JPH0567611B2 - - Google Patents

Description

[Detailed description of the invention]

"Industrial Application Field" The present invention relates to an antiviral substance obtained from a basidiomycete mycelium culture and a method for producing the same. "Prior Art" In recent years, extremely incurable diseases caused by infection with various viruses such as hepatitis B virus, AIDS virus, and herpes virus have become a serious problem. For example, the hepatitis B virus, which is a DNA virus, causes liver damage in infected individuals, progressing from acute or chronic hepatitis to cirrhosis and even liver cancer, leading to death in patients. In addition, the AIDS virus is a human RNA virus that belongs to the Retinviridae family of the Retroviridae family, but it has an affinity for T4 _- positive T cells and infects and kills them, resulting in immunodeficiency in patients.
Causes acquired immunodeficiency syndrome. Furthermore, among herpesviruses, types 1 and 2 of the herpes simplex virus are known, and the former is said to mainly cause stomatitis, and the latter to mainly cause genital herpes. At present, there are very few therapeutic drugs for such viral diseases. For example, therapeutic drugs such as adenine arabinoside and acyclovir are known, but they all have the problem of being highly toxic and having strong side effects. On the other hand, the present inventors have continued research on substances extracted from mycelial cultures of basidiomycetes such as shiitake mushrooms for many years. Furthermore, the substance extracted from the above-mentioned culture has an immunostimulatory effect (Special Publication No. 53
-23392), the molecular weight extracted from the above culture
6,000,000 to 1,500,000 (A fraction) and 1,500,000 to 800,000 (B fraction) of substances extracted from the above culture were found to be effective against chronic hepatitis B (Japanese Patent Application Laid-open No. 70532/1983). It has already been discovered that a complex of cytokinin-based active substances mainly consisting of polysaccharides and zeatin-related substances is effective as an antiviral agent (Japanese Patent Publication No. 36009/1983). ``Problem to be solved by the invention'' Substances extracted from the mycelium culture of basidiomycetes such as shiitake mushrooms are highly effective against adenine arabino in that they have almost no toxicity, no concerns about side effects, and exhibit excellent antiviral effects. It is superior to known antiviral substances such as CID and acyclovir. However, the substances extracted from the mycelium culture are composed of a complex group of substances ranging from low molecules to polymers, and it has been extremely difficult to determine their active ingredients. For this reason, it has not been possible to extract only the active ingredients and enhance their effects. Therefore, an object of the present invention is to provide a more effective antiviral substance and a method for producing the same by extracting an active ingredient exhibiting an antiviral effect from a mycelium culture of a basidiomycete such as a shiitake mushroom. "Means for Solving the Problems" As a result of intensive research to achieve the above object, the present inventors discovered that the active ingredient exhibiting antiviral activity contained in the mycelial culture of basidiomycetes such as shiitake mushrooms is derived from sugar. The present inventors discovered that the present invention is a substance whose main components are lignin, protein, and denatured water-soluble lignin. That is, the antiviral substance according to the present invention is obtained by culturing basidiomycete mycelium using a medium containing plant fiber components, and extracting a soluble fraction with an alcohol concentration of 37.5% and an insoluble fraction of 50% from a warm water extract of this culture. Separate and
This fraction is then applied to a phenylsepharose column and eluted with 75% ethylene glycol.The fraction has a molecular weight of 10,000 to 1,500,000 and contains 12 to 20 carbohydrates.
