JP2003212790A - Fat-soluble extract component derived from hericium erinaceum - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a component derived from nature, effective for preventing and/or treating neurodegenerative diseases. <P>SOLUTION: This fat-soluble extract component derived from Hericium erinaceum has cell death induction inhibitory activity, especially endoplasmic reticulum stress inhibitory activity. The composition containing the component as an active ingredient is provided. The method for screening substances having endoplasmic reticulum stress inhibitory activity is also provided. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD [0001] The present invention relates to Hericium eri which is useful for the prevention and / or treatment of neurodegenerative diseases.
naceum ) a fat-soluble extract component, a composition containing the extract component, and a screening method for identifying a substance that can be used for the prevention and / or treatment of a neurodegenerative disease using the effect of the extract component as an index.

[0002]

2. Description of the Related Art In an aging society in recent years, the ratio of the number of patients with neurodegenerative diseases is increasing, and it is expected that it will become a more serious social problem in the near future. As these neurodegenerative diseases, for example, Alzheimer's disease and Parkinson's disease are known. Neurodegenerative diseases such as Alzheimer's disease have a long incubation period, the symptoms may progress without the person's knowledge, and it is almost impossible to cure completely after the onset by current medicine. In other words, it is considered to be particularly important in the case of neurodegenerative diseases to take a habitual preventive measure before it progresses.

Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are considered to be caused by the neuronal cell death caused by endoplasmic reticulum stress, and this endoplasmic reticulum stress is caused in some of these neurodegenerative diseases. It is considered a common mechanism. It has been suggested that Creutzfeldt-Jakob disease, which is considered to be one of prion diseases including mad cow disease, which is a neurodegenerative disease of amyloid deposition similar to these, may be a disease caused by cell death derived from endoplasmic reticulum stress. . However, up to now, no substance or component having an activity of suppressing endoplasmic reticulum stress has been reported.

On the other hand, it is a generally practiced method to search for useful substances or components having a specific pharmacological action from natural products and utilize them for drug discovery. Such techniques are frequently used because isolation of such useful substances or components from natural products often leads to elucidation of the pathogenic mechanism and application to new therapeutic agents. Mushrooms are one of the most popular natural products. There are more than 10,000 kinds of mushrooms on the earth, and it is said that there are 2-3 thousand kinds in Japan alone. For example, helicenone, which induces nerve growth factor, has been isolated as a useful substance from a mushroom, Pleurotus cornucopiae.

[0005]

DISCLOSURE OF THE INVENTION It is an object of the present invention to provide a substance having an endoplasmic reticulum stress inhibitory activity capable of preventing and / or treating a neurodegenerative disease, and various uses of the substance. .

[0006]

[Means for Solving the Problems] The present inventor has conducted extensive studies in order to achieve the above-mentioned object, and as a result of paying attention to Yamabushitake mushrooms, which have been reported to have nerve growth factor inducing activity up to now, as a result, In order to complete the present invention, it was found that the fat-soluble extract component derived from the present invention has a cell death induction inhibitory activity, particularly an endoplasmic reticulum stress inhibitory activity, and that it can be used for the prevention and / or treatment of neurodegenerative diseases. I arrived.

[0007] That is, the present invention is a fat-soluble extract component derived from Pleurotus cornucopiae, which is characterized in that it has preventive and / or therapeutic activity for neurodegenerative diseases, and the fat-soluble extract. Provided is a composition for preventing and / or treating a neurodegenerative disease, which comprises a component as an active ingredient. Preferably, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease and Creutzfeldt-Jakob disease.

[0008] The present invention also comprises a fat-soluble extract component derived from Pleurotus cornucopiae, which is characterized by having a cell death induction inhibitory activity, and the fat-soluble extract component as an active ingredient. A composition for inhibiting cell death is provided.

The present invention further comprises a fat-soluble extract component derived from Pleurotus cornucopiae, characterized in that the component has endoplasmic reticulum stress suppressing activity, and the fat-soluble extract component as an active ingredient. A composition for suppressing endoplasmic reticulum stress is provided.

