JP4648682B2 - Cerebral infarction inhibitor, platelet aggregation inhibitor, chemokine gene expression inhibitor containing Yamabushitake strain and Yamabushitake as active ingredients - Google Patents

Description

  The present invention relates to a cerebral infarction inhibitor, a platelet aggregation inhibitor, and a chemokine gene expression inhibitor comprising a Yamabushitake strain and Yamabushitake as active ingredients.

Mushrooms have been used for food and medicine since ancient times. Among them, Yamabushitake is a crushed dried fruit body, which is prescribed as a “bun” in Chinese herbal medicine, and is said to be effective for gastric ulcer, gastritis, physical weakness, etc.
Yamabushitake has been reported to have a neuronal growth factor (NGF) synthesis inducer that acts to improve dementia (Patent Document 1, etc.), and has attracted attention. In addition, regarding Yamabushitake, antitumor action (Patent Document 2), blood lipid lowering action (Patent Document 3), IL-12 production promoting action (Patent Document 4), IgE production inhibitory action (Patent Document 5), etc. Has also been reported.

JP-A-9-59172 Japanese Patent Laid-Open No. 5-17366 JP-A-11-1438 JP 2003-327541 A JP 2003-28111 A

As described above, many pharmacological effects have been reported for Yamabushitake, but not all of them have been clarified. For example, cerebral infarction inhibitory action, platelet aggregation inhibitory action, chemokine gene expression inhibitory action, etc. Not reported.
Moreover, in mushrooms, even if they have the same name, it is considered that the effect varies depending on the production method or strain, and it is important to use a strain having an excellent effect.
The present invention has been made in view of the above-mentioned problems of the prior art, and the purpose thereof is a cerebral infarction inhibitor, a platelet aggregation inhibitor, a chemokine gene expression inhibitor, and a yamabushitake strain excellent in the effect. Is to provide.

In order to achieve the above object, as a result of intensive studies by the present inventors, cerebral infarction suppressing action, platelet aggregation inhibiting action, chemokine gene expression excellent in Yamabushitake, particularly Yamabushitake strain having a deposit number of FERM P-20158 It has been found that there is an inhibitory action, and the present invention has been completed.
That is, the first subject of the present invention is the Yamabushitake strain whose deposit number is FERM P-20158.
The second subject of the present invention is a cerebral infarction inhibitor comprising Yamabushitake and / or an extract thereof as an active ingredient.
The third subject of the present invention is a platelet aggregation inhibitor comprising yamabushitake and / or an extract thereof as an active ingredient.
The third subject of the present invention is a chemokine gene expression inhibitor comprising yamabushitake and / or an extract thereof as an active ingredient.
In the cerebral infarction inhibitor, platelet aggregation inhibitor, and chemokine gene expression inhibitor according to the present invention, it is preferable that Yamabushitake is the Yamabushitake strain having the deposit number FERM P-20158.

  According to the present invention, an excellent cerebral infarction inhibitor, platelet aggregation inhibitor, and chemokine gene expression inhibitor can be obtained by using Yamabushitake, particularly Yamabushitake with the deposit number FERM P-20158, and / or its extract as an active ingredient. Can be obtained. Yamabushitake has been edible for a long time, and it has a low possibility of causing side effects even with long-term continuous use and is highly safe.

Yamabushitake (Hericium erinaceum (Bull .: Fr) Pers.) Is classified as a basidiomycete and belongs to the genus Coralharitake. It is distributed in the temperate regions of the northern hemisphere (China, Japan, USA, etc.). In Japan, the fruit body (mushroom) is named “Yamabushi Kaoru” because it resembles the ornament that Yamabushi attaches to the chest of clothes.
The fruit body has a needle-like shape, and the inside of the fruit body has a spongy structure. The size is about 5 to 10 cm, and the color is white to tan.
In the present invention, Yamabushitake may be a fruiting body, an extract of the fruiting body, or a dried product of the fruiting body, and the fruiting body may be either raw or dried, but it is easy to handle, preserve and extract. In view of the above, a dried fruit body is desirable, and in order to efficiently ingest an effective amount, it is preferably used as a dried powder of the fruit body or an extract thereof.

