JPH054375B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to an anti-teratogenic agent containing a protein polysaccharide obtained from a bacterium belonging to Basidiomycetes as a main component. [Prior Art] Malformations refer to morphological and functional abnormalities that are recognized from birth. Patients have to live with severe disadvantages throughout their lives, and they often give up on these symptoms as fateful. Katta. Therefore, its specific causes or conditions for its establishment have not yet been completely clarified. Malformations in the present application include genetic diseases including morphological and functional abnormalities of external and internal organs, morphological and functional abnormalities of cells, and abnormalities at the molecular level such as chromosomal abnormalities. Currently, there are no effective inhibitors for these. Therefore, there is a strong need for a safe and effective anti-teratogenic agent. The present applicant has made numerous inventions related to protein polysaccharides obtained from basidiomycetes and their drugs [British Patent No. 1331513 (=Special Publication No. 1983-363), British Patent
1581315 (=Special Publication No. 56-46481), US Patent 4051314
(=Special Publication Showa 55-363), U.S. Patent No. 4202885 (=Special Publication Showa 55-363)
56-28152), U.S. Patent No. 4289688 (=Special Publication No. 55-
23271), U.S. Patent No. 4271151 (=Special Publication No. 55-23271),
U.S. Patent 4202969 (=Special Publication No. 56-14274), U.S. Patent
4229570 (=Special Publication No. 56-14275), US Patent 4140578
(=Special Publication No. 56-14276), U.S. Patent No. 4237233 (Special Publication No. 14276)
55-1790 and Japanese Patent Publication No. 55-4393), U.S. Patent No. 4288555
(=Special Publication Showa 55-7228), U.S. Patent No. 4162939 (=Special Publication Showa 55-7228)
55-32355), U.S. Patent No. 4159225 (= Special Publication No. 55-3235)
11318 and Japanese Patent Publication No. 60-41591), U.S. Patent No. 4268505
(=Japanese Patent Publication No. 56-39288) and U.S. Patent No. 4,663,438 (=Japanese Patent Publication No. 59-32480) have been proposed. As a result of intensive research by the present inventors in order to provide a safe and effective anti-teratogenic agent, it was discovered that a protein polysaccharide obtained from a bacterium belonging to the Basidiomycetes has a safe and anti-teratogenic effect. Based on this, the present invention was completed. [Means for Solving the Problems] The protein polysaccharide (hereinafter abbreviated as the present substance) which is the active ingredient of the anti-teratogenic agent of the present invention is obtained from a bacterium belonging to the Basidiomycetes. The bacteria used in the present invention are based on the Illustrated Encyclopedia of Japanese Fungi (Yukensha edition), co-authored by Rokuya Imaseki and Tsuguo Hongo, and the Journal of Japanese Mycology (Yokendo edition), written by Seiya Ito. Any type of basidiomycete may be used, but bacteria belonging to the genus Coriolis, Ciconia, Enokitake, and Fungus are preferred. Particularly preferred are bacteria belonging to the genus Coriolis. Examples of the genus C. versicolor include C. versicolor, M. chinensis, M. nikuusbata, M. sakazuki kawaratake, Yakifutake, e.g. The genus Enokitake includes Enokitake, and the genus Wood Fungus includes Wood Fungus, Wood Fungus, and Hidaki Fungus. This substance is an aqueous solvent extract of mushroom fruiting bodies, artificially cultured mycelia, and/or their culture medium. Artificially cultured mycelia can be obtained from basidiomycetes by inoculating mother fungi into a medium and culturing at an appropriate temperature. Generally, it is preferable to use a liquid medium in terms of handling and productivity. The culture medium composition is a formulation used for normal culture. There are known methods for obtaining protein polysaccharides from fruiting bodies, fungal systems, and culture media, such as Japanese Patent Publication No. 51
-36322, Japanese Patent Publication No. 56-14274, Japanese Patent Publication No. 56-14276, and Japanese Patent Publication No. 56-39288 are applied. The method for obtaining the protein polysaccharide will be described in detail below. The fruiting body or the artificially cultured mycelium obtained by culturing and/or the culture medium are subjected to extraction treatment. At this time, it may be dried and stored before being used as appropriate. Extraction is performed with an aqueous solvent. An aqueous solvent is a small amount of water or a water-soluble organic solvent, acid, or base.
