DE3123830A1 - "ANTIVIRUS SUBSTANCE AND METHOD FOR THE PRODUCTION THEREOF" - Google Patents

Description

Antiviral substance and method for its

Manufacturing

The invention relates to an .Antivirussubstanz with an active ingredient, which is a mixture of polysaccharides and active cytocaine substances is that mainly related to zeathin Substances are and which »extracted from the culture medium and tissue medium of Basidiomycetes, such as Lentinus edodes, and so on as they are, i.e. without breaking them down into special fractions. The invention is also to a method of manufacture directed for such a substance.

It is well known that virus is a filtering pathogen, 20 to 60 µ in size and smaller as rickettsia is that it is parasitic to organisms other than viruses and that egg can only proliferate in living cells.

Viruses are pathogens of such diseases as Japanese encephalitis, influenza and hepatitis in living things, and recently a theory has gained weight that viruses are also carcinogenic. More recently, the important paper "Mechanism of carcinogenesis" by Eiichi Soeda, a member of the National Genetics Laboratory, appeared in "Nature" (Jan. 31, 1980). After this work, cancer is not erzeugjb by incubating a carcinogenic virus Hondern a hereditary existing cancerous gene (has a normal cell cancerous gene, and about 90 ° / o of all people as) is excited by X-rays or by some chemical substances to induce a cancerous protein similar to the medium-sized cancerous protein of a cancerous virus, which is directed to carcinogenesis. Based on this theory, the invention assumes that controlling a virus also means controlling cancer.

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ORIGINAL INSPECTED

So far, several hundred types of viruses have been defined, and it Japanese encephalitis and flu vaccines are available, but so far is not a sufficient medicine has been found to treat these disorders.

According to the invention, the mycelia of edible mushrooms such as Basidiomycetes, such as Lentinus edodes, studied and many Methods of extracting the pharmacologically active components of the mycelium, and it was found that the active ingredient of the cytokainin system contained in the extract of the mycelium and that the extract acts on plant viruses.

The invention consists in a further development of these findings, and according to the invention for the first time the presence of cytokainin (zeathin, zeatin riboside, etc.) in the extract of the culture medium medium and the tissue medium of Basidiomycetes discovered.

The invention relates to an antiviral substance manufactured through cultivation of mycelia from Basidiomycetes by solid or deep cultivation, heating or homogenizing the same to form the component of the mycelium and the component MetaboLiten at the same time from the aqueous solvent to extract, and purifying the extract by filter condensation or freeze drying. Subject of Invention is also the corresponding manufacturing process.

It is already known that some polysaccharides are effective against viruses, and in part various studies have been carried out on Basidi omycetes-Tolysaccharide published. According to the invention was the plant hormone produced by Basidiomycetes is found and ascertained that it is an active cytocaine substance which consists mainly of zeathin-related substances; in addition, according to the invention found that a mixture of the polysaccharide contained in the culture medium and the tissue medium of Basidiomycetes is contained, and said active cytocaine substance acts most effectively on animal viruses.

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BATH ORIGINAL

The Basidiomycetes polysaccharide, which has already been investigated by some scientists is a component of a fruiting body or a mycelium, but was produced by the mycelium Substance, i.e. a metabolite, discarded as waste after extraction.

According to the invention, the was produced by this mycelium Substance that is a metabolite and a partial decomposition product of lignin by the mycelium, as well as the component of the mycelium examined.

According to the invention, the active ingredient consists not only of the component of the mycelium, but also of the component of the metabolite produced by the mycelium and the component contained in the partial decomposition product of the lignin, the nitrogen-containing polysaccharide in the extract being regarded as one of the antiviral components according to the invention; the polysaccharide has a fairly low molecular weight of 3,000 to 10,000. Based on the sugar composition and the amino acid composition of the extract, the ι polysaccharide does not come from the mycelium, but from that caused by the _; Mycelium produced metabolites. In addition, the polysaccharide is quite different from that derived from a fruiting body or mycelium, and has structural components different from those of the latter. The partial decomposition product of the lignin from the medium naturally has an antiviral effect.

These components seem to increase immunity as well as prevent viral infection! that is, an antiviral substance comes into contact with tissue protein, which degenerates and the virus is captured and inactivated before it causes disease. Of the metabolites, cytocaine active substances of vegetable hormone such as zeathin and zeathin riboside, and the anticytocaine active substances analogous to absidinic acid have been confirmed so far, and they can contribute to the control of DNA synthesis.

