JPH11302191A - Immunoactivator and antitumor agent containing extract from lyophyllum decastes (fr.) sing. as active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an inexpensive immunoactivator which has immunopotentiation and antitumor activities and is free from adverse effects, e.g. acute toxicity, mutagenicity and the like, by including a specific extract (purified product) from Lyophyllum decastes (Fr.) Sing. as an active ingredient. SOLUTION: This immunoacrtivator is obtained by including an extract (purified product) from Lyophyllum decastes (Fr.) Sing. which contains polymeric polysaccharides as an active ingredient. Lyophyllum decastes (Fr.) Sing. is a mushroom belonging to the genus Lyophyllum of the family Tricholomaceae. As the Lyophyllum decastes (Fr.) Sing., the fungal strain, 'Kameyama No. 1' (nursery law form registration application number 6811) of Lyophyllum decastes (Fr.) Sing. capable of artificially cultivating is preferably used. As the culture medium component for cultivation, the component containing chitins (crab shells), beer yeast lees or the like known as substances having biological response modifier-like effect is favorable. The extract from Lyophyllum decastes (Fr.) Sing. is obtained by extracting fruit bodies of Lyophyllum decastes (Fr.) Sing. with hot water. As the active ingredient, the component which is a water-soluble yellowish grown powder and contains saccharides and proteins in amounts of 30-90 wt.% and 5-15 wt.%, respectively, is favorable.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an immunostimulant / anti-tumor agent comprising a Hatakeshimeji extract or a purified product thereof as a main component,
And a method of manufacturing the same.

[0002]

2. Description of the Related Art Various polysaccharides and protein polysaccharides derived from Basidiomycetes are known to exhibit antitumor effects (Hitoshi Ito, Shin Keishiro: Antitumor polysaccharide, Infectious Disease No. 75, pp. 18-22). , 19
1984). Among these, there are substances that have been shown to have an effect of regulating the function of the biological defense mechanism mainly of the immune system, and if they exhibit an antitumor effect through this regulating action (immunomodulatory action). It is considered.

[0003] At present, these substances are regarded as one of biological response modifiers (BRM). As existing immunotherapeutic agents, “Krestin” of Kawatake mushroom extract, “Lentinan” of Shiitake mushroom extract, and “Shizophyllan” of Suehirotake mushroom extract have been approved and put to practical use. However, it is not possible to increase the immunostimulatory activity of all individuals alone and to expect a sufficient antitumor effect. Therefore, multidrug immunotherapy (using several BRMs) has been adopted. Is coming.

[0004] For the extract of Hatake shimeji, Oji Paper Co., Ltd. Forest Resources Research Institute announced the anti-cancer effect (Summary of the 17th National Tree-planting Festival Forestry Exchange Meeting: p11-13 (199)
3), Mitsui Graph 97: p8-9 (1994)).
A group of Ikegawa et al. Of Kanazawa University reported that "Yamabiko Takashimeji" (artificial cultivation of Hatakeshimeji) has antitumor activity against Sarcoma 180 transplanted cancer in the abdominal cavity of mice (Abstracts of the Japanese Cancer Society) , 1997). At the same time, the group of Ikegawa et al. Reported that oral administration of amylase-treated Hatakeshimeji to mice also had an antitumor effect.

[0005] On the other hand, a purified product of the extract of Hatake shimeji has not yet been reported on its production method or its anticancer effect. In addition, in the case of basidiomycetes of artificially cultivated products, it is known that the components in basidiomycetes vary greatly depending on the medium components and the cultivation method. Not always promising.
And it was not known that an extract of an artificially cultivated product using Hatakeshimeji “Kameyama No. 1” as a seed fungus and a purified product thereof had a very high antitumor activity.

A high-yield and efficient artificial cultivation method for Hatake shimeji has already been established by Oji Paper Co., Ltd. Forest Resources Laboratory (Japanese Patent Publication No. 5-15404, Patent 1).
No. 969534), and by using this method, artificially cultivated products using "Kameyama No. 1" of Hatake shimeji as a seed fungus can be supplied at low cost.

