GB2079150A

GB2079150A - Antiviral substance from Basidiomycetes

Info

Publication number: GB2079150A
Application number: GB8024704A
Authority: GB
Original assignee: Individual
Current assignee: Individual
Priority date: 1980-06-25
Filing date: 1980-07-29
Publication date: 1982-01-20
Also published as: JPS5712999A; FR2485373A1; GB2079150B; DE3123830A1

Abstract

An antiviral substance having an active component of a mixture of polysaccharide and cytokainine active substances mainly consisting of zeathin-relating substances is extracted from the nutrient medium and tissue-medium of Basidiomycetes such as Lentinus edodes.

Description

SPECIFICATION Antiviral substance and the manufacturing method thereof The present invention relates to an antiviral substance having the active component of a mixture of polysaccharides and cytokainine active substances mainly consisting of zeathin-relating substances which are extracted from the nutrient medium and tissue-medium of Basidiomycetes such as Lentinus edodes and formulated as it is without fractionating into special fractions, and the manufacturing method thereof.

It is well known that a virus is a filter-passage pathogen, is from 20y to 60y in size and smaller than rickettsia, is parasitic in organisms other than viruses, and can proliferate only in living cells.

Viruses are pathogens of such diseases as Japanese encephalitis, influenza and hepatitis in animals, and recently there becomes weighty a theory that a virus may be a pathogen of cancer.

Recently, an important paper on " Mechanism of carcinogensis" by Eiichi Soeda, a member of National Genetics Laboratory, appeared in "Nature" an English science magazine, dated January 31, 1 980.

According to the paper, cancer is induced not by incubation of a carcinogenic virus, but a hereditarily existing cancerous gene (a normal cell has a cancerous gene, and about 90% of persons may have it,) is excited by X-ray or some chemical substances (carcinogenic substances) to induce cancerous protein resembling middle-sized cancerous protein of cancerous virus, which relates to carcinogenesis. The present inventor has an opinion that to control of a virus is to control cancer, based on the abovedescribed theory.

There have been defined several hundreds of viruses, and vaccines of Japanese encephalitis and influenza have been used, but there has not been developed any satisfactory drug for treating these diseases.

The present inventor has researched myceliums of edible mushrooms such as Basidiomycetes like Lentinus edodes, and accomplished many inventions as to the method extracting pharmacologically active components contained in the myceliums, while the present inventor has found out that an active substance of cytokainine system is contained in the extract of the mycelium and that its extract is effective on vegetable viruses.

The present invention is accomplished by developing the above findings further and it is surely the present inventor who discovered first the presence of cytokainine (zeathin, zeathin ribocide, etc.) in the extract of nutrient medium and tissue-medium of Basidiomycetes.

The present invention relates to an antiviral substance prepared by pure-culturing of myceliums of Basildiomycetes by solid or deep cultivation, heating or homogenizing them to extract the component of the myceliums and the component of the metabolite simultaneously from the aqueous solvent, and refining the extract by filtering condensation or frozen dry, and the manufacturing method thereof.

It has been already known that some polysaccharides have antiviral activities, and some inventions of polysaccharides from Basidiomycetes are partially published; the present inventor has noticed the vegetable hormone produced by Basiadiomycetes and confirmed it is a cytokainine active substance mainly consisting of a zeathin-relating substance; moreover, the inventor has found out that a mixture of the polysaccharide contained in the nutrient medium and tissue-medium of Basidiomycetes and the said cytokainine active substance is very effective on animal viruses, and accomplished the present invention.

The polysaccharide from Basidiomycetes which has been studied by some workers is the one which a component of a fruiting body or a mycelium, but the substance produced by a mycelium, i.e. a metabolite is discarded as a waste in the extracting process.

The present inventor has noticed the substance produced by the said mycelium, which is the metabolite, and a partial decomposition product of lignin by the mycelium as well as the component of the mycelium, and accomplished the present invention.

In the present invention the active component includes not only the component of the mycelium but the component of the metabolite produced by the mycelium and the component contained in the partial decomposition produce of lignin, and the present inventor considers the nitrogen-containing polysaccharide in the extract as one of the antiviral components: the polysaccharide is of a rather low molecular weight form 3000 to 10000. Supposing from the sugar composition and amino acid composition of the extract, the polysaccharide is not the one in the mycelium but the one in the metabolite produced by the mycelium. In addition, the polysaccharide is quite different from the polysaccharide coming from a fruting body or mycelium and has structural components different from those of the latter.The partial decomposition product of lignin from the medium will of course have an antiviral activity.

