JP3943399B2 - Fat-soluble extract from Yamabushidatake - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a fat-soluble extract component of Herbium erinaceum useful for the prevention and / or treatment of neurodegenerative diseases, a composition containing the extract components, and prevention and prevention of neurodegenerative diseases using the effect of the extract components as an index. The present invention relates to a screening method for identifying substances that can be used for therapy.
[0002]
[Prior art]
In the aging society in recent years, the ratio of the number of patients with neurodegenerative diseases is increasing, and it is expected to 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, and symptoms may progress without their awareness, and it is almost impossible to cure them completely after onset. In other words, taking a path that is habitually prevented in some way before it progresses may be particularly important in the case of neurodegenerative diseases.
[0003]
Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are thought to develop when neuronal cell death is caused by endoplasmic reticulum stress, and this endoplasmic reticulum stress is a common mechanism in some of these neurodegenerative diseases. It is believed that. It has been suggested that Creutzfeldt-Jakob disease, which is considered to be a kind of prion disease including mad cow disease, is a disease caused by endoplasmic reticulum stress-induced cell death. . However, no substance or component having an endoplasmic reticulum stress inhibitory activity has been reported so far.
[0004]
On the other hand, searching for a useful substance or component having a specific pharmacological action from a natural product and utilizing it for drug discovery is a commonly practiced technique. Such techniques are frequently used because isolation of such useful substances or components from natural products often leads to elucidation of the onset mechanism and application to new therapeutic agents. Mushrooms are one of the natural products that are attracting attention. It is said that there are more than 10,000 kinds of mushrooms on the earth and 2-3 thousand kinds in Japan alone. For example, helicenone that induces nerve growth factor has been isolated from Yamabushitake, a kind of mushroom, as a useful substance.
[0005]
[Problems to be solved by the invention]
An object of this invention is to provide the substance which has the endoplasmic reticulum stress inhibitory activity which can enable prevention and / or treatment of a neurodegenerative disease, and various uses of this substance.
[0006]
[Means for Solving the Problems]
The present inventor conducted intensive research to achieve the above-mentioned object, and as a result of paying attention to Yamabushitake, which has been reported to induce nerve growth factor so far, as a result of its investigation, the fat-soluble extract component derived from Yamabushitake has been subjected to cell death. It has been found that it has induction-inhibiting activity, particularly endoplasmic reticulum stress-inhibiting activity, and has been found to be used for the prevention and / or treatment of neurodegenerative diseases, thereby completing the present invention.
[0007]
That is, the present invention is a fat-soluble extract component derived from Yamabushidatake, characterized in that the component has a preventive and / or therapeutic activity for neurodegenerative diseases, and the fat-soluble extract component is effective. Provided is a composition for preventing and / or treating a neurodegenerative disease, which is contained as a component. Preferably, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease and Creutzfeldt-Jakob disease.
[0008]
The present invention is also a fat-soluble extract component derived from Yamabushidatake, characterized in that the component has a cell death induction inhibitory activity, and a cell death containing the fat-soluble extract component as an active ingredient An inhibitory composition is provided.
[0009]
The present invention further relates to a fat-soluble extract component derived from Yamabushidatake, characterized in that the component has an endoplasmic reticulum stress-inhibiting activity, and an endoplasmic reticulum containing the fat-soluble extract component as an active ingredient A stress-suppressing composition is provided.
[0010]
Preferably, the fat-soluble extract component derived from Yamabushidatake is obtained by continuous extraction with 85% ethanol, acetone and chloroform.
[0011]
The present invention further provides a screening method for a substance having endoplasmic reticulum stress inhibitory activity, comprising at least the following steps:
a) treating a test cell with a compound that induces endoplasmic reticulum stress and a test substance;
b) a step of treating a test cell with a compound that induces endoplasmic reticulum stress and a fat-soluble extract component derived from Yamabushitake (wherein the test cell and the compound that induces endoplasmic reticulum stress are the same as those used in step a) And c) 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 endoplasmic reticulum stress inhibitory activity.