[0007]

SUMMARY OF THE INVENTION For a successful multi-drug immunotherapy capable of responding to various patients, a novel and powerful BRM is required.
In actual use, an inexpensive substance free from side effects such as acute toxicity and mutagenicity, unlike conventional anticancer drugs, has been demanded. In BRM derived from basidiomycetes,
From the viewpoint of cost, cultivation periods are short, and cultivars that can be artificially cultivated in large quantities and have high immunostimulating activity and antitumor activity have been demanded.

[0008]

Means for Solving the Problems As a result of the inventor's intensive search, Hatake shimeji, a long-time popular edible, especially Hatake-shimeji, which can be artificially cultivated in large quantities, is used as a seed fungus. It was found that the extract of the cultivated product had strong immunostimulating activity and antitumor activity. In addition, the inventors have found that a purified product of Hatake shimeji mushroom extract also has immunostimulating activity and antitumor activity, and completed the invention. Accordingly, the present invention provides an immunostimulant comprising, as an active ingredient, a Hatake shimeji extract or a purified product thereof comprising a high molecular polysaccharide. In a preferred embodiment, the present invention provides an immunostimulant wherein the Hatake shimeji is an artificially cultivated product having "Kameyama No. 1" as a seed bacterium.

In another aspect of the present invention, the active ingredient has the following properties: (a) color and form: tan powder (b) chemical ingredient: sugar content 30-90%, protein content 5-5
15% (c) Solubility: Provides an immunostimulant which is an extract having water solubility. In another embodiment of the present invention, the active ingredient has the following properties: (a) color and form: white to tan powder (b) chemical ingredient: sugar content 60 to 100%, protein content 0
(C) Solubility: Provided is an immunostimulant which is a purified product of a water-soluble extract.

[0010] The present invention also provides an antitumor agent comprising, as an active ingredient, Hatakeshimeji extract or a purified product thereof comprising a high-molecular polysaccharide. The present invention, in a preferred embodiment,
The present invention provides an antitumor agent, wherein the Hatake shimeji is an artificially cultivated product using "Kameyama No. 1" as a seed fungus. In another embodiment of the present invention, the active ingredient has the following properties: (a) color and form: tan powder (b) chemical ingredient: sugar content of 30 to 90%, protein content of 5 to 5%
15% (c) Solubility: Provides an antitumor agent which is an extract having water solubility.

In another embodiment of the present invention, the active ingredient has the following properties: (a) color and form: white to tan powder (b) chemical ingredient: sugar content 60 to 100%, protein content 0
(C) Solubility: Provides an antitumor agent which is a purified product of a water-soluble extract. The present invention further relates to a method for producing an immunostimulating agent, comprising extracting an fruit body of Hatake shimeji with hot water to obtain an extract, and further purifying the extract if desired. The present invention further relates to a method for producing an antitumor agent, which comprises extracting an fruit body of Hatake shimeji with hot water to obtain an extract, and further purifying the extract if desired.

Hereinafter, the present invention will be described in detail. Hatake shimeji (Lyophyllum decastes (Fr.) Sing.) Is a mushroom belonging to the genus Shimeji, belonging to the genus Oribatidae. Natural products occur in relatively familiar places such as gardens and fields in the form of stocks (Ikuseki Rokuya,
Tsuguo Hongo: Primary color Japanese fungi illustration book (1), Nursery, 1987
Year).

The Hatakeshimeji used in the present invention is an artificially cultivable Hatakeshimeji "Kameyama No. 1" (Seed and Seedling Variety Registration Application No. 6811, the type of agriculture, forestry and fisheries plants: Hatakeshimeji, the name of the applied variety: Kameyama No. 1) ) Is optimally used as the inoculum. Further, B as a culture medium component
It is preferable to use chitin (crab shell), brewer's yeast cake, and the like, for which an RM-like effect has been reported. Hatake shimeji is a cultivation method established by Oji Paper Co., Ltd.
It is preferable to use "Kameyama No. 1" as a seed bacterium in Japanese Patent No. -15404, Patent No. 1969534). Alternatively, Hatakeshimeji, a registered trademark of “Shakinko”, cultivated and sold in the same manner can be used as a raw material.