Preferably, the fat-soluble extract component derived from Pleurotus cornucopiae is obtained by continuously extracting it with 85% ethanol, acetone and chloroform.

The present invention further provides a screening method for a substance having an endoplasmic reticulum stress suppressing activity, which comprises at least the following steps: a) Test cells are tested with a compound that induces endoplasmic reticulum stress. A step of treating the test cell with a substance and an endoplasmic reticulum stress-inducing compound and a lipid-soluble extract derived from Phellinus linteus (wherein the test cell and the compound inducing the endoplasmic reticulum stress are the same as those in the step a). Same as that used in step 1)), and c) the degree of survival of the test cells in step a) and step b) is measured and compared to determine whether the test substance has an endoplasmic reticulum stress suppressing activity. The step of determining. Preferably, the oil-soluble extract component derived from Pleurotus cornucopiae has endoplasmic reticulum stress suppressing activity.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION As used herein, the term "lipid-soluble extract component derived from Phellinus linteus" refers to a component that can be extracted from the fruiting body of Phellinus linteus with an organic solvent, and particularly,
It refers to a component having physiological activities such as endoplasmic reticulum stress inhibitory activity and cell death induction inhibitory activity and useful for the prevention and / or treatment of neurodegenerative diseases. Various extraction methods can be used to extract the fat-soluble extract component derived from the above-mentioned Bamboo mushroom.
For example, a suitable solvent for extracting the above components is typically methanol, ethanol, acetone,
Examples include chloroform, phenol, hexane, butanol, isopropanol, benzene, diethyl ether, ethyl acetate, tetrahydrofuran, toluene, dichloromethane, trichloroethylene and the like. These solvents may be used alone, or a mixed solvent composed of several kinds of organic solvents may be used. Furthermore, the solvent and mixed solvent consisting of one of the above may be continuously used in various extraction steps. Those skilled in the art can appropriately select the type of solvent to be used and how to combine them. The amount of solvent used for extraction can be appropriately selected by those skilled in the art depending on the type of solvent used, extraction efficiency, and the like. For example, in the first step of extraction, ethanol,
Preferably, 85% ethanol is used in an amount of 5 to 20 parts by weight based on 1 part by weight of Pleurotus cornucopiae fruiting body (dry).
After that, the fat-soluble extract component can be obtained by repeating extraction with an appropriate solvent and weight ratio. In one embodiment, the above-mentioned “lipid-soluble extract component derived from Pleurotus cornucopiae” can be obtained, for example, by continuously extracting the fruit body of Pleurotus cornucopiae with 85% ethanol, acetone and chloroform, and then passing the obtained chloroform through a silica gel column. Is.

As used herein, the term "neurodegenerative disease" refers to a disease in which nerve cells undergo apoptosis (nerve cell death) and normal neurotransmission function is lost. Examples of the "neurodegenerative disease" include Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, Huntington's disease, Pick's disease, progressive supranuclear palsy, spinocerebellar degeneration, amyotrophic lateral sclerosis, and others. Various dementia diseases (for example, Binsbanger's disease, cerebrovascular dementia, normal pressure hydrocephalus, post-traumatic dementia, chronic subdural hematoma, brain tumor), preferably Alzheimer's disease, Parkinson's disease and Creutzfeldt. I have Jacob's disease.

The term "induction of cell death" used in the present specification means that information is transmitted to the inside of the cell to induce cell death by physical or chemical stimulation from the outside of the cell.

The term "endoplasmic reticulum stress" used in the present specification accumulates a protein having an abnormal structure due to functional abnormality in the rough endoplasmic reticulum, which is a place of translation of extracellular proteins on the cell surface. The stress caused by something.

