TWI610676B - Method for producing nerve cell death inhibitor, anti-Alzheimer's disease agent, anti-brain hypofunction agent, medicine having anti-Alzheimer's disease effect or anti-brain function, and nerve cell death inhibitor - Google Patents

Description

Method for producing nerve cell death inhibitor, anti-Alzheimer's disease agent, anti-brain hypofunction agent, medicine having anti-Alzheimer's disease effect or anti-brain function, and nerve cell death inhibitor

The present invention relates to a method for producing a neuronal cell death inhibitor, an anti-Alzheimer's disease, an anti-brain hypofunction agent, a pharmaceutical product having an anti-Alzheimer's disease action or an anti-brain function, and a neuronal cell death inhibitor.

In recent years, with the aging of Japan, the number of patients with dementia has increased. The number of patients with dementia is expected to continue to increase in the future as long as the epoch-making prevention and treatment of dementia is not established. Therefore, not only the medical point of view, but also the administrative point of view also seeks the prevention and treatment of dementia.

However, there are various diseases that cause dementia, Alzheimer's disease (Alzheimer's disease: Alzheimer dementia), Dementia with Lewy body And three types of vascular dementia are considered to be the majority. Among them, because of the large number of patients with Alzheimer's disease and the fundamental prevention and treatment methods have not been established, there are now various studies on Alzheimer's disease.

As a mechanism of Alzheimer's disease, amyloid-like cascade Said (amyloid cascade hypothesis) is widely supported. The amyloid cascade hypothesis refers to the accumulation of amyloid β, such as amyloid protein, in the brain. As a result, changes in Alzheimer's neurofibril occur directly or indirectly. The hypothesis of the symptoms of dementia caused by the death or detachment of nerve cells caused by the death or detachment of nerve cells. This hypothesis is widely supported and is a policy in the development of prevention and treatment methods (see, for example, Non-Patent Document 1).

Conventionally, for the treatment of Alzheimer's disease, donepezil, galanthamine, rivastigmine, and the like are used (for example, refer to Non-Patent Document 2). According to the anti-Alzheimer's agent containing the substance as described above as an active ingredient, the symptoms of dementia can be suppressed.

However, Rhinacanthus nasutus (L.) Kurz, also known as the white crane ganoderma lucidum, is an evergreen shrub belonging to the genus Ganoderma lucidum, which is believed to be native to the Deccan Plateau in southern India. It is known that the whole grass of Ganoderma lucidum has antibacterial activity against insects, anti-inflammatory, and dermatophyte (for example, refer to Non-Patent Document 3), and is mainly used as a Chinese medicine or food in China, Taiwan, and the like, and recently as a Chinese medicine or food in Japan. use. Others disclosed in the applicant's prior application include: the ability of the crane to remove active oxygen (for example, refer to Patent Document 1); to promote excretion (for example, refer to Patent Document 2); and to have an antiallergic effect (for example, refer to Patent Document 3) has an antitumor effect (for example, refer to Patent Document 4).

[Patent Document 1] Japanese Patent Publication No. 9-143091

[Patent Document 2] Japanese Patent Publication No. 9-169662

[Patent Document 3] Japanese Patent Laid-Open Publication No. 2001-10964

[Patent Document 4] Japanese Patent Laid-Open Publication No. 2002-53481

[Non-Patent Document 1] Experimental Medicine, Vol.26 No.16, October 2008, 2550 pages, Yangtu Society, October 1, 2008

[Non-Patent Document 2] The latest medical treatment for dementia, Vol.4 No. 2, April 2014 issue, 58-63 pages, published by FUJI MEDICAL, issued on April 25, 2014

[Non-Patent Document 3] Original Color Makino and Han Herb Large Illustration, 492 pages, Beilong Pavilion, 1988

However, the predetermined distribution, the predetermined fraction obtained by the predetermined method of the extract of the white crane ganoderma lucidum, or the rhinocanthin C contained in the white crane ganoderma lucidum has toxicity to the amyloid-like β. The inhibition of nerve cell death is not known.

Accordingly, it is an object of the present invention to provide a neuronal cell death inhibitor, an anti-Alzheimer's agent, an anti-brain hypofunction agent, and an anti-Alzheimer's disease or anti-brain function having excellent neuronal cell death inhibitory effects. A drug that has a reduced effect. Further, it is an object of the invention to provide a method for producing a neuronal cell death inhibitor which can produce a neuronal cell death inhibitor having excellent neuronal cell death inhibitory action.