%, protein 2-5%, modified water-soluble lignin 70-85
%. Furthermore, the method for producing an antiviral substance according to the present invention includes:
Basidiomycete mycelium is cultured using a medium containing plant fiber components, water-soluble components are extracted from this culture with hot water, and a soluble fraction with an alcohol concentration of 37.5% and an insoluble fraction of 50% is separated from this extract. The method is characterized in that this fraction is further applied to a phenylsepharose column and the fraction eluted with 75% ethylene glycol is collected. Hereinafter, the present invention will be described in more detail by citing preferred embodiments. Examples of the basidiomycetes used in the present invention include shiitake mushrooms, Kawara mushrooms, Hiratake mushrooms, Enoki mushrooms, Stone mushrooms,
Examples include various edible mushrooms and medicinal mushrooms, such as maitake mushrooms, and among these, shiitake mushrooms are particularly preferred. Although either a solid medium or a liquid medium can be used as the medium, it is necessary to include a vegetable fiber component in the medium components. Examples of the plant fiber component include bagasse, beet lees, wheat straw, rice straw, corn stems and leaves, rice bran, and wheat bran, with bagasse being particularly preferred. In the present invention, it is thought that the plant fiber components are assimilated by the mycelium of the basidiomycete, and together with the mycelium components of the basidiomycete, they form a substance that exhibits an antiviral effect. In addition, sawdust, peptone, yeast, cane molasses, etc. may be added and mixed into the medium as nutritional components. Note that the medium is preferably sterilized using an autoclave or the like. Cultivation of basidiomycete mycelia is carried out, for example, by inoculating the above-mentioned medium with mycelium pellets obtained by liquid culturing basidiomycete spores. After inoculating the mycelium, in the case of a solid medium, it is grown for 3 months to 6 months in an air-conditioned culture room with a temperature of 18 to 25 degrees Celsius and a humidity of 50 to 90%.
Cultivate for about a month. Most ideally, temperature 20-25
Culture in an air-conditioned culture room at 60% temperature for about 4 to 6 months. It is preferable that the medium in which mycelium is spread in this manner be transferred to a temperature treatment chamber and subjected to variable temperature treatment. Thermostatic treatment is performed, for example, at 32-34℃ first.
Warm for 24 to 48 hours, then transfer to a low temperature treatment room for 4 to 48 hours.
Low temperature treatment is performed at 8°C and 85% humidity for 5 to 7 days. This variable temperature treatment is preferably employed in view of stabilizing the quality of the product, but is not necessarily required.
After that, if you move the medium to the cultivation room and leave it alone, fruiting bodies will start to develop, but at this point, the cultivation will end.
The culture as described below is pulverized using a pulverizer.
On the other hand, in the case of liquid culture, aerated culture or shaking culture is used for 1 week to 1 week at a temperature of 15 to 30°C.
Cultivate for about a month. Cultivation ends when mycelium is widespread in the medium. After culturing, the mycelium is self-digested using enzymes inherent in the mycelium, and metabolites are extracted. As a preferred method, in the case of a solid medium, first, the culture after cultivation is pulverized, and the pulverized product is treated at 40 to 90° C. for about 3 to 6 hours to allow autolysis by enzymes of the mycelium. Most preferably, heating is carried out through ventilation at around 80°C for 3 to 4 hours to promote enzymatic reaction and self-digestion, while reducing water content by 3 to 4 hours.
Dry to about 5%. Then add 40 to this shatter
Extract the active ingredients by pouring hot or hot water at a temperature above ℃. The most preferred embodiment is the above-mentioned pulverized product.
Add about 5 parts water per 600g, boil for about 1 hour, and stir. By this stirring, the metabolites of the mycelium and the active ingredients contained in the mycelial cell fluid are leached into the hot water. The suspension obtained in this way is filled into a filter bag made of flannel cloth, for example, and this is pressurized and filtered, and this filtrate is further filtered with a membrane filter to sterilize it, thereby obtaining an extract containing the active ingredients. . On the other hand, in the case of a liquid medium, the mycelium is crushed if necessary and then heated to 40 to 60°C to perform autolysis to obtain a liquid suspension culture in which the mycelium is dissolved. This culture was filtered as above,