The terms "cell death induction-inhibiting activity" and "endoplasmic reticulum stress-inhibiting activity" used in the present specification refer to the ability to suppress the above-mentioned cell death induction and endoplasmic reticulum stress, respectively. Therefore, a component having “suppression of cell death induction” or “inhibition of endoplasmic reticulum stress” can be used for preventing and / or treating various diseases caused by cell death induction or endoplasmic reticulum stress.

The term "composition" used in the present specification means a substance containing a specific substance as an active ingredient, and for example, "a pharmaceutical composition for preventing and / or treating neurodegenerative diseases" means Also, it contains a fat-soluble extract component derived from Pleurotus cornucopiae as an active ingredient, and has a preventive and / or therapeutic effect on neurodegenerative diseases. Such a pharmaceutical composition is in a solid, semi-solid or liquid form containing, as an active ingredient, a lipid-soluble extract derived from Pleurotus cornucopiae as a mixture with an organic or inorganic carrier or excipient suitable for oral or parenteral application. Can be used in the form of pharmaceutical preparations. The active ingredient is, for example, a conventional, non-toxic substance for powders, tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, aerosols, sprays and other forms suitable for use. It can be combined with a pharmaceutically acceptable carrier. Further, if necessary, auxiliary agents, stabilizers, thickeners and the like may be used. These carriers and excipients may be sterilized as needed, or may be sterilized after being formulated. The therapeutically effective dose of the fat-soluble extract derived from Pleurotus cornucopiae, which is the active ingredient, is the degree of purification of the extract, the age and type of disease of the individual patient to be treated,
It also depends on the severity and depends on them.

The fat-soluble extract component derived from Pleurotus cornucopiae can also be provided as a health food. It is known that the risk of the onset of neurodegenerative diseases such as Alzheimer's disease is deeply related to the genetic predisposition, and, for example, a person who has such a genetic predisposition contains the above fat-soluble extract component. It is thought that the risk of developing such healthy foods can be reduced by daily intake. Such a health food is expected to have further effects by containing not only the fat-soluble extract component but also other active ingredients, for example.

The present invention also provides a method for screening a substance having an endoplasmic reticulum stress suppressing activity. The above-mentioned screening method comprises a) a step of treating test cells with a compound that induces endoplasmic reticulum stress and a test substance, and b) treating the test cells with a compound that induces endoplasmic reticulum stress and a lipid-soluble extract derived from Pleurotus cornucopiae. (Wherein the test cell and the compound that induces endoplasmic reticulum stress are the same species as used in step a)), and c) the degree of survival of the test cell in step a) and step b). Are measured and compared to determine whether the test substance has endoplasmic reticulum stress.

The above-mentioned "test cell" is not particularly limited as long as it is a cell that can be used for screening a substance having an endoplasmic reticulum stress suppressing activity and can induce endoplasmic reticulum stress. animal,
Preferably any cell line or primary culture of human origin,
More preferably, a nervous system cell line is mentioned. In the present invention, preferable nervous system cell lines include PC-12 and Ne.
uro2a, CHP-126, GOTO-P3, LA-
N-5, SK-N-SH, TNB-1, and C-1300 are mentioned, but PC-12 cells are particularly preferable.

[0021] The above-mentioned "compound that induces endoplasmic reticulum stress"
Is a compound capable of inducing endoplasmic reticulum stress in any cell. Examples of the above-mentioned “compound that induces endoplasmic reticulum stress” include tunicamycin, 1-deoxynojirimycin, bromoconduritol and australin, with tunicamycin being particularly preferred.

The above-mentioned "test substance" is not particularly limited as long as it is a compound required to determine whether or not it has an endoplasmic reticulum stress inhibitory activity in the above screening method,
It may be a known one or a new one.
As the “test substance” to be screened in the screening method of the present invention, any natural compound and synthetic compound such as nucleic acid, protein, peptide, lipid, saccharide, and low molecular compound can be selected.

