JP7239208B2 - Method for producing aging-related IL-1β blood level elevation inhibitor for oral intake - Google Patents

Description

The present invention relates to a method for producing an age -related IL-1β blood level increase suppressor for oral intake, containing an extract extracted from Hakutsuru Reishi mushroom with a solvent containing 30% or more of ethanol by mass in the total mass.

Rhinacanthus nasutus (L.) Kurz, also referred to as Hakutsuru Reishi grass, is an evergreen small shrub belonging to the genus Rhinacanthus and belonging to the family Vulpiaceae, said to be native to the Deccan Plateau in southern India. Whole grass of Hakutsuru Reishi is known to have anthelmintic, anti-inflammatory, and antibacterial effects against skin fungi (see, for example, Non-Patent Document 1). Used as food. In addition, in previous applications by the present applicant, Hakutsuru Reishi has the ability to eliminate active oxygen (see, for example, Patent Document 1.), has an excretion-promoting effect (see, for example, Patent Document 2.), and has an anti-allergic effect. It has been disclosed that it has an effect (see, for example, Patent Document 3) and that it has an antitumor effect (see, for example, Patent Document 4).

JP-A-9-143091 JP-A-9-169662 Japanese Unexamined Patent Application Publication No. 2001-10964 JP-A-2002-53481

Primary Color Makino Japanese and Chinese Medicinal Herb Encyclopedia, 492 pages, Hokuryukan, 1988

However, Hakutsuru Reishi extract is effective as an oral IL-1β blood concentration inhibitor, an oral amyloid-β brain concentration inhibitor, an oral hair-improving agent, and an oral anti-aging agent. Not known to be a component.

An object of the present invention is to clarify the novel properties of the Hakutsuru Reishi extract and to provide new uses based thereon.

In order to solve the above problems, the present inventors have extensively studied the new usefulness of extracts of Hakutsuru Reishi mushrooms. However, the inventors have found that orally ingesting it has an effect of suppressing an increase in IL-1β concentration in the blood, an effect of suppressing an increase in an amyloid-β concentration in the brain, an effect of improving hair and an anti-aging effect, and completed the present invention. That is, the present invention includes the following embodiments.

(1) IL-1β blood level increase suppressor for oral intake, which contains an ethanol extract of Hakutsuru Reishi mushroom as an active ingredient and suppresses an increase in blood level of IL-1β when taken orally.
(2) The IL-1β blood level increase suppressor for oral intake according to (1), wherein the ethanol extract is an extract with water-containing ethanol.
(3) IL-1β blood level increase inhibitor for oral intake according to (1) or (2), which is in the form of a pharmaceutical, food or drink, feed, or an active ingredient composition blended therein.
(4) The IL-1β blood level increase suppressor for oral intake according to (3), wherein the food or drink is a food or drink for health promotion.
(5) at least one of chronic obstructive pulmonary disease, autoinflammatory disease, rheumatoid arthritis, osteoarthritis, gouty arthritis, and arteriosclerosis by suppressing an increase in IL-1β blood level by oral intake; IL-1β blood level increase inhibitor for oral intake according to any one of (1) to (4), which alleviates, improves, suppresses, prevents or treats
(6) An amyloid-β intracerebral concentration increase inhibitor for oral intake containing an ethanol extract of Hakutsuru Reishi mushroom as an active ingredient and suppressing an increase in the intracerebral concentration of amyloid-β upon oral ingestion.
(7) The orally ingested amyloid-β intracerebral level elevation inhibitory agent according to (6), wherein the ethanol extract is an extract with water-containing ethanol.
(8) The orally ingested amyloid-β intracerebral level elevation inhibitory agent according to (6) or (7), which is in the form of a pharmaceutical, food or drink, feed, or an active ingredient composition blended therein.
(9) The orally ingested amyloid-β brain level increase inhibitor according to (8), wherein the food or drink is a food or drink for health promotion.
(10) Any one of (6) to (9), which alleviates, improves, suppresses, prevents or treats cerebral amyloid angiopathy by suppressing an increase in the brain concentration of amyloid-β by oral intake. The amyloid-β intracerebral concentration increase inhibitor for oral ingestion described above.
(11) A hair-improving agent for oral ingestion, which contains an ethanol extract of Hakutsuru Reishi mushroom as an active ingredient, and suppresses a decrease in the glossiness of hair when orally ingested.
(12) The orally ingestible hair-improving agent according to (11), wherein the ethanol extract is an extract with water-containing ethanol.
(13) The orally ingestible hair-improving agent according to (11) or (12), which is in the form of a pharmaceutical, food or drink, feed, or an active ingredient composition blended therein.
(14) The orally ingested hair-improving agent according to (13), wherein the food or drink is a food or drink for beauty or health promotion.
(15) An anti-aging agent for oral ingestion, containing an ethanol extract of Hakutsuru Reishi mushroom as an active ingredient.
(16) The anti-aging agent for oral intake according to (15), wherein the ethanol extract is an extract with water-containing ethanol.
(17) The orally ingestible anti-aging agent according to (15) or (16), which is in the form of a pharmaceutical, food or drink, feed, or an active ingredient composition blended therein.
(18) The orally ingestible anti-aging agent according to (17), wherein the food or drink is a beauty food or drink or a health promotion food or drink.
(19) Orally ingested pharmaceuticals, food and drink or used for suppressing elevation of IL-1β blood level, suppressing elevation of amyloid-β brain level, improving hair and anti-aging in humans or non-human mammals Use of an ethanol extract of Hakutsuru Reishi mushroom for producing an active ingredient composition to be incorporated therein.