The extract of the present invention is obtained by extracting the fruit body of Hatake mushroom with hot water. The fruiting body may be extracted with hot water as it is, or may be dried and extracted with hot water. Further, the fruiting body may be extracted with hot water as it is, or may be extracted with hot water by pulverizing it in advance. The heating method may be any of reduced pressure, normal pressure, and pressurization, but it is better to perform high-temperature treatment under pressure for extraction efficiency. The heating temperature can be set in the range of 70 to 260 degrees. The extraction time may be 2 to 4 hours at normal pressure, or 15 minutes to 2 hours if carried out under high temperature and pressure. After the hot water extraction treatment, the Hatake shimeji extract has a solid content of 0.5% to 10%.
It is obtained as an extract.

The Hatake mushroom extract of the present invention can be obtained by concentrating and drying the above extract. The extraction extract may be concentrated by any of the heat concentration method, the heat concentration method under reduced pressure, and the concentration method by ethanol precipitation. The concentrated extract extract may be dried by any of an air drying method, a heat drying method, a spray drying method, and a freeze drying method.

The purified product of the extract of Hatake shimeji of the present invention comprises:
What is necessary is just to carry out according to the normal purification method of a high molecular polysaccharide.
The Hatake mushroom extract is dissolved in pure water and adsorbed on an ion exchange column (Q-Sepharose, etc.). Next, the Hatake mushroom extract adsorbed on the ion exchange column can be purified by gradient elution with varying concentrations of potassium chloride. The purified product of the fractionated Hatake shimeji extract,
Further purification can be performed by fractionation by a gel filtration method utilizing a difference in molecular weight. The purified Hatake shimeji extract is concentrated and dried by the above-described method except for salts and buffers used in the purification method by dialysis, ethanol precipitation, or the like.

The Hatake shimeji extract and its purified product of the present invention can be used in a form convenient for obtaining a medicinal effect according to the symptoms of the disease, and can be used alone or as a mixture with a pharmaceutically acceptable diluent and other drugs. Can be used as The Hatake mushroom extract and its purified product can be provided in a dosage unit form. It contains an effective amount of the active ingredient, and is in the form of powder, granules, tablets, sugar-coated tablets, capsules, syrups, pills, suspensions, liquids, emulsions and the like for oral use. For parenteral use, ampules and bottles of injections can be made. It can also be used as a suppository.

The diluent may be solid, liquid or semi-solid, and includes, for example, the following. That is, excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, fragrances, preservatives, solubilizers, solvents and the like. Specifically, lactose, sucrose, sorbit,
Mannitol, starch, precipitated calcium carbonate, heavy magnesium oxide, talc, calcium stearate, magnesium stearate, cellulose or derivatives thereof, amylopectin, polyvinyl alcohol, gelatin, surfactant, water, saline, ethanol, glycerin, Propylene glycol, cacao butter, laurin butter, vaseline, paraffin, higher alcohols and the like.

The ratio of Hatakeshimeji extract and its purified product used in the composition of the immunostimulant and antitumor agent of the present invention is generally 1 to 100% by weight, and is 5 to 80 watts.
It is desirable to contain t%. The extract of Hatake shimeji and its purified product are orally or parenterally administered to humans and animals, but oral administration is preferred. Oral administration includes sublingual administration, and is performed by parenteral administration (for example, subcutaneous, intramuscular, intravenous injection, infusion) and rectal administration. The dose of the extract of Hatake shimeji and its purified product is affected by animals or humans, and is affected by age, individual differences, medical conditions, etc.Therefore, doses outside the following ranges may be administered. When targeting, weight 1kg, 1
1 mg to 10 g per day, preferably 10 mg to 10 g
0 mg is administered in 1 to 4 divided doses.

The extract of Hatake shimeji of the present invention and its purified product have no acute toxicity, no mutagenicity, immunostimulating activity,
Since it has antitumor activity, it is useful as those pharmaceuticals. The extract of Hatake shimeji of the present invention and its purified product may be used as they are, or may be mixed with excipients or other pharmaceuticals. If mixed, 30
It is preferable to mix at a ratio of ８０80 wt%.

[0021]

The present invention will be described below in detail with reference to examples.