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ORIGINAL INSPECTED

The antiviral substance according to the invention is characterized by that they are from the extract of the culture medium and the tissue medium of Basidiorayceten as it is without fractionation in special fractions.

The main object of the invention is to create an antiviral substance, which acts safely and effectively on viral diseases such as viral hepatitis, influenza and cancer and which can also be easily produced at a low cost.

In the accompanying drawing details of the invention are explained. Tn of the drawing is:

Fig. 1 is a gas chromatogram of the fraction development of active Cytocaine substance according to the invention,

Fig. 2-1 is a gas chromatogram showing the peak values of already shows known active cytocainin substances; the Underlying values are shown in this figure,

Fig. 2-2 is a diagram in which the peak of fraction II in Fig. 1 is developed by gel chromatography,

Fig. 2-3 is a diagram in which the top of fraction III in Fig.! gasChromatographiech is developed, and

Fig. 3 is a gas chromatogram of the fraction development of constituent sugar according to the invention.

The substance according to the invention is derived from the metabolite of Mycelium and the zeil solution, produced by self-digestion of the Mycelium obtained after culturing the mycelium of Basidioraycetes fungi such as Lentinus edodes in solid or liquid medium.

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ORIGINAL INSPECTED

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Lentinus edodes (Shiitake), Pleuratur ostreatus (Hiratake), Pleliata nameko (Nameko) and Lyophyl.lum aggregatum (Shimeji) serve as Bas idioraycetes mushrooms which can be used according to the invention, the extract of the mycelium of Lentinus edodes showing the greatest activity .

Both solid and liquid media can be used. Any bagasse medium can be used as a solid medium, which consists mainly of bagasse (sugar cane sediment) and which was developed according to the invention (Bagassez rice bran 10: 1). Furthermore, the paper fiber medium (beat medium), the consists mainly of paper fiber sediment (paper fiber Dregs rice bran 12: 1) and the usual wood pulp medium (Wood pulp rice bran 3 s1). Bagasse is so far most of the time it has been burned because it was not used, and so it is easily and cheaply available. as the latter serve GPY medium (a synthetic liquid medium consisting of glucose, peptone and Hofe) and MY medium (a synthetic liquid medium consisting of malt extract powder and yeast); it is appropriate to have a vegetable fiber component to add. The cultivation of the mycelium requires no special method.

The invention is characterized by cultivating the above Mycelia of Basidiomycetes Ln a solid or liquid Medium and extracting the active substance contained in the meta- bolites of the mycelium and the mycelium from the mixture of mycelium and medium is present (which latter is used here as a nutrient medium and tissue medium is referred to), without separating the mycelium and medium. Column chromatography with Sephadex LH-20 of the above nutrient medium and tissue medium revealed six active cytocaine insubstances determined by gas chromatography identified primarily as Zeathin and ZeathLnriboside and the presence of polysaccharides was also shown.

ORIGINAL INSPECTED 13QÖSS / G32Ö

The antiviral substance according to the invention has been shown to that it affects diseases caused by viruses, such as viral hepatitis, flu and cancer, and safety auditors also found to be extremely low in toxicity. In addition, the oral toxicity test of the substance of freeze-dried Powder values of 3.84 g / kg / day 90 in male rats and 7.98 g / kg / day 90 in female rats.

example 1

A solid medium consisting of 90 9 & bagasse, 5% of rice bran and 5 ⁰ Io soil materials, such as bran, was sterilized in a conventional manner and inoculated with fixed Samenmyzel vonLentinus edodes. Then, the inoculated medium was placed in an air-conditioned cultivation room having a temperature of 18 to 20 ° C. and a humidity of 60 % , and cultivation was started.

After the cultivation was completed, the medium was transferred to a cultivation room and left to stand. When fruit bodies began to grow from the medium surface, the medium was taken out of the cultivation room and ground into thumb-sized pieces in a mill. The poorly ground mediui was placed in a container into which 5 liters of pure water per 600 g of the medium was added; then was ⁱ⁴ iihren 4 to 5 hours at 60 to 130 ° C mixed. The active ingredients in the metabolite of the mycelium and the mycelial solution were released into the water by the agitation.

The suspension obtained was passed through a flannel filter bag filtered and pressed. The obtained filtrate was filtered again through a membrane filter to remove fungi, and those in the metabolite of the mycelium and the mycelial cell solution Active substance contained has been extracted. Then the extract was pressed through an ultrafilter membrane to condense and freeze-dried to give a brown powder.