According to the present invention, as novel uses of the ethanol extract of Hakutsuru Ganoderma lucidum, an orally ingestible IL-1β blood level increase inhibitor containing an ethanol extract of Hakutsuru Ganoderma lucidum, orally ingested amyloid-β intracerebral It is possible to provide concentration increase inhibitors, orally ingested hair-improving agents, orally ingested anti-aging agents, beauty foods and beverages, and health promotion foods and beverages.

FIG. 1 is a flow chart showing the manufacturing process of the active ingredient composition of the present invention. FIG. 2 is a graph showing serum IL-1β concentrations on day 133 from the start of the test in accelerated aging mice to which the active ingredient composition of the present invention was administered. FIG. 3 is a graph showing the concentration of amyloid-β in the brain homogenate supernatant of aging-accelerated mice to which the active ingredient composition etc. of the present invention was administered, 133 days after the start of the test. FIG. 4 is a graph showing hair gloss scores at 22 weeks of age of accelerated aging mice to which the active ingredient composition and the like of the present invention were administered. FIG. 5 is a graph showing changes over time in the mean value of the 11-item senescence evaluation scores of senescence accelerated mice to which the active ingredient composition and the like of the present invention were administered. FIG. 6 is a graph showing the average scores of 11 items for evaluating the degree of senescence at 22 weeks of age in aging-accelerated mice to which the active ingredient composition etc. of the present invention was administered.

Next, each embodiment of the present invention will be described with reference to the drawings. It should be noted that each embodiment described below does not limit the invention according to the scope of claims, and all of the elements described in each embodiment and combinations thereof are means for solving the present invention. is not necessarily required for

(I) Ethanol Extract of Hakutsuru Ganoderma Lucidum The active ingredient in one embodiment of the present invention is an ethanol extract extracted with ethanol from Hakutsuru Ganoderma lucidum (Rhinacanthus nasutus (L.) Kurz). In the present specification, the term "ethanol" simply means "a solvent in which ethanol accounts for 30% or more of the total mass". In other words, "ethanol" includes not only so-called absolute ethanol but also mixed solvents of ethanol and other solvents.

(II) IL-1β Blood Concentration Elevation Suppressor for Oral Intake The “IL-1β blood concentration elevation inhibitor for oral ingestion” as used herein refers to an agent that suppresses an increase in blood concentration of IL-1β. Say. IL-1β is a type of inflammatory cytokine, and sustained production of IL-1β causes various chronic inflammatory diseases in bones, joints, blood vessels, muscles, etc. (Onozaki, Interleukin 1: Cell Proliferation From control to chronic inflammatory diseases, YAKUGAKU ZASSHI, 133(6), 645-660, 2013). Specifically, IL-1β is known to be associated with diseases such as chronic obstructive pulmonary disease (emphysema), autoinflammatory disease, rheumatoid arthritis, osteoarthritis, gouty arthritis, and arteriosclerosis. In addition, "for oral intake" in the present invention means to be ingested through eating and drinking (including ingestion).

(III) Orally Ingested Amyloid-β Brain Concentration Suppressor The term “orally ingested amyloid-β brain concentration elevation inhibitor” as used herein refers to an agent that suppresses an increase in the brain concentration of amyloid-β. Say. As will be described later, amyloid-β is closely related to Alzheimer's dementia, and is also a cause of cerebral amyloid angiopathy and associated cerebral hemorrhage when deposited in the meninges and blood vessel walls in the brain (Kowa et al., Current state of stroke in the elderly 2. Current state of cerebral hemorrhage in the elderly, Journal of the Japan Geriatrics Society, Vol. 54, No. 4, 514-518, 2017).

