WO1999053937A1

WO1999053937A1 - Immunopotentiators and antitumor agents

Info

Publication number: WO1999053937A1
Application number: PCT/JP1999/002074
Authority: WO
Inventors: Masaru Shibata; Yasushi Kojima; Yuuichi Ukawa; Hiroshi Hara; Hitoshi Ito; Makoto Hisamatsu
Original assignee: Eishogen Co., Ltd.; Oji Paper Co., Ltd.
Priority date: 1998-04-20
Filing date: 1999-04-19
Publication date: 1999-10-28
Also published as: JPH11302191A

Abstract

Immunopotentiators and antitumor agents which are nontoxic and less expensive. These immunopotentiators and antitumor agents can be obtained by using as the active ingredient an optionally purified extract of Lyophyllum decastes (Fr. Sing.) which is rich in high molecular weight polysaccharides. As this fungus, in particular, use is made of those having been artificially cultivated with the use of 'Kameyama No. 1' as the seed culture.

Description

Description Immunostimulants and antitumor agents Technical field

The present invention relates to an immunostimulant and an antitumor agent containing a Hatakeshimeji extract or a purified product thereof as a main component. Related technology

Various basidiomycetes-derived polysaccharides and protein polysaccharides are known to exhibit antitumor effects (Hitoshi Ito, Shinshiro Shinkei: Antitumor polysaccharides, infectious disease 75 No. 18-22, p. 1, 1) 9 84 years). Some of these polysaccharides have a substance that regulates the functions of the body's defense mechanisms, mainly the immune system. The antitumor effect is controlled through the regulatory action (immune regulation) of this substance. It is thought to be expressed. At present, these substances are regarded as one of biological response modifiers (hereinafter, abbreviated as “BRM”).

As existing immunotherapeutic agents, “Krestin”, an extract of Agaricus versicolor, “Lentinan”, an extract of Shitake mushroom, and “Shizophyllan”, an extract of Shirohirotake, have been approved and put to practical use. However, each alone raises the immunostimulatory activity of all individuals and cannot achieve sufficient antitumor effects, so multidrug immunotherapy using several BRMs in combination has been adopted. .

On the other hand, for the extract of Hatake shimeji, Oji Paper Co., Ltd. Forest Resource Research Institute has announced that it has an anti-cancer effect. 1993), Mitsui Darafu 97: p8-9 (1994)), and the group of Kanazawa University's Ikegawa et al. Reported that “Yamabiko treasure shimeji” (artificial cultivated product of Hatake shimeji) was used for Sarcomal 80 transplantation cancer in the abdominal cavity of mice. It shows that it has antitumor activity (Abstracts of the Cancer Society of Japan, 1997). It is reported that oral administration of Hatakeshimeji mushrooms treated with lyse to mice also had an antitumor effect.

However, no method has been reported for the production of the purified extract of Hatake shimeji mushrooms or its anticancer effect. In particular, in the case of basidiomycetes of artificially cultivated products, it is known that the components in basidiomycetes vary greatly depending on the culture medium components and cultivation methods. Is not always expected. In addition, it was not known that the extract of the artificially cultivated product using Hatakeshimeji's “Kameyama No. 1” as a seed fungus and its purified product had extremely high antitumor activity.

A high-yield and efficient artificial cultivation method for Hatake shimeji has already been established by the Oji Paper Co., Ltd. Forest Resource Research Institute (Japanese Patent Publication No. 5-154404, Patent No. 196995). 34 4) By using this method, artificially cultivated products using Hatakeshimeji's “Kameyama No. 1” as a seed fungus can be supplied at low cost.

As evident from the above, the development of a new and powerful BRM is necessary for the success of multidrug immunotherapy that can respond to various patients. In actual use, it is desirable to use an inexpensive drug that does not have side effects such as acute toxicity and mutagenicity, unlike conventional anticancer drugs.

