JPH02286623A - Antiviral substance and production thereof - Google Patents

Abstract

PURPOSE:To obtain an antiviral substance, consisting essentially of a glucide, protein, inorganic substance and modified water-soluble lignin prepared by extraction of a mycelial culture of a basidiomycete and having ultrahigh safety. CONSTITUTION:Mycelia of a basidiomycete (preferably SHIITAKE mushroom) are cultured in a culture medium containing a vegetable fiber ingredient (preferably bagasse) and the resultant culture is autolyzed. Warm water at >=40 deg.C or hot water is then poured thereinto to extract active ingredients. An alcohol is subsequently added to the resultant extract solution to collect a fraction soluble in 37.5% alcohol concentration and insoluble in 50% concentration thereof. The resultant fraction is then dissolved in a phosphoric acid buffer (pH7.2) containing 1 M ammonium sulfate and chromatographed with Phenyl- Sepharose(R). A fraction passing therethrough without adsorption and a fraction eluted with the phosphoric acid buffer solution are subsequently separated to afford the objective antiviral substance, having 10000-1500000 molecular weight and containing 12-20% glucide, 2-5% proteins, 70-85% modified water-soluble lignin and P, S, Mg, Ca, K and Na as inorganic ingredients as a fraction eluted with 75% ethylene glycol.

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 difficult-to-cure 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 retrovirus and antivirus subgroup, but T4
It has an affinity for positive T cells and infects and kills them, resulting in immunodeficiency in patients and acquired immunodeficiency syndrome.

Furthermore, among herpesviruses, type 2 and type 2 of 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. The substance extracted from the culture has an immunostimulatory effect (Special Publication No. 5323392), and the molecular weight of the substance extracted from the culture is 6 million to 1.5 million (A fraction).
and 1,500,000 to 800,000 (B fraction) are effective against chronic hepatitis B (Japanese Patent Application Laid-open No. 70532/1983), and cytokinin mainly consisting of polysaccharides and zeatin-related substances extracted from the above culture. Complexes of system-active substances are effective as antiviral agents (Special Publication No. 62-36009)
etc. have already been found.

[Problem to be solved by the invention] Substances extracted from mycelial cultures of basidiomycetes such as shiitake mushrooms are
It is superior to known antiviral substances such as adenine arabinoside and acyclovir in that it has almost no toxicity, no worries about side effects, and exhibits excellent antiviral effects.

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 ingredient and enhance its effect.

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 mycelium culture of basidiomycetes such as shiitake mushrooms is derived from sugar. The present inventors have discovered that the present invention is a substance whose main components are minerals, proteins, minerals, and modified water-soluble lignin.

That is, the antiviral substance according to the present invention is obtained by culturing basidiomycete mycelia using a medium containing plant fiber components, and extracting carbohydrates, proteins, minerals, and denatured water-soluble substances from this culture. The main component is lignin.

In addition, the method for producing an antiviral substance according to the present invention involves culturing basidiomycete mycelium using a medium containing a plant fiber component,
The method is characterized in that water-soluble components are extracted from this culture to obtain substances whose main components are carbohydrates, proteins, minerals, and modified water-soluble lignin.

Hereinafter, the present invention will be described in more detail by citing preferred embodiments.

Examples of the basidiomycete used in the present invention include various edible and medicinal mushrooms, such as Shiitake, Katsura mushroom, Hiratake, Enoki mushroom, Stonewort mushroom, and Maitake mushroom, among which Shiitake mushroom is particularly preferred.

As the medium, either a solid medium or a liquid medium can be used, but 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 considered that the plant fiber components are utilized by the mycelium of the basidiomycete, and together with the mycelium components of the basidiomycete, they form a substance that exhibits an antiviral effect.

Note that other nutritional components such as sawdust, peptone, yeast, and cane molasses may be added and mixed into the medium.

Note that the medium is preferably sterilized using an autoclave or the like.

