WO2001032192A1

WO2001032192A1 - Active substance in ganoderma lucidum extract and drugs, health foods and cosmetics containing the same

Info

Publication number: WO2001032192A1
Application number: PCT/JP2000/007662
Authority: WO
Inventors: Kenji Sakamoto
Original assignee: Sakamoto Bio Co., Ltd.
Priority date: 1999-11-01
Filing date: 2000-10-31
Publication date: 2001-05-10
Also published as: JP2001131083A

Abstract

An active substance which has a hypoglycemic effect, etc. and is useful as drugs, health foods and cosmetics. Namely, an active substance obtained from Ganoderma Lucidum, an extract thereof or a water- or lower alcohol-extract thereof; and drugs, health foods or cosmetics containing the same.

Description

Specification

Kazuno Reishi Extract Active Substance and Pharmaceutical, Health Food, and Cosmetics Containing It

Technical field

The present invention relates to an active substance obtained from Kazuno Reishi or Kazuno Reishi extract, particularly an extract with water and / or an organic solvent, more preferably a water and / or lower alcohol extract, and a pharmaceutical or health food containing the same. And cosmetics.

Background technology

Perennial mushroom (Lingzhi) is a basidiomycete belonging to the family Sarcodonaceae, and has been prized as a remedy for immortality since ancient times. Kazuno Reishi is a type of Reishi, but it has the shape of a deer horn and extends in a branch without a shade. It has also been reported that other substances, including 3-D-glucan, are contained at higher concentrations than Reishi.

Disclosure of the invention

The present invention is an active substance obtained from Kazuno Reishi or an extract of Kazuno Reishi or a solvent extract of Kazuno Reishi. In the present invention, the extract is preferably an extract using water and an organic solvent such as Z or an alcohol represented by a lower alcohol in particular. The present invention also relates to a medicament containing the active substance, particularly a medicament for lowering blood glucose, lowering blood pressure, and preventing or treating tumors such as cancer. Further, the present invention is a composition for enhancing immunity and a health food containing the above-mentioned active substance. Furthermore, the present invention relates to a cosmetic containing the active substance, particularly for suppressing melanin production. Cosmetics. '' Brief description of the drawings

FIG. 1 shows the results of a blood glucose lowering test in a rat using the active substance-containing Kazuno Reishi extract of the present invention.

FIG. 2 shows the results of a blood pressure reduction test in rats using the active substance-containing Kazuno Reishi extract of the present invention.

FIG. 3 is a photomicrograph of the result of the B-16 cell melanogenesis inhibition test as a control (0.6% methanol).

FIG. 4 is a micrograph of the results of a melanin production inhibition test on B_16 cells using the active substance-containing Kazuno Reishi extract (0.6% Kazuno Reishi methanol extract) of the present invention.

FIG. 5 is a micrograph of the results of a test for inhibiting the production of melanin in B_16 cells using arbutin. BEST MODE FOR CARRYING OUT THE INVENTION Kazuno Reishi is commercially available. Kazuno Reishi can also be cultivated according to the method described in Japanese Patent Publication No. 57-39605 and Japanese Patent Application Laid-Open No. 11-146728. However, it is not limited to these methods, and these methods are not the best.

The method of cultivation described in Japanese Patent Publication No. 57-39605 is described in "Mushroom cultivation using a transparent hood that is longer than the fruit body at the completion of growth when growing the fruit body. The hood is narrowed below the stem of the fruit body to cover the culture medium surface, and the hood above the stem is expanded upward to surround the upper part of the fruit body, and only the upper surface is open above the fruit body. Child A method for cultivating ornamental mushrooms characterized by providing a physical growth space "

(Claims). In other words, when growing the perennial mushrooms, enclose the sides of the perennial mushrooms with a transparent food, create an environment for growing the perennial mushrooms in this enclosed space, and open the upper part of the hood to improve air exchange. By doing so, the perennial mushrooms are extended upward. The culture medium is mainly a mixture of beech and oak wood waste. After sterilizing the container filled with the culture medium, it is cooled and inoculated with the inoculum to culture the mycelium. Germinating fruiting bodies by adjusting temperature and humidity conditions. When the fruiting body is completely germinated, the temperature is adjusted, natural light is taken in, and water is appropriately replenished using the food as described above to grow the fruiting body.

