autoimmune encephalomyelitis is used as a representative experimental animal model to study multiple sclerosis because its clinical symptoms and pathological findings are very similar to multiple sclerosis (Steinman & Zamvil, Ann. Neurol. 60: 12- 21, 2006). Therefore, the present invention demonstrates that the composition is effective for multiple sclerosis using experimental autoimmune meningitis.

Preferably, the composition greatly alleviates the clinical symptom index and incidence of experimental autoimmune meningitis.

Preferably, the composition inhibits infiltration of CD4 + T cells, CD8 + T cells, macrophages and B cells in the central nervous system of experimental animals induced experimental autoimmune meningitis, while inhibiting myelin dropping.

Preferably, the composition reduces the expression of vascular cell adhesion molecule (VCAM) -1, which is a type of cell adhesion molecule, in the central nervous system of experimental animals induced with experimental autoimmune meningitis.

Preferably, the composition reduces the number of lymphocytes that are strongly expressed integrin-α4 in the local lymph nodes of experimental animals induced by experimental autoimmune meningitis.

The present invention also provides a preparation for preventing or treating multiple sclerosis comprising a composition comprising an extract of at least one mushroom selected from the group consisting of dung mud, wood mud and Baumi mud mushroom and a pharmaceutically acceptable carrier. to provide.

In another aspect, the present invention provides a health supplement or functional food for preventing and improving multiple sclerosis comprising a composition and a carrier comprising an extract of one or more mushrooms selected from the group consisting of dung mud, wood mud and Baumi mud to provide.

In another aspect, the present invention is a type 1 diabetes mellitus, Hashimoto thyroiditis, comprising a composition comprising an extract of one or more mushrooms selected from the group consisting of dung mud mushrooms, wood mud mushrooms and Baumi mud mushrooms and a pharmaceutically acceptable carrier, Provided are the preparations for the prevention and treatment of various immune diseases such as celiac disease, giant cell arteritis, ringworm, autoimmune pancreatitis, autoimmune hepatitis or irritable bowel syndrome.

In another aspect, the present invention is a type 1 diabetes mellitus, Hashimoto thyroiditis, celiac disease, giant cells comprising a composition and a carrier comprising an extract of at least one mushroom selected from the group consisting of dung mud, wood mud and Baumi mud It provides a dietary supplement or functional food for the prevention and improvement of various immune diseases, such as sexual arteritis, ringworm, autoimmune pancreatitis, autoimmune hepatitis or irritable bowel syndrome.

The composition comprising the extract of the dung mud mushroom, woody mud mushroom or Baumi mud mushroom of the present invention blocks the influx of immune cells into the central nervous system by inhibiting the expression of cell adhesion factors in vascular endothelial cells and immune cells, respectively. As a result, the inflammatory response and myelin drop of the central nervous system are suppressed to improve the onset and symptoms of multiple sclerosis. Therefore, the composition of the present invention can be used for the prevention and treatment of multiple sclerosis. In addition, the composition of the present invention can be used for the treatment of immune diseases such as type 1 diabetes, Hashimoto thyroiditis, celiac disease, giant cell arteritis, ringworm, autoimmune pancreatitis, autoimmune hepatitis or irritable bowel syndrome caused by immune cells infiltrating into tissues. It can be used for prevention and treatment.

Figure 1 shows the ethanol sedimentation of water extracts of horseradish fungi (D) against daily clinical symptom index (A) and incidence (B) of experimental animals with experimental autoimmune meningitis using myelin oligodendrocyte glycoprotein (MOG) 35-55. Piwep) ”is shown. MOG + Vehicle: MOG-only group, MOG + Piwep: Combined administration of MOG and Piwep.

Figure 2 shows the inhibitory effect of Piwep on perivascular inflammatory cell infiltration in the cerebral (Cbr) and cerebellum (Cbl) of experimental animals induced with experimental autoimmune meningitis using MOG. MOG + Vehicle (X400) enlarges the display area of MOG + Vehicle (X100) by 4 times. MOG + Vehicle: MOG-only group, MOG + Piwep: Combined administration of MOG and Piwep.