The present inventors have considered a nerve thinning which has direct toxicity to amyloid-like β which can be used for prevention or treatment of Alzheimer's disease. As a result of intensive research, it was found that the extracts from the extract of S. chinensis by the prescribed method, the prescribed distributions, the prescribed retractions, and the white crane Ganoderma lucidum contained in the white crane Ganoderma lucidum have nerve cell death. Inhibition is achieved to achieve the present invention. The present invention consists of the following matters.

In addition, in the following description, unless otherwise indicated, the ratio of the solvent is represented by volume.

[1] A use of a plant containing only white crane ganoderma lucidum C as an active ingredient for the preparation of a neuronal cell death inhibitor which inhibits the toxicity of amyloid β.

[2] A use of a neuronal cell death inhibitor containing the toxicity of the inhibitory amyloid β of the above [1] as an active ingredient, for use in the preparation of an anti-Alzheimer's agent.

[3] A use of the neuronal cell death inhibitor containing the toxicity of the inhibitory amyloid β of the above [1] as an active ingredient, for use in the preparation of an anti-brain hypofunction agent.

[4] Use of a neuronal cell death inhibitor containing the toxicity of the inhibitory amyloid β of the above [1] as an active ingredient for preparing an anti-Alzheimer's disease effect or an anti-brain function Pharmaceutical products.

According to the present invention, it is also known from the test examples described later that a neuronal cell death inhibitor, an anti-Alzheimer's agent, an anti-brain hypofunction agent, and an anti-Az A drug that acts on the action of Haimo or is resistant to brain function.

Moreover, according to the present invention, a method for producing a neuronal cell death inhibitor having excellent neuronal cell death inhibitory action can be provided.

Further, regarding the above [1], for example, it can also be expressed as follows: [The use of an active ingredient of the above-mentioned white crane ganoderma lucidum C for the production of a nerve cell death inhibitor containing only white crane ganoderma lucidum C as an active ingredient.

Further, regarding the above [2], for example, it can also be expressed as follows: [Use of an active ingredient for producing an anti-Alzheimer's agent as the neuronal cell death inhibitor of the above [1]. ]

Further, regarding the above [3], for example, it can also be expressed as follows: [The use of an active ingredient of the neuronal cell death inhibitor of the above [1] for producing an anti-brain hypofunction reducing agent. ]

Further, regarding the above [4], for example, it can also be expressed as follows: [The anti-Alzheimer's agent used as the above [2] for the manufacture of a pharmaceutical having an anti-Alzheimer's effect or an anti-brain function. Or the use of the active ingredient of the anti-brain hypofunction agent of the above [3]. ]

Fig. 1 is a flow chart showing a procedure for distributing and dividing a prescribed distribution and a prescribed division by a crane.

Fig. 2 is a flow chart showing the procedure for separating the white crane Ganoderma lucidum C from the white crane.

Fig. 3 is a table showing the results of a test example.

Hereinafter, the nerve cell death inhibitor of the present invention, anti-Az A method for producing a Hammer's disease agent, an anti-brain hypofunction agent, a drug having an anti-Alzheimer's disease action or an anti-brain function, and a nerve cell death inhibitor will be described.

[A nerve cell death inhibitor of the present invention, which comprises liquid-liquid partitioning of an ethanol extract of chlorinated ganoderma lucidum with hexane and methanol, and liquid-liquid partitioning of a distribution of methanol by dichloromethane and water; The specified distribution of the distribution by the dichloromethane is regarded as the active ingredient] by the method for producing a nerve cell death inhibitor of the present invention, which comprises the following sequence containing only the specified distribution As an active ingredient: a first procedure for extracting ethanol extract from ethanol, and extracting the ethanol extract with hexane and methanol to obtain a second procedure of the distribution of the methanol; And the water is obtained by liquid-liquid distribution of the distribution of the methanol-dispensing distribution to obtain a predetermined distribution of the distribution of the distribution of the dichloromethane.

The predetermined distribution product in the present invention can obtain various conditions as long as the conditions described are satisfied. Hereinafter, a method that can be used and the like will be described.

Further, in the present specification, [dispensing substance] means a purified product and is obtained by liquid-liquid distribution.

In addition, in the present specification, the phrase "containing only a predetermined distribution as an active ingredient" means that the active ingredient having a nerve cell death inhibitor has only a predetermined distribution, and does not negate the component which is not an active ingredient (various solvents). Or auxiliary additives, etc.).