The extract can be obtained by sterilization. The obtained extract can be concentrated using an ultrafiltration membrane, an evaporator, or the like, if necessary, and then freeze-dried to form a brown powder. In the present invention, the thus obtained extract or its dry powder is further subjected to alcohol fractional precipitation and chromatographic fractionation to separate its active ingredients. As this separation method, the following method can be adopted. Add preferably about 10 times the amount of water to the above dry powder to prepare a suspension at a pH of about 7.2, and add ethyl alcohol (or other lower alcohol) to this suspension to obtain a precipitate. . As is clear from the Examples described below, the growth-inhibiting effect on viruses is strongly observed in the fraction that is soluble at an alcohol concentration of 37.5% and insoluble at an alcohol concentration of 50%. Therefore, alcohol concentration 37.5% soluble, 50
Collect the % insoluble fraction. The alcohol precipitate thus obtained is applied to a phenylsepharose column to separate the active fraction. That is, the above alcohol precipitate was dissolved in a phosphate buffer (PH7.2) containing 1M ammonium sulfate, applied to a phenylsepharose column to separate the fraction that passed through, and then dissolved in a phosphate buffer (PH7.2). ) to separate the fraction that elutes, and then separate the fraction that elutes with 75% ethylene glycol. thus,
Among the obtained fractions, the last fraction eluted with 75% ethylene glycol has a more pronounced antiviral effect. An example of analysis of the fraction eluted with 75% ethylene glycol is shown below. Carbohydrate: 15.9%, Protein: 3.2% Elemental analysis values: Carbon 44.97%, Hydrogen 3.81%, Nitrogen 1.88
%, ash 5.7% Inorganic components: P0.52%, S0.34%, Mg0.03%,
Ca0.21%, K0.34%, Na2.17% Although this fraction shows strong ultraviolet absorption at 280 nm, it has a low protein content and is highly hydrophobic (adsorbed by phenylcephalose). ), is a highly unsaturated organic substance that contains almost no hydrogen or nitrogen, and has a brown color, so it is possible that the organic substance other than sugars and proteins mentioned above is water-soluble lignin. was considered. The acetyl bromide method, which is commonly used as a colorimetric method for quantifying lignin, is hardly affected by substances with aromatic rings other than lignin, such as tyrosine in proteins and hydrolyzed tannins, and is highly specific to lignin. However, as a result of quantifying the ethylene glycol elution fraction using this method, approx.
A value of 80% was obtained, which almost matched the value after subtracting sugar and protein. In addition, as a result of analyzing this substance using IR spectrum, NMR spectrum, etc., we found that the native
It gave almost the same spectrum as lignin, but
It was found that this is a modified water-soluble lignin that has more carboxyl groups than native lignin and has many condensed structures in its aromatic rings. The results of the IR spectrum are shown in Figure 1. In the figure, the solid line shows this substance, and the broken line shows the lignin of wheat straw. From these results, it was confirmed that the fraction finally eluted with ethylene glycol had the following molecular weight and composition. Molecular weight: 10,000 to 1,500,000 Chemical composition Carbohydrate: 12 to 20% Protein: 2 to 5% Denatured water-soluble lignin: 70 to 85% From the above results, the antiviral substance of the present invention
The main component is denatured water-soluble lignin obtained by assimilating dietary fiber components contained in the culture medium by basidiomycetes, and contains carbohydrates, proteins, and P, S, Mg, Ca, K,
It was found that it is made up of a combination of trace amounts of inorganic substances such as Na. "Effect" As is clear from the Examples described below, the antiviral substance of the present invention has an excellent infection prevention effect against various viruses, and since it is obtained from a natural product, it cannot be used as a synthetic chemical. There is no need to worry about side effects from drugs. The reason why the antiviral substance of the present invention has an excellent infection prevention effect against various viruses is not yet known in detail, but it is speculated that the antiviral substance of the present invention inhibits and reverses the adsorption of various viruses to host cells. Possible for inhibiting transcriptase activity. Further, according to the method for producing an antiviral extruded substance of the present invention, an active ingredient having an antiviral effect can be effectively extracted and separated. "Example" Example 1 (1) Culture of shiitake mushroom mycelium Nutrient source 5 of bagasse 90%, rice bran 5%, bran sugar
% solid medium is sterilized by a conventional method and inoculated with shiitake mushroom inoculum. Thereafter, the culture medium is transferred to an air-conditioned culture chamber at a temperature of 20 to 25° C. and a humidity of 60%, and cultured for 4 to 6 months. After the mycelium spreads in the medium, transfer it to a temperature treatment chamber and incubate at 32-34℃ for 24-24 hours.
Heat for 48 hours, then transfer to a low temperature treatment room for 5 to 8 hours.
