JP5066713B2 - Chroman derivatives and pharmaceutical compositions and foods for treating or preventing neurodegenerative diseases - Google Patents

Description

  The present invention relates to chroman derivatives, pharmaceutical compositions for treating or preventing neurodegenerative diseases, and foods.

In recent years, in response to an increase in the number of patients with neurodegenerative diseases represented by Alzheimer's disease and Parkinson's disease, development of therapeutic methods and therapeutic agents with high therapeutic or preventive effects has been promoted on a global scale.
Transdegenerative diseases such as Alzheimer's disease, Parkinson's disease and prion disease are diseases caused by the loss of nerve cells, and it is thought that endoplasmic reticulum stress triggers the death of nerve cells.
As a drug that seems to suppress endoplasmic reticulum stress, Patent Document 1 discloses dilinoleoleyl phosphatidylethanolamine. Patent Document 2 discloses a polypeptide based on a gene encoding a ubiquitin E3-linked enzyme (KF-1) having an endoplasmic reticulum stress-induced apoptosis inhibitory action. In addition, an endoplasmic reticulum stress inhibitor using a fat-soluble extract component derived from mulberry yellow, which is a type of mushroom, as an active ingredient has also been proposed (Patent Document 3).

On the other hand, it is known that many mushrooms contain substances and components having pharmacological activity effective for the human body. For example, a polysaccharide called krestin from Kawaratake, lentinan from Shiitake, and schizophyllan from Suehirotake has been isolated and used as an anticancer agent.
Yamabushitake has inductive production of nerve growth factor, helicenone and erinasin, a nerve growth factor acidity-inducing activity and an anti-bacterial activity, and a methicillin-resistant Staphylococcus aureus (MRSA) activity. A compound (Patent Document 6) and the like have been isolated.
JP 2005-247728 A Japanese Patent Laying-Open No. 2005-082557 JP 2006-342077 A JP-A-7-70168 JP 2001-259686 A JP 2005-27326 A

However, since the preventive or therapeutic effects of the above-mentioned neurodegenerative diseases may vary greatly depending on the administration subject, it is important to develop many candidate compounds that can be used for treatment or prevention. Further, from mushrooms from which various physiologically active substances have been isolated, compounds that are thought to act on the onset mechanism of neurodegenerative diseases have not been isolated yet.
Accordingly, an object of the present invention is to provide new compounds, pharmaceutical compositions and foods effective for the treatment or prevention of neurodegenerative diseases.

  The chroman derivative of the present invention is represented by the following formula (I).

The pharmaceutical composition for treating or preventing neurodegenerative diseases of the present invention comprises a chroman derivative represented by the above formula (I) as an active ingredient.
The food for treating or preventing neurodegenerative diseases of the present invention contains a chroman derivative represented by the above formula (I).

  ADVANTAGE OF THE INVENTION According to this invention, the new compound effective in the treatment or prevention of a neurodegenerative disease, a pharmaceutical composition containing this, and a foodstuff can be provided.

  The chroman derivative of the present invention is a compound represented by the above formula (I). This compound has an action of suppressing cell death due to endoplasmic reticulum stress. As a result, neurodegenerative diseases based on endoplasmic reticulum stress can be treated or prevented by using this compound.

  In the present invention, the “neurodegenerative disease” refers to a disease in which nerve cells undergo apoptosis (nerve cell death) and normal nerve transmission 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 (eg, Binsbanger disease, cerebrovascular dementia, normal pressure hydrocephalus, post-traumatic dementia, chronic subdural hematoma, brain tumor), preferably Alzheimer's disease, Parkinson's disease, Creutzfeldt Jacob disease and Huntington's disease.

In the present invention, “cell death induction” 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.
In the present invention, “endoplasmic reticulum stress” refers to stress caused by accumulation of proteins having an abnormal structure due to functional abnormalities in the rough endoplasmic reticulum, which is a place for translation of cell surfaces or extracellular proteins.
In the present invention, the terms “treatment” and “prevention” are generally appropriately expressed as “prevention” in the application before onset, but are strictly discriminated due to the nature of the disease to which this application is applied. It is not something. In particular, after onset, not only improvement of symptoms but also suppression of worsening of symptoms is encompassed by the concept of “treatment or prevention” in the present invention.