About Baihe Ganoderma lucidum, you can use the original collection at the appropriate time. After the leaves, stems, roots, flowers, etc. of the white crane ganoderma lucidum, they are left untouched or attached to a drying process such as air drying and drying, and are used as raw materials for extraction. The white crane ganoderma lucidum used as the extraction raw material is preferably used by pulverizing or chopping.

The extraction using ethanol can be carried out by a well-known extraction method generally used industrially and experimentally by using white crane ganoderma and ethanol (for example, refer to [FIGHT MEDICINE, Vol. 16, pp. 929-934, 2009]). . An ethanol extract of Ganoderma lucidum can be obtained by the extraction.

In the present specification, when it refers to only [ethanol], it means [a solvent containing ethanol as a main component (containing 50% or more of ethanol)]. That is to say, [ethanol] contains not only so-called absolute ethanol but also a mixed solvent of ethanol and other solvents. In the extraction by Ganoderma lucidum, it is preferred to use a mixed solvent of ethanol and water (aqueous ethanol), particularly 90% ethanol.

The extraction temperature is usually from 0 to 100 ° C, preferably from 5 to 50 ° C. In the case of performing extraction under the conditions of boiling on a solvent, it is preferred to perform extraction while refluxing the solvent. The extraction time is about 1 hour to 10 days, and the amount of the solvent is usually 1 to 30 times by weight, preferably 5 to 10 times by weight, per dry raw material. The extraction operation can be either agitation or impregnation only. The extraction operation may be repeated as many times as necessary. Moreover, extraction operations with different combination conditions are also possible.

Further, the operation of removing the insoluble residue by filtration or centrifugation of the crude extract obtained by the above operation is preferred.

With regard to liquid-liquid distribution, it is also possible to use industrial and experimental The well-known methods used are carried out.

The liquid-liquid distribution is a method which is also called a two-phase distribution, a two-phase solvent distribution method, a liquid-liquid extraction method, or the like, and uses a solvent having a different polarity to recover a target compound by a difference in solubility.

After the liquid-liquid distribution, it is preferred to remove the solvent to obtain a dry state. However, this is not the case where the removal of the solvent is not required for the purpose of use.

In the present specification, when it refers to only [methanol], it means [a solvent containing methanol as a main component (containing 50% or more of methanol)]. That is, [methanol] contains not only so-called anhydrous methanol but also a mixed solvent of methanol and other solvents. In the liquid-liquid partitioning, a mixed solvent of methanol and water, particularly 90% methanol, is preferred. On the other hand, in the present specification, when referring to [anhydrous methanol], it means anhydrous methanol, and does not contain a mixed solvent containing methanol.

It can be obtained by [manufacturing method of a nerve cell death inhibitor containing only a predetermined distribution as an active ingredient].

Further, the present invention [the neuron cell death inhibitor comprises liquid-liquid partitioning of an ethanol extract of chlorinated ganoderma lucidum with hexane and methanol, and liquid-liquid partitioning of the methanol-dispensed partition with dichloromethane and water. Dispensing, when the partition which is distributed by methylene chloride is attached to the ruthenium column chromatography using a solvent which consists of n-hexane, ethyl acetate and anhydrous methanol, only the ruthenium column is dissolved by the dissolution solvent. The prescribed fraction of the partition is regarded as an active ingredient] by [the method for producing a neuronal cell death inhibitor, comprising the following sequence containing the prescribed fraction as an effective ingredient Divided: the first procedure for extracting ethanol extract from ethanol and extracting ethanol from ethanol; the liquid-liquid partitioning of ethanol extract with hexane and methanol to obtain a second procedure of the distribution of the above methanol; dichloromethane and water Performing a liquid-liquid partitioning of the partition dispensed with methanol to obtain a third procedure for the partition dispensed by the dichloromethane; adding the partition dispensed by dichloromethane to the use of n-hexane, ethyl acetate and anhydrous methanol The kneading column chromatography of the formed elution solvent is obtained by a fourth procedure of a predetermined partition of the cross-section of the kneading tube column by the dissolution solvent.

The predetermined partition in the present invention can obtain various conditions as long as the conditions described are satisfied. Hereinafter, a method that can be used and the like will be described.

Further, in the present specification, the [sparing object] means a purified product obtained by division of column chromatography.