These components seem to act to increase the immunity as well as to prevent the viral infection: that is, an antiviral substance has a contact with tissue protein to degenerate it and to catch the virus before it causes the disease and inactivate it. In the metabolite, cytokainine active substances of vegetable hormone such as zeathin and zeathin ribocide and the anticytokainine active substance analogous to absidinic acid are confirmed to be present, and they may contribute to control of DNA synthesis.

The antiviral substance of this invention is characterizediby being formulated from the extract of the nutrient medium and tissue-medium of Basidiomycetes as it is, without fractionating into special fractions.

SUMMARY OF THE INVENTION The main purpose of the invention is to propose antiviral substances which are safe and effective on virus-induced diseases such as viral hepatitis, influenza and cancer.

Another purpose of the invention is to propose antiviral substances which are easily manufactured with low cost.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a gaschromatographic figure of the fraction development of a cytokainine active substance of the present invention.

Fig. 2-1 is a gaschromatographic figure showing the peak values of already known cytokainine active substance; basic values are shown in this figure.

Fig. 2-2 is the figure in which the peak of the fraction II in Fig. 1 is developed by gaschromatography.

Fig. 23 is the figure in which the peak of the fraction Ill in Fig. 1 is developed by gaschromatography.

Fig. 3 isthe fraction developing figure of the constituting sugar of the present invention by gaschromatography.

DETAILED DESCRIPTION OF THE INVENTION The substance of this invention is obtained from the metabolite of myceliums and the cellsolution produced by self digestion of myceliums after cuturing the myceliums of mushrooms belonging to Basidiomycetes such as Lentinus edodes in a solid or liquid medium.

As Basidiomycetes used in the present invention, there are Lentinus edodes (shiitake), Pleuratus ostreatus (hiratake), Pleliata nameko (nameko) and Lyophyllum aggregatum (shimeji), and the extract from the mycelium of Lentinus edodes shows the highest activity.

Either solid or liquid medium can be used. As the former, there may used any one of the bagasse medium mainly consisting of bagasse (dregs of sugar cane) developed by the present inventor (bagase:rice bran, 10:1), the beat medium mainly consisting of beat dregs (beat dregs:rice bran, 12:1), and usuall;y used shavings medium (shavings:rice bran, 3:1). Bagasse has been burnt mostly because no use of it, and thus it is easily and cheaply available. As the latter, there are GPY medium (a synthetic liquid medium consisting of glucose, peptone, and yeast) and MY medium (a synthetic liquid medium consisting of malto extract powder and yeast); it is preferable to add a vegetable fiber component. No particular method is used for culturing myceliums.

The present invention is characterized by culturing the above-described myceliums of Basidiomycetes in a solid or liquid medium, and extracting the active substance contained in the metabolite of the myceliums and the myceliums from the mixture of the myceliums and the medium (which is called as the nutrient medium and tissue-medium in this specification) without separating the myceliums and the medium. Sephadex LH-20 column chromatography of the above-described nutrient medium and tissue-medium revealed six cytokainine active substances, which were identified to be mainly consisting of zeathin and zeathin riboside by gas chromatography, and also showed the presence of polysaccharides.

The present antiviral suabstance is proved to be effective on diseases caused by viruses such as viral hepatitis, influenza and cancer, and also its toxicity was found to be extremely low in the safety test. In addition, the substance oral toxicity test of a frozen dry powder gave the values of 3.84g/kg/day x 90 in male rats and 7.98g/kg/day x 90 in female rats.

EXAMPLE 1 A solid medium consisting of 90% of bagasse, 5% of rice bran and 5% of nutritional matters such as bran was sterilized as usual, and solid seed mycelium of Lentinus edodes was inoculated on it. Then, the inoculated medium was put in a culture room kept at a temperature of 18 to 200C at a humidity of 60% by air-conditioning and the culture of the mycelium was begun.