(IV) Orally ingested hair-improving agent The term “orally-ingested hair-improving agent” as used herein refers to an agent that contributes to improvement of hair, particularly an agent that prevents or improves loss of luster of hair. As is clear from Example 2 to be described later, the orally ingested hair-improving agent of the present invention has a remarkable effect on reducing the luster of hair.

(V) Anti-Aging Agent for Oral Ingestion The term “anti-aging” is broadly defined, and is used to treat age-related diseases such as myocardial infarction, cerebral infarction, osteoporosis, rheumatoid arthritis, cataracts, age spots, It refers to alleviating, improving, suppressing, preventing or treating skin diseases such as wrinkles and dullness. Preferably, it means having an effect of alleviating, ameliorating, suppressing, preventing or treating diseases caused by chronic inflammation associated with aging or accumulation and deposition of harmful substances associated with aging. In addition, it also means to have an effect of preventing or improving skin and hair aging caused by external factors (ultraviolet rays, dry air, etc.) and aging. The term "anti-aging agent" includes such agents used for anti-aging.

(VI) Form of drug , pharmaceuticals, food and drink, or active ingredient compositions to be blended therein. When used as the active ingredient composition, not only the ethanol extract of Hakutsuru Ganoderma lucidum can be used as it is, but also the crude or purified ethanol extract of Hakutsuru Ganoderma lucidum can be used.

The active ingredient composition of the present embodiment is preferably prepared in the form of foods, beverages, pharmaceuticals, etc. as described below by appropriately blending an appropriate edible carrier (food material) and a pharmaceutically acceptable carrier. . The forms of pharmaceuticals and food/beverage products containing the active ingredient composition are specifically described below.

(medicine)
When the orally ingested IL-1β blood level increase inhibitor, orally ingested amyloid-β brain level increase inhibitor, orally ingested hair improving agent, and orally ingested anti-aging agent of the present embodiment are used as pharmaceuticals, Using an ethanol extract of Hakutsuru Reishi and a suitable pharmaceutical carrier that is pharmaceutically acceptable, it is prepared in the form of a general pharmaceutical composition for practical use. As the pharmaceutical carrier, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, etc., which are generally used in this field agents can be exemplified. These are appropriately selected and used according to the dosage unit form of the resulting preparation.

Various forms can be selected as the dosage unit form of the pharmaceutical composition, and oral administration formulations are preferred. Typical oral formulations include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules and the like.

When forming into a tablet form, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, etc. as the pharmaceutical carrier; water, ethanol , propanol, simple syrup, glucose solution, starch solution, gelatin solution, binders such as carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone; carboxymethylcellulose sodium, carboxymethylcellulose calcium, low-substituted hydroxypropylcellulose, dry starch, alginic acid Disintegrants such as sodium, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride; sucrose, stearin, cocoa butter, hydrogenated oil absorption enhancers such as quaternary ammonium salts and sodium lauryl sulfate; moisturizing agents such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; purified talc and stearin Acid salts, boric acid powder, lubricants such as polyethylene glycol, and the like can be used. Furthermore, the tablet may be a tablet coated with a conventional coating, such as a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, or a multilayer tablet, if necessary.

When forming into the form of pills, excipients such as glucose, lactose, starch, cacao butter, hardened vegetable oil, kaolin, and talc as pharmaceutical carriers; binders such as gum arabic powder, tragacanth powder, gelatin, and ethanol; Disintegrants such as laminaran and agar can be used.

Furthermore, the pharmaceutical composition may contain coloring agents, preservatives, flavoring agents, flavoring agents, sweetening agents, and other pharmaceutical agents, if necessary.

The administration method of the above pharmaceutical composition is not particularly limited, and is determined according to various formulation forms, patient's age, sex and other conditions, severity of disease, and the like. In addition, the dosage is appropriately selected depending on the usage, patient's age, sex and other conditions, degree of disease, etc., but it is usually 0.1 to 1000 mg, preferably 1 to 300 mg, more preferably 10 mg per day for adults. The dose is preferably about 150 mg, and the preparation can be administered to humans in 1 to 4 divided doses per day.