Figure 3 shows the inhibitory effect of Piwep (H & E) on perivascular inflammatory cell invasion in the spinal cord of experimental animals induced experimental autoimmune meningitis using MOG and the inhibitory effect of Piwep on plant dropout (LFB & PAS). It is shown. MOG + Vehicle (X400) enlarges the display area of MOG + Vehicle (X100) by 4 times. MOG + Vehicle: MOG-only group, MOG + Piwep: Combined administration of MOG and Piwep.

Figure 4 shows Piwep for the immunoreactivity of CD4, CD8, F4 / 80 and CD20, which are cell surface molecules for cluster differentiation (CD) in the spinal cord of experimental animals with experimental autoimmune meningitis using MOG. It shows an inhibitory effect. N: normal group, V: MOG + Vehicle group, P: MOG + Piwep group.

Figure 5 shows the inhibitory effect of Piwep on the expression of mRNA specific to the myocytes of CD4, CD8, CD11b and CD20 in the spinal cord of experimental animals induced experimental autoimmune meningitis using MOG. N: normal group, V: MOG + Vehicle, P: MOG + Pipep.

Fig. 6 shows Piwep expression of mRNA specific for vascular cell adhesion molecules (VCAM) -1 and integrin-α4, which are cell adhesion molecules, in the spinal cord of experimental animals with experimental autoimmune meningitis using MOG. Inhibitory effect is shown. N: normal group, V: MOG + Vehicle, P: MOG + Pipep.

Figure 7 shows the inhibitory effect of Piwep on cell adhesion factor integrin-α4 immune response in cervical lymph nodes of experimental animals induced with experimental autoimmune meningitis using MOG. Normal: Normal group, MOG + Vehicle: MOG alone group, MOG + Piwep: Combined administration of MOG and Piwep.

Fig. 8 shows ethanol precipitates (Plwep, A) and ethanol precipitates of water extracts of Baumi mud mushrooms for the clinical symptom index of experimental animals induced experimental autoimmune meningitis using MOG. (Pbwep, B) ". MOG + Vehicle: MOG alone group, MOG + Plwep: MOG and Plwep group, MOG + Pbwep: MOG and Pbwep group.

The present invention provides a composition that effectively acts in the prevention and treatment of multiple sclerosis by preventing the infiltration of various immune cells into the central nervous system and inhibiting inflammation and myelin dropout of the central nervous system. The present invention also provides a method for the preparation and administration of the composition.

The composition of the present invention includes an extract extracted from the mud mushrooms. In another embodiment, the compositions of the present invention also include extracts prepared from woody or mushrooms.

Horse mud mushroom extract in the present invention is i) hot water extracting the mushroom; ii) mixing the hot water extract with cold ethanol to obtain a precipitate and iii) dissolving the precipitate in distilled water followed by freeze drying.

In the present invention, fruiting body powder of dung mud mushroom is used. This is for obtaining the mushroom extract more efficiently because when the fruiting body powder is used to increase the extraction surface area, more active ingredients can be extracted during the extraction process.

Next, in order to obtain an extract of the fruiting body powder, the fruiting body powder is put in distilled water and boiled. At this time, by boiling over 3 hours at a temperature of 100 ℃ ~ 104 ℃ to ensure that the active ingredient is sufficiently extracted.

The extract thus obtained is concentrated under reduced pressure to increase the content of the active ingredient. Then, the extract concentrated under reduced pressure is mixed with cold ethanol. The ethanol mixture is allowed to settle overnight at temperature below 4 ° C.

Next, the mixture is centrifuged to take a precipitate. Finally, the precipitate is again dissolved in distilled water and then lyophilized.