Although the compound of this invention may be obtained by chemical synthesis etc., it is preferable that it is what was extracted from the Yamabushitake (Hericium erinaceum) which is a mushroom of the genus Haritakeae (Hydnaceae) coral genus (Hericium).
Examples of extraction solvents for obtaining this compound include methanol, ethanol, acetone, ethyl acetate, chloroform, phenol, benzene, hexane, butanol, isopropanol, diethyl ether, tetrahydrofuran, toluene, dichloromethane, trichloroethylene, and other organic solvents, and The mixture of these organic solvents and water is mentioned. These solvents may be used alone or in any combination of two or more. Among the above extraction solvents, ethyl acetate, ethanol and a mixture thereof with water are preferred, ethyl acetate and a mixture thereof with water are more preferred, and ethyl acetate is particularly preferred. When the mixture of the organic solvent and water is used as the extraction solvent, the mixing ratio of the organic solvent and water in the mixture is, for example, 1 to 99% by weight of water in the mixture, preferably 5 to 50% by weight, More preferably, the ratio which becomes 5 to 30 weight% is illustrated.

  In order to obtain this compound, first, the fruit body of Yamabushitake is treated as it is or after being dried, shredded, crushed, pressed or boiled as necessary with an extraction solvent. The extraction treatment can be carried out by adding 5 to 30 g, preferably about 15 to 25 g, of dry weight of the fruit body of Yamabushitake to 100 ml of the extraction solvent. The extraction treatment is usually performed at room temperature, but may be performed by heating to 30 to 90 ° C. Moreover, although an extraction process can be performed by stirring or standing the fruit body of Yamabushitake in the extraction solvent, you may reflux as needed. The extraction time is usually 2 to 24 hours, preferably 10 to 15 hours.

  Prior to the extraction treatment, the fruit body of Yamabushitake is immersed in chloroform (hereinafter simply referred to as chloroform treatment) to remove the chloroform-solubilized fraction, and the resulting solid content may be subjected to the extraction treatment. . Thus, by performing chloroform treatment as pretreatment, it becomes possible to extract the present compound into the extraction solvent with higher purity. In the chloroform treatment, the amount of Yamabushitake fruiting body added to chloroform, treatment temperature, treatment time, other conditions, and the like are the same as those for the extraction treatment.

  In addition, in the case of performing an extraction process using the raw fruit body of Yamabushitake, the extraction process can also be performed by, for example, an extraction method of the following mode: methanol, ethanol, acetone, or a mixture of these and water (For example, 80-85% ethanol aqueous solution, 80-85% methanol aqueous solution, 85% acetone aqueous solution, etc.) are used to extract the fruit bodies of Yamabushitake at room temperature to 90 ° C. for 1 to 72 hours, and then the solid content is removed. Further, ethyl acetate is mixed, and the components extracted in ethyl acetate are recovered.

After the extraction treatment, a solid content (insolubilized component) is removed from the extraction solvent, and then the solvent is evaporated by heating under reduced pressure, whereby a Yamabushitake solvent extract can be obtained. The solvent extract of Yamabushitake thus obtained contains the above chroman derivative. The present chroman derivative can be isolated by subjecting the solvent extract of Yamabushitake to various chromatographic fractionation treatments such as silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography, if necessary.
The present chroman derivative can also be obtained by subjecting the extraction solvent to the chromatographic fractionation as it is after the extraction treatment.

As described above, the chroman derivative represented by the above formula (I) of the present invention has an action of suppressing cell death due to endoplasmic reticulum stress, and thus is useful, for example, in the fields of foods and pharmaceuticals.
That is, the pharmaceutical composition for treating or preventing neurodegenerative diseases of the present invention comprises the chroman derivative of the present invention as an active ingredient. This therapeutic or prophylactic pharmaceutical composition is prepared by formulating the present chroman derivative together with a pharmaceutically acceptable base or carrier. The therapeutic or preventive pharmaceutical composition optionally includes a pharmaceutically acceptable additive such as a binder, a disintegrant, a lubricant, a wetting agent, a buffer, a preservative, and a fragrance. You may mix | blend. Although it does not restrict | limit especially as a form of this pharmaceutical composition for this treatment or prevention, A film agent, a troche, a powder, a tablet, a granule, a capsule, a syrup agent etc. can be mentioned as an example.

  The dosage of the pharmaceutical composition for treatment or prevention of neurodegenerative diseases containing the chroman derivative of the present invention as an active ingredient varies depending on the administration form, administration purpose, age and weight of the administration subject, etc. The amount of the chroman derivative of the present invention corresponding to, for example, 0.01 mg to 500 mg can be mentioned. Further, the number of administrations per day of the pharmaceutical composition for treatment or prevention of the neurodegenerative disease of the present invention depends on the age, body weight, symptom, dose per administration of the pharmaceutical composition for treatment or prevention, etc. It can be set accordingly.

The food for treating or preventing neurodegenerative diseases of the present invention is prepared by blending various foods using the chroman derivative of the present invention as a food material. Examples of foods to which the present invention can be applied include foods for specified health use, dietary supplements, functional foods, foods for the sick, particularly foods for antidementia, in addition to general foods and drinks.
The blending amount of the chroman derivative of the present invention in the food of the present invention can be appropriately set according to the kind of the food, the age, sex and expected effect of the subject. As an example, the ratio which becomes 0.01 mass%-5 weight% of the chroman derivative of this invention with respect to the total weight of the said foodstuff is mentioned.
The daily intake and the number of intakes of the food of the present invention can be appropriately set according to the age, weight and symptoms of the administration subject, the dose of the chroman derivative of the present invention per dose, and the like.