When finishing the culture, the medium was transferred into a culture room and allowed to stand there. When fruiting bodies begun to grow up from the surface of the medium, the medium was taken out of the culture room, and crushed into a thumb size by a crusher. The crushed medium was packed in a tank to which 5 liters of clean water per 600 grams of the medium were added; then, they were mixed at 60 to 1 300C for 4 to 5 hours under agitating. The active components in the metabolite of the myceliums and the mycelial cell solution were released into the water by agitation.

The resulting suspension was packed in a filtering bag of flannel and compressed to filter: the obtained filtrate was again filtered to remove fungi by a membrane filter and the active substance contained in the metabolite of the mycelium and the mycelial cell solution was extracted. Then, the extract was compressed to condense through an ultrafiltration membrane, and frozen to dry to get a brown powder.

Myceliums of flammulina uelutipes sing, Pleuratus ostreatus, Coriolur uersicolor and the like were cultured and the corresponding mycelial extracts were obtained by similar methods to the abovedescribed ones, and then cytokainine and polysaccharide were identified.

1) Separation and purification of cytokainine active substances: The brown powder obtained by freezing to dry the extract of the nutrient medium and tissuemedium of Lentinus edodes was solved in the water and allowed to stand 50C overnight, followed by discarding the insoluble part. Then, the filtrate was adjusted at pH 1.5 with Hcl and mixed with an equal amount of ethyl acetate; the ethyl acetate transferred substance was removed. The aqueous phase was adjusted to pH 8.7 by adding ammonia, extracted from an equal amount of water-saturated n-butanol; the n-butanol phase was adsorbed by Dowex 50-X 4, and a cytokainine active substance was obtained after efflution of ammonia.

The above-described active substance was treated through Sephadex LH-20 column chromatography to divide into six cytokainine active components, which were identified by gas chromatography to be mainly composed of zeathin and zeathin riboside in quantication there was obtained 50mg of zeathin and zeathin riboside per 1 gram of the powder. The results of Sephadex LH-20 column chromatography and gas chromatography are shown in Fig. 1 and Fig. 2, respectively.

The presence and absence of the activity was examined biologically with seed leaves of radish.

As seen in Fig. 1 , the cytokainine activity is recognized in the fraction 1-VI. Fig. 2-1 is a chart of the already known cytokainine; B, isopentenil adenine; C, dihydrozeathin; D, zeathin; E, isopenteil adenosine; and F, zeathin riboside. Fig. 2-2 is a chart of the fraction II in Fig. 1; as it is coincident with F in Fig. 2-1, it is defined to be zeathin riboside. Fig. 2-3 is a chart of the fraction Ill in Fig. 1, and as it is coincident with D in Fig. 2-1, it is defined as zeathin.

The cytokainine active substance in the present invention is a substance which is purified by the ion exchange treatment with Dowex 50-X 4, and is a mixture of six cytokainine active components.

2) Separation and purification of polysaccharide The frozen dry brown powder of Lentinus edodes which was similarly prepared as the above one was solved in the water, mixed with 10% trichloracetate (10% TCA) added thereto, stirred and centrifuged. To the obtained supernatant 3-fold volume of 95% ethanol was added, stirred and centrifuged. Then, the precipitate was dried, solved in the water again, and also solved in 3-fold volume of acetone: the solution was centrifuged and the resulting precipitate was dried.

Results

Inhibition Group Added quantity E. coag. value rate, % Control 0 132 Cytokainine 0.2PPM 36 72.7 Polysaccharide O.5PPM 14 89.4 Cytokainine + O.2PPM + O.5PPM 2 98.5 Polysacoharide In each treated group the viral inhibition rate was high, but was the highest in the cytokainine + polysaccharide group. In the group containing cytokainine the proliferation rate of cells tended to be higher than that in the control group.

Experiment 2 Protective activity of mice to herpes virus: Based on the result of Experiment 1 the effect of cytokainine + polysaccharide was found to be excellent, and the treatment was performed with a mixture of cytokainine and polysaccharide.

The mixture of cytokainine and polysaccharide was given intraperitoneally to 2-week old mice, and the survival rate was obtained after 14 days.

Inoculated amount of the virus: 2 x 103 PFU Dose of the mixture: 2 mg of cytokainine and 5 mg of polysaccharide A group consisting of 10 mice: the test was repeated 5 times.

Survival rate: mice/l0 groups.