The cultivation method described in Japanese Patent Application Laid-Open No. H11-114647228 discloses that a bag made of an airtight sheet is filled with sawdust and a cylindrical cap is mounted on an upper portion of the bag to open the bag. After forming a cultivation container having a portion and inoculating a perennial mushroom inoculum in the sterilized cultivation container, the cultivation container having the opening closed with a flocculent is cut off from direct sunlight and outside air. When the mycelium generated from the inoculum spreads in the cultivation container, the cotton-like body is removed, the cultivation container is covered with an airtight sheet, and the open air is kept in a closed state. A method of cultivating perennial mushrooms, which comprises extending perennial mushrooms from the outside. "(Claim 1)

As the sawdust, those generated from Quercus serrata, Quercus, Plum, Sakura etc. are mentioned as suitable, and it is described that rice bran, bran, soybean power, sugar and water may be added to these. I have. The temperature and humidity for holding the cultivation vessels are 18 to 21 and 70 to 80% in the early stage of perennial mushroom development, and 23 to 28% in the growth stage. It is desirable to set it to 0 to 90% I have. In the early stage of the outbreak: The opening of the cultivation container is closed by a cotton-like material such as cotton, and the inside is kept insulated from the outside air. Once the hypha has spread, the floc is removed and the cultivation vessel is covered with a vinyl sheet to keep it out of the open air and protected from oxygen under conditions of no direct sunlight.

The water extract of Kazuno Reishi can be produced by chopping Kazuno Reishi and adding it to water and boiling it down. In addition, the water extract of Kazuno Reishi can be produced by adding Kazuno Reishi to distilled water and allowing it to stand, adding Kazuno Reishi to distilled water and letting it to boil before standing. The lower alcohol extract of Kazuno Reishi can be produced by adding Kazuno Reishi to a lower alcohol such as methanol or ethanol and allowing it to stand. Insoluble components can be removed from these solutions by centrifugation or the like. In addition, the solution can be lyophilized to a powder. However, the method of extracting Kazuno Reishi extract is not limited to extraction using water or lower alcohol as a solvent, but also a mixed aqueous solution of water and other substances such as organic solvents, higher alcohol, alcohol and other substances. Includes extraction with mixtures, other organic solvents, any other solvent, and extraction by other methods. These contained the active substance of the present invention. This active substance can be obtained from the extract of Kazuno Reishi using known separation and purification means such as gel filtration column chromatography and liquid chromatography.

The active substance obtained from the extract of Kazuno Reishi of the present invention, in particular, an extract with water or an organic solvent, more preferably an extract with water or a lower alcohol, can be used for medicines, health foods, and cosmetics. It has a blood glucose lowering effect and can be used as a therapeutic drug for diabetes and a health food for the prevention of adult diseases. In addition, it has an anti-tumor effect such as an immunopotentiating effect and anti-cancer. It can be used as a composition for enhancing immunity and as a medicament for preventing and treating tumors such as cancer, and can also be used in health foods for enhancing immunity. Furthermore, it has a blood pressure lowering effect and can be used as a drug for treating hypertension or as a health food for preventing adult diseases.

The extract of Kazuno Reishi of the present invention, in particular, an extract using water or an organic solvent, more preferably an active substance obtained from water or a lower alcohol extract, has an action of inhibiting melanin production, and is a whitening skin cosmetic. It can be used for cosmetics such as cosmetics and bath salts.