Further, in the present specification, the phrase "containing only a predetermined fraction as an active ingredient" means that the active ingredient having a nerve cell death inhibitor action contains only a predetermined fraction, and does not negate the component which is not an active ingredient ( Various solvents or auxiliary additives, etc.).

Since the extraction and the liquid-liquid distribution have been described, the description will be omitted.

After the gel column chromatography, it is preferred to remove the dissolution solvent to obtain a dry state. However, this is not the case where the removal of the solvent is not required for the purpose of use.

The tannin column chromatography can be carried out by a well-known method generally used industrially and experimentally.

The elution solvent is preferably a dissolution solvent composed of a ratio of n-hexane:ethyl acetate:anhydrous methanol=3:4:2 (see the experimental example described later). In this case, the predetermined fraction is preferably a fraction which is eluted by using a solvent having a volume of 0.1 to 4 times the volume of the tannin used in the tantalum column chromatography, and is preferably 0.2 to 2.5 times. The fraction which is eluted when the solvent is dissolved in the volume is the best (refer to the test example described later).

The predetermined distribution and the predetermined division may be obtained by further performing the above-described purification method as long as the object of the present invention is not impaired.

It is conceivable that the solvent and the mixed solvent used for obtaining the predetermined distribution and the predetermined partition can be replaced by a solvent having the same polarity or a mixed solvent. In this case, if it can be used as a solvent, it can be used to obtain a predetermined distribution and a predetermined division. Examples of the solvent include water, various alcohols, esters such as ethyl acetate, various linear, branched, and cyclic hydrocarbons, dichloromethane, and chloroform. , acetone, various ethers, etc., can be used in accordance with the method and purpose of the usable solvent.

In addition, considering the results of the experimental examples described later, it is considered that the predetermined distribution and the predetermined division in the present invention contain the white crane ganoderma lucidum C as one component.

The white crane ganoderma lucidum C in the "inhibitor of nerve cell death containing only white crane ganoderma lucidum C as an active ingredient" of the present invention can also be obtained by extracting and finishing a white crane ganoderma lucidum into a raw material (refer to an experimental example described later). It can also be obtained from other plant bodies. Moreover, the white crane Ganoderma lucidum C can also It is obtained by synthesis.

The ganoderma lucidum 醌C refers to a compound represented by the following chemical formula (1).

The mechanism of action of the white crane ganoderma lucidum C in the body may be a white crane ganoderma lucidum 醌C, and a pharmaceutically acceptable salt may be used before administration.

The predetermined distribution, the predetermined partition, and the white crane Ganoderma lucidum C have an effect of suppressing the death of nerve cells due to the toxicity of the amyloid β, as shown in the experimental examples described later. Therefore, it is considered that the nerve cell death inhibitor of the present invention having the predetermined distribution, the prescribed partition, and the white crane ganoderma lucidum C as an active ingredient has anti-Alzheimer's disease agent and anti-brain dysfunction agent. The role.

Further, the [anti-Alzheimer's agent] in the present specification means at least one of prevention and treatment for Alzheimer's disease. Further, the [anti-brain hypofunction agent] refers to at least one of prevention and treatment that can be used for the decline of brain function.

The administration route of the nerve cell death inhibitor, the anti-brain hypofunction agent, the anti-Alzheimer's disease agent, and the pharmaceutical product (here, the drug to be taken or taken orally) of the present invention is not particularly limited, and examples thereof include, for example: Oral administration, intrarectal administration, oral administration, nasal administration, etc., mucosal administration, intravenous administration, Injection administration such as subcutaneous administration or the like. The dosage form may be in the form of a preparation suitable for the administration method, and examples thereof include a solid agent such as a tablet, a powder, a fine granule, a granule, a capsule, a powder, a pill, or a troche. Liquid preparations such as solutions, suspensions, emulsions, syrups, injections, gelatinous preparations, and the like. The nerve cell death inhibitor, the anti-brain hypofunction agent, the anti-Alzheimer's agent and the pharmaceutical of the present invention may also be administered as they are, or may be administered together with a pharmacologically acceptable excipient. . When the excipient is a monosaccharide, a disaccharide, a polysaccharide, an inorganic salt, a fat or oil, or distilled water, it can be used as a preparation. Additives such as a binder, a lubricant, a dispersing agent, a suspending agent, an emulsifier, a diluent, a buffer, an antioxidant, a bacterial inhibitor, or the like may also be used in the formulation.