Results:

Group Surv. rate 1. Group given only herpes virus 0 1 2. Group given herpes virus, cytokainine and 7 10 polysaccharide 3. Group inoculated with the virus 48 hours 9 10 after giving cytokainine and polysaccharide 4. Group given cytokainine and polysaccharide 6 8 48 hours after inoculation of the virus Treatment with cytokainine and polysaccharide could well prevent the viral infection, and also showed satisfactory therapeutic effects.

Experiment 3 The present inventor asked Prof. Nobuhiko Kanno, Biological Laboratory, Dept. of Pharmacology, Toyama Medical and Pharmacological University, to test the activity of the present antiviral substance to depress the liver cancer in rats.

Method: 1. Test animals: Wister male rats of 4 weeks of age were fed for 5 days as usual and tested.

2. Carcinogenic substance: 3'-methyl-4-dimethyl amino azobenzene (3'-Me-DAB)

3'-Me-DAB is known to have an affinity to a rat's liver and a specific and strong carcinogenic property.

The carcinogenic process is reported as that 3'-Me-DAB is connected with the protein of liver cytopiasm to initiate formation of liver cancer.

3. Test group: (1) Control group: standard food (2) L group: standard food + L (3) L + DAB group 0.06% 3'-Me-DAB containing food + L (4) DAB group 0.06% 3'-Me-DAB containing food Note: L: a frozen dry powder of the extract of the nutrient medium and tissue-medium of Lentinus edodes DAB: 3'-Me-DAB 4.Quantity and the method of giving the frozen dry powder of the extract of the nutrient medium and tissue-medium of Lentinus edodes

Period (day) Interval (day) Dose Concentration (w/v) 0 N20 2 1 .Og/ Kg 20% 21 N41 3 150mg/body 20% 42 1 OO 3 50mg/ body 6.67% 101 5 50mg/body 6.67s6 The frozen dry powder of the nutrient medium and tissue-medium of Lentinus edodes was solved in physiological saltwater, and given intraperitoneally; to the control group the same dose of physiological saltwater was similarly given.

5. Process and results: (1) Macroscopic observation: Within about 5 weeks, depilation appeared on the abdomen in some rats of the DAB group, and gradually spread and the degree of the depilation also progressed.

Within about 15 weeks, some rats began to show depilation on the nack or the side of the body.

No depilation appeared in the L x DAB group.

(2) Autopsy In the dissection of the DAB group after 14 weeks and 1 8 weeks, fine yellowish white particles which seemed to be cancerous tissue were seen all over the liver. In the L + DAB group, the liver showed no apparent abnormal finding. All of the rats which showed some abnormal findings in the liver were those which had depilation.

(3) Microscopic findings of the liver tissue The liver tissue was stained by hematoxin-eosin, fixed in wax and prepared into 8y thick section to examine microscopically.

The liver tissue of the DAB group in which yellowish white particles appeared showed severe disarrangement of cells and the glandular structure specific to liver cancer (adeno carcinoma); the nucleolus was clearly stained by eosin and was enlarged; cancerous cells were obviously seen. In the lever tissue in the L + DAB group, the cell arrangement was slightly disturbed, but adeno carcinoma was not seen nor changes of the muleolus was not; thus they were judged normal cells.

Based on the above results, the frozen dry powder of the nutrient medium and tissue-medium of Lentinus edodes inhibited clearly the carcinogenic activity of 3'-Me-DAB on rats, and has the carcinostatic activity.

(4) Six grams of the brown powder obtained by the method described in Example 1 were solved in 100 c.c. of water and given to a patient of viral hepatitis once a day before breakfast everyday, and the following therapeutic effects were obtained. Case: a patient of a 50 years old female who was a head nurse of a hospital and was diagnosed as viral hepatitis The serum transaminase activity was determined to observe the therapeutic results, which are shown below.