Further, in the screening method of the present invention, "treating" means incubating a test cell with a compound that induces endoplasmic reticulum stress and a test substance or a lipid-soluble extract component derived from Pleurotus cornucopiae in a culture medium. The test substance or the lipid-soluble extract derived from Pleurotus cornucopiae is preferably added to the culture medium containing the test cells at the same time as or before the compound that induces endoplasmic reticulum stress, but the compound that induces endoplasmic reticulum stress is also included. May be added later in the culture medium containing the test cells. The order of addition is appropriately set according to the type of test cell used, the type and activity of the compound that induces endoplasmic reticulum stress, and the type of test substance. Similarly, the treatment time and the amount of the compound and test substance that induce endoplasmic reticulum stress can be appropriately selected by those skilled in the art according to their activity and type and the type of test cell used. In the above-mentioned screening method, the test cell and the compound that induces endoplasmic reticulum stress used in steps a) and b) are required to be “homologous” for proper comparison. "Allogeneic" means that the same type of cells is used in the screening steps a) and b), for example, when PC-12 cells are used in step a), step b).
However, PC-12 cells are used.

The "degree of survival" (viability) of the test cells in the screening method of the present invention is MTT (3-
“Measured” by a live cell quantification method known in the art such as (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide) assay, neutral red method, trypan blue method. By "comparing" the values obtained from this "measurement", it is possible to "determine" whether the test substance has an endoplasmic reticulum stress suppressing activity.
can do. Specifically, the "survival degree" when the test substance was added and when the test substance was not added was compared, and a substance having a significantly increased survival rate was defined as a substance having endoplasmic reticulum stress suppressing activity,
It is preferable to select a substance that shows a value that is the same as or higher than the “survival degree” obtained when the lipophilic extract derived from Pleurotus cornucopiae (in particular, the extract having an endoplasmic reticulum stress suppressing activity) is added.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following specific examples.

[0026]

Examples (Example 1: Fractionation of fruiting bodies of Pleurotus cornucopiae) 360 g of fruiting bodies of Pleurotus cornucopiae were extracted with 85% ethanol and concentrated under reduced pressure. The concentrate was extracted with acetone, concentrated under reduced pressure, and then extracted again with chloroform to separate the chloroform layer. The chloroform layer was applied to a silica gel column (silica gel 60N, manufactured by WAKO) to obtain extracted fractions (designated as HE1 to HE14 in FIG. 1). Fig. 1 shows an outline of the Yamabushitake mushroom extraction.

Example 2 Measurement of Nervous System Cell Death Induction Inhibitory Activity of Extracts of Phellinus linteus PC-12 Cells (RIKEN Cell Development Bank, CellN) as a Model of Nervous System Cells
o. RCB0009) was used. Culture medium is 10%
Dulbecco's modified Eagle medium supplemented with horse serum and 5% fetal bovine serum was used, and the CO ₂ concentration was 5%. Amyloid β 1-40 was also used to induce cell death. Amyloid β 1-40 is a major peptide that constitutes senile plaques found in the brain of Alzheimer's disease patients. Amyloid β1-42 is also known as another main peptide that constitutes senile plaques, and these peptides are produced by limited degradation of amyloid precursor protein. Moreover, these peptides have been confirmed to induce cell death in nerve cells, and are said to be one of the causes of neurodegenerative diseases. Therefore, in order to determine the cell death-inducing suppressive activity of each of the extracts of Pleurotus cornucopiae, PC-12 cells were cultured at 37 ° C. for 48 hours in the presence of 10 μM amyloid-β 1-40 and the extract of Pleurotus cornucopiae. The cell viability is measured by MT, which is a commonly used live cell quantification method.
It was quantified by MTT assay using T (manufactured by Dojindo Co., Ltd.) (yellow crystal, MTT, is reduced when taken up by living cells, becomes blue crystal, and shows specific absorption at 570 nm). After the completion of the culture, the cells were further cultured in the presence of 250 μg / ml of MTT for 3 hours to stop the reaction in a reaction stop solution containing 20% SDS and 50% dimethylformamide, and then the product and the cells were allowed to react. Solubilized.
Then, the absorbance at 570 nm was measured to determine the viability of cells. As a control, PC-12
Cells cultured in the absence of amyloid-β 1-40 were used. The cell viability was compared with the value obtained from this as 100%. The results are shown in Figure 2. From the results of FIG. 2, it was shown that the extract fractions 6 and 9 to 14 of Pleurotus cornucopiae suppress the cell death induction by amyloid-β 1-40.