Preferably, the fat-soluble extract component derived from Yamabushidatake has an endoplasmic reticulum stress inhibitory activity.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term “Yamabushitake-derived fat-soluble extractive component” is a component that can be extracted from the fruit body of Yamabushitake using an organic solvent, and in particular, has an endoplasmic reticulum stress inhibitory activity, a cell death induction inhibitory activity, etc. A component having physiological 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 Yamabushidatake. For example, suitable solvents for extracting the above components typically include methanol, ethanol, acetone, chloroform, phenol, hexane, butanol, isopropanol, benzene, diethyl ether, ethyl acetate, tetrahydrofuran, toluene, dichloromethane, Examples include trichlorethylene. These solvents may be used alone or a mixed solvent composed of several kinds of organic solvents may be used. Furthermore, you may use the solvent and mixed solvent which consist of said 1 type continuously in various extraction processes. Those skilled in the art can also select the types of solvents to be used and how to combine them. The amount of the solvent used for extraction can be appropriately selected by those skilled in the art depending on the type of solvent used and extraction efficiency. For example, in the first step of extraction, ethanol, preferably 85% ethanol, is used in an amount of 5-20 parts by weight per 1 part by weight of Yamabushitake fruit body (dry), followed by extraction with an appropriate solvent and weight ratio. By repeating the above, a fat-soluble extraction component can be obtained. In one embodiment, the above-mentioned “Fat-soluble extract component derived from Yamabushitake” can be obtained, for example, by passing chloroform obtained after successive extraction of the fruit body of Yamabushitake with 85% ethanol, acetone and chloroform through a silica gel column. It is.
[0013]
As used herein, the term “neurodegenerative disease” refers to a disease in which neurons undergo apoptosis (nerve cell death) and normal neurotransmitter function is lost. Examples of “neurodegenerative diseases” include Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, Huntington's chorea, Pick's disease, progressive supranuclear palsy, spinocerebellar degeneration, amyotrophic lateral sclerosis, and others Various dementia diseases (for example, Binsbanger disease, cerebrovascular dementia, normal pressure hydrocephalus, post-traumatic dementia, chronic subdural hematoma, brain tumor), preferably Alzheimer's disease, Parkinson's disease and Creutzfeldt Jacob disease.
[0014]
The term “cell death induction” used in the present specification means that information is transmitted to the inside of a cell by physical and chemical stimulation from the outside of the cell to induce cell death.
[0015]
The term “endoplasmic reticulum stress” used in the present specification is caused by accumulation of proteins having an abnormal structure due to functional abnormalities on the rough endoplasmic reticulum, which is a place for translation of cell surface or extracellular protein. It means stress.
[0016]
The terms “cell death induction inhibitory activity” and “endoplasmic reticulum stress inhibitory activity” used in the present specification refer to the ability to suppress the above-described cell death induction and endoplasmic reticulum stress, respectively. Therefore, a component having “cell death induction suppression” or “endoplasmic reticulum stress suppression activity” can be used for preventing and / or treating various diseases caused by cell death induction or endoplasmic reticulum stress.
[0017]
As used herein, the term “composition” contains a specific substance as an active ingredient. For example, “a pharmaceutical composition for the prevention and / or treatment of neurodegenerative diseases” refers to an active ingredient. It contains a fat-soluble extract component derived from Yamabushidatake as a prophylactic and / or therapeutic effect for neurodegenerative diseases. Such a pharmaceutical composition is a solid, semi-solid or liquid form containing a fat-soluble extract component from Yamabushitake as an active ingredient 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 ingredients are, for example, powders, tablets, pellets, capsules, suppositories, liquids, emulsions, suspensions, aerosols, sprays and other forms suitable for use, normal, non-toxic And can be combined with a pharmaceutically acceptable carrier. Further, auxiliary agents, stabilizers, thickeners and the like may be used if necessary. These carriers and excipients may be sterilized as necessary, or may be sterilized after preparation. The therapeutically effective dose of the fat-soluble extract from Yamabushidatake, which is the active ingredient, also depends on and depends on the degree of purification of the extract, the age and type of disease and severity of the individual patient to be treated To be determined.