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In the same way, mycelia of Flammullna velutipes grew sing., Pleuratus ostreatus, Coriolus versicolor and the like cultivated, and the corresponding mycelial extracts were obtained, and then cytokainin and polysaccharide were identified.

1) Separation and purification of the active cytocainin substances:

The brown powder obtained by freeze-drying the extract of nutrient medium and tissue medium of Lentinus edodes was dissolved in water and left to stand at 5 ° C. overnight, after which the insoluble components were discarded. The filtrate was then adjusted to pH 1.5 with HCl and mixed with an equal amount of ethyl acetate; the ethyl acetate phase was discarded. The aqueous phase was adjusted to a pH of 8.7 by adding ammonia and extracted with an equal amount of water-saturated n-butanol. The n-butanol phase was adsorbed on Dowex 50-X k , and after effluent with ammonia, the active cytocaine substance was obtained.

The above-described active substance was separated by Sephadex LH-2O column chromatography into six active cytocalnine components, which were identified mainly as zeathin and zeathin riboside by gas chromatography; specifically, 50 mg of zeathin and zeathin riboside were obtained per 1 g of 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 activity was checked biologically with radish seed leaves. As can be seen from FIG. 1, cytokainin activity can be seen in fractions I to VI. Fig. 2-1 is a diagram of the previously known cytocainin; B = isopentenyladenine; C = dihydrozeathine; D = zeathin; E = isopentyladenosine; and F = zeathine riboside. Figure 2-2 is a diagram of fraction II in Figure 1; since it corresponds to F in Figure 2-1, it is defined as zeathin reboside. FIG. 2-3 is a diagram of FIG

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Fraction III in Fig. 1, and since it coincides with D in Fig. 2-1, she is defined as Zeathin.

The active cytocaine substance of the invention is one
Substance purified by ion exchange treatment with Dowex 50-X, and it is a mixture of six active substances
Cytokainin components.

2) Separation and purification of the polysaccharides

Freeze-dried brown powder of Lentinus edodes, which had been produced in the same way as the previous one, was dissolved in water, treated with 10 ° β> trichloroacetate (10% TCA), stirred and centrifuged. To the obtained supernatant solution
three times the volume of 95% ethanol was added,
stirred and centrifuged. Then the precipitate was dried, redissolved in water and in three times the volume
Dissolved acetone. The solution was centrifuged and the resulting precipitate was dried.

Attempt 1
Results:

Group Added amount of E. coag.value underdox

relationship %

comparison 0 132 Cytocainin 0.2 ppm ' 36 Polysaccharide 0.5 ppm 14th

72.7

89.4

Cytokainin + _Λ «·> _Λ ^ ο ^ o -

Polysaccharide 0.2 ppm ₊ 0.5 ppm 2 98.5

The virus suppression ratio was high in each treated group, but it was highest in the CytokaLnin +
Polysaccharide containing group. In the group containing cytokainin, the proliferation ratio of cells tended to become larger than that of the control group.

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ORIGINAL

Attempt 2

Protective activity against herpes viruses in mice Based on From the results of Experiment 1, it was found that the effect of cytokainin + polysuccharide was excellent, and the Treatment was with a mixture of cytokainin and polysaccharide carried out.

The mixture of cytocaine and polysaccharide was given intraperitoneally to 2-week-old mice, and the survival rate was determined after one- half days.

3
Inoculated amount of Viru «: 2 χ 1Cr PF ¹ I

The dose of the mixture was 2 mg of cytokainin and 5 ^m S polysaccharide. A group consisted of 10 mice, the experiment was repeated five times.

TJ survival rate χ mice / 10 groups

Results:

Group survival rate

1st group infected only with herpes virus _ ₁

2nd group infected with herpes virus
and the cytokainin + polysaccharide

was given 7 10

3rd group 48 hours after a dose
of cytokainin + polysaccharide mLt

was vaccinated with the virus 9 10

4th group, 48 hours after crapping
with the virus cytokainin + polysaccharide was given 6 8

Treatment with cytocaine and polysaccharide could
protect against viral infection and also showed sufficient therapeutic effects.

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Attempt 3

In the context of the invention, Prof. Nobuhiko Kanno, Biological Laboratory, Pharmacological Department of Medical and Toyama Pharmacological University, requested the activity the antiviral substance according to the invention on the suppression of liver cancer testing in rats.