Experimental autoimmune meningitis is very similar to multiple sclerosis in clinical symptoms and pathological findings, the present invention demonstrates that the composition containing the mud mushroom extract using experimental autoimmune meningitis is effective for multiple sclerosis.

In the present invention, the composition greatly reduces the incidence and clinical symptoms index of experimental autoimmune meningitis. In addition, the composition blocks the infiltration of inflammatory cells around blood vessels in the spinal cord of experimental animals induced experimental autoimmune meningitis and inhibits myelin dropout. Thus, the compositions dramatically alleviate the clinical symptoms and signs and pathological changes of experimental autoimmune meningitis.

In experimental autoimmune meningitis, various types of immune cells infiltrate the central nervous system and are involved in inflammation and demyelination by different mechanisms. In the present invention, the composition infiltrates various immune cells in the central nervous system induced experimental autoimmune meningitis. Observe the effect on. The composition reduces the immunoreactivity of CD4, CD8, F4 / 80, and CD20, which are surface differentiation molecules, in the spinal cord of experimental animals with experimental autoimmune meningitis and also reduces the CD4, CD8, CD11 and CD20 specific mRNAs. Significantly inhibit expression. These results demonstrate that the compositions of the present invention block CD4 + T cells, CD8 + T cells, macrophages and B cells from infiltrating the central nervous system in experimental autoimmune meningitis.

In order to move immune cells from blood vessels to parenchymal tissues in the central nervous system, cell adhesion molecules must be strongly expressed on the surface of immune cells as well as vascular endothelial cells of the central nervous system. In the present invention, we observe the effect of horseradish mushroom extract on the expression of vascular cell adhesion molecule (VCAM) -1 expressed in vascular endothelial cells and integrin-α4 expressed in immune cells. In the present invention, the composition inhibits the expression of (VCAM) -1 specific mRNA in the spinal cord of experimental animals with experimental autoimmune meningitis and also the number of lymphocytes showing strong immunoreactivity against integrin-α4 in local lymph nodes. Decrease. These results indicate that the composition of the present invention inhibits the expression of cell adhesion molecules in immune cells as well as vascular endothelial cells of the central nervous system in experimental autoimmune meningitis-induced animals, thereby blocking the infiltration of immune cells into the central nervous system. Prove it. In addition, the composition of the present invention inhibits the expression of mRNA specific for integrin-α4 in the spinal cord of experimental animals induced experimental autoimmune meningitis. These results indicate that the composition reduces the number of lymphocytes infiltrated into the parenchyma by passing blood vessels in the central nervous system in experimental autoimmune meningitis.

On the other hand, the composition containing the extract extracted from the woody mud or Baumi mud mushroom is also confirmed to lower the clinical symptoms index of experimental autoimmune meningitis.

In the detailed description of the present invention, only the examples for experimental autoimmune meningitis of the composition comprising the dung mushroom extract are described. However, the mud mud mushroom, wood mud mushroom or Baumi mud mushroom of the present invention The composition comprising the extract is not limited to the treatment or prevention of multiple sclerosis, but may be used for the prevention and treatment of other autoimmune diseases having an immunological mechanism similar to that of multiple sclerosis. These diseases include various immune diseases such as type 1 diabetes, Hashimoto thyroiditis, celiac disease, giant cell arteritis, ringworm, autoimmune pancreatitis, autoimmune hepatitis or irritable bowel syndrome. Therefore, the scope of the present invention is a horse mud mushroom, woody mud mushroom or Baumi mud mushroom It is to be understood that the composition comprising the extract also extends to the use of the prophylaxis and treatment of the various autoimmune diseases.

Meanwhile, the mud mud mushroom, wood mud mushroom or Baumi mud mushroom of the present invention The composition containing the extract may be used as an agent for preventing and treating multiple sclerosis or as a dietary supplement or a functional food.

The agent may be administered orally or parenterally. For oral use, the compositions are formulated in pill, capsule or liquid form. For parenteral use, the compositions are known to those skilled in the art, including various excipients, carriers, diluents, etc., and formulated with those that are readily selectable.