The effective administration amount of the nerve cell death inhibitor, the anti-brain hypofunction agent, the anti-Alzheimer's disease agent, and the pharmaceutical of the present invention varies depending on the administration route, the dosage form, the symptoms of the disease, the age of the subject, and the like. The predetermined distribution, the prescribed partition, or the white crane ganoderma lucidum C can be considered to be 0.1 to 1000 mg, preferably 0.5 to 300 mg, more preferably 1 to 100 mg per day for a normal adult. The content of the prescribed distribution, the prescribed fraction or the white crane ganoderma lucidum C can be set according to the form of the preparation, the effective dose, and the amount of the preparation as the amount of the active ingredient in the preparation most suitable for each administration form. .

The pharmaceutical product of the present invention may be a pharmaceutical for external use. The form of the external pharmaceutical is not particularly limited.

Examples of the form of the external pharmaceutical product include ointment, ointment, and mud. A cataplasm, a tape, an external preparation, and the like. In the external pharmaceutical product of the present invention, a predetermined distribution, a predetermined partition, or a white crane ganoderma lucidum C may contain various pharmaceutical ingredients as needed. Further, additives such as a binder, a dispersing agent, a suspending agent, an emulsifier, a diluent, a buffer, an antioxidant, a bacterial inhibitor, or the like can also be used.

The food of the present invention can be exemplified by a food in the form of a tea to which an active ingredient is added, or a food in a form of a processed food in which an active ingredient is blended.

It is preferably used as a tea, mixed with a dried product of leaves, stems or roots of Ganoderma lucidum, or mixed with other tea raw materials. As the tea raw material, if it is green tea, oolong tea, Pu'er tea, black tea, sencha, brown rice tea, eucommia tea, persimmon leaf tea, mulberry leaf tea, etc., it can be used as a tea user, and any tea raw material can be used. In addition, the dried leaves of the leaves, stems or roots of the white crane ganoderma can be baked and used in the same manner as other tea raw materials.

The form of the food containing the active ingredient is any form that can be provided as a food, such as a health drink, a jelly, a biscuit, a lozenge, a pill, a soft capsule, a hard capsule, a powder, a fine granule, or a granule. Can be used. Further, as an auxiliary raw material of the food, an additive such as an excipient, a binder, a lubricant, a dispersing agent, a suspending agent, an emulsifier, a diluent, a buffer, an antioxidant, a bacterial inhibitor or the like can be used.

The effective intake of the food of the present invention varies depending on the form of ingestion, the state of health of the subject, the age of the subject, and the like, and the predetermined distribution, the prescribed partition, or the white crane ganoderma lucidum C can be considered as a normal adult daily. It is 0.1 to 1000 mg, preferably 0.5 to 300 mg, more preferably 1 ~100mg.

The content of the predetermined distribution product, the predetermined partition, or the white crane ganoderma lucidum C in the food of the present invention varies depending on the form of the food, and is usually 0.0001 to 1% by weight, preferably 0.001 to 0.5% by weight. More preferably, it is 0.01 to 0.1% by weight.

[Examples]

Hereinafter, the present invention will be described in more detail by a predetermined distribution, a predetermined partition, and a distribution, division, single separation, or a test example relating to nerve cell death inhibition of the white crane Ganoderma lucidum C. In addition, the following experimental examples are specific examples, and the present invention is not limited by the following experimental examples.

1. Distribution, division, and single departure of the distribution product of the present invention, the partition of the present invention, and the white crane Ganoderma lucidum C

The distribution and division of the distribution of the present invention and the division of the present invention are carried out in accordance with the flow shown in Fig. 1.

First, the raw material was 2 kg of a dried product of the root of Rhinocanthus nasutus (L.) Kurz, and it was pulverized by a general grinder.

Next, extraction with 90% ethanol was carried out. The extraction was carried out by immersing the white crane ganoderma lucidum in 20 L of 90% ethanol for 3 days, and then refluxing the solvent for 1 hour. The extract and the extraction residue are separated by a filter paper. This extraction was carried out three times in total for the same raw materials, and the solvent was removed from the extract liquid under reduced pressure to obtain 77.82 g of a dry solid ethanol extract.