The normal value: GOT, 8-40; GPT, 5-35; Kunkel, 4-12.

a) at the onset on May 12, 1978, GOTh, 170; GPT, 270; Kunkel, 5 b) on hospitalization on June 7, 1978 GOT, 756; GPT, 793; Kunkel, 7 c) the administration of a daily dose of the mixture starting on July 1 5, 1978, as described above d) on determination on June 21, 1978 GOT, 230; GPT, 320; Kunkel, 7 e) on determination on July 6, 1978 GOT, 140; GPT, 230; Kunkel, 7 f) on determination on August 1, 1978 GOT, 22; GPT, 19; Kunkel, 7 g) She was completely cured and discharged on August 2, 1978, and returned on her work on the same day.

h) on determination on August 19, 1 978 GOT, 27; GPT, 25; Kunkel, 7 Case 2: The present inventor asked Nishitetsu Hospital (Fukuoka, Fukuoka Pref.) to test the present substance, which was given to a patient of chronic viral hepatitis to produce the following results.

The frozen dry powder began to be given orally with a dose of 5 grams on Sept. 25.

Date 1979 Items 8.27 9.10 9.17 9.27 10.8 10.15 10.22 11.5 11.12 11.26 12.3 12.10 Jaundice index (MG) 5 5 5 4 5 4 5 5 7 6 6 7 Serum GOT 46 43 33 44 50 65 90 119 113 143 110 136 transminase GTP 46 40 44 40 42 54 73 115 90 99 91 105 Serum Cobalt reaction colloidal T.T.T. 7.1 6.0 6.9 6.4 7.5 7.8 7.9 9.1 8.7 8.7 9.4 9.6 reaction Zn.T.T. 8.0 9.5 8.5 8.3 9.4 8.6 8.7 10.4 10.3 11.6 12.2 12.7 Total protein 7.8 7.7 7.3 7.3 8.1 7.9 7.7 7.8 7.6 7.9 7.9 8.1 Albumin 65.6 67.9 67.1 67.1 63.9 68.4 66.2 66.0 65.7 64.9 63.9 63.9 Serum Protein α1 3.1 3.0 3.0 3.3 3.5 3.2 3.1 2.4 3.3 3.4 2.6 3.9 fraction α;2 9.3 9.2 9.1 8.6 9.4 9.2 8.8 7.9 8.7 8.5 7.9 8.7 protein Globulin ss 7.1 7.9 7.1 7.3 8.0 7.1 7.3 8.0 7.5 7.3 7.4 7.3 ratio γ 14.6 11.7 13.5 13.5 14.9 11.9 14.3 15.6 14.6 15.7 18.2 16.0 A / G 1.91 2.11 2.04 2.04 1.77 2.161 1.96 1.94 1.91 1.84 1.75 1.77 AL - P 11.0 9.6 10.3 10.7 11.7 10.6 10.2 9.2 11.7 11.4 14.2 10.4 Total cholesterol 193 188 185 191 195 203 173 201 188 195 194 208 L.D.H.

L A P 215 203 185 186 193 210 215 215 219 257 250 228 γ - GPT 24.9 23.7 22.8 21.7 26.2 24.84 24.0 31.5 30.5 33.7 37.3 37.4 α - FP (+) (-) (-) (-) (-) (-) (-) (-) (-) - - (-) HBS antigen (+) (+) (+) (+) (+) (-) (-) (-) (-) - - (-)

Date 1980 Items < 12.17 12.24 1.7 1.21 1.28 2.4 2.18 Jaundice index (MG) 6 7 5 6 6 5 6 Serum GOT 104 67 57 38 38 35 34 GOT transaminase GPT 71 64 48 35 30 28 26 . colloidal T.T.T. 9.1 9.4 9.9 9.1 8.0 9.1 7.9 reaction Zn.T.T. 11.9 12.4 11.9 11.1 10.6 11.2 9.7 iotal protein Total protein 7.6 7.9 8.0 8.1 7.4 8.1 / 7.9 Albumin 63.1 1 64.8 63.8 61.8 65.6 63.5 64.9 Serum Protein a1 3.2 2.9 3.8 3.9 3.2 2.9 3.5 fraction a2 8.2 7.6 9.0 9.1 8.4 9.8 9.2 Globulin protein ratio ss 7.6 7.7 7.7 8.7 8.0 8.2 9.2 ratio y 17.6 16.7 15.4 16.3 14.5 15.3 14.1 A/G 1.71 1.84 1.76 1.61 1.91 1.74 1.85 AL - P 13.4 10.6 15.5 13.2 10.2 14.5 9.7 Total cholesterol 189 197 209 206 177 191 221 L.D.H.