  Prior to obtaining the Yamabushitake extract, it is preferable to destroy the Yamabushitake tissue. This makes it possible to extract efficiently. As a means of destroying the tissue of Yamabushitake, physical processing such as pulverization processing by various pulverization mixers such as bead mill, Waring blender, homogenizer, impact crushing processing by blasting machine, freezing processing, ultrasonic processing, sodium hydroxide, etc. There are chemical treatments such as alkali treatment with an aqueous solution of the above, treatment with an enzyme having cell wall decomposing action such as cellulase and pectinase, and osmotic pressure treatment, and these can be carried out alone or in appropriate combination. Of these tissue disruption treatments, pulverization or enzyme treatment is preferred when the fruit body of Yamabushitake is used as the raw material, and enzyme treatment is preferred when the mycelium is used as the raw material.

In the enzyme treatment, a known cell wall degrading enzyme or polysaccharide degrading enzyme can be used, and one or two of cellulase, hemicellulase, chitinase, α- and β-glucuronidase, pectinase, xylanase, α- and β-glucanase, etc. The above can be used. In order to enzyme-treat Yamabushitake, the enzyme may be added as an aqueous solution to Yamabushitake, which has been appropriately shredded or pulverized, and shaken or stirred.
In order to obtain the Yamabushitake extract of the present invention, an extraction solvent may be added to the Yamabushitake that has been subjected to tissue destruction treatment as described above, and the mixture may be appropriately stirred and extracted.
The extraction solvent is not particularly limited. For example, primary alcohol such as water, methanol and ethanol, polyhydric alcohol such as 1,3-butylene glycol and propylene glycol, lower alkyl ester such as ethyl acetate, carbonization such as benzene and hexane. Hydrogen, ethyl ether, acetone, etc. are mentioned, These can also be used in combination of 2 or more types. Preferable extraction solvents include water, methanol, ethanol, 1,3-butylene glycol, or a mixed solvent thereof. In particular, those obtained by extracting dried powder of Yamabushitake fruit body with hot water are preferred.

Moreover, although extraction operation may be performed under normal pressure and normal temperature, you may carry out under 1-5 atmospheres, 60-150 degreeC pressurization, and a heating. Specifically, for example, after extraction with hot water or the like, it is preferable to separate into an extract and a residue by centrifugation or vacuum filtration.
The extraction residue may be re-extracted under the same conditions as described above, and this extraction operation may be repeated several times.
The extract may be an extract or a dried product thereof. In the case of a dried product, the extract is removed by subjecting the extract to a treatment such as concentration under reduced pressure, sterilization, freeze drying, spray drying, or the like.
In the present invention, it is preferable to use fruit bodies, but an effect can be expected for the mycelium. As the mycelium, a raw or dried mycelium obtained by culturing inoculum in a medium containing a carbon source and a nitrogen source can be used. The dried one is convenient.

  The elongation of the mycelium of Yamabushitake is the best at around 25 ° C., and low temperature treatment at 10 to 12 ° C. is suitable for generating fruit bodies. The cultivation period takes 20 to 25 days for the hyphae to spread, and about 20 days for the subsequent fruiting body development. The harvest of about 150g can be expected with 800mL bottle cultivation. Most hardwoods are suitable for medium-based sawdust, and cedar hydrolyzed can be used.

In the present invention, commercially available Yamabushitake can be used. However, it was commissioned to the National Institute of Advanced Industrial Science and Technology on August 11, 2004, and the identification number FERM P-20158 is Yamabushitake 20031546. The Yamabushitake strain can be particularly preferably used. Its scientific properties are as follows.
1. Scientific properties ... Fungus characteristics: Forms white colonies under nutrient medium containing carbon and nitrogen sources. In addition, a clamp connection (a faint connection) is observed under an optical microscope.
2. Taxonomic position ... Basidiomycetes