After the above ethanol extract was removed as 4 g of the analysis sample, 73.82 g of the remaining ethanol extract was subjected to liquid-liquid partitioning with hexane and 90% methanol. First, add 90 mL of 90% methanol to the ethanol extract. Then 500 mL of hexane was added and shaken for 1 minute. Wait for about 10 minutes until the solvent is separated, and collect 90% methanol phase. Further, the same operation was repeated twice, and after the obtained 90% methanol phase was combined, the solvent was removed under reduced pressure to obtain 54.32 g of a dry solid.

After 4 g of the above-mentioned distribution was used as the sample for analysis, 50.32 g of the remaining amount of the distribution was liquid-liquid partitioned with dichloromethane and water. First, 500 mL of water (refined water) was added to the distribution, then 500 mL of dichloromethane was added and shaken for 1 minute. The dichloromethane phase was collected until about 10 minutes until the solvent was separated. Further, the same operation was repeated twice, and the obtained dichloromethane phase was combined, and then the solvent was removed under reduced pressure to give 27.55 g of the desired compound. In addition, in the following description, the distribution obtained here is described as [predetermined distribution A].

Next, 20.00 g of the above-mentioned predetermined distribution A was attached to a rubber tube column chromatography.

First, 20.0 g of the predetermined distribution A was mixed with 60 g of silicone rubber (common article of 0.063 to 0.2 mm). Then, it is prepared to fill a 500 g column (6 cm in diameter, 34 cm in length, and an open column with a volume of about 204 mL) of tantalum (0.040 to 0.063 mm) to dissolve the solvent (n-hexane: ethyl acetate). : Anhydrous Methanol = 3:4:2) After equilibration, a predetermined mixture of the distribution A and the silicone was placed, and elution was carried out by using a dissolution solvent. Further, after using 1500 mL of a dissolution solvent, the tantalum adsorbate was eluted by passing through a column in the order of 500 mL of acetone and 500 mL of absolute ethanol.

In the gel column chromatography, about 20 mL each was collected in a test tube, and dissolved by TLC (thin-layer chromatography). The pattern of the precipitate. By the form of the approximate dissolution, the first to sixth test tubes are used as the partition 1-1 (the amount of the solvent is about 0 to 120 mL), and the 7th to 24th test tubes are used as the The fraction 1-2 (the amount of the solvent is about 120-480 mL) is used as the partition 1-3 (the amount of the solvent is about 480-780 mL) by the 25th to 39th test tubes, and the 40th to 70th The root tube tester was used as the partition 1-4 (the amount of the solvent was about 780 to 1400 mL), and was obtained as the partition 1-5 by the other eluting solvent, acetone, and absolute ethanol.

Further, the above-described partitions 1-1 to 1-4 are predetermined partitions.

The separation of the white crane Ganoderma lucidum C is carried out according to the flow shown in Fig. 2. The method of obtaining the predetermined partition A is different from the method of the tannin column chromatography, and therefore only the part different from the above method will be described.

First, a predetermined distribution A2.50g was mixed with 10 g of silicone (0.063 to 0.2 mm). Then, prepare a 200 g column (6 cm in diameter, 30 cm in length, and a hollow column with a volume of about 60 mL) filled with silicone (0.040 to 0.063 mm) to dissolve the solvent (n-hexane: ethyl acetate = 4:1). After the balance is made, a predetermined mixture of the distribution A and the silicone is placed, and elution by the elution solvent is performed. Further, the amount of the solvent to be dissolved was 500 mL.

In the gel column chromatography, about 10 mL each was collected in a test tube, and the form of dissolution was observed by TLC. By the pattern of the approximate dissolution, the first test tube is used as the partition 2-1 (the amount of the solvent is about 0 to 10 mL), and the second to the third test tube is used as the division. The product 2-2 (the amount of the solvent is about 10 to 30 mL) is used as the partition 2-3 (the amount of the solvent is about 30 to 100 mL) by the fourth to ten test tubes.

Then, the fractions 2-1 to 2-3 were constructed by comparing the data of ¹ H NMR and ¹³ C NMR with the literature values (Journal of Natural Products, Vol. 59, pp. 808-811, 1996). After analysis, it was confirmed that the partition 2-2 was white crane Ganoderma lucidum C. The amount of the obtained white crane ganoderma lucidum C was 444.2 mg.

Further, a JEOL JNM-GSX500 type nuclear magnetic resonance spectrum apparatus (manufactured by JEOL Ltd.) was used as a nuclear magnetic resonance spectrum apparatus.