Test procedure:

1. Test animals: Male Wister rats, 4 weeks old, became Fed and tested as usual for 5 days.

2. Carcinogenic substance ι 3 -'-methyl-4-dimethylaminoazobenzene (3 ι-Me-DAB) of the formula

{3 - * - O

3'-Me-DAB is known to have an affinity for the Rat liver and specific and potent carcinogenic properties.

The carcinogenic process is reported to be such that 3'-Me-DAB is attached to the protein of the liver cytoplasm which initiates the formation of liver cancer.

3. Test groups ι

1) Comparison group ι standard food

2) L group: standard food + L

3) L + DAB group: food containing 0.06% 3'-Me-DAB + L k) DAB group: food containing 0.06% 3'-Me-DAB

Note: L = freeze-dried powder of the extract of culture medium and tissue medium of Lentinus edodes according to the invention DAB - 3'-Me-DAB COPY

13 0 0 6 6/0820> ORIGINAL INSPECTED

dose I.
Concentration ' (Weight - <} !,) 1.0 g / kg 20 io 150 mg / body 20% 50 mg / body 6.67% 50 mg / body 6.67 ° / o

k. Quantity and method of administration of freeze-dried powder of the extract of nutrient medium and tissue medium of Lentinus edodes according to the invention

Period (days) Interval L (days)

0-20 2

21-41 3

k2- 100 3

- 5th

The freeze-dried powder of nutrient medium and tissue medium from Lentinus edodes was dissolved in physiological saline and administered intraperitoneally; the comparison group received the same amount of physiological saline solution given in the same way.

5. Process and results;

1) Macroscopic observations:

Within 5 weeks of some rats, the DAB group observed hair loss on the abdomen, which gradually spread and progressed.

Within 15 weeks, some rats showed hair loss on the neck and on the sides of the body. No hair loss was observed in the L + DAB group.

2) autopsy:

At the autopsy of the DAB group after 14 weeks and For eighteen weeks, fine yellowish-white particles, which were apparently cancerous tissues, were observed throughout the liver. In the L · ♦ · DAB group, the liver did not show any abnormal ones Finding. All rats that showed something abnormal were those that experienced hair loss' was. ·.

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ORIGINAL INSPECTED

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3) Microscopic findings of the liver tissue: The liver tissue was stained with hematoxin-eosin, in Wax bedded and prepared to 8 / u thick sections that microscopically examined.

The liver tissue of the DAB group in which the yellowish-white particles were observed showed severe Disorder of the cells and the glandular structure, which is specific for liver cancer (adenocarcinoma); the cell nuclei were clearly stained and enlarged by the Eos Ln; kanzerö cells were clearly seen. In the liver tissue of the In the L + DAB group, the cell order was slightly disturbed, but neither adenocarcinoma nor changes in the Muleolus are observed; thus they were judged to be normal cells.

These results indicate that the freeze-dried Powder of Nälirboden Medium and Tissue Medium from Lentinus edodes clearly shows the carcinogenic effect of 3'-Me-DAB and that it is carcinostatic.

4) 6 g of the brown powder based on the in Example 1 The method described above was dissolved in 100 cc of water and given to a person suffering from viral hepatitis Give to patients, once a day before breakfast The following therapeutic effects have been achieved:

Case: patient 50 years old, female, head nurse in a hospital. It was diagnosed with viral hepatitis wot the.

To observe the therapeutic results, was the serum brantiaminase activity is determined. The results are compiled below.

BATH ORIGINAL

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- 15 normal values : GOT 8 to kO, GPT 5 to 35, Kunkel k to

a) Beginning on May 12, 1978: GOT 170, GPT 270, latch L 5

b) When admitted to the hospital on June 7, 1978: GOT 756, GPT 793, Kunkel 7.

c) Administration of a daily dose of the mixture began on I5. July 1978, as explained above »

d) Determination on June 21, 1978:

GOT 23O, GPT 320, Kunkel 7.

e) Determination on July 6, 1978: GOT 14O, GPT 23O, KunkeL 7.

f) Determination on August 1, 1978 * GOT 22, GPT 19, Kunkel 7.

g) The patient was discharged as fully cured on August 2, 1978 and returned to hers on the same day Work back.

h) Determination on August 19, 1978: GOT 27, GPT 25, Kunkel 7.