Cultivation of basidiomycete mycelium is carried out, for example, by inoculating the above-mentioned medium with mycelial pellets obtained by liquid culturing basidiomycete spores. After inoculating the mycelium, in the case of a solid medium, it is cultured for about 3 to 6 months in an air-conditioned culture room, for example, at a temperature of 18 to 25° C. and a humidity of about 50 to 90%. Most ideally, the culture is carried out for about 4 to 6 months in an air-conditioned culture room with a temperature of 20 to 25° C. and a humidity of 60%. It is preferable that the medium in which mycelium is spread in this way be transferred to a temperature treatment chamber and subjected to variable temperature treatment. Change temperature treatment is 1. For example, initially 32-34°C.
Heat for 24 to 48 hours, then transfer to a low-temperature treatment chamber for 4 hours.
Low temperature treatment is performed for 5 to 7 days at ~8° C. and 85% humidity. 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 a cultivation room and leave it alone, fruiting bodies will begin to develop.
At this point, the culture is terminated, and the culture is pulverized using a pulverizer as described below. On the other hand, in the case of a liquid medium, the culture is carried out at a temperature of 15 to 30° C. for about one week to one month by aerated culture or shaking culture. 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. In the case of a solid medium, the preferred method is to first pulverize the culture after cultivation, and then heat the pulverized product at 40 to 90°C for 3 to 30 minutes.
Treat for about 6 hours to allow self-digestion by mycelium enzymes. Most preferably, it is aerated and heated at around 80° C. for 3 to 4 hours to promote enzyme reaction and self-digestion, and is dried to a moisture content of about 3 to 5%. Next, add 4
Extract the active ingredients by pouring hot water or hot water above °C. In the most preferred embodiment, water at about 5° C. is added to 600 g of the pulverized material, and the mixture is boiled and stirred for about 1 hour. 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 through a membrane filter to sterilize it to obtain 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° C. to 60° C. to perform autolysis, thereby obtaining a liquid suspension culture in which the mycelium is dissolved. This culture can be filtered and sterilized in the same manner as above to obtain an extract. The obtained extract may 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 extract obtained in this way or its dry powder is further subjected to alcohol fractional precipitation and chromatographic fractionation,
The active ingredient is separated. As this separation method, the following method can be adopted.

Adding preferably about 10 times the amount of water to the above dry powder,
The suspension is adjusted to a pH of about 7.2, and ethyl alcohol (or other lower alcohols may be used) is added to the suspension to obtain a precipitate. As is clear from the examples described below, the growth-inhibiting effect on viruses is found at alcohol concentration of 37.
It is strongly observed in the fraction that is soluble at 5% alcohol concentration and insoluble at 50% alcohol concentration. Therefore, alcohol concentration 3
It is particularly preferred to collect a 75% soluble and 50% insoluble fraction.

Further, it is preferable to further apply the alcohol precipitate thus obtained to various chromatography columns to separate an active fraction. As the chromatography column, a phenyl Sepharose column is particularly preferably employed. Particularly preferably, the alcohol precipitate is dissolved in a phosphate buffer 'Ip containing 1M ammonium sulfate.
jf pH 7,2), passed through a phenyl sepharose column to separate the fraction, and then passed through a phosphate buffer (pH 7,2) to separate the eluted fraction,
Furthermore, a fraction eluted with 75% ethylene glycol is collected. Among the fractions thus obtained, the fraction eluted last with 75% ethylene glycol has a more pronounced antiviral effect.

An analysis example of the fraction eluted with 75% ethylene glycol is shown below.

Carbohydrates: 15.9%, Protein = 32% Elemental analysis values Carbon 44.97%, Hydrogen 3.81%, Nitrogen 1.88%, Ash 57% Inorganic components: PO, 52%, SO, 34% , M
g 0.03%, Ca 0.21%, K O, 34
%, Na 2.17% Although this fraction shows strong ultraviolet absorption at 280 nm, it has a low protein content, is highly hydrophobic (adsorbed by phenyl Sepharose), and contains almost no hydrogen or nitrogen. Since it has characteristics such as being a highly unsaturated organic substance and exhibiting a brown color, it was thought that the organic substance other than sugar and protein mentioned above may be water-soluble lignin.

