JPH0249732A - Antiteratogenic agent - Google Patents

Abstract

PURPOSE:To obtain a safe and effective antiteratogenic agent containing a protein polysaccharide obtained from a microorganism belonging to Basidiomycetes as an active ingredient. CONSTITUTION:The aimed antiteratogenic agent containing a protein polysaccharide which is an extract of fruit body of microorganism belonging to Basidiomycetes, preferably microorganism belonging the genus KAWARATAKE, Ganoderma, Flammulina or Auricularia, artificial cultured mycelium thereof and/or culture medium thereof by an aqueous solvent as an active ingredient. The above-mentioned substance has the following properties: (i) Phenol sulfuric acid color reaction and color reaction by Rolly-Forlin method, positive; (ii) elemental analysis: C, 20-55%; H, 3-9%; H, >0 and <16%; (iii) infrared absorption spectrum, noticeable absorption in 3600-3200cm<-1>, 1700-1600cm<-1>; (iv) soluble in water and insoluble in chloroform, benzene and ether; (v) average molecular weight by gel filtration chromatography, 1X10<4>-1X10<6>. The daily doze of the substance is preferably orally 20-600mg/kg and parenterally 1-250mg/kg.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to Basidiomycetes (3asidiomycetes).
The present invention relates to an anti-teratogenic agent whose main ingredient is a protein polysaccharide obtained from a bacterium belonging to .

[Prior Art] Malformations refer to morphological and functional abnormalities that are recognized from birth, and patients have to live with unfavorable conditions, and it is difficult to give up on these symptoms as fateful. There were many. Therefore, the specific cause 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 safe and effective anti-teratogenic agents.

The present applicant has made numerous inventions relating to protein polysaccharides obtained from basidiomycetes and their drugs [British Patent No. 133
1513 (=Special Publication No. 55-363), British Patent 15
81315 (-Japanese Patent Publication No. 56-46481>, U.S. Patent 4051314 (-Japanese Patent Publication No. 55-363), U.S. Patent No. 4202885 (-Japanese Patent Publication No. 56-281!i2)
, U.S. Patent No. 4289688 (=Special Publication No. 55-2327L
), U.S. Patent No. 4271151 (=・Special Publication No. 55-23
271), U.S. Patent 4202969 (=Special Publication No. 1983-
14274), U.S. Patent No. 4229570 (=Special Publication No. 5
6-14275), U.S. Time r+4140578 (-Special Publication No. 56-14276), U.S. Patent No. 4237233 (
Special Publication No. 55-1790 and Special Publication No. 55-4393), U.S. Patent No. 4288555 (= Special Publication No. 55-7228),
U.S. Patent No. 4162939 (-Special Publication No. 55-32355
), U.S. Patent No. 4159225 (-Special Publication No. 55-11
318 and Japanese Patent Publication No. 6O-41591), U.S. Patent No. 42
68505 (=Special Public Service No. 56-39288) and rice (l
Patent No. 4,663,438 (-Japanese Patent Publication No. 59-32480) has 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. The present invention was completed based on the above.

[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 primary color Japanese fungi diagram @ (nursery formal wear) co-authored by Rokuya Imaseki and Tsuguo Hongo, and the Japanese Mycological Journal (1! Kendo edition) 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, C. nigra, N. nikuusbata, 1. nakazuki kawaratake, Yakifutake, e.g. The Enokitake genus includes F4:take, and the Enokitake genus includes Wood ear ear, Wood ear fungus, and Hidaki wood ear.

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.

Methods for obtaining protein polysaccharides from fruiting bodies, mycelia, and culture media include known methods, 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. Furthermore, it is preferable to cut into pieces prior to the extraction step in order to increase extraction efficiency.

Extraction is performed with an aqueous solvent. An aqueous solvent is one or a combination of two or more selected from water or an aqueous solution containing a small amount of a water-soluble organic solvent, an acid, or a base, for example, about 10% or less. be. After removing insoluble portions, the extract is transferred to the next fli production process.