LAP 219 245 223 193 180 199 183 y - GPT 36.3 37.7 33.6 29.2 26.1 27.1 ' 23.4 a - FP = - - - - (-) (-) HBA antigen ~ = = (-) (-) A dose of 5 grams of the frozen dry powder was orally given every day, and as seen in the above results, the HBs antigen test turned to negative after about 20 days, and then the patient cured completely.

The present inventor asked Nagahama Red Cross Hospital (Nagahama, Siga Pref.) to test the present substance; it was given to a a patient of acute viral hepatitis (Case 3 and a patient of severe viral hepatitis (Case 4) and the following results were obtained.

Case 3: (acute vital hepatitis)

Name Date AlP GOT GPT LDH A 42 years old male, who was discharged in the early March OM 1/17 27.5 561 622 876 1/25 Oral administration of a dose of 5 grams of a frozen dry powder of an extract of the nutrient medium and tissue-medium of Lentinus edodes was begun.

1/28 26.0 533 471 780 2/4 22.7 96 122 362 2/11 21.1 79 90 477 2/18 16.7 89 93 435 2/25 13.5 80 64 358 3/3 13.8 38 38 340 Case 4: (Severe viral hepatitis)

Name | Date | AIP | GOT | GPT | LDH A 45 years old male in hospital EM 1/16 19.1 | 4550 3730 3832 1/19 18.6 4420 3690 3660 1/25 Oral administration of a dose of 5 grams of a frozen dry powder of an extract of the nutrient medium and tissue-medium of Lentinus edodes was begun.

1/26 18.9 3460 3720 3560 2/2 13.8 391 471 - 604 2/9 15.0 380 129 352 2/16 19.2 43 73 311 2/23 14.9 36 40 278 3/1 11.9 25 1 31 270 3/8 10.1 28 29 292 As clearly seen in the above cases, several drugs after the administration, GOT and GPT both decreased, and particularly in the patient of severe viral hepatitis who was dangerous when being hospitalized, both values decreased within several days. Now, both of the patients have been completely cured, indicating an astonishingly high therapeutic effect of the substance.

Case 5: a 74 years old male of serum hepatitis In March in 1979 he was attacked by cerebral infarct and then by deglutive hepatitis to be subject to trachectomy on September 20. Because the oral excretion was heavy and could not removed, the nasal feeding was begun from Sept. 29. Anemia occurred due to shortage of calories, and the blood was often transfused. Then, the values of GOT 30, GPT 26.5 and Al-P 7.0 in September in 1979 increased to 62, 131, and 34.2 on December 3, respectively; he began to vomit on every nasal feeding (800 cal.

of fruit juice + tang juice, etc.) through he had not vomitted before: then, the liquid transfusion was increased to 1,500 ml (daily) and the condition became intractable. Then, a daily dose of 5 grams of the frozen dry powder of the nutrient medium and tissue-medium of Lentinus edodes was solved in water and given through the nose from December 19 to February 1 8. On December , the laboratory test results were improved as TTT 1.8, ZnTT 9.1, GOT 39, GPT 45.5 and Al-P 17, and simultaneously vomiting disappeared, and the general conditions were gradually improved. On January 16, the values were GOT 28, GPT 22, and Al-P 14, and on February 13, they were GOT 26, GPT 14, and Al-P 12: then, they increased and the subjective symptoms disappeared.

Case 6: a 73 years old male of serum hepatitis Past anamnesis: In September, 1944 he was diagnosed as rectal cancer and subject to the surgical operation of artificial anus in 1945. On August 28, 1978, he was attacked by cerebral infarct and admitted by Miyake Hospital, Toshincho, Omuta, Fukuka Pref. On January 25, 1 979, he had a complication of cancerous peritonitis, (with intestinal paralysis), but recovered without surgical treatment. Besides, he had intractable urinary infection and senile cutaneous pruritis, and in October in 1979 he had acute pneumonia. He was blood-transfused because of anemia, and soon CRP became 5+; On November 1 9 in 1979, there were obtained TTT 4.7, ZnTT 8.9, GOT 192, GPT 230, B-TP 7.08/dl, A/G 0.55% and gamma-globulin 40.00%.The T cells were 847/1,284 = 64%. A daily dose of 5 grams of the frozen dry powder of the nutrient medium and tissue-medium of Lentinus edodes was given for 2 months from November 30, 1979. The test results were T cells 847/1,284=64% on November 21, T cells 590/1,311=45% on December 12, TTT 6.5, ZnTT1 9.8, GOT21.5, GPT 21.5, gamma-globulin 36.33%, and A/G 0.64 on December 1 8. Dysorexia of a subjective symptom disappeared, and he could take much meal orally. He showed TTT 3.9, ZnTT 20, GOT 17.0, GPT 10, A/g=0.67, gamma-globulin 33.33%, and T cells 773/1,120=69% on January 22, and TTT 4.2, ZnTT 18, GOT 18.5, and GPT9.5 on February 19. Then, he showed no exaggeration further, and did not complain subjective symptoms particularly.The patient was being given 2 tablets of KW every other week, which caused such general conditions described above and the increase of GOT and GPT; he could be recovered from a dangerous state caused by poor general conditions.