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 quite specific for lignin. As a result of quantifying the ethylene glycol elution fraction according to this journal, a value of about 80% was obtained, which was almost the same as the value after subtracting sugar and protein.

In addition, as a result of analyzing this substance by IR spectrum, NMR spectrum, etc., it gave almost the same spectrum as native lignin obtained from plants, but it has more carboxyl groups than native lignin and has a fused structure in the aromatic ring. It turned out to be a modified water-soluble lignin with a large amount of lignin. The results of the IR spectrum are shown in FIG. 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 0 to 85% From the above results, the antiviral substance of the present invention is contained in the medium. The main component is denatured water-soluble lignin obtained by assimilating dietary fiber components by basidiomycetes, and it is made up of carbohydrates, proteins, and inorganic substances such as P, S, Mg, Ca, K, and Na bound to it. I found out that there is.

"Effect" As is clear from the examples described later, 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. This is thought to be due to inhibition of photoenzyme activity.

Further, according to the method for producing an antiviral substance of the present invention, an active ingredient having an antiviral effect can be effectively extracted and separated.

"Example" Example fl) Culture of Shiitake Mushroom Mycelium A solid medium containing 90% bagasse, 5% rice bran, and 5% nutrient source such as bran is sterilized by a conventional method, and inoculated with shiitake mushroom inoculum. After that, the culture medium was heated at a temperature of 20-25℃ and a humidity of 60℃.
% in an air-conditioned culture room and cultured for 4 to 6 months. After the mycelium spreads in the medium, it is transferred to a temperature treatment room for 32 to 30 minutes.
The sample is heated at 34° C. for 24 to 48 hours, then transferred to a low temperature treatment chamber and subjected to low temperature treatment at 5 to 8° C. and 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 through 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%. About 512 g of water is added to 600 g of this crushed material, and the mixture is boiled and stirred for about 1 hour.

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 was filled into a flannel cloth filter bag,
This is pressurized and filtered to obtain a filtrate. This filtrate is further 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%, others: 12.2%. Note that sugar is phenol/
The amount was determined by the sulfuric acid method, the protein was determined by the semi-microkeltal method, and the lignin was determined by the acetyl bromide method.

In addition, as a result of quantifying the sugar composition of the polysaccharide component, which is the main component of LEM, by gas chromatography, glucose: 18.1%, galactose: 5.4%, mannose,
7.7%, xylose 299%.

Arabinose: 376%, fucose + rhamnose.

It was 12%.

The safety of this LEM was tested and the results were as follows.

fa) Acute toxicity + LDI, omg/kg) ■Metal loa day mouse ♂19.600 ♀17.7 (10 rats
♂16.400 ♀15.600■ Intraperitoneal mouse ♂5.500 ♀4.920 Rat ♂2.
490 ♀2.270 (bl subacute toxicity (oral 90 days) maximum no effect N fmg/kg) Mouse ♂6.740 ♀9,100 Rat ♂3.
840>♀ 7,910 (3) Fractionation of LEM Add 10 times the amount of water to LEM and adjust the suspension to pH 7.2.
Adjust to.

(2) Ethyl alcohol was added to this suspension to make the alcohol concentration 80%, and the resulting precipitate was separated to form LAP.

■Also, add ethyl alcohol to the above suspension to make the alcohol concentration 50%, separate the generated precipitate, and P-L
It was named EM.

■Furthermore, ethyl alcohol was added to the above suspension to make the alcohol concentration 37.5%, and after removing the formed precipitate,
Ethyl alcohol was further added to make the alcohol concentration 50%, and the generated precipitate was separated to give neoPPT.

■Dissolve n e o P P T in a phosphate buffer (pH 7.21) containing 1M ammonium sulfate, apply this to a phenyl Sepharose CL-4B column, and collect the fraction that passes through the Pl
And so.

(2) A phosphate buffer solution (pH 7.21) was passed through the column, and the eluted fraction was designated as P2.