[0018]
The fat-soluble extract component from Yamabushidatake can also be provided as a health food. It is known that neurodegenerative diseases such as Alzheimer's disease are deeply related to the genetic predisposition, for example, those who have such genetic predisposition contain the above fat-soluble extract components. It is thought that the risk of developing the disease can be reduced by daily intake of healthy foods. 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.
[0019]
The present invention also provides a screening method for a substance having an endoplasmic reticulum stress inhibitory activity. In the screening method, a) a test cell is treated with a compound that induces endoplasmic reticulum stress and a test substance; b) the test cell is treated with a compound that induces endoplasmic reticulum stress and a fat-soluble extract component derived from Yamabushitake. (Wherein the test cell and the compound that induces endoplasmic reticulum stress are the same as those 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.
[0020]
The “test cell” is not particularly limited as long as it is a cell that can be used for screening for a substance having an endoplasmic reticulum stress inhibitory activity and can induce endoplasmic reticulum stress. Is any cell line derived from human or primary cultured cells, more preferably a nervous system cell line. Preferred nervous system cell lines in the present invention include PC-12, Neuro2a, CHP-126, GOTO-P3, LA-N-5, SK-N-SH, TNB-1, and C-1300. -12 cells are particularly preferred.
[0021]
The “compound that induces endoplasmic reticulum stress” refers to a compound that can induce endoplasmic reticulum stress in any cell. Examples of the “compound that induces endoplasmic reticulum stress” include tunicamycin, 1-deoxynojirimycin, bromoconduritol, and australin, with tunicamycin being particularly preferred.
[0022]
The “test substance” is not particularly limited as long as it is a compound that is required to determine whether or not it has an endoplasmic reticulum stress-inhibiting activity in the screening method. It doesn't matter. 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, low molecular weight compound and the like can be selected.
[0023]
In the screening method of the present invention, “treatment” refers to incubating a test cell in a culture medium together with a compound that induces endoplasmic reticulum stress and a fat-soluble extract component derived from a test substance or Yamabushitake. The fat-soluble extract component derived from the test substance or Yamabushidatake is preferably added to the culture medium containing the test cells at the same time or earlier than the compound that induces endoplasmic reticulum stress, but the compound that induces endoplasmic reticulum stress It may be added later to 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 amount used of the compound that induces endoplasmic reticulum stress and the test substance 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 screening method, the test cell and the compound that induces endoplasmic reticulum stress used in steps a) and b) are required to be “same species” in order to appropriately compare them. “Allogeneic” indicates that the same type of cells are used in screening steps a) and b). For example, if PC-12 cells are used in step a), PC- 12 cells are used.
[0024]
The “degree of survival” (survival rate) of the test cells in the screening method of the present invention is determined by MTT (3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide) assay, It is “measured” by a live cell quantification method known in the art, such as the neutral red method or the trypan blue method. By “comparing” the values obtained from this “measurement”, it is possible to “determine” whether or not the test substance has endoplasmic reticulum stress-inhibiting activity. Specifically, the “survival degree” between the addition and non-addition of the test substance is compared, and the substance having a significantly enhanced survival degree is defined as a substance having an endoplasmic reticulum stress-inhibiting activity. A substance having a value equal to or higher than the “degree of survival” obtained when a component (particularly, the extracted component having endoplasmic reticulum stress suppressing activity) is added is selected.
[0025]
EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited to the following specific Examples.
[0026]
【Example】
(Example 1: Fractionation of Yamabushidatake fruiting body)
The fruit body of Yamabushidatake was extracted with 85% ethanol and concentrated under reduced pressure. The concentrate was extracted with acetone and concentrated under reduced pressure, and then extracted again with chloroform to separate the chloroform layer. This chloroform layer was applied to a silica gel column (silica gel 60N, manufactured by WAKO) to obtain an extract fraction (shown as HE1 to HE14 in FIG. 1). An outline of Yamabushitake extract is shown in FIG.