The present invention will be described in detail through the following examples. However, this is only to aid the understanding of the invention, the present invention should not be considered to be limited thereto.

Example 1

[Preparation of Ethanol Precipitates of Water Extracts from Dung Mushrooms]

The fruiting body powder of the dung mud mushrooms was put in distilled water 10 times by weight, boiled for 3 hours, filtered to remove solid components, and the extract was concentrated under reduced pressure. The concentrated extract was mixed with twice the capacity of cold ethanol (-20 ° C.) and left overnight at 4 ° C. or less. The ethanol mixture was centrifuged to take a precipitate, which was dissolved in distilled water again, and then lyophilized, which was named "ethanol precipitate (Piwep) of water extract of dung mushroom".

[Induction of Experimental Autoimmune Meningitis and Piwep Administration]

C-7BL / 6 mouse females, 6-7 weeks old, were adapted to controlled room and humidity conditions. After dissolving Myelin oligodendrocyte glycoprotein (MOG) 35-55 at a concentration of 200 μg / 100 μl in phosphate buffered saline, the mixture was mixed with the same amount of complete Freund's adjuvant (containing 400 μg of Mycobacterium tuberculosis H37RaA) and 200 μl of the mixture was added to the experimental animals. The chest was subcutaneously injected into two sites. Pertussis toxin was dissolved in PBS at a concentration of 400 ng / 200 μl and 200 μl of the lysate was injected intraperitoneally once every 0 and 2 days after the first MOG injection. Seven days after MOG injection, 200 μl of a mixture of MOG and complete Freund's adjuvant was further inoculated. The ethanol precipitate (Piwep) of the water extract of Dung mud mushrooms was dissolved in physiological saline, sterilized using a filter, and then injected into the abdominal cavity by 100 mg / kg once daily at 0 days after the first injection of MOG. It was.

Evaluation of Clinical Symptoms in Animals with Experimental Autoimmune Meningitis

The animals were observed daily after the first injection of MOG, and the clinical symptoms of experimental autoimmune meningitis were evaluated and indexed according to the following criteria.

Grade 0: Normal

Grade 1: sagging tail

Grade 2: sagging tail, weakness of hind legs

Grade 3: sagging tail, partial paralysis of hind legs, gait disturbance

Grade 4: sagging tail, complete paralysis of both hind legs

Grade 5: Death or Death

1 shows the effect of Piwep on the clinical symptoms of experimental autoimmune meningitis. When MOG was administered to induce experimental autoimmune meningitis, the clinical symptoms first appeared on the 13th day after the first injection of MOG, and the clinical symptoms index reached its highest on the 20th day. The effects of Piwep on the maximum clinical symptoms index and incidence of experimental autoimmune meningitis are shown in Table 1 below.

Table 1

Table 1 confirms the effect of Piwep on the maximum clinical symptoms index and the maximum incidence of experimental autoimmune meningitis. In the MOG + Vehicle group that only induced experimental autoimmune meningitis, the maximum clinical symptom index was 3.15 ± 0.39 and the incidence was 90% (18 out of 20). On the other hand, in the MOG + Piwep group with Piwep-induced experimental autoimmune meningitis, the maximum clinical symptom index was 0.20 ± 0.09 and the incidence was 20% (4 out of 20 animals), which was significantly lower than the MOG + Vehicle group. . These results indicate that Piwep significantly reduces the clinical symptoms and the incidence of MOG-induced experimental autoimmune meningitis.

Pathological Study of the Central Nervous System

The effects of Piwep on inflammatory cell infiltration and myelin detachment in the central nervous system of experimental animals with experimental autoimmune meningitis were as follows.