(2) 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) Culture of AIDS virus (IIV) Hi-T
Effect of LEM on inhibiting infection in cell lines ■ Experimental method HTLV-1-positive MT-4 cells (cultured human T cell line) were infected with HIV at a multiplicity of 0.001 (1000 cells).
1 part virus) to reduce the number of cells by 10%.
FC5710RPM 2 x 10 in l-1640 culture medium
After adjusting the volume to 5/ml, the solution is dispensed into predetermined wells of a 24-well tissue culture notebook.

P-LEM (50% EtO of LEM) 1 precipitate fraction)
Final concentration 0.1 mg/ml, 0.2 mg/ml
．． 0.3n+g/mL0, 4+B/ml, 0.5mg/
Each sample was injected into a predetermined well of the above plate in an amount of 1.5 ml, and maintained at 37°C in humidified air containing 5% CO7. The medium was replaced every 3 days, and P-LEM was added to the medium each time. l(
The number of cells expressing the IV antigen was measured and compared with a control group to which P-LEM was not added.

■Experimental Results The experimental results are as 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 addition, in the figure, -★- is the control group,
-△- is 0.1mg addition group, ○- is 0.2mg addition group,
-Mu 1 is a 0.3 mg addition group, -1 is a 0.4 mg addition group,
-Low represents the 0.5 mg addition group.

In the control group, 90% of the cells became HIV antigen positive on the 3rd day of culture, and most of them died on the 9th day of culture, but P-LEM showed a dose-dependent effect on HIV infection of MT-4 cells. An inhibitory effect was observed.

This infection inhibition effect could be almost completely inhibited for 12 days at a concentration of 0.2 mg/ml.

(Experiment on the anti-AIDS virus effect of 21P-LEM, P-2, P-3 Next, I-) IV sustained production cells + MOLT-4/1
lrVl and HIV-uninfected cells (MOLT-4)
= 1 and tested 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 × 1057 ml, inoculated into individual wells of a tissue culture plate, and samples were added to various concentrations, and incubated at 37 °C in humidified air with 5% CO2. Cultured. 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, more than 95% cell fusion was observed in the control without sample addition, but with P-LEM, 200μg/cell fusion was observed.
ml showed 86% inhibition of cell fusion. In addition, P2 (phenyl sepharose column with phosphate buffer (pH 7, 2)
) is 6.25 μg/ml.
14.5% at 12.5 μg/ml, 70% at 25 μg
/m1 or more showed cell fusion inhibition of 82.6% or more. Furthermore, P3 (the fraction eluted by flowing 75% ethylene glycol through a phenyl sepharose column) is 6.
65.2% at 25 u g/ml, 12.5 LLg/m
1 or more showed 90% or more inhibition of cell fusion.

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 (inhibitory effect) of purified avian myeloblastosis virus (AMV). R
everse transcriptase.

The inhibitory effect on RTI activity was tested.

■Experimental method P-LEM was added to reverse transcriptase (RT) prepared from purified avian myeloblastosis virus (AMV) at 0 to 500 μg/m
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 the concentration of P'-L E H.

From this result, it is considered that the antiviral substance of the present invention exerts its antiviral effect by inhibiting the activity of reverse transcriptase, which transcribes the RNA genome into DNA in retrovirus host cells.

Test Example 2 Next, L E, M and LAP (80% EtO of LEM
The antiviral effect against herpes simplex virus type 1 was investigated using the H precipitate fraction).

■Experimental method 229 cultured HeLa cells derived from human cancer cells were grown in a 24-well plate for tissue culture, and herpes simplex virus l
Multiplicity of infection (m, o, i) for type (H5V-1) 0.1
~0.01 (10 to 100 cells for 1 virus)
After 1 hour of infection at room temperature (percentage of
Cultured in S supplemented medium for 4 days and then grown again.