The medicament of the present invention can be administered orally or parenterally, and can be in the form of a pharmaceutical preparation suitable for each. Examples of the pharmaceutical preparations include liquid preparations such as syrups, injections, inhalants and emulsions, solid preparations such as tablets, powders, granules and capsules, and external preparations such as suppositories. Various additional components such as excipients, stabilizers, wetting agents, emulsifiers, absorption enhancers, pH adjusters or surfactants can be used in pharmaceutical preparations. These additives include, for example, sugars such as starch and lactose, magnesium sulfate, talc, gelatin, cellulose derivatives such as hydroxypropylcellulose, vegetable oils such as soybean oil, sesame oil, animal oil or synthetic oil, rubber, Examples thereof include water and alcohols such as polyalkylene alcohol.

The pharmaceutical preparation may contain 0.01 to 100% by mass of the active ingredient, preferably 0.1 to 30% by mass for injection, and 25 to 98% for tablets and the like. Active ingredient content in% by weight. Dosage will be varied as appropriate based on the age and weight of the patient, the route of application, the course of the disease and the concurrent treatment. It can be administered once a day or divided into two or three times a day, but is not limited thereto. Health foods are manufactured, for example, as confectionery, beverages such as soft drinks, vegetable or fruit supplements, livestock meat products, seasonings, etc. Its form is powder, solid product, solution and the like. The content of the active ingredient can be appropriately changed depending on the purpose, product form and the like.

Cosmetics are manufactured, for example, as skin cosmetics such as mouth lotions, emulsions, and creams, packs, and bath additives. The amount of the active ingredient to be added can be appropriately changed according to the purpose and the form of the product. Other additives include hydrocarbons such as petrolatum, higher alcohols such as jojoba oil, olive oil, and ethanol, antioxidants such as tocopherol, propyl gallate, and butylhydroxytoluene, surfactants, and ultraviolet absorption. Agents, fragrances, lower alcohols such as ethanol, water, coloring agents, organic acids such as citric acid, inorganic acids such as phosphoric acid, thickeners, sequestering agents, water-soluble polymer substances, etc. Can be. As the ultraviolet absorber, there can be used, for example, paramethoxysilicic acid-2-ethoxyhexyl, paramethoxysilicic acid ethylhexyl, salicylic acid-2-ethylhexyl, paraaminobenzoic acid and the like. Other whitening ingredients may be further added, and examples thereof include ascorbic acid or a derivative thereof, kojic acid or a derivative thereof, licorice extract, hydroquinone derivative, and daltathione.

Example

Production of active substances obtained from Kazuno Reishi extract

In Examples 1 to 7 below, “Kakuno Reishi” (trademark, manufactured by Sakamoto Bio Farm Co., Ltd.) was used as Kazuno Reishi.

Example 1

10 g of Kazuno Reishi was chopped, added to 200 ml of water, and boiled for about 1 to 2 hours to 100 ml. Insoluble components due to centrifugation The extract of Kazuno Reishi containing the active substance was obtained.

Example 2

10 g of Kazuno Reishi was chopped, added to 200 ml of water, and boiled for about 1 to 2 hours to bring it down to 100 ml. The insoluble components were removed by centrifugation, and the obtained Kazuno Reishi extract was lyophilized to remove water, thereby obtaining a powder containing the active substance.

Example 3

2 g of Kazuno Reishi was chopped, added to 200 ml of water, boiled for about 1 to 2 hours, and boiled down to 100 ml. The insoluble components were removed by centrifugation to obtain a Kazuno Reishi extract containing the active substance.

Example 4

10 g of Kazuno Reishi was weighed, 30 ml of distilled water was added thereto, and the mixture was allowed to stand for 30 minutes. This solution was centrifuged (40000 rpm, 20 minutes), and the supernatant was filtered through a filter paper to remove insoluble components, thereby obtaining a Kazuno Reishi extract containing the active substance.

Example 5

10 g of Kazuno Reishi was removed, 30 mL of distilled water was added thereto, and the mixture was boiled up and left to stand for 30 minutes. This solution was centrifuged (40000 rpm, 20 minutes), and the supernatant was filtered through a filter paper to remove insoluble components, thereby obtaining a Kazuno Reishi extract containing the active substance.