Therefore, an object of the present invention is to provide an immunostimulant and an antitumor agent which have a short cultivation period, can be artificially cultivated in large quantities, and have high immunostimulatory activity and antitumor activity, for BRM derived from basidiomycete. . Disclosure of the invention

As a result of the inventor's intensive research, strong immunity has been gained for the extract of Hatake shimeji, a long-famous edible product, and in particular, artificially cultivated products using Hatake-shimeji, Kameyama No. 1 that can be artificially cultivated in large quantities and efficiently. Activating activity · It was found to have antitumor activity. Further, the present inventors have found that a purified product of Hatake-shimeji mushroom extract also has immunostimulatory activity and antitumor activity, and thus completed the present invention.

9 (1) The first aspect of the present invention is an immunostimulant comprising a hatake shimeji extract containing a high molecular weight polysaccharide or a purified product thereof as an active ingredient.

In a preferred embodiment, the present invention provides an immunostimulant, 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;

(B) Color and form: tan powder

(π) Chemical composition: Sugar content 3! ] ~ 90%, protein content 5 ~ 15%

(C) Solubility: water-soluble

The present invention provides an immunostimulant which is an extract having the following.

The present invention provides, in another aspect, the active ingredient has the following properties:

(B) Color and form: white to tan powder

(Mouth) Chemical composition: Sugar content 6 (! ~ 100%, protein content 0 ~ 10%

(C) Solubility: water-soluble

An immunostimulator which is a purified product of an extract having

In the present invention, the extract of Hatake shimeji is obtained by extracting the fruit body of Hatake shimeji with hot water and, if desired, the extract is further purified.

(2) The second aspect of the present invention provides an antitumor agent comprising a hatake mushroom extract containing a high molecular weight polysaccharide or a purified product thereof as an active ingredient.

In a preferred embodiment, the present invention is an antitumor agent, wherein the Hatake shimeji is an artificially cultivated product using “Kameyama No. 1” as a seed fungus.

(B) Color and form: tan powder

(π) Chemical composition: sugar content 30 ~ 90%, protein content 5 ~ 15%

(C) Solubility: water-soluble

An antitumor agent which is an extract having

The invention provides, in another aspect, the active ingredient has the following properties:

(B) Color and form: white to tan powder (^) Chemical composition: sugar content 60 ~ 100%, protein content 0 ~ 10%

(C) Solubility: water-soluble

An antitumor agent which is a purified product of an extract having the following formula:

In the present invention, the extract of Hatake shimeji is obtained by extracting the fruit body of Hatake shimeji with hot water and, if desired, the extract is further purified. BEST MODE FOR CARRYING OUT THE INVENTION

Haphyshimeji mushroom (Yophyl lum decastes (Fr.) Sing.) Is a mushroom belonging to the genus Shimeji, belonging to the genus Shimeji, and is the closest mushroom to Hon-shimeji, which is said to be better than Matsutake mushrooms. . Natural products occur in the form of plants in relatively familiar places such as gardens and fields (Rokuya Imaseki, Tsuguo Hongo: Japanese Fungus Encyclopedia of Primary Colors (1), Nursery, 1987).

Hatake shimeji used in the present invention is an artificially cultivable Hatake shimeji,

It is preferable to use No. 1 (Seed and Seed Method Variety Registration Application No. 6 811, Type of Agriculture, Forestry and Fisheries Plant: Hatake Mushroom, Name of Applied Variety: Kameyama No. 1) as the inoculum. Further, it is preferable to use, as a cultivation medium component, chitin (force husk), brewer's yeast cake, etc., for which a BRM-like effect has been reported. Hatakeshimeji is a cultivation method established by Oji Paper Co., Ltd. Mori Forest Resources Research Institute (Japanese Patent Publication No. 5-154404, Patent No. 19)

It is desirable to use “Kameyama No. 1” as the inoculum. Alternatively, Hatake-shimeji, a Japanese registered trademark “Shakinko”, cultivated and marketed in the same manner can be used as a raw material.

The extract of Hatake mushroom used in the present invention can be 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 in advance.

The heating method may be any of decompression, normal pressure, and pressurization, but high-temperature treatment under pressure provides better extraction efficiency. The heating temperature can be carried out in the range of 7 Q to 26 D ° C. You. The extraction time is 2 to 4 hours at normal pressure, and 15 minutes to 2 hours if performed under high temperature and pressure.