Low temperature treatment is performed at 85% humidity for 5 to 7 days. Thereafter, the medium is transferred to a cultivation room and left to stand, and when fruiting bodies begin to appear on the surface of the medium, the medium is taken out and crushed using a crusher. (2) Extraction of active ingredients from culture The crushed product is heated with ventilation at around 80° C. for 3 to 4 hours to promote enzyme reaction, autolyze the mycelium, and dry to a moisture content of 3 to 5%. Add about 5 parts of water to 600g of this crushed material, and add about 1 part of water to 600g of this crushed material.
Boil for an hour and stir. By this stirring, the metabolites of the mycelium and the active ingredients contained in the mycelial cell fluid are leached into the water. The suspension thus obtained is filled into a flannel cloth filter bag, which is then pressurized and filtered to obtain a filtrate.
This filtrate is filtered through a membrane filter to remove bacteria and obtain an extract. This extract is concentrated under pressure using an ultrafiltration membrane or the like, and a brown powder is obtained by freeze-drying or the like. Hereinafter, this powder will be referred to as LEM. As a result of analyzing the components of the above LEM, sugar: 34.0
%, protein: 10.8%, water-soluble lignin: 43.0
%, other: 12.2%. Note that sugar was determined by the phenol/sulfuric acid method, protein was determined by the semi-micro Kjeldahl method, and lignin was determined by the acetyl bromide method. In addition, the sugar composition of the polysaccharide component, which is the main component of LEM, was quantified by gas chromatography, and the results showed that glucose: 18.1%, galactose:
5.4%, Mannose: 7.7%, Xylose: 29.9
%, arabinose: 37.6%, fucose + rhamnose: 1.2%. The safety of this LEM was tested and the results were as follows. (a) Acute toxicity (LD ₅₀ mg/Kg) Oral 7 days Mouse ♂19600 ♀17700 Rat ♂16400 ♀15600 Intraperitoneal mouse ♂5500 ♀4920 Rat ♂2490 ♀2270 (b) Subacute toxicity (Oral 90 days) Max. Effect amount (mg/Kg) Mouse ♂6740 ♀9100 Rat ♂3840 ♀7910 (3) Fractionation of LEM Add 10 times the volume of water to LEM, and add the suspension to PH
Adjust to 7.2. Ethyl alcohol was added to this suspension to give an alcohol concentration of 80 ml, and the resulting precipitate was separated to form LAP. Further, ethyl alcohol was added to the above suspension to make the alcohol concentration 50%, and the generated precipitate was separated to obtain P-LEM. Further, ethyl alcohol was added to the above suspension to make the alcohol concentration 37.5%, and after removing the generated precipitate, ethyl alcohol was added to make the alcohol concentration 50%, and the generated precipitate was separated to obtain neoPPT. neoPPT was dissolved in a phosphate buffer (PH7.2) containing 1M ammonium sulfate, and the solution was applied to a phenylsepharose CL-4B column, and the fraction that passed through was designated as P1. A phosphate buffer (PH7.2) was passed through the column, and the eluted fraction was designated as P2. Furthermore, 75% ethylene glycol was passed through the above column, and the eluted fraction was designated as P3. The results of analyzing the molecular weight and composition of this P3 are as described above. Test Example 1 (1) Effect of LEM on inhibiting infection of AIDS virus (HIV) in a cultured human T cell line Experimental method HTEV-1 positive MT-4 cells (cultured human T cell line) were infected with HIV at a multiplicity of infection of 0.001 ( After infecting cells at a ratio of 1 part virus to 1000 cells and adjusting the cell number to 2 x 10 ⁵ /ml with RPMI-1640 culture medium supplemented with 10% FCS, this was placed in the designated wells of a 24-well composition culture plate. Dispense each. On the other hand, P-LEM (50% of LEM
EtOH precipitate fraction) to a final concentration of 0.1 mg/ml, 0.2
mg/ml, 0.3 mg/ml, 0.4 mg/ml, and 0.5 mg/ml, respectively, into the designated wells of the above plate at 37°C in humidified air with 5% _CO2 .