(food and drink)
When the orally ingestible IL-1β blood level increase inhibitor, orally ingestible amyloid-β brain level increase inhibitor, orally ingestible hair improving agent, and orally ingestible anti-aging agent of the present embodiment are used as foods and drinks , food in the form of tea to which the active ingredient composition is added, and food in the form of processed food to which the active ingredient composition is blended. As tea, it is preferable to use Hakutsuru Reishi itself by mixing it with dried leaves, stems or roots, or by mixing it with other tea raw materials. As a tea raw material, any tea such as green tea, oolong tea, pu-erh tea, black tea, roasted tea, brown rice tea, eucommia tea, persimmon leaf tea, mulberry leaf tea, etc. can be used as long as they are usually used for tea. can be done. The dried leaves, stems, or roots of Hakutsuru Reishi may be roasted and used in the same manner as other tea ingredients.

Examples of other forms of food and drink include pickles, miso, fermented tea, fermented foods such as bread, baby food, powdered milk, infant foods such as baby food, foaming preparations, gum, gummy, confectionery such as pudding, noodles, Nutritional supplements such as capsules, granules, powders and tablets, fermented milk, lactic acid bacteria drinks, fermented vegetable drinks, fermented fruit drinks, fermented soymilk drinks and the like can be mentioned. “Fermented milk” refers to pasty or liquid paste or liquid obtained by fermenting milk or dairy products with lactic acid bacteria, bifidobacteria, or yeast. Therefore, the fermented milk includes the yogurt form as well as the drink form. The term "lactic acid bacteria drink" refers to a drink prepared by diluting a paste or liquid obtained by fermenting milk or dairy products with lactic acid bacteria, bifidobacteria, or yeast as the main raw material with water.

The term "food and drink" as used herein includes all forms that are orally used exclusively for eating and drinking (e.g., beverages are also included), and even in forms such as tablets, As long as it is used in, it is included in the food and drink in this specification. For example, health foods, health supplements, and patients with the concept of suppressing an increase in IL-1β blood level, suppressing an increase in amyloid-β concentration in the brain, improving hair or anti-aging, and labeling them as necessary. Foods for food, dietary supplements, or foods with health claims (foods for specified health use, foods with nutrient claims) defined by the Ministry of Health, Labor and Welfare are also included in the food and drink products herein.

Preferred embodiments include health-enhancing food and beverage products. Health-enhancing foods and drinks refer to those that improve the physical condition of subjects by alleviating, for example, chronic inflammation associated with aging. As used herein, "health promotion" means enhancing the state of "health", which is a state of complete physical, mental and social well-being, not merely being free from disease and infirmity. .

A preferred embodiment also includes food and drink for beauty care. The term "cosmetic use" refers to the use for suppressing the progress of aging of the subject and maintaining or improving the aesthetic appearance, including the improvement of the aesthetic appearance of the skin and hair. be In particular, in the examples described later, aging indicators such as fur coat, hair luster, and periocular skin conditions of aged mice orally ingested with the active ingredient of the present embodiment are improved, so aging of the subject is suppressed. It is expected to improve the aesthetic appearance, that is, to be used as food and drink for beauty. In addition, the beauty food and drink of the present embodiment further includes a skin (skin) anti-aging (preventive) agent for oral intake, a skin (skin) elasticity improving agent, an anti-wrinkle agent, a skin quality improving agent, and a hair beauty improving agent. It can also be used as an agent, laxative, physical condition improver, and constitution improver.

In addition to the above active ingredients, the health-promoting food and drink and beauty food and drink may contain physiologically active substances, (food) raw materials, (food) additives, etc., as appropriate. . Examples of additives and raw materials include those used by addition, mixing, infiltration and other methods in the production, processing and preservation of foods, etc. Examples include excipients, extenders, thickeners and binders. , lubricants, stabilizers, colorants, emulsifiers, solubilizers, colorants, flavors, seasonings, spices, sweeteners, preservatives, and fragrances that are commonly used.

The content of the active ingredient composition in the food or drink according to the present embodiment is not particularly limited, and can be determined as appropriate. From the viewpoint of exhibiting the effects of suppressing elevation of IL-1β blood concentration, suppressing elevation of amyloid-β brain concentration, improving hair, or anti-aging, for example, 0.01% by mass or more is added to the total weight of each food and drink. It is preferably 0.1% by mass or more, more preferably 1% by mass or more. On the other hand, the upper limit of the content of the active ingredient composition in food and drink is not particularly limited. can be appropriately adjusted according to the form of

(VII) Method for Producing an Active Ingredient Composition In another aspect of the present invention, an effective ingredient having an IL-1β blood level increase inhibitory action, an amyloid-β intracerebral level elevation inhibitory action, a hair improvement action and an anti-aging action A method of making a component composition is provided. FIG. 1 is a flow diagram showing the manufacturing process of the active ingredient composition. This method includes a pulverization step (S01) of pulverizing Hakutsuru Ganoderma lucidum, an extraction step (S02) of extracting from Hakutsuru Ganoderma lucidum with ethanol, and a solvent removal step (S03) of removing the solvent (ethanol) used in the extraction. and including.