Example 6

10 g of Kazuno Reishi was removed, 30 ml of ethanol was added thereto, and the mixture was left for 30 minutes. This solution was centrifuged (4,000 rpm, 20 minutes), and the supernatant was filtered through a filter paper to remove insoluble components, thereby obtaining a Kazuno Reishi extract containing the active substance. 10 g of Kazuno Reishi was weighed, and 3 O ml of methanol was added thereto and left for 30 minutes. This solution was centrifuged (40000 rpm, 20 minutes), and the supernatant was filtered through a filter paper to remove insoluble components, thereby obtaining a Kazuno Reishi extract.

The active substance can be obtained by separating and purifying the extract and powder (powder) of Kazuno Reishi extract obtained in the above example by gel filtration column chromatography and reverse phase chromatography.

Test Example 1 Blood glucose lowering effect

The blood glucose-lowering effect of the Kazuno Reishi extract containing the active substance produced in Example 1 was evaluated in rats (Wistar, 5 animals per group, body weight 250-400 g, average body weight 300 g). ). Oral administration of 1.2 ml (in order to increase blood glucose) of 3 ml of Kazuno Reishi extract and 50% solution of glucose to the rat, and the blood glucose level with time before administration and up to 2 hours after administration The change was measured. Blood glucose was measured by collecting blood and measuring blood glucose using a blood glucose measuring device "Antosen" (trade name: manufactured by Sankyo Co., Ltd.). The control group received water containing no Kazuno Reishi extract. The results are shown in FIG. As is clear from FIG. 1, it was confirmed that the active substance-containing Kazuno Reishi extract of the present invention has an action of suppressing an increase in blood glucose level.

Test Example 2 Immunity enhancing and anticancer effects

The anticancer activity of the Kazuno Reishi extract containing the active substance produced in Example 2 was tested using macrophages derived from rat (Wistar species). That is, the anti-cancer effect of macrophage on L929 cells, which are cancer cells, was tested. It is believed that the anti-cancer effect of macrophages is due to the production of TNFα, a substance having an anti-cancer effect. Macrophages are bone extracted from the rat femur. The pith was obtained by culturing the pith in RPMI 1640 (FBS · 5%) and separating the cells attached to the culture flask. Dilute Kasumi Reishi extract with lmg / m 1, and as a positive control, dilute the anticancer drug picibanil (a substance that enhances macrophage TNFα production) with the medium to 0.1 mgZml and react with macrophages for 6 hours. The culture medium supernatant thus obtained was used as a sample. As a negative control, the same amount of water was added to the medium, and the medium supernatant reacted with macrophages for 6 hours was used as a control.

The L929 cells were treated with actinomycin D to select malignant cells and cultured in E-MEM (5% FBS). Samples and controls were diluted (4-, 16-, 64-, 256-, and 124-fold dilutions), and the malignant cells were reacted with the selected L929 cells. After 18 hours, the viable cells of the L 9 29 cells were stained with crystal violet, and the amount of viable cells was evaluated based on the amount of the stained dye. The anticancer effect was determined by calculating the dilution ratio at which 50% of the cells were killed based on the curve in the graph of the dilution ratio of the sample and the control and the amount of living cells of L929 cells. 50. Table 1 shows the results.

Table 1

As is evident from Table 1, the Kazuki Reishi extract containing the active substance has the same level of anticancer activity as Picibanil against cancer cells. This effect is considered to be due to TNF produced in the culture medium by macrophages, and since this anticancer effect can be said to be due to enhancement of macrophage function, it also has an immunopotentiating effect and an antitumor effect. It can be understood that it is.