3. Culture conditions (1) Medium name ... SMYA medium (S: Saccharose, M: Malt extract, Y: Yeast extract, A: Agar)
S, A → commercial product M, Y → Difco (Difco)
(2) Medium composition: per 1000 ml of medium
1% sucrose 10g
1% malt extract 10g
0.4% yeast extract 4g
2% agar 20g
(3) pH of medium: 5.0 to 7.0 (optimum pH 5.5)
(4) Sterilization condition of medium ... 121 ° C 20 minutes (5) Medium temperature ... 28 ° C
(6) Culture period: 10 days (7) Oxygen requirement ... Aerobic

4). Storage conditions Can be stored by freezing method.
(1) Freezing conditions: -80 ° C
(2) Protective agent: 10-20% glycerin aqueous solution (optimum 20%)
(3) Restoration rate after freezing: 100% in 1 year and 99% in 3 years
5. Conditions of survival test (1) Microorganism restoration ... 40 ℃
(2) Inoculation / culture / confirmation method: Same as the culture conditions.

  In the present invention, a cerebral infarction inhibitor is produced using Yamabushitake. The cerebral infarction inhibitor of the present invention can be administered orally and applied for the prevention of cerebral infarction. The amount of ingestion when used as a cerebral infarction inhibitor varies depending on individual differences and is not particularly limited, but ingests 1 to 20 g, particularly 5 to 20 g, optimally 16 to 20 g in terms of dry weight of yamabushitake per day for adults If you do this, you can expect the effect.

In the present invention, Yamabushitake is used to produce a platelet aggregation inhibitor and a chemokine gene expression inhibitor. The platelet aggregation inhibitor and chemokine gene expression inhibitor of the present invention can be administered orally and applied to prevent or improve thrombus formation, improve blood flow, prevent or improve inflammatory or allergic diseases, and the like. The oral intake when used as a platelet aggregation inhibitor or a chemokine gene expression inhibitor varies depending on the symptoms and is not particularly limited, but it is 1-20 g, particularly 5-20 g, optimally converted to Yamabushitake dry powder per day for adults. If 16 to 20 g is ingested, a sufficient effect can be expected.
Moreover, if the platelet aggregation inhibitor and chemokine gene expression inhibitor of the present invention are administered transdermally as an external preparation for skin, a preventive / improving effect on inflammatory or allergic skin diseases can be expected.

When the cerebral infarction inhibitor, platelet aggregation inhibitor, or chemokine gene expression inhibitor of the present invention is used internally, powders, tablets, capsules, granules, teas, suspending agents, flow extract agents are used by conventional methods. , Liquids, syrups and the like. In the formulation, usual formulation carriers such as excipients, binders, disintegrants, lubricants, coloring agents, flavoring agents, and flavoring agents may be used as necessary. it can. Moreover, a coating can also be given with a suitable coating agent etc. as needed.
In addition, these can be ingested not only as a pharmaceutical use but also as a functional food / beverage product, a health food / beverage product, and a supplement. These are taken in various forms, such as jelly, candy, gummi, crackers, juice, and others.

  Moreover, when using it as a skin external preparation, the Yamabushitake extract may be mix | blended with a well-known skin external preparation base. In addition to the above essential components, the external preparation for skin of the present invention is usually used for external preparations for skin such as cosmetics and pharmaceuticals, for example, whitening agents, moisturizers, antioxidants, oily components, ultraviolet absorbers, surfactants. Agents, thickeners, alcohols, powder components, color materials, aqueous components, water, various skin nutrients, and the like can be appropriately blended as necessary.

  In addition, edetate disodium, edetate trisodium, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid and other metal sequestering agents, citric acid, glycolic acid, lactic acid, salicylic acid, sodium sulfite, caffeine, tannin, Pantothenic acid and derivatives thereof, nicotinic acid and derivatives thereof, tranexamic acid and derivatives thereof, licorice extract, various plant extracts such as glabrizine, maroni and escin, vitamin E derivatives such as vitamin E and tocopherol acetate, glycyrrhizic acid and derivatives thereof Or drugs such as salts thereof, placental extract, ascorbic acid and magnesium ascorbate phosphate, ascorbic acid derivatives such as ascorbic acid glucoside, arbutin, kojic acid, ellagic acid, chamomile oil, 4-n-butylreso Whitening agents such as sinol, vitamin A derivatives such as vitamin A and retinol, sugars such as glucose, fructose, xylitol, mannose, sucrose, trehalose, moisturizers such as petrolatum, glycerin, urea, heparin, various types such as serine, arginine Lipids such as amino acids, ceramides, and cholesterols can also be appropriately blended.