The fruit body of Hatake mushroom subjected to the hot water extraction treatment is an extract having a solid content of about 5.5% to 10%. Next, the extract is concentrated and dried to obtain a Hatake mushroom extract used in the present invention.

Here, the extraction extract may be concentrated by any of a heating concentration method, a reduced pressure heating concentration method, and a concentration method by ethanol precipitation. The concentrated extract extract may be dried by air drying, heat drying, spray drying, or freeze drying.

The Hatake mushroom extract may also be a purified product thereof. This purified product can be obtained by purifying according to an ordinary method for purifying a high molecular polysaccharide. For example, a Hatake shimeji extract is dissolved in pure water and adsorbed on an ion-exchange column (such as Q-Sepharose). Then, the Hatake shimeji extract is adsorbed on the ion-exchange column, and a gradient elution is performed with a different chloride concentration. By doing so, it can be purified. In addition, the purified product of the fractionated Hatake shimeji extract can be further purified by fractionation by a gel filtration method using a difference in molecular weight. The Hatakeshimeji extract thus purified is concentrated and dried by removing salts and buffers used in the purification method by dialysis or ethanol precipitation.

The Hatake mushroom extract and its purified product used in the present invention can be used in a form convenient for obtaining a medicinal effect according to the symptoms of the disease, and used alone or as a mixture with a pharmaceutically acceptable diluent and other drugs. it can.

The feature of these Hatakeshimeji extract and its purified product is that it can be provided in the form of a dosage unit. For example, oral forms such as powders, granules, tablets, dragees, capsules, syrups, pills, suspensions, solutions, emulsions, or injections containing an effective amount of the active ingredient, or injection solutions Parenteral dosage forms such as ampules and vials. It can also be used as a suppository. The diluent may be solid, liquid, or semi-solid, and examples thereof include the following. That is, excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, fragrances, preservatives, solubilizers, solvents, etc. Lactose, sucrose, sorbite, mannite, starch, sedimentable carbonate calcium, heavy magnesium oxide, talc, calcium stearate, magnesium stearate, cellulose or its derivatives, miromibectin, polyvinyl alcohol , Gelatin, surfactants, water, physiological saline, ethanol, dariserin, propylene glycol, cocoa butter, laurin butter, cellulose, paraffin, higher alcohols and the like.

The immunostimulant and the antitumor agent of the present invention may be used in these compositions. The ratio of the extract of Hatake mushroom and its purified product is 1 to 100 ^ 1%, preferably 5 to 100%. 80 wt%.

The above-described extract of Hatake mushroom and its purified product are orally or parenterally administered to humans and animals, but preferably are administered by hand. Oral administration includes sublingual administration, and is performed by parenteral administration (eg, subcutaneous, intramuscular, intravenous injection, infusion), or rectal administration. The dose of Hatake-shimeji extract and its purified product depends on the animal or human, and depends on the age, individual difference, medical condition, etc.Therefore, the dose outside the following range may be administered. When used as a subject, 1 mg to 10 g, preferably 10 111 to 100 111, of body weight lkg per day is administered in 1 to 4 divided doses.

The extract of Hatake mushroom and its purified product used in the present invention have no acute toxicity, no mutagenicity, and have immunostimulatory activity and antitumor activity, so that they are useful as pharmaceuticals thereof. The extract of Hatake-shimeji mushroom and its purified product may be used as they are, or may be mixed with excipients or other pharmaceuticals. When mixing, it is preferable to mix at a rate of 3 Q to 8 Q wt% _c Example

Example 1 Artificial cultivation of Hatake shimeji

Bark compost (manufactured by Chu Nippon Agricultural Products Co., Ltd.): Rice bran: Force husks mixed at an absolute dry weight ratio of 100: 20: 4, and then a 850 ml 1 volume culture medium with a water content of 62%. A cultivation bottle made of polypropylene was filled with 62 g. To supply air to the entire culture medium in the bottle and improve the growth of mycelia, make a hole about 2 cm in size from the opening to the bottom of the bottle and pressurize the autoclave (120 ° C). , 1 hour). After cooling the temperature of the culture medium to 25 ° C or less, Hatakeshimeji “Kameyama No. 1” was inoculated in a clean room.