For example, it consists of one kind or a combination of two or more kinds selected from aqueous solutions containing about 10% or less of them. After removing insoluble portions, the extract is transferred to the next purification process. The purification process is carried out by one or more methods such as salting out, dialysis, ultrafiltration, reverse filtration, gel filtration, and precipitation using an organic solvent. Industrially, ultrafiltration, which is a pressure-based membrane separation method, and reverse filtration treatment, either alone or in combination, are particularly preferred. In addition, these treatments may be carried out after the salting-out step, if necessary. Water is removed from the purified substance by spray drying, freeze drying, etc., and the product is made into a product. This substance shows positive results in the phenol-sulfuric acid reaction and the color reaction by the Lory-E-Follin method. As a result of elemental analysis, carbon 20-55%, preferably 35-55
%, hydrogen 3 to 9%, nitrogen 0% to less than 16%, preferably 0.1 to 10% as components. The sugar components of this substance include at least glucose, galactose, and mannose, and the protein components include at least aspartic acid, glutamic acid, and lysine. The pH of this substance is 6.0 to 7.5, and when its infrared absorption spectrum is measured, absorption of hydroxyl groups can be seen around 3600 to 3200 cm ^-1 and absorption derived from amide groups can be seen around 1700 to 1600 cm ^-1 . This substance is soluble in aqueous solvents and insoluble in organic solvents. The aqueous solvent is water or a solvent mainly composed of water and contains water-soluble alcohols, acids, bases, etc., and the organic solvent is chloroform, benzene, ether, etc. This substance is white or brown in color and has an average molecular weight of 1×10 ⁴ to 1 as determined by gel filtration chromatography.
× ¹⁰⁶ . Rat (dragon type) 4-5 weeks old, weight 100-150g
Orally administer 1000mg/Kg of this substance using
All the animals were observed alive for 7 days. Therefore, this substance is a safe substance with extremely low toxicity. This substance has anti-teratogenic effects. Teratogenicity here includes morphological abnormalities and functional abnormalities, but even if the morphological and functional abnormalities become apparent after birth, if the cause is determined before birth, they are included in the teratogenicity of the present application. Teratogenicity includes those with known abnormalities at the molecular level, such as external and internal morphological and functional abnormalities, cellular morphological and functional abnormalities, and chromosomal abnormalities, as well as those in which such abnormalities have not been identified. This includes genetic diseases. The first cause of these teratogenic properties is due to specific genetic factors, the second is due to environmental factors, and the third is due to the combination and interaction of the first and second factors. In particular, environmental factors include ionizing radiation as a physical factor, chemicals as a chemical factor,
Examples include 5-azacytidine and chlorambucil. Biological factors include toxoplasma and rubella virus. This substance was found to be effective in preventing teratogenicity caused by environmental factors, chemical and physical. When this substance is used as an anti-teratogenic agent, it can be made into any dosage form. Administration can also be carried out by various routes. The anti-teratogenic agent of the present invention is administered to humans and animals orally or parenterally, with oral administration being preferred. The oral dosage of this substance is 1 kg body weight, 10 ~
The dose is 1000 mg, preferably 20 to 600 mg, divided into 1 to 3 doses. The parenteral dosage is 0.1 to 500 mg, preferably 1 mg to 250 mg per kg of body weight per day. [Effects of the Invention] This substance has the effect of suppressing the expression of teratogenicity caused by chemical or physical factors that are environmental factors. For example, experiments were conducted to examine the teratogenicity of pregnant mice or rats caused by chemotherapeutic agents as a chemical factor, and the teratogenicity caused by irradiation of pregnant mice with doses of radiation that caused teratogenicity as a physical factor. We observed the fetuses using teratological methods, and further investigated the teratogenic effects of chemotherapeutic agents on pregnant mice using behavioral and functional teratological methods.
As a result of observation of newborn pups, this substance suppresses the expression of morphological abnormalities and functional abnormalities. EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples. Example 1 Cinnamon mushroom (strain CM-359,
No. 6060) 100 g of dried bacterial cells were cut into small pieces, placed in a stainless steel tank with a capacity of 3, and 2000 ml of water was added, and the temperature was maintained at 90 to 95° C. while stirring. After extraction for 3 hours, it was cooled to room temperature. The extraction slurry was separated into an extract and a residue using a centrifuge. Furthermore, add 2000ml of 0.5N-NaOH to the residue.
After extraction at 90-95°C for 3 hours, the mixture was cooled to room temperature, the pH was adjusted to 7.0 with 2N-HCl, and the mixture was centrifuged to separate into an extract and a residue. The extract was collected, concentrated to 400 ml using a vacuum concentrator, and further subjected to ultrafiltration (Dow Chemical Co., Ltd.).