The pulverization step (S01) can be carried out, for example, using a commonly used pulverizer or the like. For Hakutsuru Reishi, the raw material, Hakutsuru Reishi, leaves, stems, roots, flowers, etc., are harvested at an appropriate time and then left as they are, or subjected to a drying process such as normal ventilation drying to be used as raw material. can be used. Which part of the Hakutsuru Reishi mushroom to use as the raw material for extraction and how finely it should be pulverized can be appropriately determined according to the state of the Hakutsuru Reishi mushroom and the form of the product to be manufactured. Since pulverization of Hakutsuru Ganoderma lucidum is performed to facilitate extraction, the same effect can be obtained by shredding Hakutsuru Ganoderma lucidum instead of pulverizing it. Further, if a sufficient ethanol extract can be obtained without pulverizing Hakutsuru Reishi, the pulverization step may be omitted.

The extraction step (S02) is a known extraction method that is usually used industrially and experimentally as long as Hakutsuru Reishi mushroom and ethanol are used (see, for example, Phytomedicine, Vol. 16, pp. 929-934, 2009). ). When extracting from Hakutsuru Reishi, it is preferable to use hydrous ethanol (a mixed solvent of ethanol and water) as a solvent, and more preferably to use 90% (w/w) ethanol.

The extraction temperature is generally 0-100°C, preferably 5-80°C. When the extraction is performed under boiling conditions of the solvent, it is preferable to perform the extraction while refluxing the solvent. The extraction time is about 1 hour to 10 days, and the amount of solvent is usually 1 to 30 times the weight, preferably 5 to 10 times the weight of the dry raw material each time. The extraction operation may be stirring or simple immersion. The extraction operation may be repeated multiple times as needed. Also, extraction operations with different conditions may be combined. Furthermore, it is preferable to remove insoluble residues from the crude extract obtained by the above operation by filtration or centrifugation.

In the solvent removal step (S03), the solvent (ethanol) is removed by vacuum drying, freeze drying, spray drying, drum drying, or the like, and the ethanol extract of Hakutsuru Reishi can be recovered as a dried solid (dried solid). can. The ethanol extract of Hakutsuru Reishi mushroom thus obtained can be used as the active ingredient composition of the present invention. In addition, when it is not necessary to remove the solvent at this stage in order to finally make it into a drug or food and drink (for example, when it is preferable to include ethanol as a material of the drug or food and drink), the solvent removal step is omitted. You may

EXAMPLES Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. Incidentally, in the following examples, the unit % of numerical values indicating the addition amount of various components means % by mass.

(Example 1) Production of Ethanol Extract of Hakutsuru Reishi First, 2 kg of dried roots of Hakutsuru Reishi (Rhinacanthus nasutus (L.) Kurz) were prepared as a raw material and pulverized with a general-purpose pulverizer. Subsequently, Hakutsuru Reishi was immersed in 10 L of 90% (w/w) ethanol, and the solvent was refluxed at 80° C. for 1 hour for reflux extraction to obtain an extract. The extract and extraction residue were separated by filter filtration to obtain a filtrate. Qualitative filter paper No. manufactured by Advantech Toyo Co., Ltd. was used for filtration. 2 was used. The extraction was performed a total of 3 times on the same raw material. The solvent was removed from the three filtrates under reduced pressure to obtain 77.8 g of solid dry matter of ethanol extract of Hakutsuru Reishi. In order to facilitate handling in the experiment, 9 times the amount (700.2 g) of dextrin was added to the ethanol extract of Hakutsuru Reishi mushrooms together with an appropriate amount of 90% (w/w) ethanol, and after mixing well, the The solvent was removed under reduced pressure to obtain an experimental active ingredient composition (a mixture of 10% ethanol extract of Hakutsuru Reishi and 90% dextrin). Pine Index #2 manufactured by Matsutani Chemical Industry Co., Ltd. was used as the dextrin.