The cells were cultured for 50 days in a room adjusted to a room temperature of 23 ° C and a humidity of 70% (RH), and the mycelium was sufficiently spread on the culture medium. After the bacteria were removed 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 bin was cultured for 7 days in a room at room temperature of 23: and humidity of 95% (RH). Next, the covering part of the surface layer where the hypha did not penetrate was removed, and cultivation was continued in a room adjusted to a room temperature of 17 ° (: 95% humidity (RH) and an illuminance of 150 lux). After cultivation for 85 days after inoculation, 120 g of fruiting bodies were harvested per bottle.

The harvested artificially cultivated Hatakeshimeji mushroom fruit body 1QQg was placed in a hot pot, in a pot, 1 L of water was added, and the mixture was heated and extracted for 3 hours to remove a Hatakeshimeji mushroom extract, thereby obtaining a brown Hatakeshimeji extract extract. An equal amount of ethanol was added to the Hatakeshimeji extract, the precipitate was collected, 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 varied slightly depending on the state of the mushroom, but were generally as follows. (A) Color and form: tan powder

(π) Chemical composition: sugar content 70%. Protein content 10%

Solubility: water soluble Example 2.

The Hatake mushroom extract of Example 1 was dissolved in pure water and adsorbed on an ion exchange column (Q-Sepharose, etc.). Next, the Hatakeshimeji mushroom extract adsorbed on the ion exchange column was purified by eluting it with stepwise changing the concentration of chlorinated lime from 5 mM to 35 OmM. The purified fraction of Hatake Shimeji extract was fractionated by a gel filtration method using a difference in molecular weight to further purify into 11 fractions. The purified Hatake Shimeji extract was dialyzed, the salts and buffers used in the above purification method were removed, and the mixture was concentrated by heating under reduced pressure and freeze-dried. Table 1 shows the physicochemical properties of the purified product purified into 11 fractions.

table 1

Example 3. Activation test of Mac π phage

Using the Hatake-shimeji extract extract (EX) of Example 1 and the purified product of the Hatake-shimeji extract (F-5) of Example 2 as samples, 0.2% dextrose in physiological saline was used. A solution of Strand was used as a control.

Next, the above-mentioned EX (0.5%). F-5 (0.1%) was dissolved in physiological saline, and 0.3 ml was administered intraperitoneally to each mouse. The mice used were 15-week-old mice (IC RZSLC system, early). Two hours later, blood was collected under anesthesia. Thereafter, PBS (3 ml) was injected into the abdominal cavity of the mouse, rubbed well, and partially ablated to collect peritoneal exudate cells (mainly macrophages). Then, the peritoneal exudate cells of the mouse were washed and suspended in a serum medium, and the number of cells was counted. Then, after 2 hours at C0 ₂ b Nkyubeta one at 37, the c the results of quantification of C 3 antigens by EL ISA method shown in Table 2. Controls showed little release of C3 antigen, but mice receiving Hatakeshimeji extract (EX) and its purified product (F-5) increased C3 antigen release up to 15-fold.

In addition, macrophages stimulated with substances that induce an alternative pathway (an alternative pathway of the immune response; an immune mechanism initiated by the reaction of polysaccharides with complement) stimulate C3 antigen release rapidly as macrophages are activated. And the strength of this activity was found to be consistent with the strength of the antitumor activity. Table 2

Example 4. Antitumor activity test

The Hatakeshimeji extract extract (EX) of Example 1 and the purified product of the Hatakeshimeji extract (F-1 to 11) of Example 2 were used as samples, and physiological saline was used as a control group. In the test, 6 to 11 5-week-old mice (ICR / SLC system, early) were used as a group. After transplanting to this? Sarcoma 130 cancer cells (5 × 10 5) collected from the mice on the day were transplanted. From 24 hours after transplantation, when a fixed amount of each sample was injected, the sample was administered intraperitoneally (ip) once a day for 1 Q day. In the case of oral administration (P. 0.), administration was performed every morning * twice in the evening using a gastric sonde.