Skin external preparations can be applied as pharmaceuticals, quasi drugs, and cosmetics, and the dosage form is not particularly limited as long as it is conventionally used for skin external preparations such as ointments, creams, emulsions, lotions, packs, bath preparations, etc. Not.
Hereinafter, the present invention will be described with reference to specific examples, but the present invention is not limited thereto.

Platelet aggregation inhibition test It is becoming clear that arachidonic acid and its metabolites leukotriene, thromboxane and prostaglandin are involved in the development of various inflammatory and allergic skin diseases and rough skin. For example, in psoriasis, an inflammatory abnormal keratotic disease, high arachidonic acid metabolites are found in the affected epidermis, and arachidone is also present in allergic skin diseases such as atopic dermatitis, contact dermatitis, and eczema. The involvement of acid metabolites has been suggested. In addition, since prostaglandins and thromboxanes aggregate platelets, compounds that suppress the synthesis of arachidonic acid to prostaglandins and thromboxanes have antithrombotic effects, that is, blood flow improving effects. it is conceivable that.

Therefore, Yamabushitake was examined for the inhibitory effect of PAF and sodium arachidonic acid-induced platelet aggregation.
(1) Preparation of Yamabushitake Extract A dried fruit body of Yamabushitake was pulverized and passed through a 16 mesh sieve to obtain a powder. As a comparative example, dried fruit bodies of shiitake mushroom and maitake were also made into powder. 10 g of each powder was extracted in 200 ml of hot water (90 ° C.) for 2 hours. After filtration, the filtrate was concentrated under reduced pressure to obtain a hot water extract of a dried fruit body.
In addition, as Yamabushitake, in addition to FERM P-20158 strain, three kinds of commercially available Yamabushitake (domestic A, domestic B, overseas) were used.
For comparison, shiitake and maitake were similarly processed.

(2) Test method Human blood was centrifuged at 1100 rpm at room temperature for 20 minutes to collect platelet-rich plasma (PRP), and then centrifuged at 3000 rpm for 5 minutes to collect platelet poor plasma (PPP). After pre-warming 223 μL of PRP at 37 ° C., 2 μL each of 2% DMSO solution of the methanol extract or ion-exchanged water as a control example was added and further incubated at 37 ° C. for 3 minutes. 25 μL of a certain PAF aqueous solution (500 nM) was added.
The induced aggregation was measured using an aggregometer (manufactured by MCM Hematracer MCM Medical Co., Ltd.), and the maximum aggregation rate of the test sample (maximum value obtained from the aggregation curve of the test sample with the PPP value being 100) The inhibitory effect on the PAF-induced platelet aggregation of the test substance was evaluated by comparing with the maximum aggregation rate of the control example.
In addition, the inhibitory action against platelet aggregation on arachidonic acid, which is a causative agent for the occurrence of inflammation and allergies, was similarly evaluated by using an aqueous sodium arachidonic acid solution (500 μM) instead of the above PAF aqueous solution.

(3) Results FIG. 1 shows the inhibitory effect on platelet aggregation by PAF induction and FIG. 2 by arachidonic acid induction. As can be seen from FIGS. 1 and 2, Yamabushitake has a higher platelet aggregation inhibitory action than the shiitake or maitake extract of the comparative example, and in particular, an excellent platelet aggregation inhibitory action was observed in the FERM P-20158 Yamabushitake strain.
Therefore, it is considered that ingestion of Yamabushitake or its extract can suppress platelet aggregation, and the effects such as improvement / prevention of inflammatory / allergic diseases, antithrombosis, and improvement of blood flow are exhibited. In addition, application to the skin is considered effective for skin diseases.