The purification process includes one or two of salting out, dialysis, ultrafiltration, reverse filtration, gel filtration, and precipitation using an organic solvent.
It is done by more than one method. Industrially, ultrafiltration, which is a pressure-based membrane separation method, and reverse filtration treatment, either alone or in combination, are particularly preferred. Further, depending on the case, a salting-out step or other treatment may be performed.

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-Forin method. As a result of elemental analysis, carbon 20-55%, hydrogen 3-9%, nitrogen O% exceeds 1
Contains less than 6% as a component.

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 6.0-7°5, and its infrared absorption spectrum is 3600-3200c#+-
'Absorption of water M block near 1700~1600c#r-
Absorption derived from the amide group can be observed near 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 the molecule j has an average molecular weight of 1 x 10 to 1 x 10 by gel filtration chroma lubricant.

To Rat 1 (capacity type) 4-5 weeks old, weight 100-1500
This substance was orally administered at ioooom(+/l(g) to animals and observed for 7 days, but all the animals survived. Therefore, this substance has extremely low toxicity and almost no side effects. It is a safe substance.

This substance has anti-teratogenic effects. Teratogenicity here includes morphological abnormalities and functional abnormalities, but even if 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, and chemical substances as a chemical factor, such as 5-azacytidine and chlorambucil. Biological factors include toxoplasma and rubella virus.

This substance was found to be self-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.

Oral dosage of this substance is 10 kg/day.
1000 mg, preferably 20 to 6001 g, is administered in one to three divided doses. Parenteral dosage is body? I Kg
, 0.1-500 mg per day, preferably lll1
There are g ~ 250 mg.

[Effect of the Invention 1 This substance has the effect of suppressing the expression of teratogenicity caused by chemical or physical factors that are environmental factors. For example, the expression of teratogenicity in pregnant mice or rats due to chemotherapeutic agents as a chemical factor, and the expression of teratogenicity due to irradiation of pregnant mice in the wA field, where teratogenicity of radiation occurs as a physical factor. We observed the fetuses according to experimental teratological methods, and also observed the teratogenicity of pregnant mice using chemotherapeutic agents.As a result of observing the newborn pups according to the vJ functional teratological method, we found that this substance caused morphological and functional abnormalities. suppress.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples.

115.
No.) 100 g of dried bacterial cells were cut into small pieces, placed in a stainless steel tank with a capacity of 31, and 2000 d 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, 0.5N-N a OH2000d was added 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-HCρ, and the mixture was centrifuged to separate into an extract and a residual solution.

The extracts were collected, concentrated to 400 d using a vacuum concentrator, and further subjected to ultrafiltration ([) ow Ch (3miCal
After removing low-molecular-weight substances using G0゜1-IFD), the product was freeze-dried to obtain 13.4 g of dried product.

Cut 100 g of dried enokitake mushroom (CM-601 strain, Fiber Science and Technology No. 3045) into small pieces, place in a stainless steel tank with a volume of 1 x 3 Jl, add 2,000 ae of water, and reduce the temperature to 90 - 90 ml while stirring. It was kept at 95°C. After extracting for 3 hours,
It was cooled to V temperature.

The extraction slurry was separated into an extract and a residue using a centrifuge. Furthermore, add 1000-degree water to the leftovers and heat to 90-95℃.
After extraction for 3 hours, the mixture was cooled to room temperature, centrifuged, and separated into an extract and a residual solution.

The extract was collected, concentrated to 400 ml by vacuum concentration gi, and ultrafiltered ([)ow Chemical C.

HFD)1, and was further dried by freeze-drying to obtain 21 g of dried product.

[1] Yuki jellyfish (strain CM-886, Microtechnological Research No. 1763)
100 Q of dried bacterial cells were cut into pieces, placed in a stainless steel tank with a capacity of 1 l, and 2000 d of water was added thereto, and the temperature was maintained at 90 to 95°C while stirring. After extraction for 3 hours, it was cooled to room temperature.

Centrifuge the extracted slurry for 111 minutes! The extract was separated into an extract and a residue using fi. Furthermore, 0.5N-N a OH20 was added to the residue.
After adding 00d and extracting at 90-95℃ for 3 hours,
After cooling to room temperature and adjusting the pH to 7.0 with 2 N-HCρ, the mixture was centrifuged to separate into an extract and a residue.

The extract was collected, concentrated to 400 d using a vacuum concentrator, and ultrafiltered ([)ow Chemical C0HF.
After removing low molecular weight substances using D>, lyophilization was performed to obtain 15.8 g of dried product.

Tomo LL4 Cut 100 eJ of dry bacterial cells of Kawaratake (strain CM-101, FEIKEN Bacteria No. 2412) into small pieces, put them in a stainless steel tank with a capacity of 13 N, add 1800 m of water, and adjust the temperature to 93 to 98°C while stirring. I kept it. After extraction for 3 hours, it was cooled to room temperature.

Centrifuge the extracted slurry! The extract was separated into an extract and a residue using one machine. Furthermore, 0.4N-N a OH2000 for residual soaking
- and extracted at 90-95°C for 3 hours, then air temperature gusset cold Eft L,,2N-)-tcfIF pHHI
After adjusting for 3 o'clock, it was centrifuged and separated into an extract and a residue.

The extract was collected, concentrated to 400 m using a vacuum concentrator, and subjected to ultrafiltration (Dow Chemical Co.).

After removing low molecular weight substances using 1-IF D ), freeze-drying was performed to obtain a dried product of 191Q.

The physicochemical properties of the substances obtained in Examples 1 to 4 are shown in the table below.
They are summarized in 1. In Table 1, the phenol-sulfuric acid color reaction shows the presence of saccharides, and the Lory-e-Forin method shows the presence of peptide bonds. Regarding the molecular function, the average molecular weight was determined by gel filtration method.

Effect of this substance (Nα4) on the development of finger and heel abnormalities in rat fetuses due to administration of 5-azacytidine, a chemotherapeutic agent.

5-azacytidine, a chemotherapeutic agent with teratogenic effects, was dissolved in physiological saline to make a 0.6% physiological saline solution,
This substance (No.
, 4) 50 mg/Kg of each io% physiological saline solution
, 9 or 200 for 100u/! 1/Ky was administered subcutaneously. The average weight of live fetuses and needle abnormalities were observed according to experimental teratological methods. The fingernail abnormality rate was expressed as the ratio (%) of the number of animals with fingernail abnormalities to the number of fetuses.

Furthermore, for comparison, 5-azacytidine and this substance (k4)
Group not administered any of the following, 5-azacytidine 0.6I1
g 7Kg only administration group and this substance (No4) 200+1
1 (17 kg only) was also observed. The results are shown in Table 2.

From the above results, this substance (No, 4 > 50ma/K
In the group administered y or more, a decrease in fingertip abnormalities was observed in a dose-dependent manner compared to the group administered 0.6 mg/kg of 5-7 zacytidine alone. In particular, in the 2001n (II/l (g) administration group of this substance (Nn4), a rapid decrease in the occurrence of finger stop abnormalities was observed.
The average weight of live fetuses was 5-azacytidine monoadministration group;
No difference was observed in the group administered in combination with this substance (order 4) and 5-azacytidine.

As described above, it has been found that the present substance (order 4) is extremely useful as an inhibitor of morphological abnormalities caused by chemical factors.

Disaster: The effect of this substance (Nα4) on the weight of the tail and limbs of mouse fetuses by administering chlorambutyl, a chemotherapeutic agent, once a day.

Chlorambutyl, a chemotherapeutic agent with teratogenic effects, was dissolved in sesame oil to make a 6% solution, and a dose of 61110/9 was orally administered once to mice on the 10th day of pregnancy using a gastric needle, and at the same time this substance was administered. A 10% physiological saline solution of (Nα4) was administered subcutaneously at a dose of 200 ffl (1/9), and the fetus was observed for short and short tails, curved tails, short and oligodactyly limbs, etc. according to experimental teratological methods. Comparison. as chlorambutyl 611
1(+/l(9) Observations were made for the single administration group and the non-administration group. The abnormality incidence rate is not expressed as the ratio (%) of the number of abnormality expression 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 (Nn4) and chlorambucil, the incidence of abnormalities in the tail or limbs was 79.8% and 100%, respectively. 70.5% and 95.2% respectively
A decrease in abnormal expression was observed in both sites.

Effect of this substance (Nα1) on the development of fetal abnormalities that occur in response to irradiation with 11 units of radiation.

Two groups of 39 pregnant mice were irradiated once with 200 R of radiation, and one group was simultaneously administered a 10% physiological saline solution of ``real'' (NQl) subcutaneously at a dose of 200 mQ/Kg, and an additional 6001 g. ,"*g was administered orally every day for 10 days, and according to experimental teratology, the fetuses were treated with 9 short tails and 1 long tail.
The incidence rate (%) of malformations such as cleft palate was determined.

In the group irradiated with radiation only, the malformation rate was 82%, but in the group administered with this substance (N01), the malformation rate was 48%.

As described above, the effect of this substance (NQI) on suppressing the appearance of morphological abnormalities due to physical factors was confirmed.

Effect of this substance (Na2 or Ni3) on abnormal locomotor function in mouse pups due to administration of chlorambutyl, a chemotherapeutic agent.

Chlorambutyl, a chemotherapeutic agent with teratogenic effects, was dissolved in sesame oil to make a 6% solution, and one sip was administered to each group of 82 mice on day 10 of pregnancy at a dose of 6n+a/Kg using a gastric tube. At the same time, a 10% physiological saline solution of this substance (Nα2 or Na3) was administered subcutaneously at a dose of 200 mg 7 kg, and the born pups were observed according to behavioral and functional teratological techniques. For comparison, observations were made on the chlorambutyl 6m (1/Kg alone administration group) and the non-administration group.

In the group administered with chloramacyl alone, the incidence of abnormalities in gait function was 765%, but in the group administered with this substance (Nα2) and chloramacyl, the incidence of abnormalities in gait function was 58.1%.
Met.

The incidence of abnormal walking function in the group administered with this substance (No. 3) and chloramacyl was 59.2%.

In the non-administration group, the incidence of abnormal walking function was 0%.

Using a pressure-type automatic filling VA machine, this substance (
330 mg of k4) was filled to prepare capsules.

Procedural amendment Commissioner of the Patent Office 1) Takeshi Bunka 1.Display of the incident 1986 Patent Application No. 201093 2. Name of the invention anti-teratogenic agent 3. Thoughts on making corrections Relationship with the incident

Claims (6)

[Claims] (1) An anti-teratogenic agent containing a protein polysaccharide obtained from a bacterium belonging to the basidiomycete as an active ingredient. (2) The protein polysaccharide showed positive results in the phenol-sulfuric acid color reaction and the Lowry-Folin method, and the elemental analysis showed 20 to 55% carbon, 3 to 9% hydrogen, and more than 0% to less than 16% nitrogen. and 360 in the infrared absorption spectrum
0~3200cm^-^1 and 1700~1600cm
Absorption was observed in ^-^1, soluble in water, chloroform,
Benzene and ether are unnecessary, and the average molecular weight by gel filtration chromatography is 1 x 10^4 to 1 x 10^
6. The anti-teratogenic agent according to claim 1, wherein the anti-teratogenic agent is (3) Claim No. 1 in which the teratogenicity is morphological abnormality
) or (2). (4) Teratogenicity is a functional abnormality in claim No. (1)
) or (2). (5) The anti-teratogenic agent according to claim (1), wherein the basidiomycete is selected from bacteria belonging to the genus Corsicolor, the genus Stonecroceae, the genus Enokitake, and the genus Wood fungus. (6) The anti-teratogenic agent according to claim (1), wherein the basidiomycete is selected from bacteria belonging to the genus Corsicolor.