2. Test examples of the prescribed distribution, the prescribed division, and the inhibition of nerve cell death by the white crane Ganoderma lucidum C

In the test examples, tests were conducted on the cytotoxicity caused by amyloid-like β, the predetermined distribution, the predetermined fraction, and the nerve cell death inhibitory effect of G. glabra L. For the evaluation of the neurocytotoxicity of amyloid-like β, amyloid-like β (25-35), which is known as a center of action of neurotoxicity of amyloid-like β, was used.

In this test example, PC12 cells (derived from a mouse accessory kidney brown cell tumor cell line) which are widely used in the evaluation of the action (toxicity or drug effect) on nerve cells were used. PC12 cells were obtained using BCRC (Bioresource Collection and Research Center: Bioresource Conservation and Research Center, Taiwan).

The obtained PC12 cells were treated with 5% fetal bovine serum, 10% horse serum, 100 units/ml penicillin, and RPMI-1640 medium (both obtained from Invitrogen Gibco) supplemented with 100 mg/ml streptomycin. The culture was carried out at 37 ° C under a saturated humidity in the presence of 5% carbonic acid gas.

The cytotoxicity of amyloid beta (25-35) was evaluated using a 96-well microplate coated with poly-L-lysine. 2×10 ⁴ /100 μl of PC12 cells per well were disseminateed into the microplate, and cultured at 37 ° C and saturated humidity in the presence of 5% carbonic acid gas.

After 24 hours of culture, 0.1% DMSO (manufactured by JT Baker Co., Ltd.) was added to the control area, each sample was added to the evaluation area (see below), and 10 μM hydrobromide galantamine was added to the positive control area. Galantamine hydrobromide (known as an anti-Alzheimer's agent with neuroprotective effects, obtained from Sigma-Aldrich).

With respect to all the test areas, 15 μM of amyloid-β (25-35) (obtained by Sigma-Aldrich Co., Ltd.) and two non-added persons were tested (described later). 72 hours after the start of the culture, the cell survival rate of each test zone was calculated by measuring the absorbance at 570 nm of absorbance by a predetermined method using MTT (manufactured by Molecular Probes).

In this test example, first, the cell survival rate was calculated, controlled, and sampled in the state where the amyloid-β was not added. Next, the cell survival rate was calculated for each sample under the same conditions except for the addition of the amyloid-like β. Then, the value of the cell survival rate when amyloid β is added is divided by the value of the cell survival rate when no amyloid β is added, and the value calculated by the calculation is multiplied by 100 times and expressed in % units. This subtracts the controlled cell survival rate from the latter as a cell survival recovery rate.

The cell survival recovery rate is expressed in the cell survival rate given to the sample. In the state where the effect is corrected, the index of recovery of a few percent of the cell survival rate is restored.

Use the predetermined distribution A (dichloromethane distribution), the division 1-1 of the predetermined division, the division 1-2 of the predetermined division, and the division 1 of the specified division -3. Specimen 1-4 of the specified division and white crane Ganoderma lucidum C (sector 2-2) are taken as samples. Further, screening for cytotoxicity or the like is performed in advance on the concentration of each sample, and the appropriate concentration is determined.

Further, since the experiment on the positive control and the experiment on each sample of the present invention were performed separately, as shown in Fig. 3, the values of the cell survival rates in the respective controls were different.

The results of the test examples are shown in Fig. 3. Fig. 3(a) is a table showing test results regarding positive control, and Fig. 3(b) is a table showing test results for each sample of the present invention.

As shown in Fig. 3, it was first apparent that amyloid-like β was highly toxic to PC12 cells (refer to the controlled item of Fig. 3). Further, it is apparent that the prescribed distribution, the prescribed division (the divisions 1-1 to 1-4), and the white crane Ganoderma lucidum C show a high cell survival recovery rate.

The cell survival recovery rate achieved by the fraction 1-1 and the fraction 1-2 is comparable to galantamine hydrobromide.

Moreover, the concentration was as low as 1 μM, and the cell survival recovery rate achieved by the white crane Ganoderma lucidum C was 30.2%. The cell survival recovery rate achieved by 10 μM galantamine hydrobromide was 17.2%, so it was found that the white crane Ganoderma lucidum C showed a particularly high recovery rate of cell survival.

As a result of the above test, it was confirmed that the nerve cell death inhibitor of the present invention has an excellent effect of suppressing the death of nerve cells caused by the toxicity of amyloid β, that is, an excellent nerve cell death suppressing effect. Therefore, it is considered that the nerve cell death inhibitor of the present invention having the predetermined distribution, the prescribed partition, and the white crane ganoderma lucidum C as an active ingredient has anti-Alzheimer's disease agent and anti-brain dysfunction agent. The role.