Embodiment 1 Artificial cultivation of Hatake shimeji mushroom bark compost (manufactured by Nakanihon Agricultural Products Co., Ltd.): After mixing rice bran: crab shell in a ratio of absolute dry weight of 100: 20: 4, 850 ml of a culture medium having a water content of 62% is mixed. 620 g was filled in a polypropylene cultivation bottle. In order to supply air to the whole culture medium in the bottle and improve the growth of mycelia, a hole with a diameter of 2 cm was made from the opening to the bottom of the bottle, and sterilized in a high-pressure sterilization pot (120 ° C, 1 hour). . After the temperature of the culture medium was cooled to 25 ° C. or lower, Hatakeshimeji “Kameyama No. 1” was inoculated in a clean room.

The cells were cultured in a room adjusted to a room temperature of 23 ° C. and a humidity of 70% (RH) for 50 days, and the mycelium was sufficiently spread on the culture medium. After the bacteria were scraped and water was supplied, the opening was covered with the above-mentioned bark compost so as to have a thickness of 1 to 2 cm.
Further, the coated culture bottle is placed at room temperature 23 ° C. and humidity 95% (R
The cells were cultured in the room H) for 7 days. Next, the covering portion of the surface layer where the hypha did not enter was removed, and the room temperature was 17 ° C and the humidity was 95% (R
H), the cultivation was continued in a room adjusted to the condition of 150 lux of illuminance, and 120 days per bottle in the culture 85 days after inoculation of the inoculum.
g fruit bodies were harvested.

100 g of the harvested artificially cultivated Hatakeshimeji mushroom fruit body was placed in an enamel pot, and 1 L of water was added thereto.
Extraction was performed by heating for a period of time to remove a Hatakeshimeji mushroom residue, thereby obtaining a brown Hatakeshimeji extract extract. An equal amount of ethanol was added to the Hatake mushroom extract, the precipitate was recovered, and distilled water was added to the precipitate to dissolve again. Next, the mixture was concentrated by heating under reduced pressure to obtain a concentrated extract having a solid content of 15%.
The concentrated extract was freeze-dried to obtain 2 g of a tan extract powder. The physicochemical properties of the extracted extract vary slightly depending on the state of the mushroom, but are generally as follows.

(A) Color and form: yellow-brown powder (b) Chemical components: sugar content 70%, protein content 10% (c) Solubility: water-soluble

Embodiment 2 FIG. The Hatake shimeji extract of Example 1 was dissolved in pure water, and ion-exchange column (Q-Sepharose, etc.) was used.
Was adsorbed. Next, the Hatake mushroom extract adsorbed on the ion exchange column was subjected to a potassium chloride concentration of 5 mM to 350 m.
Purification was performed by eluting stepwise to M. The fractionated purified product of the Hatake shimeji extract was further fractionated by a gel filtration method utilizing a difference in molecular weight to be further purified into 11 fractions. The purified product of the purified Hatake shimeji extract was dialyzed to remove salts and buffers used in the above-mentioned purification method. The physicochemical properties of the 11 fractions are shown in Table 1.

[0027]

[Table 1]

Embodiment 3 FIG. Macrophage activation test Using the Hatakeshimeji extract extract (EX) of Example 1 and the purified product of the Hatakeshimeji extract (F-5) of Example 3 as samples, 0.2% dextran was dissolved in physiological saline. Those were used as a control group.

The mice were 15-week-old mice (ICR / SL).
C system, ♀) was used. EX (0.5%) in physiological saline,
F-5 (0.1%) was dissolved in the mouse intraperitoneally in a volume of 0.1% each.
3 ml was administered. Two hours later, blood was collected under anesthesia. PBS (3 ml) was injected into the peritoneal cavity, rubbed well, and partially opened to collect peritoneal exudate cells (mainly macrophages). After washing the peritoneal cells, the cells were suspended in a serum medium and counted. Next, in a 37 ° C CO2 incubator,
After culturing for an hour, C3 antigen was quantified by ELISA.

The results are shown in Table 2. In the control, the release of C3 antigen was hardly observed, but the release of C3 antigen was increased up to 15-fold in mice to which Hatakeshimeji extract (EX) and its purified product (F-5) were administered. In general, the alternative pathway.
Macrophages stimulated with a substance that induces an immune mechanism initiated by the reaction of polysaccharides with complement) are found to rapidly increase the release of C3 antigen with macrophage activation. Has been found to be consistent with the strength of the antitumor activity.

[0031]

[Table 2]

Embodiment 4 FIG. Anti-tumor activity test Hatakeshimeji extract (EX) of Example 1 and purified products of Hatakeshimeji extract of Example 2 (F-1 to 11)
Was used as a sample, and physiological saline was used as a control group.