Case 2:

As part of the invention, the Nishitetsu-Hospltal (Fukuoka, Fukuoka Pref.) Requests the substance according to the invention to testing. It was given to a patient with chronic viral hepatitis with the following results:

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The freeze-dried powder became off 25 September given orally in a dose of 5 g

"——- ~ ^ ____ ^ Di turn
Measured variable ~ ——-—. GOT albumin Λ / G 1979
27.8. 10.9 . 17.9 27.9. 8. 10 .15. Jaundice Index (MG) GPT I. AL - P 5 5 5 4th 5 I. Serum- Cobalt> reaction Globulin Γ
\ ß Total choles tcrin 46 33 44 50 6! transaminase T.T.T. \ ^Y L.D.H. 46 44 40 42 S e rum— Zn.T.T. LAP olloid Go.total pro toin γ ~ GPT 7.1 6.0 6.9 6.4 7.5 ψ
I. reaction l'ro to ί η— 0 (-FP 8.0 9.5 8.5 8.3 9.4 ^? IIB5 antigen 7.8 7.7 7.3 7.3 8.1 r This way-* fraction 65.6 67.9 67.1 67.1 63.9 G\ Proportions 3.1 3.0 3.0 3-3 3.5 - pro tein 9.3
7.1 9.2
7.9 9.1
7.1 8.6
7.3 9.4
8.0 ψ 14.6 11.7 13.5 13.5 14.9 ι: 1.91 2.Il 2.04 2.04 1.77 2.κ IL. 0 9.6 10.3 10.7 11.7 10, 193 188 IS ₅ 191 195 2 ( 215 203 185 186 193 2Y 24.9 23.7 22.8 21.7 26.2 24. U) (-) (-) <-) (-) (■ C ₊ ) <♦> ω (♦> <+> (-

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ORIGINAL INSPECTED

~~ "———_ ^ ___ ^^ date
Measured variable '~~~ GOT albumin β A / G I. 22. 1 C ►, 5.11. 12.1 1 11/26 .3.12. 10.12. Jaundice-Tndex (MG) GPT Y AL-P 5 5 7th 6th 6th 7th Serum- Cobalt reaction Globulin Total cholesterol 90 119 113 143 110 136 transaminase T.T.T. L.D.H. 73 115 90 99 91 105 Serum- LAP lcolloid Zn.T.T. y- GPT 7.9 9.1 8.7 8.7 9.4 9.6 reaction Total pro t ο i η Ot- FP 8.7 10.4 10.3 11.6 12.2 12.7 I'rotein- HBn antigen 7.7 7.8 7.6 7.9 7.9 8.1 Serum- 66.2 66.0 65.7 64.9 63.7 63.9 fraction 3.1 ..1.4 2.6 pro tein Proportions 8.8 7.9 8.7 8.5
7.3 7.9 8.7
7.3 7.3 8.0 7.5 15.7 7.4 16.0 14.3 15.6 14.6 1.84 18.2 1.77 1.96 • 1.94 1.91 11.4 1.75 10.4 10.2 9.2 11.7 195 14.2 208 j 173 201 188 194 χ 257 228 ' 215 215 219 33.7 250 37.4) 24.0 31.5 30.5 - 37.3 (-) (-) (-) - (-) U) (-)

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ORIGINAL INSPECTED COPY

"~~~" —-__ ^ _____ ^ date
Measured variable "^ ~ ——- ^ _ ^ GOT albumin Globulin dz
β A / G December 17 24. 12 1980
1.7 21. 1 . 28 January 2.4. Jaundice Index (MG) GPT j
\ oli r AL-P 6th 7th 5 6th 6th serum Cobalt reaction Total cholesterol 104 67 57 38 38 35 transaminase T.T.T. L.D.H. 71 64 48 35 30th 28 serum Zn.T.T. LAP colloid Total protein r- GPT 9.1 9.4 9.9 9.1 8.0 9.1 reaction Protein" C * - FP 11.9 12.4 11.9 11.1 10.6 11.2 HB ₆ antigen 7.6 7.9 8.0 8.1 7.4 8.1 serum fraction. 63.1 64.8 63.8 61.8 65.6 63.5 Proportions 3.2 2.9 3.8 3.9 3.2 2.9 pro tein 8.2 7.6 9.0
7.7 9.1
8.7 8.4
8.0 9.8 7.6 7.7 15.4 16.3 14.5 8 _e 2 17.6 16.7 1.76 1.61 1.91 15.3 1.71 1.84 15.5 13.2 10.2 1.74 13.4 10.6 209 206 177 14.5 189 197 191 223 193 180 219 245 33.6 29.2 26.1 199 36.3 37.7 - - - 27.1 - - - - <-) - -