[Examples]

The active ingredient of the nerve cell death inhibitor of the present invention, the active ingredient of the anti-Alzheimer's agent or the anti-brain hypoactive agent of the present invention, the pharmaceutical and food of the white crane Ganoderma lucidum C by the following examples The modulation method is described. In addition, it is of course possible to change the item of the white crane Ganoderma lucidum C to a predetermined distribution or a predetermined division.

(1), tablets

Using a white crane ganoderma lucidum C, a tablet was prepared as follows.

(modulation method)

Ganoderma lucidum C (0.2 g), dried corn starch (2 g), talc (1.8 g), and calcium stearate (0.2 g) were added to lactose (95.8 g) for mixing. Next, a tablet was produced by a usual method using a single stroke tablet press.

(2), hard capsules

Using a white crane ganoderma lucidum C, a hard capsule (360 mg per capsule) was prepared in the following manner.

(modulation method)

Lactose (220 g) and corn starch (110 g) were added to and mixed with Shiraishi Ganoderma lucidum C (5 g), and an aqueous solution of hydroxypropylcellulose (25 g) was added thereto and kneading was carried out. Next, pellets were produced by a usual method using an extrusion granulator. A hard capsule is prepared by filling the granules into a gelatin hard capsule.

(3), soft capsules

Using a white crane ganoderma lucidum C, a soft capsule (170 mg per capsule) was prepared under the following prescription.

(modulation method)

White Crane Ganoderma Lucidum C (0.5 g) was added to soybean oil (169.5 g) and mixed. Next, a soft capsule is prepared by filling into a soft capsule according to a usual method by using a rotary soybean type automatic molding machine.

(4), pills

Using Baihe Ganoderma Lucidum C, make a pill with the following prescription (each 100mg).

(modulation method)

The mixed raw materials were mixed as described above, and water was added in an appropriate amount. Then, a homogenous kneaded product was produced by a kneader, and the obtained kneaded product was rolled, pelletized by a pelletizer, and dried to prepare a pellet.

(5), powder

Using a white crane ganoderma lucidum C, a powder (1000 mg per pack) was prepared by a usual method using the following prescription.

(6), jelly

Using the white crane ganoderma lucidum C, jelly (100 g) was prepared by a usual method using the following prescription.

Water 77.998g

(modulation method)

The above ingredients were mixed and heated to 90 °C. After confirming the dissolution of the gelatin, it is filled in a container and cooled. Jelly is made by curing gelatin.

(7), ointment

Using a white crane ganoderma lucidum C, an ointment (100 g) was prepared by a usual method using the following prescription.

(oil phase component)

(aqueous phase component)

(modulation method)

The oil phase component and the water phase component were separately heated to 80 ° C and made uniform, and the aqueous phase was added to the oil phase while stirring, and the mixture was emulsified and cooled to prepare an ointment.

(8), tape agent

Using a white crane ganoderma lucidum C, a tape (100 g) was prepared by a usual method using the following prescription.

(adhesive solvent)

(medicinal ingredients)

(percutaneous absorption enhancer)

(modulation method)

The adhesive solvent and the medicinal ingredient are uniformly dispersed, and the medicinal ingredient and the percutaneous absorption enhancer are added to the adhesive solvent, and the composition is stirred at room temperature. The composition was stretched on a polyester film subjected to a silicone treatment, dried and cooled at 120 ° C, and then the adhesive layer was transferred to a polyethylene film to prepare a tape. Agent.

Claims (4)

The invention relates to a method for preparing a nerve cell death inhibitor which inhibits the toxicity of amyloid β by using only white crane ganoderma lucidum C as an active ingredient. A use of a neuronal cell death inhibitor containing the toxicity of the inhibitory amyloid beta according to claim 1 of the patent application as an active ingredient, for the preparation of an anti-Alzheimer's agent. The use of a neuronal cell death inhibitor containing the toxicity of the inhibitory amyloid beta of claim 1 of the patent application as an active ingredient is for the preparation of an anti-brain hypofunction agent. A medicine comprising a neuronal death inhibitor which inhibits the toxicity of amyloid β according to claim 1 of the patent application as an active ingredient, and is used for preparing a medicine having anti-Alzheimer's disease effect or anti-brain function Product.