(Example 2) Evaluation of IL-1β Blood Concentration Suppression Action, Amyloid-β Brain Concentration Suppression Action, Hair Improvement Action and Anti-Aging Action Using Aging Animal Model Selective Inbreeding of AKR/J Strains have generated senescence accelerated mice (SAMs). This mouse exhibits premature aging phenomena such as senile amyloidosis, senile osteoporosis, cataract, decreased immune response, learning and memory impairment. Senescence-accelerated mouse prone 8 (SAMP8) is a model that is widely used especially in dementia research because brain atrophy begins early and is accompanied by somnolence, memory impairment, and emotional disturbance (T. Takeda, et al., A new murine model of accelerated sensitivity, Mech. Aging Dev. 17 (1981) 183-194). The lifespan of SAMP8 is almost half that of the control strain SAM/resistant1 (SAMR1). Senescence-accelerated mouse SAMP8 is known to become a pathological model of Alzheimer's dementia while further accelerating aging and developing type II diabetes by giving a high-fat diet (Mehla et al., Experimental Induction of Type 2 Diabetes in Aging-Accelerated Mice Triggered Alzheimer-Like Pathology and Memory Deficits. Journal of Alzheimer's Disease 39:145-162, 2014).

3-1. Mice Male SAMP8/TaHsd (senescence accelerated mice) were obtained from Envigo RMS B. V. (Netherlands). According to the vendor's housing policy, SAMP8 mice are to be single-housed at 6 weeks of age. Mice were 4-7 weeks old upon arrival. 6-7 week old SAMP8 mice were single-housed prior to shipment and were assigned as Batch 1. 4-5 week old SAMP8 mice were single-housed at 5-6 weeks of age (post-quarantine) and assigned as Batch 2. All mice were housed singly in 365 x 207 x 140 mm cages (without caps) at a temperature of 23 ± 2°C with a 12 hour light/dark cycle from 7:30 am. Batch 2 mice began the experiment two weeks later than Batch 1 mice and were age-matched. For acclimatization, all mice were given a control diet (LFD, D12450Ji, Research Diets) from 17 days before the start of the test. Some mice in SAMP8 were switched to a high fat diet (HFD, D12492i, Research Diets) at 10-11 weeks of age at the start of the study. HFD and LFD were fed three times a week. Animal health was monitored daily. Water and food were given ad libitum. Daily food consumption was measured weekly. Water consumption was measured as 24-hour water intake every 2-3 weeks. Body weight was measured twice weekly for the duration of the study. The percentage change in body weight of mice was calculated. All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee of the Taiwan Institute of Industrial Technology (ITRI-IACUC-2019-069V1).

3-2. Administration method 10-11 week old male SAMP8 mice were divided into 3 groups (each group n=9, consisted of both batch 1 and batch 2, batch 2 had 4-5 animals/group). (1) LFD (low-fat diet) group, (2) vehicle (high-fat diet) group, and (3) RN30 (high-fat diet) group. Vehicle (high-fat diet) group and RN30 (high-fat diet) group (300 mg/kg/day of the experimental active ingredient composition prepared in Example 1, that is, 30 mg/kg/day of the ethanol extract of Hakutsuru Reishi) The group administered in ) was fed with a high-fat diet from day 0 of the start of the test, and the LFD group was fed with a low-fat diet. In addition, the LFD (low-fat diet) group and the vehicle (high-fat diet) group were administered 1500 mg/kg/day of dextrin, and the RN30 (high-fat diet) group was administered an experimental active ingredient composition of 300 mg/kg/day. Dextrin was administered at 1200 mg/kg/day along with day (30 mg/kg/day of ethanolic extract of Hakutsuru Reishi). As for the administration method, 5 mL/kg/day was orally administered using a sonde every day for 122 days (0-121 days after the start of the test). From day 122 to day 132, the study was continued without administration of the test substance, and the mice were sacrificed on day 133.