It was maintained. The medium was changed every 3 days and P-
LEM was added to the medium each time. The number of cells expressing HIV antigen was measured every 3 days by indirect fluorescent antibody method and compared with the irradiated group to which P-LEM was not added. The experimental results are shown in FIG. In FIG. 1, the vertical axis represents the percentage (%) of fluorescence-positive cells, and the horizontal axis represents the number of days after infection.
In the figure, -★- is the control group, -△- is 0.1 mg
Addition group, -○- is 0.2mg addition group, -▲- is 0.3mg
Addition group, -■- is 0.4mg addition group, -□- is 0.5mg
Represents the addition group. In the control group, 90% of the cells were present on the third day of culture.
Although the cells became HIV antigen positive and almost died on the 9th day of culture, P-LEM was found to have a dose-dependent inhibitory effect on HIV infection of MT-4 cells. This infection prevention effect is 0.2
Almost complete inhibition was achieved for 12 days at a concentration of mg/ml. (2) Experiment on the anti-AIDS virus effect of P-LEM, P-2, and P-3 Next, we conducted an experiment on the anti-AIDS virus effect of P-LEM, P-2, and P-3.
HIV) and HIV-uninfected cells (MOLT-4) were mixed at a ratio of 1:1 to conduct a test for inhibition of giant cell formation (infected cells fuse due to the virus and induce multinucleated giant cells). . Experimental method The above cell mixture was adjusted to 5 × 10 ⁵ /ml,
Inoculating individual wells of a tissue culture plate and adding samples to various concentrations;
Cultures were incubated at 37°C in humidified air with 5% _CO2 . In addition, as a control, the cells were cultured in the same manner without adding the sample. After culturing, the cells were observed with a microscope over time, the number of living cells was counted, and the cell fusion inhibition rate of the sample-added group was calculated. Experimental results After 20 hours of culture, the control without sample addition
More than 95% cell fusion was observed, but P-
LEM showed 86% inhibition of cell fusion at 200 μg/ml. In addition, P2 (the fraction eluted by flowing phosphate buffer (PH7.2) through the phenyl-Sepharose column) was 6.25 μg/ml, 14.5%,
70% at 12.5 μg/ml, 82.6 at 25 μg/ml or more
% or more of cell fusion inhibition. Furthermore, P3
(Fraction eluted by running 75% ethylene glycol through a phenylsepharose column)
showed cell fusion inhibition of 65.2% at 6.25 μg/ml and 90% or more at 12.5 μg/ml or more. These results indicate that the P3 fraction in particular has a strong antiviral effect. (3) Inhibitory effect on reverse transcriptase Next, in order to elucidate the mechanism of action of the antiviral substance of the present invention against retroviruses such as AIDS virus, we investigated the reverse transcriptase ( Reverse
The inhibitory effect on transcriptase (RT) activity was tested. Experimental method P-LEM was added to reverse transcriptase (RT) prepared from purified avian myeloblastosis virus (AMV).
was added at various concentrations from 0 to 500 μg/ml, and the activity of reverse transcriptase (RT) at each concentration was measured. Experimental results The experimental results are shown in Figure 3. The vertical axis of the figure represents the inhibition rate of reverse transcriptase activity, and the horizontal axis represents P-
It represents the concentration of LEM. From this result, the antiviral substance of the present invention is effective for retrovirus host cells.
It is thought that it exerts its antiviral effect by inhibiting the activity of reverse transcriptase, which transcribes RNA genomes into DNA. Test Example 2 Next, the antiviral effect against herpes simplex virus type 1 was investigated using LEM and LAP (80% EtOH precipitated fraction of LEM). Experimental method: 229 cultured HeLa cell lines derived from human cancer cells were grown in a 24-well plate for tissue culture, and herpes simplex virus type 1 (HSV-1) was infected at a multiplicity of infection (moi) of 0.1 to 0.01 (virus 1 to virus 1). After infection at room temperature for 1 hour so that the number of cells was 10 to 100, the cells were cultured for 4 days in MEM (2% FBS supplemented) medium supplemented with various concentrations of LEM or LAP. After 4 days, the HeLa cells were frozen and thawed, the resulting suspension was centrifuged at 3000 rpm for 10 minutes, and the amount of virus in the supernatant was measured by plaque formation on the HeLa cells. Plaque formation was determined by adsorbing the virus in the centrifuged supernatant to HeLa cells for 1 hour, and then placing the above-mentioned solution containing 1% agar in a plastic Petri dish with a diameter of 35 mm.