Example 3 Measurement of Oxidative Stress Suppression of Extracts of Pleurotus cornucopiae Saccharomyces The mechanism of suppressing neuronal cell death shown in Example 2 was further investigated. Focusing on oxidative stress inhibition as a mechanism of cell death inhibition, nitric oxide (N
O) Donor sodium nitroprusside (WAKO
Was manufactured in the same manner as in Example 2 above. P
C-12 cells were cultured at 37 ° C. for 24 hours in the presence of 50 μM sodium nitroprusside and Phellinus linteus extract fractions, and cell viability was measured by the MTT assay in the same manner as in Example 2 to suppress the induction of cell death in each fraction. The activity was determined. As a control, PC-1
Two cells cultured in the absence of NO were used, and the cell viability was compared with the value obtained from this as 100%.
The results are shown in Fig. 3. From the results in FIG. 3, it was shown that the extract fractions 1 to 12 of Pleurotus cornucopiae suppress the cell death induction by NO.

(Example 4: Measurement of endoplasmic reticulum stress inhibitory activity of the extract of Phellinus linteus) Since endoplasmic reticulum stress has been reported as a cause of neuronal cell death, the extract of Phellinus linteus has an activity of inhibiting endoplasmic reticulum stress. Was examined. The endoplasmic reticulum stress was induced by treatment with tunicamycin, which is an inhibitor of endoplasmic reticulum sugar chain biosynthesis. 0.25 μM tunicamycin (WAKO)
In addition, cell death was induced at 37 ° C. for 24 hours in the presence of the extract of Phellinus linteus, and the cell viability was the same as in Example 2.
The cell death induction inhibitory activity of each fraction was determined by the T assay. As a control, PC-1
Two cells cultured in the absence of tunicamycin were used, and the cell viability was compared with the value obtained from this being 100%. The results are shown in Fig. 4. From the results in FIG. 4, it was shown that the extract fractions 9 to 11, 13 and 14 of Physcomitrella patense var.

Example 5 Dose Dependence Test of Endoplasmic Reticulum Stress Suppressing Activity of Extract of Boletus edulis Fraction 9, 10 and 13 of the fractions in which cell death induction was suppressed in Example 4 above.
Further experiments were performed on 14. In the same manner as in Example 4, cell death induction was carried out at 37 ° C. for 24 hours in the presence of 0.25 μM tunicamycin, the cell viability was measured by MTT assay, and the cell death induction inhibitory activity of each fraction was determined.
In addition, the content of each of the extract fractions of Pleurotus cornucopiae in the plate used in the MTT assay is 2.5 μg /
100 μl, 5 μg / 100 μl, 10 μg / 100 μ
The experiment was carried out by changing the amount to 50 μg / 100 μl. As a control, PC-12 cells cultured in the absence of tunicamycin were used, and the cell viability was compared with the value obtained from this as 100%. Results are shown in FIG. From the results of FIG. 5, it was shown that, among the extracts of Phellinus linteus extract fractions 9, 10, 13, and 14, in particular, fractions 9 and 10 suppress the cell death induction by endoplasmic reticulum stress in a dose-dependent manner.

[0031]

EFFECTS OF THE INVENTION The fat-soluble extract derived from Physarum edulis has excellent ER stress-inhibiting activity and cell death-inducing inhibitory activity, and therefore can prevent various diseases caused by ER stress and cell death, especially prevent neurodegenerative diseases. And / or is useful in therapy. Further, the fat-soluble extract component of the present invention is a natural product, especially derived from edible mushrooms, and can be safely used, and can be used not only as a medicine but also as a habitual health food for the prevention of various diseases. it can. Furthermore, the lipophilic extract derived from Pleurotus cornucopiae is also useful as a tool for elucidating the mechanism of nerve cell death.