After removing low molecular weight substances using HFD), the product was freeze-dried to obtain 13.4 g of dried product. Example 2 Enokitake (strain CM-601, Microtechnical Research Institute No. 3045)
No.) 100 g of dried bacterial cells were cut into small pieces, placed in a stainless steel tank with a capacity of 3, and 2000 ml of water was added, and the temperature was maintained at 90 to 95°C while stirring. After extraction for 3 hours, it was cooled to room temperature. The extraction slurry was separated into an extract and a residue using a centrifuge. Furthermore, add 1000ml of water to the residue and
After extraction at ~95°C for 3 hours, the mixture was cooled to room temperature and centrifuged to separate into an extract and a residue. The extract was collected, concentrated to 400 ml using a vacuum concentrator, and subjected to ultrafiltration (Dow Chemical Co., HFD).
This was further dried by freeze-drying to obtain 21.0 g of dried product. Example 3 Wood ear fungus (CM-886 strain, Microtechnical Research Institute No. 1763)
100 g of dried bacterial cells were cut into small pieces, placed in a stainless steel tank with a capacity of 3, and 2000 ml of water was added thereto, and the temperature was maintained at 90 to 95° C. while stirring. After extracting for 3 hours,
Cooled to room temperature. The extraction slurry was separated into an extract and a residue using a centrifuge. Furthermore, add 2000ml of 0.5N-NaOH to the residue.
After extracting at 90 to 95°C for 3 hours, the mixture was cooled to room temperature, the pH was adjusted to 7.0 with 2N-HCl, and the mixture was centrifuged to separate into an extract and a residue. The extract was collected, concentrated to 400 ml using a vacuum concentrator, and subjected to ultrafiltration (Dow Chemical Co., HFD).
After removing low molecular weight substances, freeze-drying was performed to obtain 15.8 g of dried product. Example 4 Kawaratake (strain CM-101, Microtechnical Research Institute No. 2412)
No.) 100 g of dried bacterial cells were cut into small pieces, placed in a stainless steel tank with a capacity of 3, and 1800 ml of water was added, and the temperature was maintained at 93 to 98°C while stirring. After extraction for 3 hours, it was cooled to room temperature. The extraction slurry was separated into an extract and a residue using a centrifuge. Furthermore, add 2000ml of 0.4N-NaOH to the residue.
After extracting at 90 to 95°C for 3 hours, the mixture was cooled to room temperature, the pH was adjusted to 7.0 with 2N-HCl, and the mixture was centrifuged to separate into an extract and a residue. The extract was collected, concentrated to 400 ml using a vacuum concentrator, and subjected to ultrafiltration (Dow Chemical Co., HFD).
After removing low molecular weight substances, freeze-drying was performed to obtain 19.1 g of dried product. The physicochemical properties of the substances obtained in Examples 1 to 4 are summarized in Table-1. In Table-1,
The phenol sulfuric acid color reaction shows the presence of sugars, and the Rollie-Follin method shows the presence of peptide bonds. Regarding the molecular weight, the average molecular weight was determined by gel filtration method.

[Table] Example 5 This substance (No.
4) Effect. 5-azacytidine, a chemotherapeutic agent with teratogenic effects, was dissolved in physiological saline to form a 0.6% physiological saline solution, and 15 mice per group became pregnant at a dose of 0.6 mg/Kg.
On the 13th day, rats were administered once intravenously, and at the same time, 50% each of this substance (No. 4) in 10% physiological saline solution was administered.
mg/Kg, 100 mg/Kg or 200 mg/Kg was administered subcutaneously. The mean weight of live fetuses and abnormalities of fingers and toes were observed according to experimental teratological methods. The rate of abnormal fingers and toes was expressed as the ratio (%) of the number of abnormal fingers to the number of fetuses. Furthermore, for comparison, a group receiving neither 5-azacytidine nor this substance (No. 4), a group receiving only 0.6 mg/Kg of 5-azacytidine, and a group administering this substance (No. 4) 200
The group administered only mg/Kg was also observed. The results are shown in Table-2. From the above results, it was observed that the group administered 50 mg/Kg or more of this substance (No. 4) had a dose-dependent decrease in finger and toe abnormalities compared to the group administered 0.6 mg/Kg of 5-azacytidine alone. In particular, in the 200 mg/Kg administration group of this substance (No. 4), a rapid decrease in the occurrence of abnormalities in fingers and toes was observed. There was no difference in the average body weight of live fetuses between the group administered with 5-azacytidine alone and the group administered in combination with this substance (No. 4) and 5-azacytidine. As described above, this substance (No. 4) was found to be extremely useful as an inhibitor of morphological abnormalities caused by chemical factors.