Test Example 3 Blood pressure lowering effect

The blood pressure lowering effect was tested using the Kazuno Reishi extract containing the active substance obtained in Example 3. Rats (SHR species, 10 animals per group, body weight 250-408, average body weight 300 g) were orally administered with 3 ml of the above Kazuno Reishi extract, and administered. The time course of blood pressure before and up to 2 hours after administration was measured. Blood pressure was measured in the tail artery using a KN-210 rat tail artery, pulse meter (Natsume Seisakusho). The average of the measured values was taken as the blood pressure value. Water was administered to the control group. The results are shown in FIG. As is clear from FIG. 2, it was confirmed that the Kazuno Reishi extract containing the active substance of the present invention has a blood pressure lowering effect. Test Example 4 Melanin production inhibitory effect

With respect to the Kazuno Reishi extract containing the active substance obtained in Examples 4 to 7, the melanin production inhibitory effect was tested using B-16 cells, which are melanin producing cells. B- 16 cells were reacted with Kazuno Reishi extract and the melanin production was observed. For B-16 cells, wash 70% confluent cells with PBS, add trypsin to detach them, add the same amount of trypsin to serum-containing medium (E-MEM + 10% FBS), and collect cells. Centrifuge (800 rpm, 5 minutes). Counting the number of cells were adjusted to about ^{1 0 4 ce 1 1 s /} m 1 (E -MEM + 1 0% FBS), and the cell suspension. To dilute the samples (water extract, hot water extract, ethanol extract, and methanol extract in Examples 4 to 7, respectively), dispensed 201 sterile PBS into cell culture plates (for adherent cells). And Take 20 // 1 of each extract and dilute it twice (extract 10% Z 5% Z 2.5% / 1.25% / 0.625% / 0.33% / 0% . 156% / 0.0778%). As controls, arbutin (500 / xM), PBS, ethanol, and methanol were similarly diluted. The cell suspension was dispensed to the plate containing the diluent at a rate of 800 1. Furthermore, the cells were cultured at 37 ° C in a C〇2 incubator for 72 hours, and melanin production in B-16 cells was observed under a microscope. B- 16 cells appear black when melanin is produced. Micrographs of the results are shown in FIGS.

As shown in FIGS. 3 to 5, the Kazuki Reishi extract containing the active substance has an inhibitory effect on melanin production on B-16 cells. As shown in the photomicrographs in Figs. 3 to 5, in the case of Kazuki Reishi extract containing the active substance, the blackness of B-16 cells was suppressed more than in the case of arbutin, indicating excellent inhibition of melanin production. It was found that it had an effect. Industrial applicability

According to the present invention, there is provided an active substance obtained from an extract of Kazuno Reishi, especially an extract with water or an organic solvent, more preferably an extract with water or a lower alcohol, which is useful as a medicine, a health food, and a cosmetic. The active substance obtained from the Kazuno Reishi extract of the present invention has a blood glucose lowering effect, an immunopotentiating effect, an antitumor effect including anticancer, a blood pressure lowering effect, a melanin production inhibitory effect, a therapeutic agent for diabetes, and an immunopotentiating effect. It is useful as an agent, a prophylactic / therapeutic agent for cancer, a blood pressure lowering agent, a health food for enhancing immunity, a health food for preventing adult diseases, a whitening cosmetic, and a bathing agent.

Claims

The scope of the claims

1. Active substance obtained from Kazuno Reishi or Kazuno Reishi extract or Kazuno Reishi solvent extract, respectively.

2. The active material according to claim 1, wherein the solvent is water, Z or an organic solvent.

3. The active substance according to claim 2, wherein the organic solvent is an alcohol.

4. The active substance according to claim 3, wherein the alcohol is a lower alcohol.

5. A medicament comprising the active substance according to claims 1 to 4.

6. The medicament according to claim 5, which is used for lowering blood glucose.

7. The method according to claim 5, which is for preventing or treating a tumor including cancer.

8. The medicament according to claim 5, which is used for lowering blood pressure.

9. An immunopotentiating composition comprising the active substance according to claims 1 to 4.

10. A health food comprising the active substance according to claims 1 to 4. 1 1 A cosmetic comprising the active substance according to claims 1 to 4.

12. The cosmetic according to claim 11, which is used for suppressing melanin production. 1 3 Claims One or more substances that constitute active substances 1 to 4