229 HeLa cells were layered and solidified in MEM medium,
The cells were cultured in a carbon dioxide gas incubator for 2 days. Furthermore,
A MEM agar medium containing 0.005% neutral red was overlaid, and the plaques formed after 24 hours of culture were counted. Experimental Results The experimental results are shown in Figure 4. The vertical axis of the figure is
The number of plaques obtained from cells cultured in a medium containing LEM or LAP is divided by the number of plaques obtained from cells cultured in a medium not containing LEM or LAP. Indicates concentration. Both LEM and LAP suppressed the proliferation of herpes simplex virus type 1 in a concentration-dependent manner, especially
LAP showed less than 40% growth inhibition at a concentration of 10 μg/ml. Test Example 3 Using LEM, the in vivo growth inhibitory effect on herpes simplex virus type 1 was tested. Experimental method: The left eye of a male golden hamster (5 to 6 weeks old, weight approximately 60 g) was incised with a needle, and HSV was detected.
−1 to 8×10 ⁵ PFU (plaque forming units)/10μ
The animals were infected for 11 days. LEM was orally administered twice a day in the morning and evening using a probe, and the number of surviving animals after 11 days was compared with the control group. Experimental Results The experimental results are shown in Table 1. The animals in the LEM administration group were significantly more dependent than the control group, confirming that LEM has an antiviral effect when administered orally to animals.

[Table] Test Example 4 Effect of administration of LEM on Utsudochiyatsk hepatitis virus (WHV) Marmota monax is a rodent and squirrel that grows widely from the eastern United States to Canada. ), which belongs to the same hepadnavirus group as HBV. WHV has very similar biological characteristics to HBV, and has recently attracted attention as an experimental model for human hepatitis B. Therefore, we decided to use Utsudochiyak (weight 2-4 kg, estimated age 1-3 years) who was persistently positive for WHV.
LEM was dissolved in water and 3g per day was orally administered. Two animals were administered continuously for 18 weeks and 40 weeks,
Blood is collected at appropriate intervals and serum biochemical tests and
WHV virological markers (DNA polymerase, WHV-DNA) were measured. As a result, in the group treated with LEM for 18 weeks, liver function abnormalities improved, DNA polymerase and WHV-DNA
A slight decrease was observed. In addition, in the 40-week administration group, a significant decrease in DNA polymerase and WHV-DNA was observed. The results of the 40-week administration group are shown in Figure 5. In the figure, □-□ and ○-○ represent the measured values of DNA polymerase of Utsudochik No. 1 and No. 2,
□…□ and ○…○ are Utsudochik No.1 and No.
2 shows the measured value of WHV-DNA. These results support that oral administration of LEM can be expected to be sufficiently effective against chronic hepatitis B in humans. Test Example 5 66 chronic hepatitis B patients who were positive for e-antigen [HBeAg(+)] were orally administered 6 g of LEM per day, 3 g in the morning and evening for 16 consecutive weeks, and subjective and objective symptoms and blood biochemical tests were evaluated. , investigated virological testing. As a result, among the results judged by the attending physician, the degree of general improvement was found to be significant in 13 cases (19.7%) and improvement in 5 cases (7.6%).
%), 13 cases (19.7%) showed mild improvement, and 47% showed some improvement. Some improvement in liver function was observed in 31 cases (47%), including 11 cases (16.7%) with marked improvement. In addition, as a result of statistical analysis of the viral e-antigen in the blood at the start of administration and after administration in 47 patients who could be analyzed, the e-antigen was 4.77 at the start of administration.
After 16 weeks (expressed as cut of idex), it was 3.64, which was a significant decrease. Similarly, as a result of analyzing the e-antibody in 46 cases, it was 9.28% (expressed as inhibition rate) at the start of administration, but it significantly increased to 19.30% after 16 weeks, and the overall judgment shown above confirmed. Regarding side effects, 2 patients (3%) had mild abdominal bloating and indigestion symptoms.