The size of the cancer was measured on the first and second transplants, and the cancer suppression rate (%) was measured in comparison with the control group. On the 28th day after transplantation, the complete disappearance rate of the cancer and the survival rate of the mice were compared with those of the control group. Table 3 shows the results.

Table 3

i. P .: Intraperitoneal injection, P. 0 .: Oral administration, not measured Example 5. Powder preparation of Hatakeshimeji extract and its purified product

Hatakeshimeji extract and its purified product are concentrated and dried to form a powder composed mainly of a permanent soluble polysaccharide, but have a very high hygroscopicity and absorb moisture when left indoors, and become sticky. However, there is a problem that the surface is solidified. Therefore, after adding 33% of dextrin (Pinedettas manufactured by Matsutani Chemical Co., Ltd.) as a shaping agent to the above purified product and concentrating and drying by spray drying, the Hatakeshimeji extract extract which suppressed deterioration due to moisture absorption and the powdered product of the purified product Could be manufactured. Industrial applicability

As described above, the immunity 2 activator and antitumor agent of the present invention have strong antitumor activity against Sarcomal80 transplanted cancer and are useful as antitumor agents. In particular, this antitumor activity is due to the immunostimulatory effect (activation of macula phage) and is also effective against various diseases caused by reduced immune activity. In addition, Hatakeshimeji mushroom extract, which is commonly used as food, and its purified product are safer without toxicity as compared with conventional anticancer drugs. In addition, the artificially cultivated product can be used as an inexpensive and stable quality raw material for producing Hatake shimeji extract and its purified product.

Claims

Claim scope

1. An immunostimulant comprising, as an active ingredient, a Hatakeshimeji extract containing a high-molecular-weight polysaccharide or a purified product thereof.

2. The immunostimulant according to claim 1, wherein an artificially cultivated product using "Kameyama No. 1" as a seed fungus is used as the Hatakeshimeji.

3. The active ingredient has the following properties:

(B) Color and form: tan powder

(π) Chemical composition: sugar content 30 ~ 90%, protein content 5 ~ 15%

(C) Solubility: water-soluble

The immunostimulator according to claim 1 or 2, which is an extract having the following.

4. The active ingredient has the following properties:

(B) Color and form: white to tan powder

(Mouth) Chemical composition: sugar content 60 ~ 100%, protein content (! ~ 10%

(C) Solubility: water-soluble

The immunostimulator c according to claim 1 or 2, which is a purified product of an extract having

5. The immunostimulant according to claim 1, wherein the Hatakeshimeji extract is obtained by extracting a fruit body of Hatakeshimeji with hot water.

6. The immunostimulant according to claim 1 or 2, wherein the Hatake shimeji extract is a product obtained by extracting a fruit body of Hatake shimeji mushroom with hot water and further purifying the fruit body.

7. An antitumor agent comprising a hatake mushroom extract containing a high molecular weight polysaccharide or a purified product thereof as an active ingredient.

8. The antitumor agent according to claim 7, wherein an artificially cultivated product using "Kameyama No. 1" as a seed fungus is used as the Hatakeshimeji.

9. The active ingredient has the following properties:

(B) Color and form: tan powder

(π) Chemical composition: Sugar content 30-9Q%, protein content 5-15% (C) Solubility: water-soluble

9. The antitumor agent according to claim 1 or 8, which is an extract having:

10. The active ingredient has the following properties:

(B) Color and form: white to tan powder

(π) Chemical component: sugar content 60-1 10%, protein content (3-10%)

(C) Solubility: water-soluble

9. The antitumor agent according to claim 7 or 8, which is a purified product of an extract having:

11. The antitumor agent according to claim 7, wherein the Hatakeshimeji extract is obtained by extracting a fruit body of Hatakeshimeji with hot water.

1

12. The antitumor agent according to claim 7 or 8, wherein the extract of Hatake mushroom is obtained by hot water extraction of the fruit body of Hatake mushroom.