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OftenlÖIHAt INSPECTED

~~ "- ■ Da t um
Measured variable ~~ ——. GOT albumin r A / G 18.2. • • - ----- ! Jaundice Index (MG) GPT AL-P 6th j Serum- Cobalt reaction *
Globulin Gesarat cholesterin 3h transaminase T, T.T. L.D.H. 26th Serum- Zn.T.T. LAP k olloid- Total log Y- GPT 7.9 reric tion l'rotcin_ rf- FP 9.7 ΗΒλ antigen 7.9 Scrura fraction 64.9 Vernaltn. 3.5 protein 9.2
9.2 14.1 1.85 9.7 221 183 23.4 (-) (-)

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ORIGINAL INSPECTE »COPY

A dose of 5 g of the freeze-dried powder was given orally daily, and as the above results show, the HH antigen test was negative after about 20 days and then the patient was completely cured.

As part of the invention, the Nagahama Red Cross Hospital (Nagahama, Siga Pref.) Requests the substance of the invention to test; it was given to a patient with acute viral hepatitis (Case 3) and to a patient with severe viral hepatitis (Case 4), whereby the following results were obtained:

Case 3 * (acute viral hepatitis) 42 year old male Patient discharged in early March.

Date AIP GOT GPT LDH

17.1. 27.5 561 522 876

25.1. A daily dose of 5 S of the freeze-dried powder of an extract of culture medium and tissue medium of Lentinus edodes was started.

28 January 26.0 533 471 780 4.2. 22.7 96 122 362 11 .2. 21.1 79 90 477 18.2. 16.7 89 93 435 25.2. 13.5 80 64 358 3.3. 13.8 38 38 340

BAD ORfGiNAL

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Case 4ι (severe viral hepatitis), 45 year old male patient

Date AIP GOT GPT LDH

16.1. 19.1 4550 3730 3832

19.1. 18.6 4420 369O 366O

25.1. It was started to give a daily dose of 5 S a freeze-dried Powders of an extract of Nährbodenmediutn and tissue medium of Lentinus edodes.

26.1. 18.9 3460 3720 3560 2.2. 13.8 391 471 6o4 9-2. 15.0 380 129 352 I6.2. 19.2 43 73 311 23.2. 14.9 36 40 278 1.3. 11.9 25th 31 270 8.3. 10.1 28 29 292

As these two cases show, after a few doses the GOT and GPT values were lower, and especially with the Patients with severe viral hepatitis to whom on admission When the patient was in danger of death, both values were lowered after a few days. Both patients were complete cured, which indicates the astonishingly high therapeutic effect of the substance.

Case 5: 74 year old male patient with serum hepatitis

In March 1979 he fell ill with cerebral infarction and then with deglutive hepatitis, resulting in trachectomy. required on September 20th made. Because the oral excretion was difficult and could not be removed, it was started on September 29th to feed the nose. The low calorie intake resulted in anemia and frequent blood transfusions

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will. Danryerhöhfcen calibrates the values of GOT 30, GPT 26.5 and Al-P 7.0 from September 1979 to 62, 131 and 34.2 respectively on December 3rd; the patient began to vomit after each nasal feed (800 cal as fruit juice + seaweed juice, etc.), which had not been the case before. Then the liquid transfusion was increased to 1500 ml daily and the condition became untreatable. Then a daily dose of 5 S of the freeze-dried powder of nutrient medium and tissue medium of Lentinus edodes dissolved in water was given through the nose from December 19th to February 18th. On December 21, the laboratory values were improved to TTT 1.8, ZnTT 9.1, GOT 39, GPT 45.5 and AIP 17, at the same time the vomiting stopped and the general condition 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: 73 year old male patient with serum hepatitis

Past History: Rectal cancer was diagnosed in September 1944 and an artificial anus was surgically created in 19 ^ 5. On August 28, 1978, the patient suffered a cerebral infarction and was admitted to Miyake Hospital, Toshinocho, Omuta, Fukuoka Pref. On January 25, 1979, a complication of cancerous peritonitis (with intestinal paralysis) occurred, but it was surgically removed. There was also an untreatable urinary infection and old age skin itching, and in October 1979 acute pneumonia. A blood transfusion was performed because of the anemia and soon the CRP was 5+. On November 19, 1979, TTT 4.7, ZnTT 8.9, GOT 192, GPT 230, β-TP 7.08 / dl, A / G 0.55 # and gamma globulin 40.00 ⁰ Jo were obtained. The T cell count was 847/1284 = 64%. A daily dose of 5 g of the freeze-dried powder of culture medium and tissue medium of Lentinus edodes was given for two months from November 30, 1979 on. The laboratory values were as follows: T cells 847/1284 = 64 96