[Brief description of drawings]

FIG. 1 is a diagram showing an outline of extraction from dried fruit bodies of Physcomitrella patens. Each extracted fraction (HE-1 to H shown in FIG. 1
The number of mg described under E-14) indicates the dry weight of each fraction.

FIG. 2 is a diagram showing the effect of a fraction derived from Pleurotus cornucopiae on amyloid β 1-40 toxicity.

FIG. 3 is a graph showing the effect of a fraction derived from Pleurotus cornucopiae on NO toxicity.

FIG. 4 is a graph showing the effect of the Physcomitrella patens-derived fraction on the toxicity of tunicamycin.

FIG. 5 is a graph showing the dose-dependence of the effect of the fraction derived from Pleurotus cornucopiae on the toxicity of tunicamycin.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 39/06 A61P 39/06 43/00 107 43/00 107 C12Q 1/02 C12Q 1/02 G01N 33 / 15 G01N 33/15 Z 33/50 33/50 Z // A23L 1/30 A23L 1/30 BF term (reference) 2G045 AA40 BB20 CB01 4B018 MD82 ME14 MF01 4B063 QA18 QR41 QR77 QX10 4C088 AA02 AC17 BA08 BA10 CA04 CA09 NA ZA15 ZA16 ZB22 ZC37

Claims (12)

[Claims] 1. A fat-soluble extract component derived from Pleurotus cornucopiae, which has a preventive and / or therapeutic activity for neurodegenerative diseases. 2. The neurodegenerative disease is Alzheimer's disease,
The fat-soluble extract component according to claim 1, which is selected from the group consisting of Parkinson's disease and Creutzfeldt-Jakob disease. 3. A fat-soluble extract component derived from Pleurotus cornucopiae, which has a cell death induction inhibitory activity. 4. A fat-soluble extract component derived from Pleurotus cornucopiae, which has an endoplasmic reticulum stress suppressing activity. 5. The fat-soluble extract component derived from Pleurotus cornucopiae is a component obtained by continuous extraction with 85% ethanol, acetone and chloroform.
4. The fat-soluble extract component according to any one of 4 above. 6. A pharmaceutical composition for preventing and / or treating a neurodegenerative disease, which comprises, as an active ingredient, a fat-soluble extract component derived from Pleurotus cornucopiae. 7. The pharmaceutical composition according to claim 6, wherein the degenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease and Creutzfeldt-Jakob disease. 8. A cell death induction-inhibiting composition containing a fat-soluble extract component derived from Pleurotus cornucopiae as an active ingredient. 9. An endoplasmic reticulum stress-suppressing composition containing, as an active ingredient, a fat-soluble extract component derived from Pleurotus cornucopiae. 10. The fat-soluble extract component derived from Pleurotus cornucopiae is a component obtained by continuous extraction with 85% ethanol, acetone and chloroform.
The composition according to any one of 1 to 9. 11. A method for screening a substance having an endoplasmic reticulum stress suppressing activity, comprising at least the following steps: a) a step of treating a test cell with a compound that induces endoplasmic reticulum stress and a test substance. B) a step of treating the test cell with an endoplasmic reticulum stress-inducing compound and a lipid-soluble extract derived from Pleurotus cornucopiae (wherein the test cell and the compound that induces an endoplasmic reticulum stress are those used in step a)) The same species), and c) a step of measuring and comparing the degree of survival of the test cells in the steps a) and b) to determine whether or not the test substance has an endoplasmic reticulum stress suppressing activity. 12. The method according to claim 11, wherein the fat-soluble extract component derived from Pleurotus cornucopiae is the component according to claim 4 or 5.