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 occurs when the virus in the centrifuged supernatant is transferred to HeLa cells.
After adsorption for an hour, HeLa cells were placed in a plastic Petri dish with a diameter of 35 mm in the above MEM medium containing 1% agar.
Nine strains were solidified in layers and cultured in a carbon dioxide brooder for 2 days. Furthermore, MEM containing 0.005% neutral red
An agar medium 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 shows the percentage of the number of plaques obtained from cells cultured in a medium containing LEM or LAP divided by the number of plaques obtained from cells cultured in a medium not containing LEM or LAP, The horizontal axis indicates the concentration of the sample.

Both LEM and LAP suppressed the proliferation of herpes simplex virus type 1 in a concentration-dependent manner, and LEM in particular suppressed the proliferation of herpes simplex virus type 1.
It showed less than 40% growth inhibition at a concentration of Lg/ml.

Test Example 3 In vitro test against herpes simplex virus type 1 using LEM
The antiproliferative effect in vivo was tested.

■Experiment method Male golden hamster (5-6 weeks old, weight 60g)
)'s left eye was incised with a syringe needle, and 8X1 injection of HS V-1 was made.
The mice were infected with an amount of 0.05PF former plaque forming unit)/1oLL and reared 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. Animals in the LEM administration group survived significantly more than the control group, and oral administration to animals showed that LEM
It was confirmed that EM has an antiviral effect.

(Hereinafter, blank space) Table 1 Test Example 4 LEM against woodchuck hepatitis virus FW1m
The woodchuck (Marmota monax) is an animal of the squirrel family that lives widely from the eastern United States to Canada. WHV has biological characteristics extremely similar to HBV, and has recently attracted attention as an experimental model for human hepatitis B. , estimated age 1~
(3 years old), LEM was dissolved in water and administered orally at 3 g per day.

Two animals each were administered continuously for 18 weeks and 40 weeks, and blood was collected at appropriate intervals for serum biochemical tests and WHV virological markers (DNA polymerase, WHV-DN).
Al was measured.

As a result, in the group treated with LEM for 18 weeks, an improvement in liver function abnormalities and a slight decrease in DNA polymerase and Wl (V-DNA) were observed.
Polymerase, Wl (a significant decrease in V-DNA was observed).

The results of the 40-week administration group are shown in FIG. In the figure, Rollo and ○-○ represent the measured values of DNA polymerase for woodchucks No. 1 and No. 2, and mouth...mouth and ○...○ represent woodchuck No. 1 and No. 2.
Wl (represents the measured value of V-DNA).

These results also indicate that LEM is effective against human chronic hepatitis B.
This confirms that sufficient effects can be expected with oral administration.

Test Example 5 Chronic hepatitis B patient 6 with e-antigen positive group BeAg (l)
Six patients were orally administered 6 g of LEM, 3 g in the morning and evening, and 6 g per day for 16 consecutive weeks, and their subjective and objective symptoms, blood biochemical tests, and virological tests were investigated.

As a result, among the results of the attending physician's assessment, the degree of overall improvement was as follows:
Significant improvement in 13 cases (19.7%), improvement in 5 cases (7.6%)
Of the 13 patients (197%) with mildly modified M, 47% showed some improvement. Regarding the degree of improvement in liver function, 31 cases showed some improvement, including 11 cases (16.7%) with marked improvement.
It was observed in 47% of cases.

In addition, as a result of statistical analysis of the viral e-antigen in the blood at the start of administration and after administration for 47 patients who could be analyzed, the e-antigen was found to be 477 (cut of
After 16 weeks, 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 nJ1 arrest rate) at the start of administration, but it significantly increased to 19.30% after 16 weeks, which was higher than the above. The overall judgment given was reversed.

Regarding side effects, only mild abdominal distension and indigestion symptoms were observed in 2 patients (3%), indicating a very high safety rate.

These results show that the antiviral substance of the present invention is effective in sufficiently improving the symptoms of hepatitis B.

"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, so in many cases we had to abandon their practical application, but the substance of the present invention is extracted from 100% natural products. Therefore, it is extremely safe. Furthermore, since the substance of the present invention inhibits the activity of viral reverse transcriptase even at low concentrations, it can be expected to suppress the proliferation of retroviruses and have excellent preventive or therapeutic effects against various viral diseases. .