EXAMPLE 2 A GPT medium was mixed with a boiled bagasse solution and packed in a container and sterilized in an autoclave at 121 OC for 30 minutes. A plutinum loop of seed hyphae of Lentinus edodes was inoculated on the said medium, shaked by a shaker 120 times per minutes in a culture room at 250C, and subject to deep culture for 7 to 8 days. Then, myceliums grew in the medium to be like pellet, indicating the myceliums grown well.

One liter of the nutrient medium and tissue-medium obtained as described above was homogenized and the resulting suspension was filtered and extracted. The extract was then filtered to condense through an ultrafilter membrane to get an extract, and the resulting extract was frozen to dry to obtain brown powder. The yield was 1 0 grams of the brown powder per liter of the extract.

From the material obtained as described above, the cytokainine active substance and polysaccharide were separated and purified as described in Example 1, and then it was identified that zeathin and zeathin riboside of cytokainine system were contained in the said material.

The antiviral effect of the material obtained as described above was determined similarly as in Example 1, and almost similar results were obtained.

The present inventor examined the anti-tumoral effect of the powder obtained as described above, and got excellent therapeutic results.

Tumoral cells of zarcoma 1 80 were transplanted into the abdomen of a mouse, and when they sufficiently proliferated after 8 days, 107 of the cells were transplanted subcutaneously in the lower arm of another mouse to produce a solid tumor; from 24 hours after transplantation the above-described powder was given to the mouse. It was given intraperitoneally with a dose 1 Omg/kg, i.e., 0.2mg/20g (the body weight of the mouse) once a day 12 times every other day; orally it was given with a dose of 1 Omg/kg, i.e., 0.2mg/20g (the body weight of the mouse) once a day 23 times every day.

As the result, the antiviral substance (the brown powder described above) of the present invention was found to have en excellent antiviral activity, and to be effective as an anti-cancerous drug.

Case 1: a 65 years old male of a practitioner, who was diagnosed as esophageal cancer a) 1974: subjective symptoms: smart on drinking cold fluids. He received the above-described brown powder of the present invention from some of our patients and took it once a day before breakfast with a dose of 3 grams.

b) December,1976: He was subject to X-ray examination in some findings in the esophagus.

Immediately he was examined endoscopicly in Tokyo Women's Medical College and diagnosed as esophageal cancer because atypical cells were found in the esophagus.

c) February, 1977: He refused the surgical operation: he had the weight of 38kg, the length of 1 52 cm and complained severe fatigue with dullness and loss of appetite.

d) February, 1977: The lesion was found to be restricted in the regenerated epithelial cells by the endoscopic examination.

e) December, 1977: The appetite abruptly reduced and the patient and his family were very anxious about it.

f) January, 1978: A dose of the brown powder was increased to 6 grams once a day. The appetite was improved 1 week after it.

g) March, 1978: He was improved and showed an increase of body weight.

i) September, 1 978: X-ray and endoscopic examination revealed no abnormal finding in the esophagus.