  Examples of the present invention will be described below, but the present invention is not limited thereto. Further,% in the examples is based on weight (mass) unless otherwise specified.

[Example 1]
Isolation of chroman derivative 3.8 kg of dried fruit body of Yamabushitake was added to 20 L of chloroform, homogenized, and allowed to stand at 25 ° C. for 12 hours. Subsequently, solid content was collect | recovered, 3.7 kg of this solid content (dry weight) was added to 20 L of ethyl acetate, and it left still at 25 degreeC for 12 hours.

  40 g of the obtained chloroform layer was subjected to column chromatography using a silica gel column (silica gel 60N, manufactured by Kanto Chemical Co., Inc.). At this time, chloroform / methanol = 9: 1, 8: 2, 7: 3, 6: 4, 5: 5, and 0:10 were each used as a developing solvent so that the polarity increased, and 40 mL A total of 150 fractions were obtained. Among these, chloroform / methanol = 8: 2 elution fraction was further subjected to silica gel column chromatography using chloroform / methanol = 8: 2, 6: 4, 0:10 1 L each as a developing solvent. A total of 75 fractions were obtained. Subsequently, the obtained 21st to 28th fractions were subjected to thin layer chromatography (TLC) using chloroform / methanol = 8: 2 as a developing solvent, and the fractions in which spots were detected were scraped off. Further, the collected fraction was subjected to HPLC (column; CAPCEL PAK AQ φ4.6 × 250 mm, flow rate: 5 mL / min, solvent 95% methanol aqueous solution) to obtain 1.2 mg of an oily substance. This was named YAT-1.

[Example 2]
Characteristics of YAT-1 Next, physicochemical properties and structural analysis of YAT-1 obtained in Example 1 were performed.
The obtained compound exhibits the following physicochemical properties and structural analysis results.
(1) Molecular weight: 586.7991 (C ₃₅ H ₅₄ O ₇ )
(2) Property: Oily (3) Solubility in solvent: Soluble in methanol, ethanol, acetone, ethyl acetate, slightly soluble in water (4) Basic, neutral, acidic: Neutral (5) ¹ H-NMR and ¹³ C-NMR (in CDCl ₃ ): as shown in Table 1.
In addition, this compound was found to absorb a hydroxyl group at 3446 cm ⁻¹ in IR. Optical rotation was measured, but no optical activity was observed.
From these results, YAT-1 has the molecular formula C ₃₅ H ₅₄ O ₇ , 8-formyl-3-hydroxy-5-methoxy-2-methyl-3- (4′-methyl-2) as shown in Formula I. '-Oxo-3'-pentenyl) -7-chromanylmethyl palmitate (8-formyl-3-hydroxy-5-methoxy-2-methyl-3- (4'-methyl-2'-oxo-3'-) pentenyl) -7-chromanylmethyl palmitate).

[Example 3]
Endoplasmic reticulum stress-induced neuronal cell death inhibitory effect of YAT-1 In order to confirm the wide-ranging neurodegenerative disease preventive effect of the chroman derivative YAT-1 whose structure was confirmed in Example 2, endoplasmic reticulum stress-induced cells in neurodegenerative diseases Whether YAT-1 suppresses the cytotoxicity of tunicamycin (TM: inhibition of sugar chain biosynthesis) and thapsigargin (TG: depletion of endoplasmic reticulum calcium), which are widely used as a model of death, to nervous system cells (Neuro2a) Was examined. Tunicamycin and thapsigargin are models of intracellular signal transduction systems involved in neurodegenerative diseases. YAT-1 was dissolved in a small amount of ethanol, and a suspension in which water (medium for cell culture) was added was prepared and subjected to the following test.

The mouse neuroblastoma cell line Neuro2a (IFO50081) is cultured for 24 hours in a 96-well plate at a density of 5,000 cells / well. The culture uses Dulbecco's modified Eagle medium (D-MEM) supplemented with 10% fetal bovine serum (FBS) and penicillin 100 units / ml and streptomycin 100 μg / ml as antibiotics, at a temperature of 37 ° C. and a CO ₂ concentration of 5%. Performed under conditions.
After culturing for 24 hours, 0.5 μg / ml tunicamycin (manufactured by Wako Pure Chemical Industries) or 10 nM thapsigargin (manufactured by Wako Pure Chemical Industries) and 0.1 μg / ml, 1 μg / ml, 10 μg / ml were added to the above medium without FBS. The culture was further continued for 24 hours in a medium supplemented with ml of YAT-1.
After culturing, the number of living cells was quantitatively analyzed by the 3- (4,5-dimethyl-2-thiazolyl) 2,5-diphenyl-2H-tetrazolium butamide (MTT) method. Specifically, the medium was replaced with the above serum-free medium containing 250 μg / ml of MTT, and further cultured for 2 hours. The reaction stop solution (20% (w / v) SDS, 50% (v / v) Add the same amount of dimethylformamide aqueous solution) as the medium to stop the reaction. After solubilization of the cells and product, the number of living cells was quantitatively evaluated by measuring absorbance at 570 nm with a microplate reader.
The results are shown in Table 2 and FIG.