Chemokine gene expression suppression test In recent years, it has been revealed that chemokines (Chemokine, Chemokine) that control the migration and activation of inflammatory cells are strongly involved in the development of various inflammatory and allergic skin diseases. Chemokine is a general term for basic proteins having a molecular weight of 8 to 10 kDa and having heparin binding properties. It plays a central role in the migration and activation of various cells involved in inflammation in the living body and is important for inducing inflammation. It is thought to play a role.

In skin diseases, inflammatory cells such as neutrophils and eosinophils migrate and activate in inflammatory sites such as contact dermatitis and atopic dermatitis, and are thought to be involved in the generation and amplification of inflammation. Yes. In this case, it is known that chemokines such as interleukin-8 (IL-8, interleukin-8) are produced from cells such as skin keratinocytes and fibroblasts. In psoriasis, which is an inflammatory abnormal keratotic disease, it is known that IL-8 is present in a large amount in the affected skin area.
Therefore, suppressing the expression of chemokines such as IL-8 is considered effective in improving or preventing these inflammatory or allergic disease symptoms.
Then, the chemokine gene expression inhibitory effect in Yamabushitake was examined.

(1) Preparation of Yamabushitake Extract A dried fruit body of Yamabushitake was pulverized and passed through a 16 mesh sieve to obtain a raw powder. 10 g of the raw powder was added to 200 ml of hot water (90 ° C.), extracted for 2 hours, filtered, concentrated with an evaporator, and the residue was dried under reduced pressure. This was dissolved in an appropriate amount of DMSO and used in the following experiments.

(2) Measurement of chemokine gene expression suppression action Human dermal fibroblasts are cultured in a 6 cm diameter culture dish in DMEM medium (Dulbecco's modified Eagle medium) containing 10% fetal bovine serum until they become confluent. It used for experiment.
Yamabushitake extract or hydrocortisone (positive control) was added to the test medium as a test sample. The final concentrations of Yamabushitake extract and hydrocortisone were 0.0025 to 0.04% (dry mass) and 10 ⁻⁷ M, respectively.

Furthermore, tumor necrosis factor TNF-α (1 ng / ml), which is known to promote chemokine gene expression, was added and cultured at 37 ° C. for 6 hours.
Next, RNA was isolated from cells and cDNA was synthesized according to a conventional method, and then the expression level of IL-8 gene was measured by a quantitative PCR method (TaqMan PCR method). GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene was used as an internal standard gene, and the obtained data was corrected.
Human skin fibroblasts can be obtained as a commercial product, for example, from Kurashiki Boseki Co., Ltd. Human fibroblasts may be cultured according to a conventional method for culturing animal cells, but a DMEM medium containing 10% fetal bovine serum is particularly preferable.

(3) Results FIG. 3 shows the gene expression level of IL-8 at each concentration of FERM P-20158 Yamabushitake strain extract. The expression level of DMSO alone was 0.47. Yamabushitake extract suppressed the gene expression of IL-8 in human skin fibroblasts. The effect of the Yamabushitake extract concentration increased from 0.02 to 0.03% with increasing concentration, but the inhibitory effect tended to decrease with increasing concentration.
Hydrocortisone has a strong inhibitory effect, but because it is a steroid hormone, it may cause side effects such as inflammation, tissue damage, and ulceration. In contrast, Yamabushitake extract is not as effective as hydrocortisone, but it can be used for a long time because there are no side effects.

Cerebral infarction suppression test When sufficient blood is not supplied to the brain tissue, the supply of oxygen is ischemic, resulting in cell death and eventually cerebral infarction. In addition, if excessive blood is supplied to the brain tissue that is in a cerebral infarction state, a serious tissue disorder will occur in the vicinity of the infarct (penumbra region) between the life and death due to the generation of a large amount of active oxygen. I understand that it will happen. This is called cell death (apoptosis), and it has been attracting attention as an extremely important clinical phenomenon not only in cerebral infarction but also in myocardial infarction and stress ulcer.
Therefore, the effect of Yamabushitake on the onset of cerebral infarction associated with such a blood flow disorder was examined using a cerebral infarction model caused by transient middle cerebral artery occlusion (MCA).