Case 2: a 45 years old male a) April, 1977: He was attacked by severe pain in the left abdomen, found to have two masses of a fist size, and diagnosed as malignant migrating cancer and pancreatic cancer based on X-ray, blood and urine examinations.

b) May, 1977: A dose of 6 grams of the above-described brown powder of the present invention was given once a day before breakfast every day.

c) June, 1977: Several days after the beginning of the administration, the flatus was released to shothe the compressive feeling and the malaise in the left abdomen. Only the powder of this invention was given and the other drugs were ceased. He had bowel movements and felt better.

d) July, 1977: He felt no pain but malaise in the abdomen, and sometimes had prickling. He was told not to be anxious about it because it was due to beginning of the movement of juodenum and small intestine. He had grumbling and flatus frequently. The blood, X-ray and other examinations indicated the cure of cancer. After vomiting, much flatus was released every day; the cause of vomiting was considered to be in the intestine.

e) August, 1977: He was surgically operated, and the suppuration of diaphragm was found. The pus was discharged by suction through a tube every day. The administration of the powder which had been stopped for a while before and after the operation was begun.

f) September, 1977: He began to feel starved and was well as showing the body weight increasing; he began to train walking.

g) October 1977: He was discharged and. visited the hospital an out-patient. Then, he was completely cured and since then he was not visited the hospital.

EXAMPLE 3 The mycelium of flammulina uelutipes sing were cultured as described in Example 1, and an extract containing the above-described active component was obtained from the nutrient medium and tissue-medium similarly, filtered to condense, frozen to dry to produce a powder.

The material obtained as described above was examined on the antiviral activity and the antitumoral activity, and gave the results almost similar to those in the previous examples.

EXAMPLE 4 Pleliata nameko was cultured as described in Example 1, and an extract containing the abovedescribed active component was obtained from the nutrient medium and tissue-medium similarly, filtered to condense, and frozen to dry to produce a powder.

The material obtained as described above was tested on the antiviral and antitumoral activities, and gave the results similar to those in the previous examples.

Safety of the antiviral substance of this invention: The antiviral substance produced according to Example 1 was analyzed by Nomura Consolidated Laboratory for evaluating the safety: the results are as follows: (1) Acufe toxicity (oral) LDsOg/kg (body weight) Rats (male) 16.5 Rats (female) 1 5.6 Mice (male) 19.6 Mice (female) 1 7.7 (2) Toxicity on fish TLM Carps, 48 hours ca 1.02% Carps, 72 hours 1.07% Water flea, 3 hours 0.56% Results: TLM is corresponding to LD50 for acute toxicity (rats and mice).

Usual synthetic fertilizers show 1 Oppm in carps and also show values ppb levels in water flea: thus, the present antiviral substance is confirmed to show extremely low toxicity.

Claims

1. An antiviral substance which is a nutrient medium and tissue-medium of one of Basidiomycetes selected from Lentinus edodes, Pleuratus ostreatus, Plaliata nameko, Lyophyllum aggregatum, Cariolus uersicolor and the like has an active component of a mixture of polysaccharide and cytokainine active substance mainly consisting of zeathin-relating substances which are produced by the nutrient medium and tissue-medium.

2. The antiviral substance described in Claim 1, in which the mixture of polysaccharide and the cytokainine active substance mainly consisting of zeathin-relating substances which are produced by the nutrient and tissue-medium is given orally.

3. The antiviral substance described in Claim 1, which is effective on viral hepatitis, cancer and influenza.

4. The antiviral substance described in Claim 1, which has the molecular weight of 3,000 to 10,000 and the cytokainine active substance consisting of zeathin and zeathin riboside.

5. A method of manufacturing of an antivirai substance consisting of a process to culture hyphae of Basidiomycetes in a solid or liquid medium, a process to mix the nutrient medium and tissue-medium obtained in the said process with an aqueous solvent and extract the active component, and a process to filtrate the extract.

6. The method of manufacturing of the antiviral substance described in Claim 4, in which the materials are mixed and agitated at 60-1 300C for 4-5 hours in the process to extract the active component of the nutrient medium and tissue-medium.

7. The method of manufacturing of the antiviral substance described in Claim 4, in which water is used as the aqueous solvent.

8. The method of manufacturing of the antiviral substance described in Claim 4, in which the extract of the nutrient and tissue-medium is compressed and filtered by an ultrafiltering membrane.

9. The method of manufacturing of the antiviral substance described in Claim 4, in which bagasse is used as the main material of the solid or liquid medium.

10. The method of manufacturing of the antiviral substance described in Claim 4, in which the filtered extract is frozen to dry.