On the 21st day after the first injection of MOG, experimental animals were anesthetized, and the brain and spinal cord were fixed by perfusion through the heart with PBS solution containing 4% paraformaldehyde. The brain and spinal cord were extracted and postfixed in the same fixative. Brain and spinal cord were embedded with paraffin, and 5 µm thick serial sections were prepared. Brain sections were stained with Hematoxylin-Eosin (H & E) method according to the conventional method, and the infiltration state of inflammatory cells was observed. Spinal cord sections were stained with Hematoxylin-Eosin (H & E) or Luxol Fast Blue (LFB) -Periodic Acid Schiff (PAS) to observe the invasive state of the inflammatory cells and the dropout state of myelin.

Figure 2 shows the effect of Piwep on peritoneal inflammatory cell infiltration in the cerebral (Cbr) and cerebellum (Cbl) of experimental animals with experimental autoimmune meningitis. Inflammatory cells infiltrated around the blood vessels were observed in the MOG + Vehicle group administered only MOG, but not in the MOG + Piwep group administered Piwep. These results suggest that Piwep significantly inhibits inflammatory cell infiltration in the central nervous system associated with experimental autoimmune meningitis.

Figure 3 shows the effect of Piwep on inflammatory cell infiltration and myelin detachment in the spinal cord of experimental animals with experimental autoimmune meningitis. In the MOG + Vehicle group treated with MOG alone, there was very severe inflammatory cell infiltration around the blood vessels, but no such infiltration was observed in the MOG + Piwep group treated with Piwep. In addition, very severe myelin dropping was observed in the MOG + Vehicle group, but no dropping was observed in the MOG + Piwep group. These results indicate that Piwep inhibits the development of experimental autoimmune meningitis by inhibiting inflammatory cell infiltration and myelin detachment in the central nervous system associated with experimental autoimmune meningitis.

[Immunohistochemical Study of Spinal Cord]

The effect of Piwep on spinal cord infiltration of various immune cells was examined using immunohistochemistry for cell surface molecules for cluster differentiation (CD).

Spinal cord fixed by perfusion of 4% paraformaldehyde was post-fixed in the same fixative and washed with phosphate buffered saline containing 30% sucrose for 2 days at 4 ° C. Spinal cord frozen sections of 30 µm thickness were used as monoclonal antibodies against CD4, CD8, F4 / 80 or polyclonal antibodies against goats against CD20 as the first antibody and anti-mouse IgG or anti-goat IgG antibody as the second antibody, respectively. Used as. Immune response was developed by 0.06% 3,3'-diaminobenzidine after incubation in ABC reagent according to a conventional method.

4 shows spinal cord stained by immunohistochemistry. The immunoreactivity of CD4, CD8, F4 / 80, and CD20 was significantly increased in the spinal cord of experimental autoimmune meningitis-induced animals, and Piwep significantly suppressed the increase of IGF-surface factors. These results indicate that Piwep inhibits infiltration of CD4 + T cells, CD8 + T cells, macrophages, B cells, etc. into central nervous system tissues during experimental autoimmune meningitis.

[Analysis of the Expression of CD4, CD8, CD11b and CD20 Specific mRNAs in the Spinal Cord]

To determine the effect of Piwep on spinal cord infiltration of various immune cells, mRNA levels of cell surface molecules for cluster differentiation specifically expressed on the surface of each immune cell were analyzed by reverse transcription-polymerase chain reaction, RT-PCR) was measured as follows.

Three weeks after the first injection of MOG, the animals were anesthetized, perfused with PBS through the heart, and spinal cords were collected. Total RNA was extracted from the spinal cord using Trizol reagent, and then the expression of specific mRNAs for CD4, CD8, CD11b and CD20 was measured by conventional RT-PCR. The first cDNA strain was synthesized from 1 μg RNA using the cDNA synthesis kit. 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 A-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'. 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 isolated on agarose gel, stained with ethidium bromide and the amount of RNA was measured using Image J.