[0027]
(Example 2: Measurement of inhibitory activity on induction of nervous system cell death of extract of Yamabushidatake)
PC-12 cells (RIKEN Cell Development Bank, Cell No. RCB0009) were used as a model of nervous system cells. The culture medium used was Dulbecco's modified Eagle medium supplemented with 10% horse serum and 5% fetal calf serum, and the CO ₂ concentration was 5%. Amyloid β 1-40 was also used to induce cell death. Amyloid β 1-40 is the main peptide that constitutes senile plaques found in the brains of Alzheimer's disease patients. Other amyloid β 1-42 is known as a main peptide constituting senile plaques, and these peptides are produced by limited degradation of amyloid precursor protein. These peptides have been confirmed to induce cell death in neurons, and are said to be one of the causes of neurodegenerative diseases. Therefore, in order to determine the cell death induction inhibitory activity of each Yamabushitake extract, PC-12 cells were cultured at 37 ° C. for 48 hours in the presence of 10 μM amyloid-β 1-40 and Yamabushitake extract. MTT assay using MTT (manufactured by Dojindo), a commonly used method for quantifying living cells, to determine cell viability (yellow crystals, MTT is reduced to blue crystals when incorporated into living cells) Specific absorption). After completion of the above culture, the cells are further cultured for 3 hours in the presence of 250 μg / ml MTT, the reaction is stopped in a reaction stop solution containing 20% SDS and 50% dimethylformamide, and then the product and cells are allowed to pass. Solubilized. Thereafter, absorbance at 570 nm was measured to determine cell viability. 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 FIG. From the results of FIG. 2, it was shown that Yamabushitake extract fractions 6 and 9-14 suppress cell death induction by amyloid-β 1-40.
[0028]
(Example 3: Measurement of suppression of oxidative stress of extract of Yamabushidatake)
It was decided to further investigate the nerve cell death suppression mechanism shown in Example 2 above. Focusing on the suppression of oxidative stress as a mechanism of cell death suppression, experiments were conducted in the same manner as in Example 2 above using sodium nitroprusside (manufactured by WAKO), which is a nitric oxide (NO) donor, as a model of oxidative stress. PC-12 cells were cultured at 37 ° C. for 24 hours in the presence of 50 μM sodium nitroprusside and Yamabushidatake extract fractions, and the cell viability was measured by MTT assay in the same manner as in Example 2 to suppress cell death induction in each fraction. Activity was determined. As a control, PC-12 cells cultured in the absence of NO were used, and the cell viability was compared with the value obtained therefrom as 100%. The results are shown in FIG. From the result of FIG. 3, it was shown that the Yamabushidatake extract fractions 1-12 suppress the cell death induction by NO.
[0029]
(Example 4: Measurement of endoplasmic reticulum stress inhibitory activity of Yamabushitake extract)
Since endoplasmic reticulum stress has been reported as a cause of neuronal cell death, it was investigated whether or not the above extract of Yamabushidatake has endoplasmic reticulum stress-inhibiting activity. Endoplasmic reticulum stress was induced by treatment with tunicamycin, an endoplasmic reticulum sugar chain biosynthesis inhibitor. Cell death was induced at 37 ° C. for 24 hours in the presence of 0.25 μM tunicamycin (manufactured by WAKO) and Yamabushitake extract, and the cell viability was measured by MTT assay in the same manner as in Example 2. Induction inhibitory activity was determined. As a control, PC-12 cells cultured in the absence of tunicamycin were used, and the cell viability was compared with the value obtained therefrom as 100%. The results are shown in FIG. From the results of FIG. 4, it was shown that Yamabushitake extract fractions 9 to 11, 13 and 14 suppress cell death induction by tunicamycin.