[Table] Example 6 Effect of this substance (No.
4) Effect. Chlorambucil, a chemotherapeutic agent with teratogenic effects, is dissolved in sesame oil to make a 6% solution, and 6mg/
A dose of Kg was orally administered once to mice on the 10th day of pregnancy using a gastric needle, and at the same time a 10% saline solution of this substance (No. 4) was administered subcutaneously at a dose of 200 mg/Kg.
Experimental teratological techniques were used to observe short and short tails, curved tails, short and oligodactyl limbs, etc. of the fetuses. For comparison, a group administered with 6 mg/Kg of chlorambutyl alone and a group without administration were observed. The abnormality incidence rate is expressed as the ratio (%) of the number of abnormalities at the site to the number of fetuses. In the group administered with chlorambucil alone, the incidence of abnormalities in the tail and limbs was 79.8% and 100%, respectively, whereas in the group administered with this substance (No. 4) and chlorambucil, the incidence of abnormalities in the tail and limbs was 70.5%, respectively.
% and 95.2%, a decrease in abnormal expression was observed in both sites. Example 7 Effect of this substance (No. 1) on fetal abnormalities occurring in response to radiation irradiation. Radiation 200R was administered to two groups of 39 pregnant mice.
At the same time, a 10% physiological saline solution of this substance (No. 1) was administered subcutaneously to group 1 at a dose of 200 mg/Kg, and then orally administered at a dose of 600 mg/Kg every day for 10 days. The incidence rate (%) of fetal malformations such as short tail, curved tail, and cleft palate was determined according to teratological methods. In the group receiving only radiation, the malformation rate was 82%, but in the group receiving this substance (No. 1), the malformation rate was 48%. As described above, the effect of this substance (No. 1) on suppressing the appearance of morphological abnormalities due to physical factors was confirmed. Example 8 This substance (No. 2
Or the effect of No. 3). Chlorambucil, a chemotherapeutic agent with teratogenic effects, is dissolved in sesame oil to make a 6% solution, and 6mg/
The substance (No. 2
Or No. 3) 10% physiological saline solution was administered subcutaneously at a dose of 200 mg/Kg, and the born pups were observed according to behavioral and functional teratological techniques. For comparison, a group administered with 6 mg/Kg of chlorambutyl alone and a group without administration were observed. In the group administered with chlorambucil alone, the incidence of abnormal gait function was 76.5%, but in the group administered with this substance (No. 2) and chlorambucil, the incidence of abnormal gait function was 58.1%. The incidence of abnormal walking function in the group administered with this substance (No. 3) and chlorambucil was 59.2%. In the non-administration group, the incidence of abnormal walking function was 0%. Example 9 Using a pressure-type automatic filling machine, 330 mg of this substance (No. 4) was filled into No. 000 hard capsules to produce capsules.

Claims (1)

[Scope of Claims] 1. An anti-teratogenic agent containing a protein polysaccharide obtained from a bacterium belonging to Basidiomycetes as an active ingredient. 2. The protein polysaccharide shows a positive color reaction by the phenol sulfuric acid color reaction and the Rollie-Follin method, and the elemental analysis shows 20 to 55% carbon, 3 to 9% hydrogen, and more than 0% to less than 16% nitrogen. , 3600-3200 cm ^-1 and 1700-1600 cm ^-1 in the infrared absorption spectrum
It is soluble in water, insoluble in chloroform, benzene, and ether, and has an average molecular weight of 1×10 ⁴ to 1× by gel filtration chromatography.
Claim 1 characterized in that 10 ⁶
Anti-teratogenic agents as described in Section. 3. The anti-teratogenic agent according to claim 1 or 2, wherein the teratogenicity is morphological abnormality. 4. The anti-teratogenic agent according to claim 1 or 2, wherein the teratogenicity is a functional abnormality. 5. The anti-teratogenic agent according to claim 1, wherein the basidiomycete is selected from bacteria belonging to the genus Corsicolor, genus Cinnamon, genus Enokitake, and genus Wood fungus. 6. The anti-teratogenic agent according to claim 1, wherein the basidiomycete is selected from bacteria belonging to the genus Kawarateke.