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ORIGINAL INSPECTION9

November 21, T cells 590/1311 = ^ 5 <> /, on December 12, TTT 6.5, ZnTT 19.8, Gi ¹ T 21.5, gamma globulin 36.33 ° J> and A / G 0.64 on December 18. The dysoiexia of a subjective symptom disappeared and the patient could ingest normal amounts of food through the mouth. He showed TTT 3.9, ZnTT 20, GOT 17.0, GPT 10, A / G 0.67, Gammag] obulin 33.33 fo and T cells 773/1120 = 69 io on January 22nd, and TTT 4 , 2, ZnTT 18, GOT 18.5 and GPT 9.5 on February 19th. Then no exaggerated values were determined and the patient did not show any particular subjective symptoms. The patient was given 2 tablets of KW every additional week, which produced the above-mentioned general condition and the increase in GOT and GPT. I The danger to life, caused by the poor general condition, was averted.

Example 2

A GPT medium was mixed with a boiled bagaese solution and placed in a container and sterilized in an autoclave at 121 ° C for 30 minutes. The medium was with a platinum loop inoculated with seed hyph'in from Lentinus edodes, Shaken 120 times per minute in a cultivation room at 25 C and deep cultivated for 7 to 8 days. Then the mycelia grew like a pellet in the medium, indicating good mycelial growth.

1 1 nutrient medium and tissue medium to which the above-described In the same manner, it was homogenized, and the resulting suspension was filtered and extracted. The extract was then filtered through an ultra-liter membrane to condense to obtain a P, 'extract which is freeze-dried to a brown powder became. 10 g of brown powder were obtained per liter of IOxtrakc th.

This material became active oytocainin substance and polysaccharide separated and purified as in Example 1, and then has been identified as d-ate zeathin and zeathin riboside were contained in the material.

ORIGINAL INSPECTED

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The antiviral effect of the material thus obtained was determined as in Example 1 determined, with almost the same results were obtained.

According to the present invention, the anti-tumor effect of the thus obtained Powder tested and excellent therapeutic results were obtained.

Tumor cells of Sarcoma 180 were transplanted into the abdomen of a mouse and, after a sufficient Proliferationsz "8 days 10 cells were transplanted subcutaneously in the forepaw of another mouse, zx produce a solid Tumur. After Zh hours after the transplantation powder of the invention that is 0.2 ms / 2O g (body weight of mouse) was intraperitoneally / kg in Einei dose of 10 mg in the mouse, given once daily for another 12 days. The substance was administered orally at a dose of 10 mg / kg, ie 0.2 mg / 20 g (body weight of the mouse) once a day for 23 additional days.

It was found that the antiviral substance (the brown above described powder) according to the invention had an excellent antiviral activity and that it was used as an anti-cancer agent worked.

Case 1: 65 year old male general practitioner, at who had been diagnosed with esophageal cancer

a) 197 ^ subjective symptoms: pain when drinking cold Liquids. He received the brown powder according to the invention and took it once a day before breakfast at a dose of 3 ß ·.

b) In December 1976 he had an X-ray examination done: to confirm his own diagnosis, and received some finding on the esophagus. Immediately an endoscopic

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I did study ¹ at Tokyo Women's Medical College and diagnosed esophageal cancer because atypical cells were found on the esophagus.

c) In February 1977 the patient refused the surgical operation. He had a weight of 38 kg with one height of 152 cm and suffered from severe weariness and indifference as well as loss of appetite.

d) In February 1977 »By endoscopic examination a Decrease in damage in the regenerated epithelial cells was noted.

e) In December 1977ί the appetite decreased abruptly and in worrying manner Way off.

f) In January 1978 the dose of brown powder was reduced to 6 g increased per day. A week later, the appetite improved.

g) In March 1978 there was improvement and an increase in body weight.

h) In September 1978, x-ray and endoscopic examination revealed no abnormal finding of the esophagus.

Case 2: 45 year old male patient

a) In April 1977 severe pain appeared in the left abdomen, two masses the size of a fist were found and based on X-ray, biu and urine examinations as malignant migratory Cancer and pancreatic cancer diagnosed.

b) In May 1977, 6 g of the aforementioned brown powder according to the invention were added once a day before breakfast given.