Figure 5 shows the results of observing the expression of specific mRNA for CD4, CD8, CD11b and CD20 in the spinal cord of animals induced experimental autoimmune meningitis. In the spinal cord of experimental autoimmune meningitis-induced animals, the expression of specific mRNAs for CD4, CD8, CD11b and CD20 was significantly increased, and Piwep significantly suppressed the increase of mRNA expression specific for these HFCS factors. These results indicate that Piwep inhibits the infiltration of CD4 + T cells, CD8 + T cells, macrophages and B cells into the central nervous system tissues involved in experimental autoimmune meningitis. It fits well.

[Analysis of mRNA Expression of Adhesion Molecules in Spinal Cord]

In order for immune cells to pass through blood vessels, vascular cell adhesion molecule (VCAM) -1, to which the cell adhesion molecules of immune cells bind, must be expressed on the surface of vascular endothelial cells. Observed as follows. Integrin-α4 was also expressed in the immune cells infiltrated into the central nervous system, and the effects of Piwep on the infiltration of immune cells were measured by measuring the expression level of integrin-α4 in the spinal cord.

Anesthesia was anesthetized 21 days after the first injection of MOG and blood was removed by perfusion of cold (4 ° C) physiological saline through the heart, followed by spinal cord collection, and vascular cell adhesion molecules (VCAM) among the cell adhesion molecules in the spinal cord tissue). The expression of mRNAs specific for -1 and integrin-α4 was measured by RT-PCR method.

The first cDNA strain was synthesized from 1 μg RNA using the cDNA synthesis kit. Oligonucleotide primers are as follows: vascular cell adhesion molecule (VCAM) -1 forward 5′-AGG CAC AGC TGC AGG ATG CC-3 ′, reverse 5′-GGA GGG GGC GGG GCT GTA AT-3 ′; integrin-α forward 5′-CCA CTA CGA TCG CTC CGC CTG T-3 ＇, reverse 5′-CCA CTA CGA TCG CTC CGC CTG T-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 isolated on agarose gel, stained with ethidium bromide and the amount of RNA was measured using Image J.

Figure 6 shows the results of observing the expression of specific mRNA for VCAM-1 and integrin-α4 in the spinal cord. Expression of specific mRNAs for VCAM-1 and integrin-α4 in the spinal cord of animals with experimental autoimmune meningitis was elevated, and Piwep significantly reduced the expression of specific mRNAs for these cell adhesion molecules. These results demonstrate that Piwep inhibits the increased expression of vascular cell adhesion molecules in central nervous system vascular endothelial cells, thereby preventing the infiltration of immune cells into the central nervous system. In particular, the decrease in the expression of mRNA specific for integrin-α4 by Piwep means that the number of immune cells infiltrated into the central nervous system by Piwep was reduced.

Immunohistochemical Observation on Integrin-α4 Expression in Lymph Nodes

In order for the immune cells to pass through blood vessels, the cell adhesion molecules must be expressed on the cell surface and combined with vascular cell adhesion molecules such as VCAM-1. Therefore, the effect of Piwep on the expression of integrin-α4, a cell adhesion molecule of immune cells, in the cervical lymph nodes Observation was as follows.

Three weeks after the first injection of MOG, cervical lymph nodes were fixed in neutral formaldehyde solution for 2 days. 5 μm sections were embedded with Paraffin and then stained by immunohistochemistry using anti-integrin-α4 monoclonal antibody and anti-mouse IgG antibody as first and second antibodies, respectively. Immune responses were developed in 0.06% 3,3'-diaminobenzidine after incubation in ABC reagent.

7 shows immunohistochemical findings for integrin-α4 in cervical lymph nodes. In lymphocytes of animals with experimental autoimmune meningitis, the appearance of lymphocytes with strong immunoreactivity against integrin-α4 was increased. Lymphocytes with strong immunoreactivity against integrin-α4 were not observed with Piwep. These results indicate that Piwep inhibits the integrin-α4 expression of lymphocytes in lymph nodes during experimental autoimmune meningitis.