The safety factor was extremely high. These results show that the antiviral substance of the present invention is effective in sufficiently improving the symptoms of hepatitis B as well. Example 2 Contains 90% corn stems and leaves and 10% rice bran,
A solid medium prepared to have a moisture content of 70% was sterilized, inoculated with the mycelium of C. chinensis, and then incubated at 25℃ for 4 hours.
Cultured for several months. After culturing, the medium was crushed and dried at 80°C for 3 hours.
Water was added and extracted at 80°C for 6 hours, and the filtrate was freeze-dried. A suspension was prepared by adding 10 times the amount of water to this powder, and ethyl alcohol was added to this suspension to make the alcohol concentration 37.5%, and the formed precipitate was removed. After further adding ethyl alcohol to make the alcohol concentration 50%, the resulting precipitate was separated to obtain a soluble fraction with an alcohol concentration of 37.5% and an insoluble fraction with an alcohol concentration of 50%. This fraction was dissolved in phosphate buffer (PH) containing 1M ammonium sulfate.
7.2) and dissolve it in phenylcepharose CL.
−4B column to 75% as in the previous example.
An ethylene glycol elution fraction was obtained. Analysis of this material revealed that it contained 18.1% carbohydrates, 4.6% protein, and 73.4% denatured water-soluble lignin. Test Example 6 Regarding the substance obtained in Example 2 above, AIDS virus (HIV), herpes simplex virus type 1,
Antiviral effects against herpes simplex virus type 2, poliovirus, and measles virus were investigated. Regarding HIV, an experiment was conducted using the method described in Test Example 1 (2) above, and the cell fusion inhibition rate was measured. For other viruses, Vero (derived from African green monkey kidney) is used instead of HeLa cells.
An experiment was conducted in the same manner as in Test Example 2, except that cells were used, and the plaque formation rate was measured. These results are shown in Table 2.

【table】

[Table] From the results in Table 2, it can be seen that the substance obtained in Example 2 has an excellent antiviral effect against various viruses. "Effects of the Invention" As explained above, it has been confirmed that the antiviral substance of the present invention has the effect of suppressing the proliferation of various viruses. Additionally, what is noteworthy about the present invention is that conventional substances with antiviral effects have strong toxicity and side effects, and in many cases had to be abandoned for practical application. The substance is extracted from 100% natural products, so it is extremely safe. The substance of the present invention inhibits the activity of viral reverse transcriptase even at low concentrations, thereby suppressing the proliferation of retroviruses and can be expected to have excellent preventive or therapeutic effects against various viral diseases. .

[Brief explanation of the drawing]

Figure 1 shows the relationship between the substance of the present invention and the lignin of wheat straw.
Figure 2 shows the IR spectrum, Figure 2 shows the infection suppressing effect of LEM on the AIDS virus, Figure 3
Figure 4 shows the inhibitory effect of P-LEM on the reverse transcriptase activity of purified avian myeloblastosis virus.
The figure shows the infection suppressing effect of LEM and LAP against herpes simplex virus type 1, and Figure 5 shows WHV.
DNA polymerase, WHV- when LEM was continuously administered for 40 weeks to persistently positive Utsudochika
FIG. 3 is a diagram showing measured values of DNA.

Claims (1)

[Claims] 1. Cultivating basidiomycete mycelium using a medium containing plant fiber components, and separating a soluble fraction with an alcohol concentration of 37.5% and an insoluble fraction with an alcohol concentration of 50% from a warm water extract of this culture. This fraction is then applied to a phenylsepharose column and eluted with 75% ethylene glycol.The fraction has a molecular weight of 10,000 to 1,500,000, contains 12 to 20% carbohydrates, 2 to 5% protein, An antiviral substance comprising a substance containing 70 to 85% of modified water-soluble lignin. 2 Cultivate basidiomycete mycelia using a medium containing plant fiber components, extract water-soluble components from this culture with hot water, and extract alcohol concentration of 37.5 from this extract.
1. A method for producing an antiviral substance, which comprises separating % soluble and 50% insoluble fractions, applying this fraction to a phenyl-Sepharose column, and collecting the fraction eluted with 75% ethylene glycol.