(1) Preparation of Yamabushitake Suspension A dried fruit body of Yamabushitake was pulverized and passed through a 16 mesh sieve to obtain a raw powder. This raw powder was suspended in distilled water and used for the following experiments.

(2) Experimental animals and breeding method ddY male mice (Kiwa Laboratory Animal Research Institute, Wakayama) weighing 25-30 g were used as experimental animals. Mice were housed in plastic cages in an animal room with room temperature 23 ± 2 ° C., humidity 60 ± 2%, and a 12 hour light / dark cycle (7:00 AM lighting). In addition, water and food (CE-2; Nippon Claire Co., Ltd., Tokyo) were allowed to be ingested freely. The handling of experimental animals was in accordance with the Fukuoka University Animal Animal Care and Use Committee.

(3) Preparation procedure of MCA occluded mouse Anesthesia was introduced into the mouse by inhalation of 2% halothane, and then the anesthesia was maintained by inhalation of 1% halothane and fixed on the operating table. Under the surgical microscope (Olympus OMK-1F), the center of the neck was incised, and the left common carotid artery and external carotid artery were ligated. The common carotid artery was incised, and 9 mm was inserted from the bifurcation of the internal carotid artery and external carotid artery via the internal carotid artery so that the embolus reached the beginning of the MCA. Reperfusion was performed by pulling the embolus forward. As the embolus, an 8-0 nylon monofilament (Ethylon; Ethicon, NJ, USA) having a length of 11 mm was coated with silicon resin (Xantopren; Bayer Dental, Osaka, Japan) 4 mm. The diameter of the coating part was selected as appropriate depending on the body weight.

(4) 2,3,5-triphenyltetrazole chloride (TTC) staining After occlusion for 4 hours by the above operation, the embolus was withdrawn and reperfused. After 24 hours from the start of reperfusion, the mouse cervical spine was dislocated and immediately decapitated. The removed brain was cooled well with cold water, and pre-cut slices of the brain containing the cerebral cortex were prepared at intervals of 2 mm. Sections were incubated at 37 ° C. for 30 minutes in 2% 2,3,5-triphenyl-tetrazolium chloride (TTC, Sigma) solution. And it arranged on the slide glass and image | photographed with the digital still camera (MVC-FD91, SONY).

(5) Measurement of infarct volume The Yamabushitake powder was suspended in 3, 10 or 30 mg per 1 ml of distilled water, and 0.1 ml was orally administered to 10 g of mouse body weight (0.3 mg, 1 mg or 3 mg · 10 BW administration) group). Distilled water was orally administered to the control group. There were 6 mice in each group.
Yamabushitake was orally administered once a day for 14 consecutive days, and the next day was loaded with MCA occlusion. On the next day, the area of the infarct was determined by TTC staining.
The infarct volume was calculated by measuring the area of the infarct from the photograph of the cleavage plane using image analysis software (NIH Image 1.62).

(6) Results FIG. 4A is a photograph of a brain cross section of a mouse in a control group (Yamabushitake non-administered group). 24 hours after the blood flow in the left middle cerebral artery of the mouse was temporarily stopped and the blood flow was reperfused, a significant cerebral infarction occurred in most of the temporal lobe and parietal lobe of the left brain dominated by the middle cerebral artery. Happened (the part on the left side of the photo that looks white).
FIG.4 (b) is a brain cross-sectional photograph of the mouse | mouth of a Yamabushitake (FERM P-20158) administration group. When the Yamabushitake aqueous solution was taken every day for 14 days, the cerebral infarction level after 24 hours after reperfusion was suppressed in proportion to the dose of Yamabushitake and the formation of cerebral infarction foci was suppressed.