Example 2

[Preparation of Ethanol Precipitates of Water Extracts from Wood Mud Mushrooms or Baumi Mud Mushrooms]

Plwep (wood mud mushrooms) or Pbwep (baumy mud mushrooms), which are ethanol precipitates of water extracts, were obtained from wood mud mushrooms or Baumi mud mushrooms in the same manner as in Example 1.

[Effect of Wood Mud Mushroom Extract and Baumi Mud Mushroom Extract on Experimental Autoimmune Meningitis]

The effect of Plwep and Pbwep extracts from the woody mud mushrooms belonging to the same genus as dung mud mushrooms was examined on the clinical index of experimental autoimmune meningitis.

8 shows the effects of Plwep and Pbwep on the clinical symptoms of animals induced with experimental autoimmune meningitis. Both Plwep and Pbwep markedly inhibit the clinical index of experimental autoimmune meningitis. These results imply that wood mud extract and Baumi mud mushroom extract significantly improved clinical symptoms of experimental autoimmune meningitis as well as horse mud extract.

Claims

Composition for the prevention and treatment of multiple sclerosis, characterized in that it comprises at least one mushroom extract selected from the group consisting of dung mud mushrooms, wood mud mushrooms and Baumi mud mushrooms.
In claim 1,

The extract is

i) hydrothermal extraction of one or more mushrooms selected from the group consisting of dung mud mushrooms, wood mud mushrooms and Baumi mud mushrooms;

ii) mixing the hot water extract with cold ethanol to obtain a precipitate, and

iii) a composition for the prevention and treatment of multiple sclerosis, characterized in that obtained from the step of dissolving the precipitate in distilled water and freeze drying.
In claim 1,

The composition is a composition for the prevention and treatment of multiple sclerosis, characterized in that the central nervous system inhibits inflammation and myelin detachment.
In claim 1,

The composition inhibits the expression of cell surface molecules CD4, CD8, CD11 (or F4 / 80) and CD20 in the central nervous system or inhibits infiltration of CD4 + T cells, CD8 + T cells, macrophages and B cells into the central nervous system. Composition for the prevention and treatment of multiple sclerosis, characterized in that.
In claim 1,

The composition is a composition for the prevention and treatment of multiple sclerosis, characterized in that to inhibit the expression of vascular cell adhesion molecule (VCAM) -1 in the central nervous system.
In claim 1,

The composition is a composition for the prevention and treatment of multiple sclerosis, characterized in that by inhibiting the expression of integrin-α4 in the central nervous system.
In claim 1,

The composition is a composition for preventing and treating multiple sclerosis, characterized in that to inhibit the expression of integrin-α4 in lymphocytes.
An agent for the prevention and treatment of multiple sclerosis comprising the composition of claim 1 and a pharmaceutically acceptable carrier.
In claim 8,

The agent is a pill, capsule or oral or parenteral preparation for the prevention and treatment of multiple sclerosis, characterized in that the liquid.
A dietary supplement or functional food for preventing and improving multiple sclerosis comprising the composition of claim 1 and a carrier.
Prevention and treatment of immune diseases of type 1 diabetes mellitus, Hashimoto thyroiditis, celiac disease, giant cell arteritis, ringworm, autoimmune pancreatitis, autoimmune hepatitis or irritable bowel syndrome, comprising the composition of claim 1 and a pharmaceutically acceptable carrier Dragon sanctions.
In claim 11,

The agent is a pill, capsule or liquid for oral or parenteral type 1 diabetes, Hashimoto thyroiditis, celiac disease, giant cell arteritis, ringworm, autoimmune pancreatitis, autoimmune hepatitis or irritable bowel syndrome Sanctions for the prevention and treatment of diseases.
A dietary supplement for the prevention and improvement of immune diseases of type 1 diabetes mellitus, Hashimoto thyroiditis, celiac disease, giant cell arteritis, ringworm, autoimmune pancreatitis, autoimmune hepatitis or irritable bowel syndrome comprising the composition and carrier of claim 1 or Functional food.