Statistical analysis is performed by analysis software EZR (R version 4.0.3, R Commander 2.7-1), FIG. 2 is repeated measures analysis of variance (Repeated measures ANOVA), FIGS. 3 to 6 are one-way ANOVA and Dunnett's Post-hoc analysis was performed. **p<0.01, ***p<0.001

3-3. Tissue Harvest Mouse serum and brain tissue were preserved when the mice were sacrificed. Mice were deeply anesthetized by inhalation of 2.5% isoflurane using an induction box and mask, and anesthesia was confirmed by toe pinch reflex. After thoracotomy, 0.35 mL of whole blood was collected from the right ventricle with a syringe (25G needle) and used as serum. For intracardiac perfusion, 15 to 20 mL (10 mL/min) of ice-cold artificial cerebrospinal fluid (ACSF) was injected from the left ventricle immediately after the right atrium was incised using a peristaltic pump. ACSF consisted of 124 mM NaCl, 2.5 mM KCl, _1.2 mM _NaH2PO4 , ₂₄ mM NaHCO3, 5 _mM HEPES, _12.5 mM glucose, ₂ mM MgSO4.7H2O, ₂ mM CaCl2.2H2O. . The ACSF pH was adjusted to 7.4 after oxygenation with carbogen (95% O ₂ /5% CO ₂ ) at room temperature. ACSF was stored at 4°C or on ice and reoxygenated with carbogen for 20 minutes before use. After intracardiac perfusion, the brain was rapidly harvested and separated into two hemispheres. The left hemisphere was snap frozen in liquid nitrogen and stored at -80°C. Whole blood samples were allowed to clot at room temperature for 2 hours and then centrifuged at 1000 xg for 10 minutes. Serum was collected and stored at -80°C.

3-4. Measurement of serum inflammatory factor interleukin-1β (IL-1β) It is known that the production of inflammatory cytokines such as IL-1 is increased in the elderly (Isobe et al. Geriatrics, 48:205-210, 2011, and Licastro et al., Innate immunity and influence in aging: a key for understanding age-related diseases. Immunity & Aging 2:8, 2005). An ELISA was performed to examine whether the administration of ethanol extract of Hakutsuru Reishi alters the levels of IL-1β in SAMP8 mice. Serum samples from each group were pooled, and IL-1β was detected using Abcam's High Sensitivity IL-1β ELISA Kit (abl97742). The results are shown in FIG. As shown in FIG. 2, IL-1β levels were significantly elevated (Dunnett, p<0.001) in the vehicle (high-fat diet) group compared to the LFD (low-fat diet) group, whereas RN30 (high-fat diet) The increase in IL-1β was significantly suppressed (Dunnett, p<0.001) in the vehicle (high-fat diet) group compared to the vehicle (high-fat diet) group.

3-5. Measurement of intracerebral amyloid-β Frozen (−80° C.) left hemisphere brain was treated with 800 μL of T-PER ^™ Tissue Protein Extraction Reagent (78510, Thermo Fisher Scientific) to which a protease inhibitor cocktail (K266-100, BioVison) was added. was homogenized with The homogenate was then centrifuged at 10,000 xg for 5 minutes. Supernatants were collected and stored at -80°C. The total concentration of amyloid-β _1-42 in the hemisphere was measured using an ELISA kit (Mouse amyloid beta peptide 1-42 ELISA Kit, CSB-E10787m, CUSABIO) according to the manufacturer's instructions.

SAMP8, an accelerated aging model mouse, is known to be a model of Alzheimer's disease whose etiology is increased oxidative stress due to high expression of amyloid-β (Morley et al., The sensitivity accelerated mouse (SAMP8) as a model for oxidative stress and Alzheimer's disease. Biochimica et Biophysica Acta 1822:650-656, 2012). FIG. 3 shows the results of evaluating the effect of the RN30 (high-fat diet) group on amyloid-β in the brain of SAMP8 mice. As shown in Figure 3, levels of amyloid-β in brain homogenate supernatants were significantly (Dunnett, p<0.01) elevated in the vehicle (high-fat diet) group compared to the LFD group, whereas RN30 ( The increase in amyloid-β was significantly suppressed in the high-fat diet) group compared to the vehicle (high-fat diet) group (Dunnett, p<0.01).

3-6. Hair Glossiness When the mice reached 22 weeks of age, they were observed for hair glossiness. The observation was visually performed and evaluated by a four-level score from 0 to 4 (the lower the score, the better). FIG. 4 shows the glossiness of fur scores at 22 weeks of age in each administration group. As shown in Figure 4, the vehicle (high-fat diet) group showed significantly higher scores than the LFD (low-fat diet) group (Dunnett, P<0.001). On the other hand, the RN30 (high-fat diet) group significantly suppressed this increase compared to the vehicle (high-fat diet) group (Dunnett, P<0.001).

3-7. Measurement of Aging Index In order to evaluate the degree of aging of SAMP8 mice, the degree of aging was obtained by summing the scores of 11 items (see Table 1) representing changes in behavior and appearance, including the glossiness of the hair, from 0 to 4. Evaluation scores were used. FIG. 5 shows the time course of the mean value (total aging index) of the 11-item senescence evaluation scores of senescence accelerated mice to which the active ingredient composition etc. of the present invention was administered.