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c) In June 1977: a few days after the start of administration
the flatus was derived to relieve the feeling of pressure and the broom in the left abdomen. Only the powder according to the invention was given and other drugs were discontinued. The patient had viscera dislocations and was feeling
better.

d) July Ί 977 ί He felt no pain, but felt discomfort ii abdomen and sometimes tingling. This was due to the beginning of the: displacements of the duodenum and small intestine. He had rumbling and frequent winds. Blood, x-ray, and other examinations indicated the cure for the cancer. After vomiting, numerous winds went off every day; the cause of
the vomiting was seen in the intestines.

e) August 1977: The patient was operated on and suppuration of the diaphragm was found. The pus was sucked out daily through a tube. Administration of the powder that
had been stopped for a while before and after the operation started again.

f) September 1977: The patient strengthened himself, and his body: weight increased. Kr started walking.

g) October 1977: * ^; r was released from the hospital and
Continued on an outpatient basis. He was completely healed.

Example 3

The mycelium of Flammulina velutipes sing was like in example! cultured, and also became the one described above
Extract containing active component from culture medium mediura to obtain tissue medium, filtered to condense and freeze-dried to a powder.

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The material thus obtained was tested for antiviral activity and antitumor activity, with almost the same results were obtained as in the previous examples.

example k

Pleliata nameko was cultivated in the manner described in Example 1, and an active component mentioned above extract containing extract was also obtained from culture medium and tissue medium, filtered to condense and freeze-dried to obtain a powder.

The material thus obtained was tested for antiviral and antitumor activity were checked to obtain the same "Cr results" as in the previous examples.

Security of the antiviral substance according to the invention:

The anti-flu substance produced according to Example 1 was from Nomura Consolidated Laboratory analyzes to evaluate safety. The following results were obtained:

1. Acute toxicity (perorally)

Rats (male) rats (female) mice (male) mice (female)

LD ₀ (g / kg body weight)

16.5

'15.6 19.6 17.7

2. Toxicity to fish:

Carp, '' f8 hours Carp, 72 hours Vasserflohe, 3 hours

TLM

approx. 1.02 "/ ο 1.07 ⁰ Jo 0.56 Io

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Results:

TLM is equivalent to LD- in terms of acute toxicity (rats and mice. Common synthetic fertilizers have one Value of 10 ppm for carp and values in the ppb range for water bottles. It is thus ensured that the antiviral substance according to the invention has an extremely low toxicity.

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Claims (10)

Claims 1. Antivirus substance from culture medium and tissue medium of Basidiomycetes of the group Lentinua edodes, Pleuratus ostreatus, Pleliata nameko, Lyophyllum aggregatum, Coriolus Vereicolor and the like, where the active ingredient is a mixture of Polysaccharide and active cytocaine substance is the consists mainly of zeathin-related substances, which are produced by nutrient medium and tissue medium. 2. antiviral substance according to claim 1, characterized in that the mixture of polysaccharide and the active cytokainin substance, which consists mainly of zeathin-related substances, which are produced by the culture medium and tissue medium produced, is administered orally. 3. Antiviral substance according to claim 1, characterized in that that it interacts with viral hepatitis, cancer, and flu. MUNICH: TELEPHONE (089) 225585 CABLE: PROPINDUSTE LEX O5 24 244 IN: TELEPHONE (030) 8312Ο88 EL: PROPINDUS -TELEX 01 84087 4. Antiviral substance according to claim 1, characterized in that that the polysaccharide has a molecular weight of 3,000 to 10,000 and that the active cytokainin substance consists of Consists of zeathin and zeathin riboside. 5. Method of obtaining an antiviral substance after a or more of claims 1 to 4, characterized in that one hyphae of Basidiomycetes in solid or cultured liquid medium by mixing nutrient medium and tissue medium with an aqueous solvent and the active ingredient is extracted and that the extract is filtered « 6. The method according to claim 5 »characterized in that the materials are 4 to 5 hours at 60 to 130 ° C mix and stir to extract the active ingredient from the culture medium and tissue medium. 7. The method according to claim 5 »characterized in that the aqueous solvent used is water. 8. The method according to claim 5 »characterized in that the extract of nutrient medium and tissue medium is pressed through an ultrafilter membrane and filtered. 9. The method according to claim 5 »characterized in that the main material for the solid or liquid medium Bagasse used. 10. The method according to claim 5 »characterized in that the filtered extract is freeze-dried. 130066/0820 ORiGlNAL INSPECTED * COPY