As shown in the results, the survival rate of Neuro2a cells administered with tunicamycin was 48.91 ± 2.15% of the control without addition, but 0.1 μg / ml, 1 μg / ml, 10 μg / ml of YAT- The survival rates of the cells to which 1 was added were 52.80 ± 1.61%, 56.13 ± 5.17%, and 65.17 ± 2.46%, respectively. As a result of analysis by t-test, a significant difference of p <0.05 was observed in the 0.1 μg / ml and 1 μg / ml addition groups, and p <0.01 in the 10 μg / ml addition group.
On the other hand, in the thapsigargin administration system, the survival rate was 51.37 ± 3.66% by thapsigargin alone, but the cells to which 0.1 μg / ml, 1 μg / ml, 10 μg / ml YAT-1 was added The survival rates were 51.99 ± 2.68%, 56.90 ± 3.69%, and 57.35 ± 3.08%, respectively. As a result of analysis by t-test, a significant difference of p <0.05 was observed in the groups added with 1 μg / ml and 10 μg / ml.
From the above results, it is considered that YAT-1 has an inhibitory effect on a wide range of endoplasmic reticulum stress-induced neuronal cell death. Ingestion of YAT-1 can be expected to suppress or alleviate symptoms of neurodegenerative diseases.

[Example 4]
Effect of YAT-1 on Alzheimer's Disease Model The rat pheochromocytoma cell line PC-12 (JCRB0733) is cultured in a 96-well plate at a density of 10,000 cells / well for 24 hours. The culture was performed using Dulbecco's modified Eagle medium (D-MEM) supplemented with 10% horse serum (HS) 5% fetal bovine serum (FBS) and penicillin 100 units / ml and streptomycin 100 μg / ml as antibiotics at a temperature of 37 ° C. And under the condition of a CO ₂ concentration of 5%.
After culturing for 24 hours, the above medium without FBS was supplemented with 10 μM amyloid β1-40-peptide (SEQ ID NO: 1: 4307-v, Peptide Institute) and 0.1 μg / ml YAT-1. The culture was further performed for 48 hours.

After culturing, the number of living cells was quantitatively analyzed by the 3- (4,5-dimethyl-2-thiazolyl) 2,5-diphenyl-2H-tetrazolium butamide (MTT) method. Specifically, the medium was replaced with the above serum-free medium containing 250 μg / ml of MTT, and further cultured for 2 hours. The reaction stop solution (20% (w / v) SDS, 50% (v / v) Add the same amount of dimethylformamide aqueous solution) as the medium to stop the reaction. After solubilization of the cells and product, the number of living cells was quantitatively evaluated by measuring absorbance at 570 nm with a microplate reader.
The results are shown in Table 3 and FIG.

The survival rate of PC-12 cells to which tunicamycin was administered was 76.29 ± 7.9% of the control without addition, whereas the survival rate of cells to which 0.1 μg / ml YAT-1 was added was 90.43. It was ± 5.62%. As a result of analysis by t-test, a significant difference of p <0.05 was observed in the 0.1 μg / ml added group.
From the above results, it is considered that YAT-1 has an inhibitory effect on neuronal cell death caused by amyloid-β-peptide, which is considered to be the cause of Alzheimer's disease, and the symptoms of Alzheimer's disease by ingesting YAT-1 Suppression or mitigation can be expected.

  Thus, according to the present invention, a chroman derivative having an effect of treating or preventing a neurodegenerative disease can be provided, and a pharmaceutical for treating or preventing a neurodegenerative disease can be provided by blending this chroman derivative with a carrier or the like according to the application field. Compositions as well as food products can be provided.

It is a graph which shows the result of the endoplasmic reticulum stress induction nerve cell death suppression test concerning the Example of this invention. It is a graph which shows the result of the nerve cell death suppression test by the amyloid (beta) 1-40-peptide concerning the Example of this invention.

Claims (3)

The chroman derivative represented by the following formula (I).
  A pharmaceutical composition for treating or preventing a neurodegenerative disease comprising the chroman derivative according to claim 1 as an active ingredient.   A food for treating or preventing a neurodegenerative disease comprising the chroman derivative according to claim 1.