[Brief explanation of the drawing]

Figure 1 shows the IR spectra of the substance of the present invention and the lignin of wheat straw, and Figure 2 shows the LEM spectrum for the AIDS virus.
Figure 3 shows the inhibitory effect of P-LEM on the reverse transcriptase activity of purified avian myeloblastosis virus. Figure 4 shows the inhibitory effect of P-LEM on the reverse transcriptase activity of purified avian myeloblastosis virus. Figure 4 shows the inhibitory effect of P-LEM on the infection of herpes simplex virus type I. Figure 5 shows the suppressive effect on DNA polymerase and WHV-DN when LEM was continuously administered to WHV persistently positive woodchucks for 40 weeks.
It is a figure showing the measured value of A. Patent applicant Noda Shokubyou Kogyo Co., Ltd. Agent
Patent Attorney Shigeru Matsui 1K. Su - State Attendance Procedure Neho Seisho (Spontaneous) 1990 1, Display of the Case 1999 Patent Application No. 108116 2, Title of Invention Anti-virus Substance and Process for Producing the Same 3゜Amendment Relationship with the case of a person who does Target (1) Claims of the specification (2) Detailed description of the invention column of the specification March 13, 6, Contents of amendment (1) Page 1 of the specification, lines 5 to 2, The claims column on the second line is amended as shown in the attached sheet. (2) In the first line of page 5 of the specification, 1 "Protein, inorganic matter and modified water-soluble lignin" is corrected to "Protein and modified water-soluble lignin". (3) "Proteins, minerals, and modified water-soluble lignin" on page 5, line 7 of the specification is corrected to "proteins and modified water-soluble lignin." (4) "Proteins, minerals, and modified water-soluble lignin" on page 5, line 12 of the specification is corrected to "proteins and modified water-soluble lignin." (5) On page 12 of the specification, line 10, "bonded by an inorganic substance" is corrected to "bound by a trace amount of an inorganic substance." Attachment above (amended scope of claims) (1) Carbohydrates obtained by culturing basidiomycete mycelium using a medium containing plant fiber components and extracting from this culture, Protein! and an antiviral substance whose main component is modified water-soluble lignin. (2) Molecular weight is 10,000 to 1.5 million, carbohydrates are 12 to 20%,
Claim 1 containing 2-5% protein and 70-85% modified water-soluble lignin; iiE! Antiviral substances. (3) Cultivate basidiomycete mycelium using a medium containing plant fiber components, extract water-soluble components from this culture, and extract substances whose main components are carbohydrates, proteins, and modified water-soluble lignin. A method for producing an antiviral substance characterized by obtaining. (4) From the hot water extract of the culture, an alcohol concentration of 3
4. The method for producing an antiviral substance according to claim 3, wherein a 7.5% soluble and 50% insoluble fraction is separated, and this fraction is further applied to a phenyl Sepharose column to separate the active ingredient.

Claims (4)

[Claims] (1) Cultivate basidiomycete mycelium using a medium containing plant fiber components, and extract from this culture an antimicrobial agent whose main components are carbohydrates, proteins, minerals, and modified water-soluble lignin. Viral material. (2) Molecular weight is 10,000 to 1.5 million, carbohydrates are 12 to 20%,
The antiviral substance according to claim 1, which contains 2 to 5% protein, 70 to 85% modified water-soluble lignin, and contains P, S, Mg, Ca, K, and Na as inorganic components. (3) Cultivate basidiomycete mycelium using a medium containing plant fiber components, extract water-soluble components from this culture, and create a substance whose main components are carbohydrates, proteins, minerals, and modified water-soluble lignin. A method for producing an antiviral substance, characterized in that it obtains. (4) From the hot water extract of the culture, an alcohol concentration of 3
5. The method for producing an antiviral substance according to claim 4, wherein a 7.5% soluble and 50% insoluble fraction is separated, and this fraction is further applied to a phenyl Sepharose column to separate the active ingredient.