The mice were 5 week old mice (ICR / SLC
System, ♀) 6 to 11 animals were used as a group in the test. Sarcoma18 collected from the mouse 7 days after transplantation
0 cancer cells (5 × 10 ⁵ ) were transplanted. 24 hours after transplantation, when a fixed amount of each sample was injected, once a day,
Administered intraperitoneally (ip) for 10 days. In the case of oral administration (po), administration was carried out twice daily in the morning and evening using a gastric tube. The size of the cancer was measured 21 days after transplantation, and the cancer suppression rate (%) was measured as compared with the control group. In addition, the complete disappearance rate of the cancer and the mouse survival rate on day 28 of the transplantation were compared with those of the control group. Table 3 shows the results.

[0034]

[Table 3]

Embodiment 5 FIG. Hatake Shimeji extract extract and
The powdered extract of Hatake-shimeji mushroom extract and its purified product become water-soluble polysaccharides as the main component by concentrating and drying, but have very high hygroscopicity. There is a problem that it absorbs moisture, sticks, or solidifies the surface. Therefore, 33% of dextrin (Paindex, manufactured by Matsutani Chemical Co., Ltd.) was added as a shaping agent, followed by concentration and drying by spray drying to produce a Hatakeshimeji extract extract and a purified powdered product, which suppressed deterioration due to moisture absorption.

[0036]

The extract of Hatake mushroom and its purified product of the present invention have strong antitumor activity against Sarcoma180 transplanted cancer and are useful as an antitumor agent (new BRM). This antitumor activity is due to the immunostimulatory effect (activation of macrophages) and is also effective against various diseases caused by decreased immunological activity. In addition, the extract of Hatake shimeji, which is commonly used as a food, and the purified product thereof are safe without toxicity as compared with conventional anticancer agents. Further, the artificially cultivated product can be used as an inexpensive and stable quality raw material for producing Hatake mushroom extract and its purified product.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yuichi Ukawa 98 Kushibiki, Fukaya-shi, Saitama Pref., Nagasaki Gen Research Institute Co., Ltd. (72) Inventor Hitoshi Ito 2-3-10 Shiroyama, Tsu-shi, Mie Prefecture

Claims (10)

[Claims] 1. An immunostimulant comprising, as an active ingredient, a Hatake shimeji extract or a purified product thereof comprising a high molecular polysaccharide. 2. The immunostimulant according to claim 1, wherein the Hatake shimeji is an artificially cultivated product using “Kameyama No. 1” as a seed bacterium. 3. The active ingredient has the following properties: (a) color and form: tan powder (b) chemical ingredient: sugar content 30-90%, protein content 5-5
The immunostimulator according to claim 1 or 2, which is an extract having a solubility of 15%. 4. The active ingredient has the following properties: (a) color and form: white to tan powder (b) chemical ingredient: sugar content 60-100%, protein content 0
The immunostimulator according to claim 1 or 2, which is a purified product of an extract having solubility: water solubility. 5. An antitumor agent comprising a Hatakeshimeji extract or a purified product thereof comprising a high molecular weight polysaccharide as an active ingredient. 6. The antitumor agent according to claim 5, wherein the Hatake shimeji is an artificially cultivated product using “Kameyama No. 1” as a seed fungus. 7. The active ingredient has the following properties: (a) color and form: tan powder (b) chemical ingredient: sugar content 30-90%, protein content 5-5
The antitumor agent according to claim 5 or 6, which is an extract having 15% (c) solubility: water solubility. 8. The active ingredient has the following properties: (a) color and form: white to tan powder (b) chemical ingredient: sugar content 60-100%, protein content 0
The antitumor agent according to claim 5, which is a purified product of an extract having solubility: water solubility. 9. The method for producing an immunostimulant according to any one of claims 1 to 4, wherein the fruiting body of Hatakeshimeji is extracted with hot water to obtain an extract, and the extract is further purified as required. A method comprising: 10. The method for producing an antitumor agent according to any one of claims 5 to 8, wherein a fruiting body of Hatake-shimeji mushroom is extracted with hot water to obtain an extract, and the extract is further purified as required. A method comprising: