JPS63316734A - Antiviral agent - Google Patents

Abstract

PURPOSE:To obtain an antiviral agent containing polysaccharide or protein polysaccharide produced from fungus belonging to Basidiomycetes as an active ingredient, having antiviral infection action as well as safeness and excellent effect. CONSTITUTION:A polysaccharide or protein saccharide which is produced from a fungus belonging to the genus Ganoderma Flammulina or Auricularia and is an extract from fruit body of mushroom, artificial culture fungus cell and culture medium thereof by an aqueous solvent is contained as an active ingredient. The active ingredient is especially effective to AIDS, since it inhibits absorption to human-derived lymph based cell by human immune disorder virus and successive infection thereof and inhibits reverse transcriptase activity, especially infection of retrovirus having reverse transcriptase. The above-mentioned substance is administered orally in doses of 0.1-1,000mg, preferably 1-100mg/kg/ day 1-3 times.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the Basidiomycota class (Ba5ldi011VC(!te
A polysaccharide or protein polysaccharide extracted from a bacterium belonging to the Heterobidiomycetes or Heterobasidiomycetes belonging to s) may be abbreviated as the present substance hereinafter. ) as an active ingredient.

There is. Until now, viral diseases have been dealt with through vaccination, and smallpox, yellow fever, and polio have been brought under control. However, vaccines alone cannot fight against diseases such as AIDS where persistent infection or latent infection is a problem, and there are currently high hopes for the development of a safe and highly effective antiviral agent. be. Therefore, the present inventors conducted intensive studies on the 8-second drug and surprisingly found that the polysaccharide or protein polysaccharide of the present invention has a pharmacological effect of antiviral infection action. This led to the invention.

The bacteria used in the present invention are based on the Illustrated Encyclopedia of Japanese Fungi (Yukensha edition) co-authored by Rokuya Ima and Tsuguo Hongo and the Journal of Japanese Mycology (Yokendo edition) by Seiya Ito. .

This substance is an aqueous solvent extract of mushroom fruiting bodies, artificially cultured bacterial cells, and their culture medium.

To obtain artificially cultured mycelia from basidiomycetes, a part of a plant on which the fungus is attached, or tissues of fruiting bodies developed on the plant, or spores are transplanted onto an appropriate agar medium. This is obtained by culturing at an appropriate temperature, repeating this culture procedure 2 to 3 times, and confirming that there is no contamination with contaminants, then inoculating the culture medium as a mother fungus and culturing at an appropriate temperature. It is. Generally, it is preferable to use a liquid medium in terms of handling and productivity.

As for the medium composition for culturing, a formulation used for normal culturing is sufficient, as long as it contains the various nutrients necessary for the growth of the above-mentioned bacteria. That is, carbon sources include, for example, glucose, maltose, lactose, sucrose, starch, and nectar; nitrogen sources include organic compounds such as peptone, meat extract, WI mother extract, Ill, cornstarch liquor, ammonium salts, and urea; Inorganic nitrogen compounds can be used.

Also, as a mineral component, 'W4IS! Inorganic salts such as di-salts, magnesium salts, iron salts, calcium salts, etc. may also be added. In addition, vitamins necessary for growth may be added as appropriate.

The culture conditions are initial pu2-7.Culture is usually carried out at 20-35°C for 2-30 days. p114 in this inner cultured strawberry
5. A temperature of 25 to 30°C is particularly preferred. When performing aeration agitation culture, the aeration rate is 0.1-2. Oj! /1/1n,
It is appropriate to carry out the stirring at a stirring speed of 30 to 800 r, p, m.

The fruiting body or the artificially cultured mycelium obtained by the above culture is then transferred to an extraction step. At this time, after washing, it may be subjected to drying treatment such as air drying or freeze drying, and then stored and used as appropriate. In addition, it is preferable to cut the material into small pieces prior to the extraction step in order to enhance the extraction effect.

Extraction is performed using an aqueous solvent. An aqueous solvent is a small amount of water or a water-soluble organic solvent, acid, or base, for example, 10
% or less, or a combination of two or more thereof. As the organic solvent, methanol, ethanol, isopropyl alcohol, etc. are mainly used. Examples of acids include hydrochloric acid, sulfuric acid, and acetic acid.

Examples of the base include ammonia, caustic soda, caustic acid, and soda carbonate. For extraction, use a 5- to 200-fold suspension of bacterial cell material (dry J standard), and usually 4
The treatment is carried out at a temperature of 120°C to 120°C for 20 minutes to 20 hours.

The purification process includes one or two of salting out, dialysis, ultrafiltration, reverse permeation treatment, gel passage, and precipitation treatment with an organic solvent.
This means removing low molecular weight substances by applying more than one method. (2) From a scientific point of view, the ultra-e method, which is a membrane separation method using pressure, the GDR method, or a combination of the reverse permeation treatment method, is particularly preferred. In addition, these treatments may be carried out after the salting-out step, depending on the case.

Salting-out agents used in the salting-out step include ammonium sulfate, common salt, potassium chloride, barium carbonate, etc., but ammonium sulfate is most preferably used. Further, as a minor treatment in the salting-out step, any one of dialysis, ultrafiltration, gel filtration, reverse permeation treatment, etc., or a combination of two or more of these steps is required.

Dialysis is usually performed using a semipermeable membrane such as a cellophane membrane or a collodion membrane. Gel filtration is performed using a column packed with an adsorbent such as dextran or polyacrylamide gel. Fillers sold under the names Cef/Dex and Biogel are commonly used.

Both the ultraviolet osmosis method and the reverse osmosis method are methods of fractionation using a membrane under pressure. The former is usually carried out at 0.5-5Kg/cd1, and the latter at 20-35Ky/l:d.

In the precipitation method using an organic solvent, methanol, ethanol, isopropanol, acetone, etc. are generally used. Further, in addition to the above operations, ion exchange treatment may be performed as necessary.

After the above purification operation is completed, water is removed by spray drying, freeze drying, etc., and then the product is manufactured.

If a cultured medium is used, only the purification step may be used.

Furthermore, it is more preferable to treat this substance with an alkaline aqueous solution because the antiviral effect will be enhanced. 5 to 200 times the amount of this substance from 0.01N to 5N,
Preferably 40% in an alkaline aqueous solution of 0.1N to 2N.
C. to 250.degree. C., preferably 60.degree. C. to 200.degree. C., most preferably 100.degree. C. to 150.degree. C. for 5 minutes to 2 hours, preferably 10 minutes to 1 hour.

Next, the treatment liquid is neutralized and a purification treatment step is performed.

The purification process can be carried out by applying one or more of the above-mentioned methods such as salting out, dialysis, ultrafiltration, permeation treatment, gel filtration, and precipitation treatment with an organic solvent. The conditions are the same as above.

, After the purification operations described in F are completed, water is removed by spray drying, freeze drying, etc., and then the product is manufactured.

In addition, this substance may be used in an ion exchange cell such as diethylaminoedyl (DEAE)-cellulose [1-sgel etc. are more preferred.

This substance is a carbon 1
The main components are 51 to 55%, hydrogen 3 to 9%, and nitrogen 0 to 16%.

1 The sugar components of this substance include at least lucose, galactose, and mannose, and the protein components include at least aspartic acid, glutamic acid, and ginsin.
Do it.

When the infrared spectrum is measured, it is 3600-3200α-
Absorption of hydroxyl group near 1 and or -1100 to 1600α
In the vicinity of -1, absorption derived from the amide group could be observed.

This substance is soluble in aqueous solvents and insoluble in organic solvents. Aqueous solvents are water or water-based alcohols,
It contains acids, bases, etc., and organic solvents include chloroform, benzene, ether, etc.

The substance is white or brown in color and has a molecular weight of 103-3X10G as determined by gel filtration chromatography.

This substance was orally administered at 1000 Jv/Kg to rats (drinking turtle type), 4 to 5 weeks old and weighing 100 to 1509 kg.
All the animals were observed alive for 7 days.

This substance is a safe substance with extremely low toxicity and almost no 01 effect.

It is generally known that viruses are replicated through the process of adsorbing to target cells, injecting the viral nucleic acid into the cell, and further inserting it into the cell's chromosomal genome. In addition, especially regarding the Reto [1 virus],
Before being inserted into the chromosomal genome of 1111, a process is required in which RNA, which is a virus-derived gene, is transcribed into DNA by the action of reverse transcriptase.

The present invention shows that the polysaccharide and polysaccharide protein of the present invention inhibit the adsorption of human immunodeficiency virus (HIV) to human-derived lymphoid cells and subsequent infection, and inhibit reverse transcriptase activity. I found out what to do. That is, after treating HIV with this substance at a concentration of 5 to 1000 μg/d for 2 hours at 0°C, the HIV was washed, added to MT-4 cells, infected, and cultured for 3 days. When we investigated the effect of this substance using a method of measuring 1-1V antigen-positive cells in 3-III infection, we found that with pretreatment with this substance, almost no H[V antigen-positive cells were detected, and it was found that 1-1V antigen-positive cells in HIV were detected. A strong adsorption inhibitory effect on derived lymphoid cells was observed. On the other hand, when the effect of this substance on reverse transcriptase activity was measured using rat liver whole metsuhinge p-RNA as a template, strong inhibition of reverse transcriptase activity was observed with the addition of 500i/ttdl of this substance.

These facts indicate that this substance has the effect of inhibiting viral infection, especially retrovirus infection that has reverse transcriptase, and is especially effective against AIDS caused by HIV infection. This indicates that

Azide-3° already used as an antiviral agent
- In the case of deoxygumidine (AZ T >, side effects that inhibit division are also observed on normal cells, but the polysaccharide or protein polysaccharide of the present invention has extremely low acute severity;
It is a safe substance and is useful as an antiviral infectious disease agent because it shows the effect of inhibiting viral infections, especially retrovirus infections.

That is, it is effective against viral infections, particularly retrovirus infections, ie, AIDS.

The polysaccharide or protein polysaccharide of the present invention can be made into any dosage form when used as an antiviral infectious disease agent. Moreover, administration is also performed by each route. Furthermore, the drug of the present invention is
The above-mentioned A used as a 1-hour treatment for viral infections
Even when used in combination with ZT etc., the efficacy will not decrease,
Combination with these drugs can be used as an effective means.

In the case of oral administration, tablets, granules,
Powders, capsules, etc. may contain additives commonly used in pharmaceutical formulations such as binders, encapsulating agents, fillers, lubricants, disintegrants, and wetting agents in their compositions. In addition, when used as a liquid preparation for good days, oral water preparation, ff11
It may be in the form of a mixture, suspension, emulsion, syrup, or as a dry product that is redissolved before use. Additionally, such liquid formulations may contain any commonly used additives and preservatives.

If for injection, the composition may contain additives such as stabilizing agents, buffering agents, preservatives, tonicity agents, etc., and may be presented in medium-dose ampoules or multi-dose 5-dose containers. be done. It should be noted that the composition may be in the form of an aqueous solution, suspension, solution, emulsion in an oily or aqueous vehicle, while the active ingredient may be in the form of a powder which is redissolved in water before use. Good too.

The antiviral infectious disease agent of the present invention is administered orally or parenterally to humans and animals. Oral administration includes sublingual administration. Parenteral administration includes injections such as subcutaneous, intramuscular, intravenous, infusion, and the like. The dosage of the antiviral infectious disease agent of the present invention depends on whether it is an animal or a human, and is influenced by age, individual differences, medical conditions, etc. Therefore, in some cases, it may be necessary to administer an amount outside the following range, but in general, it may be administered to humans. In the case of oral administration of the active substance of the present invention, the oral dosage of the active substance of the present invention is 0.1 to 1100 OR, preferably 1 to 1002 g per kg per day.
Administer in 3 to 3 divided doses.

Examples are shown below.

Example 1 Cinnamon mushroom (strain CM-359, Microtechnical Research Institute No. 6060)
No.) Dried bacterial cells 1009 are cut into pieces and the capacity is 3I! The mixture was placed in a stainless steel tank, and 2000 d of water was added thereto, and the temperature was maintained at 90 to 95°C while stirring. After about 3 hours of extraction,
Cooled to room temperature.

The extraction slurry was separated into an extract and a residue using a centrifuge. Furthermore, 0.4N-NaOll 2000 d for residual soaking
After extracting at 90-95°C for 3 hours, I)H was adjusted to 1.0 with cold UIL, 2N-Hcfl to room temperature, and then centrifuged to separate the extract and the residue.

The extracts were combined and concentrated to 400 m using a vacuum concentrator, and after removing low molecules by ultrafiltration, they were freeze-dried to obtain 12.5 g of dried product.

Example 2 100 g of dried Enokitake mushroom (strain CM-601, FIKEN Bacteria No. 3045) was cut into small pieces, placed in a stainless steel tank with a capacity of 131, and 2000 d of water was added, and the temperature was raised to 90 to 95 with stirring. It was kept at ℃. After extraction for about 3 hours, it was cooled to room temperature.

The extraction slurry was separated into an extract and a residue using a 9111 centrifuge. Furthermore, add 2,000 ae of water to the leftovers and bring to 90~
After extraction at 95° C. for 3 hours, the mixture was cooled to room temperature and centrifuged to separate the extract and the residue.

The extracts were combined, concentrated to 400 d using a vacuum concentrator, and further dried by freeze-drying to obtain 283 as a dry product.

Example 3 Wood ear fungus (CM-886 strain, Microtechnical Research Institute No. 1763)
Cut 100g of dried bacterial cells into small pieces and use 3 volumes! 200011 in a stainless steel tank! of water was added and the humidity was maintained at 90 to 95° C. while stirring frequently. After extraction for about 3 hours, it was cooled to room temperature.

The extraction slurry was separated into an extract and a residual solution using a centrifuge. Furthermore, 0.4N-Na0112000 m was left behind!
Add 90-9! After Sr1 extraction at It℃,
After cooling to room temperature and adjusting the pH to 7.0 with 2N-HCIl, the mixture was centrifuged to separate into an extract and a residue.

The extracts were combined and concentrated to 400 d using a vacuum concentrator, and after removing low molecular weight substances by ultrafiltration, freeze-drying was performed to obtain a dry product of 15.19.

Example 4 5 g of the hot water extract derived from enokitake obtained in Example 2 was
00d in 1.0N-Na011, 120℃, 2
Heat treatment was performed for 0 minutes. After heating treatment, cool to room temperature 7
After adjusting the pH to 7.0 with 41 L/GN-HCρ, dialysis was performed in running water using a VISKING cellulose tube for 2 days to remove low-molecular substances, and 3.8 g of dried material was obtained by freeze-drying. Obtained.

Table 1 shows the physicochemical properties of the substances obtained in Examples 1 to 4.
are summarized in the following. On the wood surface, phenol sulfur M! 2
51u color reaction indicates the presence of sugars and towrey-ro
The lin method shows the presence of peptide bonds. For molecular products, both components, which are present in average abundance, are listed using the gel filtration method.

Example 5 Regarding the substances obtained in Examples 1 to 4, the degree of inhibition of reverse transcriptase, which is specifically retained by retroviruses, was measured by the following method.

All test substances are freeze-dried products (10 Rg) mixed with 10 Rg of sterile distilled water.
d (concentration: 1/-).

1 scrap of 20 mHD, T, T, (dithiothreitol: manufactured by Sigma), 5p1 50 concentration enzyme reaction solution (250
mHTriS-11cj (1)I+ 8.3)-25
0mHKCj −・401118 H(lc12)1ρ
3d NTP solution (Tp-1a + HG-1 to 1 mHd)
'-1n dcho TP+manufactured by Sigma), 100 layers of 2 compartments/
d oligo (d ■) 12-18 (PL-biochemi
Cals Inc., 11iIl message p-RNA
(derived from 4 normal rat organs: 1 line//ii), 0.5 adult R
Nase Inhibitor (16 units/d
: Takara Shuzo) and 1111 [α-32P] dC
TP (approximately 800 ci/mmol, 10 μCi/i
11: manufactured by Amajam Japan Co., Ltd.) was added to a 1.5 m capacity Etzbendorf tube and placed in a 37°C water path.

After 5 minutes, test substance 12.
Add 57J to the reaction chicove and further reverse transcription of 1 product!
Y element (7 units//It: Takara Shuzo Co., Ltd., RouSas
(derived from associated virus), and the final reaction volume was set to 25, and the reaction was carried out at 37°C.

After 1 hour, the 5-component reaction solution was transferred to a 2 ctn x 2 cm D
After soaking in EAE paper (manufactured by Toyo Roshi Co., Ltd.) and air-drying, each filter paper was soaked in 10 m of 0.5H-Na HP O4 aqueous solution and shaken to remove the [α-32P] that was not used for DNA synthesis on the filter paper. ] dCTP was washed (this operation was performed 5 times at 5 minute intervals).

The above D'EAE paper was placed in a glass vial containing liquid scintillation cocktail (manufactured by Ama Jam Japan Co., Ltd.) of [107], and the radioactivity (c, p, m,)
was counted.

The inhibition rate (%) was determined by the following formula.

0-O8 Inhibition rate (χ)=　　　　　　　X100O Co: Radioactivity without addition of test substance O8 = radioactivity of test substance addition Table 2 shows the reverse transcriptase activity inhibition effect of each test substance.

Table 2 Reverse transcriptase activity inhibition effect +: 40%<, ”: 40%<>80
%, +++: 80%<Example 6 Inhibition of adsorption of HIV (AIDS virus) to human lymphocytes by the test substance was carried out by the following method (all operations were performed under sterile conditions).

HI V (lluman Im*unodcfici
virus) suspension 1- and test substance solution (8
00 g/ad Ld was placed in a test tube and left standing in water. After 2 hours, the virus suspension of 1-1 was taken from the test tube and added to the human lymphocyte-derived cell line MT-4 [Jpn, J.
Cancer Rcs, (Gann), 28°219-229 (1982) J.
,1,)-2 to adsorb the virus. Centrifugation (2 per minute,
000 revolutions.

After 10 minutes), the supernatant was poured into the precipitated MT-41 cells in RPMI 1640 (GIBC) containing 20% FC8.
OLaborato-rics, NY) 1;, II
IItraflFJ was floated at 2x10"/mA.

Transfer the above M'T-4 cell suspension to a 9G well plate at 100%
The cells were divided into portions and cultured under conditions of 5% CO2 and 37°C. At the 31st day of culture, the number of HIV-infected cells and non-infected cells was calculated by indirect fluorescent antibody method.

That is, MT-4 cells were treated with methanol to avoid reaction with oceanate-conjugated rabbit anti-human IQG (immunoglobulin) at 37°C.

500 MT-4 cells under a fluorescence microscope? M'lX, and fluorescence-positive cells were counted as HIV-infected cells.

Table 3 shows the inhibitory effect of the test substance on adsorption of HIV to human lymphocytes.

Table 3 Example 7 of Adsorption Inhibition Effect Using a pressure-type automatic filling IITItl, a No. 0 hard capsule was filled with 330η of this substance to create a capsule.

Representative Patent Attorney: Itoshi Nakamura, Procedures Available TiE 81, Title of Invention: Antiviral Agent 3, Relationship to the Amendment Case Name of Patent Applicant: (110) Kureha Chemical Industry Co., Ltd. 4, Agent: Shinjuku-ku, Tokyo Shinjuku 1-chome 1M+4 Yamada Building (and 1 other person) Details Old 1, Name of the invention Antiviral agent 2, Claims (1) Basidiomycete g14
An antiviral agent whose active ingredient is a polysaccharide or protein polysaccharide produced by bacteria belonging to the genus tes.

■ Polysaccharides or protein polysaccharides are found to be positive or slightly positive in α-bunotol sulfuric acid reaction, indole sulfuric acid reaction, 7sulon sulfuric acid reaction, phenol sulfuric acid reaction and/or Lowry-e-Forin reaction, and nipidrine reaction after hydrochloric acid hydrolysis. System analysis shows 20-55% carbon water, 3-9% hydrogen, and 16% nitrogen.
The carbohydrate portion contains at least glucose, galactose, and mannose, and the protein portion contains at least b-aspartic acid, glutamic acid, and lysine, and has an infrared absorption spectrum of 3600 to 3200 cffi-' and or 1700 to 1600 Crn-1. It is soluble in water, insoluble in chloroform, bempine, and 1-Al, and the molecular bottle obtained by gel filtration chromatography is 10 to 3x.
106. The antiviral agent according to claim 1, wherein the antiviral agent is 106.

0. The antiviral agent according to claim 1, wherein the Basidiomycota is a Basidiomycota or a Heterobidiomycota.

(4) The anti-1 virus agent according to claim 1, wherein the Basidiomycota class is selected from bacteria belonging to the genus Stonecarp, the genus Ino4tau, and the genus Fungus.

(5) An anti-AIDS virus agent containing the polysaccharide or protein polysaccharide according to claim 1 as an active ingredient.

(■ An anti-AIDS virus agent containing the polysaccharide or protein polysaccharide of claim 1 as an active ingredient.

3. Detailed Description of the Invention The present invention relates to Basidiomycetes.
This invention relates to an antiviral agent whose active ingredient is a polysaccharide or protein polysaccharide (hereinafter sometimes abbreviated as the substance) extracted from bacteria belonging to the homobasidiomycetes and heterobasidiomycetes.

Hepatitis B, adult hypocellular leukemia, and even AIDS (＾qu
ired Imm + undeficiency Sy
ndrome), and viral diseases have become a hot topic of discussion in recent years.

Until now, viral diseases have been dealt with through vaccination, and smallpox, yellow fever, and polio have been brought under control. However, vaccines alone cannot fight against diseases such as Δ[O8, where persistent infection or latent infection is a problem, and there are high expectations for the development of antiviral drugs that show poor efficacy. is the current situation. Therefore, the inventors of the present invention conducted intensive studies on various drugs and surprisingly found that the present substance does not have the antiviral and infectious effects, leading to the present invention.

The bacteria used in the present invention are based on the primary color Japanese fungi homonymous (Yuken + edition) co-authored by Rokuya Imaseki and Tsuguo Hongo, and the journal of Japanese mycology (Yokendo edition) by Seiya Ito.

Any type of basidiomycete or heterobasidiomycete may be used, but
Bacteria belonging to the genus Stonemanthus, the genus F1take, and the genus Ephthalmia are preferred.

This substance is an aqueous solvent extract of mushroom fruiting bodies, artificially cultured bacterial cells, and their 18 culture medium.

To obtain artificially cultured mycelia from basidiomycetes, a part of a plant in which the fungi are growing, tissue of fruiting bodies developed on the plant, or spores are transplanted onto an appropriate agar medium. , which can be obtained by culturing at an appropriate temperature, repeating this culture procedure 2 to 3 times, confirming that there is no contamination by bacteria, and then attaching this to a medium as a mother fungus and culturing at an appropriate temperature. It is. Generally, it is preferable to use a liquid medium in terms of handling and productivity.

As for the culture medium composition, a formulation used for normal culture is sufficient, as long as it contains the various nutrients necessary for the cultivation of the above-mentioned bacteria. That is, carbon sources include, for example, glucose, maltose, lactose, sucrose, starch, blackstrap sugar, and nitrogen sources include, for example, peptone, potter's 4-sugar, 15m extract, FtHn, cornstarch liquor, ammonium salts, urea, etc. Organic and inorganic nitrogen compounds can be used.

Further, as mineral components, inorganic salts such as phosphates, magnesium salts, iron salts, calcium salts, etc. may be added. In addition, vitamins necessary for growth may be added as appropriate.

The culture conditions are initial p]12-7.Culture is usually carried out at 20-35°C for 2-30 days. The pH during this internal culture is 4~
5. Conditions of temperature of 25 to 30°C are particularly preferred. When performing aerated agitation culture, the aeration rate is 0.1 to 2.01/i/n+in, although it varies slightly depending on the shape of the culture tank.
, ffl is suitably carried out at a stirring speed of 30 to 800 r, p, s.

The fruiting bodies or artificially cultured mycelia obtained by monofertilization are then transferred to an extraction step. At this time, after washing, it may be subjected to drying treatment such as air drying or freeze drying, and then stored and used as appropriate. Furthermore, it is preferable to cut into small pieces prior to the extraction process in order to enhance the extraction effect.

Extraction is performed using an aqueous solvent. An aqueous solvent is a small amount of water or a water-soluble organic solvent, acid, or base, for example, 10
It consists of one type or a combination of two or more types selected from aqueous solutions containing less than %. As the organic solvent, methanol, ethanol, isopropyl alcohol, etc. are mainly used. Examples of acids include hydrochloric acid, sulfuric acid, and acetic acid.

Examples of the base include ammonia, caustic soda, caustic acid, and soda carbonate. For extraction, use an extract that is 5 to 200 times stronger than the bacterial material (dry basis), and usually at 4℃.
The treatment is carried out at a temperature of 20 minutes to 20 hours at a temperature of 120°C to 120°C.

The purification process includes one or two of salting out, dialysis, ultrafiltration, reverse filtration, gel filtration, and precipitation using an organic solvent.
This means removing low molecular weight substances by applying more than one method. From an engineering standpoint, ultrafiltration, which is a membrane separation method using pressure, and reverse permeation treatment, either alone or in combination, are particularly preferred. In addition, these treatments may be carried out after the salting-out step, depending on the case.

Salting-out agents used in the salting-out step include ammonium sulfate, common salt, sodium chloride, barium carbonate, etc., and ammonium sulfate is most preferably used. Further, as a post-treatment after the salting-out step, any one of dialysis, ultrafiltration, gel filtration, reverse permeation treatment, etc., or a combination of two or more of these steps is required.

Dialysis is usually performed using a semipermeable membrane such as a cellophane membrane or a collodion membrane. Gel filtration is performed using a column packed with an adsorbent such as dextran or polyacrylamide gel. Fillers sold under the names of Sephadex and Biogel are commonly used.

Ultrafiltration and reverse osmosis are both methods of fractionation using a membrane under pressure. The former is 0.5-5Kg/cIr1,
The latter is usually carried out at 20 to 35 Kg/cd.

In the precipitation method using a specific solvent, methanol, ethanol, isobutylene, acetone, etc. are generally used. Further, in addition to the above operations, ion exchange treatment may be performed as necessary.

After the above purification operation is completed, water is removed by spray drying, freeze drying, etc., and then the product is manufactured.

If a cultured medium is used, only the purification step may be used.

Furthermore, it is more preferable to treat this substance with an alkaline aqueous solution because surprisingly, the antiviral effect is enhanced. 0.018 to 5N of this substance on a skewer 5 times to ¥1200
, preferably in an alkaline aqueous solution of 0.1N to 2N.
0°C to 250°C, preferably 60°C to 200°C, most preferably 100°C to 150°C for 5 minutes to 2 hours.
Preferably, the treatment is carried out for 10 minutes to 1 hour.

Next, the treatment liquid is neutralized and a purification treatment step is performed.

The purification process includes the above-mentioned salting out, dialysis, ultrafiltration, reverse permeation treatment, gel F' filtration, precipitation y+ treatment with an organic solvent, etc.
It can be carried out by applying one or more methods. The conditions are the same as above.

After the above purification operation is completed, water is removed by spray drying, freeze drying, etc., and then the product is manufactured.

In addition, this substance is further purified by chromatography using ion-exchange cellulose such as diethylaminoethyl(r)EΔE)-cell [1-s], and the adsorbed substance is eluted with salt, alkali, etc. It is more preferred because it provides a strong antiviral effect.

This substance shows positive or slightly positive results in α-naphthol sulfuric acid reaction, indole sulfuric acid reaction, Ansu[1-n sulfuric acid reaction, phenol sulfuric acid reaction, and/or Lowry-e-Forin method, and ninhydrin reaction after hydrochloric acid hydrolysis. As a result of elemental analysis, it contains 20 to 55% carbon, 3 to 9% hydrogen, and less than 16% nitrogen as main components.

The sugar components of this substance include at least glucose, gunctose, and mannose, and the protein components include at least aspartic acid, glutamic acid, and lysine.

p)-1 indicates 6.0 to 7.5.

When the infrared absorption spectrum is measured, it is 3600-320
Absorption of water M group near 0 cm-' and or 1700 to 16
Absorption derived from the amide group could be observed near 00υ-1.

This substance is soluble in aqueous solvents and insoluble in organic solvents. Aqueous solvents are water or water-based alcohols,
It includes acids, bases, etc., and the two solvents on the right include chloroform, benzene, ether, etc.

The substance is white or brown in color and has a molecular weight of 103-3x106 as determined by gel filtration chromatography.

This substance was orally administered at 1000 av/Ky to rats (6 dragons), 4 to 5 weeks old and weighing 100 to 1,509 kg, and all of the rats survived when observed for 7 days.

This substance is a safe substance with extremely low toxicity and almost no side effects.

It is generally known that a virus is replicated through a process in which it adsorbs to a target cell, and the viral nucleic acid is injected into the cell and roughly inserted into the chromosomal genome of the MA vacuole. In addition, especially for retroviruses, before they are inserted into the chromosomal genome of the cell, DNA is converted from RNA, which is a gene derived from the virus, by the action of reverse transcriptase.
It is necessary to have an excess culm that is transferred to the plant.

The present inventors believe that this substance is human immunodeficiency virus (HI).
V) adsorption to human-derived lymphoid cells and subsequent infection, and reverse transcriptase activity of 4
I found out that it causes harm. That is, 1" IV to 5
1000Ii! After treating with this substance at a concentration of i/d for 2 hours at 0°C, HIV is washed, added to MT-4g cells and adsorbed, and cultured for 3 days and then measured for HIV antigen-positive cells. When the effect of this substance was investigated, it was found that almost no HIV antigen-positive cells were detected by surface treatment with this substance, and a strong adsorption inhibitory effect of HIV on human-derived lymphoid cells was observed. On the other hand, when the effect of this substance on reverse transcriptase activity was measured using rat liver whole messenger RNA as a template, strong inhibition of reverse transcriptase activity was observed by the addition of this substance 500IJ9/d.

These facts indicate that this substance has the effect of inhibiting viral infection, especially retrovirus infection that has reverse transcriptase, and is especially effective against AIDS caused by HIV infection. It is.

Azide-3- already used as an antiviral agent
In the case of deoxydemidine (AZT), an n1 effect that inhibits division is also observed on normal cells, but this substance has extremely low acute toxicity, is a safe substance, and is effective against viral infections, especially retrovirus infections. It is useful as an antiviral agent because it exhibits an inhibitory effect, that is, it is effective against viral infections, particularly retroviral infections, ie, AIDS.

When this substance is used as an antiviral agent, it can be made into any dosage form. Moreover, administration is also performed by each route.

Furthermore, the drug of the present invention does not reduce its efficacy even when used in combination with the above-mentioned AZT, which is used as an antiviral agent, and can be used as an effective means in combination with these other drugs.

In the case of oral administration, tablets, granules,
Powders, capsules, etc. may contain additives commonly used in pharmaceutical formulations such as binders, encapsulating agents, excipients, lubricants, disintegrants, wetting agents, etc. When used as a liquid preparation for daily use, it may be in the form of an internal solution, a shaken mixture, a suspension, an emulsion, or a syrup.
It may also be in the form of a dry product that is redissolved before use. Additionally, such liquid formulations may contain any commonly used additives and preservatives.

For injection, the compositions may contain additives such as stabilizers, buffers, preservatives, tonicity agents, and are presented in unit-dose ampoules or multi-dose suspended containers. . It should be noted that the compositions may be in the form of aqueous solutions, suspensions, solutions, emulsions in oily or aqueous vehicles, wherein the active ingredient is dissolved in a suitable vehicle, e.g. a pyrogen-free, sterile vehicle, before use. It may also be a powder that is redissolved in water.

The antiviral agent of the present invention is administered orally or parenterally to humans and animals. Oral administration includes sublingual administration. Parenteral administration includes injections such as subcutaneous, intramuscular, intravenous, infusion, and the like. The dosage of the antiviral infectious disease agent of the present invention depends on whether it is an animal or a human, and is influenced by age, individual differences, medical conditions, etc. Therefore, in some cases, ω may be administered outside the following range, but in general, humans For subjects, the oral dosage of the active substance of the invention is 0.1 Kg per day at rest.
~1000I1g, preferably 1-100 #I! Administer J in 1 to 3 divided doses.

Examples are shown below.

Example 1 Cinnamon mushroom (strain CM-359, Microtechnical Research Institute No. 6060)
No. 100!7 dried bacterial cells were cut into pieces, placed in a stainless steel tank with a capacity of 31, and 2,000 ml of water was added, and the temperature was maintained at 90 to 95°C while stirring. After about 3 hours of extraction,
Cooled to room temperature.

The extraction slurry was separated into an extract and a residue using a centrifuge. Furthermore, 0.48-Na012000 was added to the remaining solution and extracted at 90-95°C for 3 hours, cooled to room temperature, adjusted to pH 7.0 with 2N-1-(Cj!), and centrifuged. It was separated into an extract and a residue.

The extracts were combined and reduced to 400d using a vacuum concentrator.
i+, . . . After removing low-molecular weight substances by ultrafiltration, the product was freeze-dried to obtain a dry product of 12.5 jJ.

Example 2 Cut 100 g of dried enokitake (strain CM-601, Microtechnical Research Institute No. 3045) into small pieces, put it in a stainless steel tank with a capacity of 31, add 2000 mQ of water, and raise the temperature to 90-95 with stirring. It was kept at ℃. After about 3 hours of extraction,
Cooled to room temperature.

The extraction slurry was separated into an extract and a residue by centrifugation. Furthermore, add 2000 d of water to the remaining water and bring it 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 extracts were combined and concentrated at 400 mi! using a vacuum concentrator. The mixture was concentrated to 447 g, and further dried by freeze-drying to yield 28 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 pieces, placed in a stainless steel tank with a capacity of 3j2, and 20,007 g of water was added thereto, and the temperature was maintained at 90 to 95° C. while stirring. After about 3 hours of extraction, it was cooled to room temperature.

The extraction slurry was separated into an extract and a residual solution using a centrifuge. Furthermore, after adding 0.48-Na0112000 d to the residue and extracting at 90-95°C for 3 hours, it was cooled to room temperature, and after adjusting the pH to 7.0 with 2Nl-10ρ, it was centrifuged to separate the extract and the residue. separated.

The extracts were combined and concentrated to 400 m using a vacuum concentrator, and after removing low molecular weight substances by ultrafiltration, lyophilization was performed to obtain 15.1 g of dried product.

Example 4 5 g of the hot water extract derived from enokitake obtained in Example 2 was
00#! 1! Dissolved in 1.0N-Na leaf and heated to 120℃
, heat treatment was performed for 20 minutes. After the heat treatment was completed, the mixture was cooled to room temperature, adjusted to pH 7.0 with 6N-H(J), and then dialyzed in flowing water using a Wiesking cellulose tube for 2 days to remove low-molecular substances, and then frozen. By drying, 3.8 g of dried product was obtained.

Table 1 shows the physicochemical properties of the substances obtained in Examples 1 to 4.
are summarized in. On the wood surface, phenolic RA acid color reaction indicates the presence of sugars, and Lowrey-Folin
The method shows the presence of a peptide bond. Regarding the molecular weight, both components, which are present in large amounts on average, are listed based on the glue filtration method.

Example 5 Regarding the substances obtained in Examples 1 to 4, the degree of inhibition of reverse transcriptase, which is characteristically retained by retroviruses, was measured by the following method.

All of these substances were lyophilized by dissolving 10 η in 10 d of sterile distilled water (11 degrees: 11 g/d).

1 part of 20 mHD, T, T, (dithiothreitol: manufactured by Sigma), 5 times the concentration of enzyme reaction solution (250 n)
+tlTriS-11c12 (1)H8,3)-25
011+HKC4) -40mHH(lcj 2 )1
Waste 3d NTP solution (1mHdATP-1mHdGT
P-1mHdTTP: manufactured by Sigma), 2d 100/
j19/ml! Oligo (d■) 12-18 (PL-bi
(manufactured by Ochemicals), 1-sei Metsusenger R
NA (derived from normal rat liver: 1111/ld'), 0
．． 5III1 RNask Inhibitor (
16 units/m: Takara Shuzo Co., Ltd.) and 1111 [α-
32P] dCTP (about 800ci/I!1llo
l.

10μC1/pJl: manufactured by Amersham Javan Co., Ltd.) at 1
．． 5ad! In addition to the Eppendorf tube, 37
It was placed in a ℃ water bath.

After 5 minutes, add the previously prepared IItg/d 8 degrees of this substance 1
2.5III was added to the reaction duplex, and 1 unit of reverse transcriptase (1 unit//11: Takara Shuzo Co., Ltd., Rous
associated virus) was added thereto, the final reaction volume was 25, and the reaction was carried out at 37°C.

The reaction solution of Shun 5 was soaked into 2 am x 2 ctpr DEAE paper (manufactured by Toyo Roshi Co., Ltd.) for 1 hour, and after air-drying, 5H-N a 2 HP O was applied to each filter paper at 10 ml of water.
(4) The filter paper was immersed in an aqueous solution and shaken to wash the [α-32P]dCTP that was not used for DNA synthesis on the filter paper.This operation was performed 5 times for 5 minutes each time).

After that, the DEAE paper was placed in a glass bicelle bottle containing 10a1 liquid scintillation cocktail (Amajamushi 1!Pan Co., Ltd.), and the radioactivity (c, p, m,
) was counted.

The reverse transcriptase activity inhibition rate m was determined by the following formula.

Radioactivity of C8 dual substance addition Table 1 shows the inhibition rate of reverse transcriptase activity of each substance.

Example 6 Inhibition of adsorption of HIV (AIDS virus) to human lymphocytes by this substance was carried out by the following method (all operations were performed under sterile conditions).

HI V (Truman Immunodeficiency)
ency Virus) suspension liquid 11n1 and this substance solution (
Put 800ILi/atri 11rt1 into a test tube,
His head lay still in the water. 11I+1 from a 2 hour invasive test tube! Take the virus suspension and transform it into human lymphocyte-derived cell line MT-4.
[Jpn, J, Cancer Res, (
Gann), 28°219-229 (7982
)] was adsorbed with the virus at a multiplicity of infection (H, 0, 1,) -2. , centrifugation at 2,000 revolutions per minute.

After 10 min), the supernatant was washed with Cl-precipitated MT-4 cells, and the MT-4 cells were incubated with RPMI 1640 (GI) containing 20% FC3.
The cells were suspended in BCO Laboratorics, NY) at a cell concentration of 2×10 5 /d.

100 MT-41 cell suspensions were dispensed into 96-well plates and cultured at 37° C. and 5% CO2 in the air. On the third day of culture, HIV-adsorbing cells and non-adsorbing cells were excluded by indirect fluorescent antibody method.

That is, MT-4 cells were fixed by methanol treatment and reacted with anti-HIV infected patient serum at 37°C. 3
After 0 minutes, the cells were washed with PBS and reacted with full A rescein isobuocyanate-conjugated rabbit anti-human IgG (immunoglobulin) at 37°C.

Five hundred MT-4 cells were observed under a fluorescence microscope, and fluorescence-positive cells were excluded as T11V-adsorbed cells.

Table 1 shows the rate of inhibition of adsorption of this substance to lymphocytes (1).
It was shown to.

+-+ i v Adsorption blockage rate (%) was determined using the following formula.

HIV absorption 6 inhibition rate (%) Example 7 Using a pressure-type automatic filling machine, 3301119 doses of the present substance of Example 1 were filled into No. 0 hard capsules to prepare Cabrel.

Claims (3)

[Claims] (1) An antiviral agent containing a polysaccharide or protein polysaccharide produced from a bacterium belonging to the Basidiomycetes as an active ingredient. (2) An antiretroviral agent containing the polysaccharide or protein polysaccharide according to claim 1 as an active ingredient. (3) An anti-AIDS virus agent containing the polysaccharide or protein polysaccharide according to claim 1 as an active ingredient.