[0030]
(Example 5: Dose dependence test of endoplasmic reticulum stress inhibitory activity of Yamabushitake extract)
Of the fractions in which cell death induction was suppressed in Example 4, fractions 9, 10, 13, and 14 were further experimented. Similarly to Example 4, cell death was induced at 37 ° C. for 24 hours in the presence of 0.25 μM tunicamycin, and the cell survival rate was measured by MTT assay to determine the cell death induction inhibitory activity of each fraction. The experiment was conducted by changing the content of each extract of Yamabushitake in the plate used in the MTT assay to 2.5 μg / 100 μl, 5 μg / 100 μl, 10 μg / 100 μl, and 50 μg / 100 μl, respectively. As a control, PC-12 cells cultured in the absence of tunicamycin were used, and the cell viability was compared with the value obtained therefrom as 100%. The results are shown in FIG. From the results of FIG. 5, it was shown that fractions 9, 10 of Yamabushitake extract fractions 9, 10, 13, and 14 suppress cell death induction by endoplasmic reticulum stress in a dose-dependent manner.
[0031]
【The invention's effect】
Yamabushitake mushroom-derived fat-soluble extract has excellent endoplasmic reticulum stress inhibitory activity and cell death induction inhibitory activity, and thus is useful for the prevention and / or treatment of various diseases caused by endoplasmic reticulum stress and cell death, particularly neurodegenerative diseases. It is. In addition, the fat-soluble extract component of the present invention is derived from natural products, particularly edible mushrooms, and can be used safely, and can be used not only as a medicine but also as a customary health food for the prevention of various diseases. it can. Furthermore, this Yamabushidatake-derived fat-soluble extract component is useful as a tool for elucidating the mechanism of neuronal cell death.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing an outline of extraction from dried Yamabushida fruit bodies. The mg number described under each extraction fraction (HE-1 to HE-14) shown in FIG. 1 indicates the dry weight of each fraction.
FIG. 2 is a graph showing the effect of a fraction derived from Yamabushitake on amyloid β 1-40 toxicity.
FIG. 3 is a graph showing the effect of a fraction derived from Yamabushitake on NO toxicity.
FIG. 4 is a graph showing the effect of a fraction derived from Yamabushitake on tunicamycin toxicity.
FIG. 5 shows the dose dependence of the effect of the fraction derived from Yamabushitake on tunicamycin toxicity.

Claims (8)

  A cell death induction inhibitory composition comprising, as an active ingredient, a fat-soluble extract component obtained by continuously extracting fruit bodies of Yamabushidatake with ethanol, acetone and chloroform.   The cell death induction inhibitory composition according to claim 1, wherein 85% ethanol is used as ethanol.   An oxidative stress-inhibiting composition containing, as an active ingredient, a fat-soluble extract component obtained by continuously extracting fruit bodies of Yamabushidatake with ethanol, acetone and chloroform.   The composition for suppressing oxidative stress according to claim 3, wherein 85% ethanol is used as ethanol.   An endoplasmic reticulum stress-inhibiting composition containing, as an active ingredient, a fat-soluble extract component obtained by continuously extracting fruit bodies of Yamabushidatake with ethanol, acetone and chloroform.   The endoplasmic reticulum stress-suppressing composition according to claim 5, wherein 85% ethanol is used as ethanol. A screening method for a substance having endoplasmic reticulum stress inhibitory activity, comprising at least the following steps:
a) treating a test cell with a compound that induces endoplasmic reticulum stress;
b) a step of treating a test cell with a compound that induces endoplasmic reticulum stress and a fat soluble extract component derived from Yamabushitake, which is obtained by successively extracting the fruiting body of Yamabushitake with ethanol, acetone and chloroform (wherein The test cell and the compound that induces endoplasmic reticulum stress are the same as those used in step a)), and c) measure and compare the degree of survival of the test cell in step a) and step b). The step of determining whether or not the fat-soluble extract component derived from Yamabushitake has endoplasmic reticulum stress-inhibiting activity. The method according to claim 7 , wherein 85% ethanol is used as ethanol.

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