Compared with the Yamabushitake administration group and the control group for the infarct areas at 4 mm, 6 mm and 8 mm in the temporal lobe, parietal lobe, and linear body regions, where the influence of blood supply from the middle cerebral artery is strongest Then, Hericium erinaceus 14.01Mm ² each mean a non-administration group, 21.63Mm ² and to 15.91Mm ² there was the, Hericium erinaceus 3 mg / 10 g 7.75 mm ² in each average body weight administered group, 13. a 25 mm ² and 9.54mm ^2, infarct size was markedly decreased by Hericium erinaceus administration.

FIG. 5 shows the cerebral infarct volume by the NHI image measurement method. In the cerebral infarct volume, as in the case of the infarct area, the higher the dose, the more pronounced the inhibitory effect, and a statistically significant inhibitory effect was observed in the 3 mg / 10 g BW administration group (p <0.05).
FIG. 6 shows the infarct area and the infarct volume of each mouse in the Yamabushitake 3 mg / 10 g BW administration group, but the brains of 2 of the 6 test mice were maintained in a normal state after the infarction.
These results revealed that Yamabushitake has the effect of protecting brain cells from cerebral ischemia and suppressing cerebral infarction. If cerebral infarction can be suppressed, it is possible to prevent motor dysfunction, memory impairment, emotional disorder, etc., as seen in cerebral infarction sequelae. In this test, mice are used, but these effects can be sufficiently expected in humans.

Genetic characteristics of FERM P-20158 Yamabushitake strain In general, strains of cultivars (strains) are obtained by directly obtaining binuclear hyphae from the natural world by separating the fruit bodies, or crossing mononuclear hyphae from spores Artificially created by cross breeding. They are used through the stabilization procedure as varieties, but often lead to deterioration during repeated cultures from mycelia. Each grower has a unique strain, and uses a fungus bed suitable for each cultivation form to select a medium base material and a mixed nutrient and obtain fruit bodies.
The character of fruiting body is influenced by many factors such as fungus bed components and cultivation environment. Among them, the most important is the strain, and the shape of the fruiting body varies greatly depending on the strain. It is also reflected in the functional ingredients.
Therefore, the gene characteristics of the FERM P-20158 Yamabushitake strain having a very high function as described above were compared with other varieties by molecular biological techniques.

<Anti-culture (band formation) test>
FERM P-20158 Yamabushitake strain and commercially available cultivars Yamabushitake A strain and B strain were countercultured with each other. In this test method, the homology between the two can be easily determined by observing the presence or absence of a band line (zone line) at the contact surface of both fungus algae (colony).
As a result of counterculture of the FERM P-20158 strain and the commercially available A strain and B strain, band lines were formed (FIG. 7).
Therefore, the FERM P-20158 strain has low homology with both the commercially available A strain and B strain, and can be said to be a different variety.

<RAPD analysis>
The genetic characteristics of the FERM P-20158 strain were further analyzed at the DNA level by the RAPD (Randam Amplified Polymorphic DNA) method.
FIG. 8 shows a conceptual diagram of the RAPD method. First, a specific primer (about 10 bases) is used to perform polymerase chain reaction (PCR) using genomic DNA as a template to specifically amplify the DNA. After the amplified DNA is stained, it is electrophoresed on an agar gel to obtain a DNA band pattern. When there are differences between individuals on the base sequence of genomic DNA, the amplified DNA fragments differ in type and size, and show different band patterns. Therefore, by comparing the band patterns, the homology at the DNA level can be examined.

1. Test Method (1) Yamabushitake mycelium was crushed with liquid nitrogen.
(2) 500 μl of citrate phosphate buffer (pH 6.0) containing 2% sarkosyl was added and mixed.
(3) Lysis treatment was performed at 65 ° C. for 30 minutes.
(4) After centrifugation at 15,000 rpm for 30 minutes, the supernatant was recovered.
(5) An equal amount of phenol-chloroform (1: 1) was added to the collected supernatant and mixed by vortexing.
(6) After centrifugation at 15,000 rpm for 15 minutes, the upper layer was recovered.