The average score of each administration group gradually increased from 9 weeks of age to 25 weeks of age. Although there was no significant difference up to 15 weeks of age, there was a clear difference after 22 weeks of age (Fig. 5) (Repeated measures ANOVA; RN30 vs. vehicle: P < 0.01, LFD vs. vehicle: P <0.001). FIG. 6 shows the total average senescence evaluation score of each group at 22 weeks of age. As shown in Figure 6, the vehicle (high-fat diet) group showed significantly higher scores than the LFD (low-fat diet) group (Dunnett, P<0.001). On the other hand, the RN30 (high-fat diet) group significantly suppressed this increase compared to the vehicle (low-fat diet) group (Dunnett, P<0.001). The changes in scores were mainly centered around the categories of glossiness and density, which are indicators of hair appearance (see Table 2).

(Prescription example 1 tablet)
According to a conventional method, the following ingredients were mixed to produce tablets. The solid dried product of the ethanol extract of Hakutsuru Reishi obtained in Example 1 was used as the active ingredient composition.
Composition Active ingredient composition (Example 1) 150 mg
Cellulose 80mg
20 mg starch
Sucrose fatty acid ester 2mg
The above ingredients were mixed and tableted to obtain tablets.

(Prescription Example 2 Capsules)
According to a conventional method, the following ingredients were mixed to obtain soft capsules. The solid dried product of the ethanol extract of Hakutsuru Reishi obtained in Example 1 was used as the active ingredient composition.
Composition Active ingredient composition (Example 1) 100 mg
Beeswax 10mg
Grape seed oil 110mg
The above ingredients were mixed and filled into a gelatin and glycerin mixed capsule base to obtain a soft capsule.

(Prescription example 3 Beauty drinking water)
According to a conventional method, the following ingredients were mixed to prepare a cosmetic drinking water with a total weight of 100 g. The solid dried product of the ethanol extract of Hakutsuru Reishi obtained in Example 1 was used as the active ingredient composition.
Composition Active ingredient composition (Example 1) 500 mg
Coenzyme Q10 500mg
Vitamin B2 5mg
Vitamin B6 5mg
Nicotinamide 10mg
Vitamin E 100mg
L-ascorbic acid 100 mg
10g brown sugar corn syrup
1 g of citric acid
Perfume 100mg
Pigment 100mg
97.5 g of water

The application of a composition containing an ethanol extract of Hakutsuru Reishi mushroom has been shown to have inhibitory effects on blood level increases of IL-1β and brain levels of amyloid-β in evaluations using high-fat diet-administered accelerated aging model mice. , it was confirmed that it exhibits a hair-improving action and an anti-aging action. Therefore, the orally ingestible IL-1β blood level increase inhibitor, orally ingestible amyloid-β brain level increase inhibitor, orally ingestible hair-improving agent, and orally ingestible anti-aging agent of the present invention can be used as pharmaceuticals, foods and drinks. In particular, there is a possibility that it can be used as a food and drink for health promotion.

Claims (5)

including the step of obtaining an extract extracted from Hakutsuru Reishi mushrooms with a solvent in which the mass of ethanol is 30% or more of the total mass, containing the extract as an active ingredient and orally ingesting IL-1β caused by aging A method for producing an age-related IL-1β blood level elevation inhibitor for oral intake that suppresses an increase in the blood level of IL-1β. 2. The method for producing an age-related IL-1β blood level increase suppressor for oral intake according to claim 1, wherein said extract is an extract with water-containing ethanol. 3. The aging-related IL-1β for oral intake according to claim 1 or 2, wherein the agent for suppressing an increase in blood concentration of aging-related IL-1β for oral intake is in the form of a pharmaceutical, a food or drink, a feed, or an active ingredient composition blended therein. A method for producing a 1β blood level elevation inhibitor. 4. The method for producing an age-related IL-1β blood level increase suppressor for oral intake according to claim 3, wherein the food or drink is a food or drink for promoting health. The age-related IL-1β blood level increase inhibitor for oral intake suppresses the increase in blood level of IL-1β due to aging by oral intake, thereby reducing chronic obstructive pulmonary disease, autoinflammatory disease, and rheumatoid arthritis. , osteoarthritis, gouty arthritis and at least one of arteriosclerosis . A method for producing a blood level increase inhibitor.

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