(7) After repeating operations 5 and 6 twice, 1/10 amount of 3M sodium acetate and 2.0 to 2.5 times the amount of ice-cold 100% ethanol were added and stirred.
(8) Cooled at −80 ° C. for 20 minutes.
(9) After centrifugation at 15,000 rpm for 15 minutes, the supernatant was discarded.
(10) The precipitate was washed with 70% ethanol.
(11) Air-dried for 20 minutes.
(12) The precipitate was dissolved in 500 μl TES solution (30 mM Tris / HCl, 50 mM NaCl, 5 mM EDTA pH 8.0).
13) 10 μl of RNase (5 mg / ml, Sigma) and α-amylase (10 mg / ml, Sigma) were added and treated at 37 ° C. for 1 hour.

(14) Phenol-chloroform extraction was performed again.
(15) Ethanol precipitation and washing were performed.
(16) After air drying, it was dissolved in TE buffer (10 mM Tris / HCl, 1 mM EDTA pH 7.5) to obtain a DNA solution. DNA concentration was measured by absorbance at 260 nm. The DNA solution was stored at -20 ° C.
(17) PCR was performed using 5 ng of DNA as a template. PCR conditions, PCR reaction solution composition and primer base sequence are as follows.

(PCR conditions)
――――――――――――――――――――――――――――――――――
94 ° C 30 sec
94 ° C (denaturation) 30 sec
42 ° C (annealing) 2min 45cycles
72 ° C (extension reaction) 3 min
72 ° C 7min
4 ℃ ∞
――――――――――――――――――――――――――――――――――

(PCR reaction solution composition)
――――――――――――――――――――――――――――――――――
10 × PCR-buffer 1 (μl)
2.5 mM dNTP 0.8
Primer 10 μM 0.5
Template 5ng 1
Taq 0.05
(TaKaRa Ex Taq Hot start version)
Water 6.65
――――――――――――――――――――――――――――――――――
Total 10
――――――――――――――――――――――――――――――――――

(Primer sequence)
Primer A01: tgcactacaaca
Primer A12: ttcggacgaata

2. Results FIGS. 9A and 9B show the results of RAPD analysis of FERM P-20158 Yamabushitake and commercially available Yamabushitake A to C strains using primers A01 and A12 as primers, respectively. It can be seen that the FERM P-20158 strain and the commercially available AC strains all show different band patterns.
Thus, the FERM P-20158 strain was different from the commercially available strain at the DNA level, and it was speculated that the high functionality of the FERM P-20158 strain is related to such genetic specificity.

It is a figure which shows the inhibitory effect with respect to the PAF induced platelet aggregation of Yamabushitake. It is a figure which shows the inhibitory effect with respect to arachidonic acid induced platelet aggregation of Yamabushitake. It is a figure which shows the chemokine gene expression inhibitory effect of Yamabushitake (FERM P-20158). It is a mouse | mouth cerebral infarction brain cross-section photograph of (a) control group (Yamabushitake non-administration) and (b) Yamabushitake (FERM P-20158) administration group. It is a figure which shows the change of a mouse | mouth cerebral infarction foci volume by a Yamabushitake (FERM P-20158) dosage.

It is a figure which shows the cerebral infarction foci area and the cerebral infarction foci volume in each mouse | mouth of a Yamabushitake (FERM P-20158) 3 mg / 10BW administration group. It is a figure which shows the opposing test result (band formation) with a FERM P-20158 strain | stump | stock, and a commercially available A strain | stump | stock and B strain | stump | stock. It is a conceptual diagram which shows the principle of RAPD method. It is a figure which shows the result of having analyzed DNA by RAPD method, using (a) primer A01 or (b) primer A12 as a primer about FERM P-20158 Yamabushitake strain and commercially available Yamabushitake strains AC.

Claims (3)

Yamabushitake strain with a deposit number of FERM P-20158. A platelet aggregation inhibitor comprising Yamabushitake and / or an extract thereof having a deposit number of FERM P-20158 as an active ingredient . A chemokine gene expression inhibitor comprising, as an active ingredient, Yamabushitake with a deposit number of FERM P-20158 and / or an extract thereof .

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