DE2050714C2 - Polysaccharide with anti-tumor activity, process for its preparation and its use as an active ingredient in anti-tumor agents - Google Patents

Description

The invention relates to a polysaccharide with antitumor activity and the process for its preparation and its use as an active ingredient in anti-tumor drugs.

In the following, the term "polysaccharide" not only refers to the chemically pure polysaccharide, but also denotes the polysaccharide protein (mucopolysaccharide).

The substance containing the polysaccharide according to the invention is from the mycelium of the fungi of the family Polyporaceae of the genus Basidiomycetes or from a broth culture of the family Polyporaceae belonging species extractable.

Some methods of extracting active substances with antitumor activity from the basidiofruit pods of species belonging to the genus Basidiomycetes have already become known earlier; B. from the Published Japanese Patent Applications 18 196/1962, 16 048/1968 and 25 563/1968. These procedures however, suffer from a number of disadvantages. For example, the production of the antitumor active ingredient on a commercial scale is difficult because of the use of the basidiofruit stands as the starting material. rig. In addition, the physico-chemical properties of the extracted anti-cancerous Substance present active substance are not specified. Accordingly, the processes for cleaning the end product are still so inadequate that these active ingredients are only minimally absent.

In the JA patent publication 18 196/1962 extraction is carried out with alcohol and the filtered alcohol extract _A.

dried directly under reduced pressure and used as an active ingredient. In addition, there is still the extraction g |

shown with ether, chloroform, acetone, benzene and methanol. In the JA patent publication 16 048/1968 $ occurs

purification by first subjecting the extract to gel filtration or dialysis and then by itself ΐ

known further steps deproteinized and then at the very end the active ingredient with a water |

miscible organic solvent is precipitated. In the JA.-Palentpublikation 25 563/1968 the fungus is: ■

Fisiolins hcpatica Fr. of the family Fistulinaceae used as starting material and the aqueous extract according to Concentration mixed with ammonium sulfate, the precipitate formed was redissolved in water and then dialyzed, after concentration with glacial acetic acid and then treated with anion exchanger, dialyzed again and then with ethanol But you can also swap the process steps as you like. As the comparative experiments that follow later show, Fistolina hepatica gives only very low yields and the material is less effective than a material derived from Polyporaceae

A job in Japan. J. Exp. Med, VoL 38.6, pp. 437-442 (1968) shows that a strain of Basidomycetes extracellular polysaccharide produced during submerged culture A strain designated B-145 and designated as Crepidotus sp. was identified, was cultured for several days and the broth was then centrifuged and precipitated from the supernatant by repeated precipitation with acetone and then further purified by dialysis There is no reference to fungi of the Polyporaceae family either

The object of the invention is to provide a method for obtaining a sufficiently pure anti-tumor agent in on an industrial scale and to provide this active ingredient itself.

The subject matter of the invention is defined by the preceding claims

In particular, it is proposed that the polysaccharide be obtained from cultures of fungi of the Polyporaceae family grown in meat broth.

As an active ingredient, the polysaccharide has no toxicity whatsoever, in particular to microorganisms.

In connection with this invention, the term “cultivated nutrient medium” is used for a broth, in particular for a meat broth, but also in general for any culture medium that is intentionally used has been mixed with the corresponding species of fungus for the purpose of incubation

The types of Polyporaceae used in the context of the invention are in the event that their mycelium as Starting material is to be used, compiled in Tables 1 to 4 and are for the event that the The culture medium cultivated with them is to be used as the starting material, summarized in Tables 5 to 12. If the mycelium is used as a starting material, it can be of natural origin as well as artificially grown cultures. For use in the context of this invention, the Mycelium is present both unprocessed and preserved by washing with water and subsequent drying or subsequent freeze-drying. If a breeding ground cultivated with species of Polyporaceae is used as a starting material, the breeding ground of a common :) culture of this species itself can be used will.

To isolate the active substance from these starting maerials, either the mycelium is extracted with warm water or a warm aqueous solvent, or the cultivated nutrient solution is used Filtration of the mycelium recovery. Both starting materials can also be used mixed together.

The temperature of the solvent used for extraction is not critical below a temperature of 120 ⁰ C. Temperatures between 60 and 100 ^° C. are preferably used. The extraction is promoted by previously pulverizing the mycelium, but such prior pulverization is not essential. In general, the extraction is carried out with warm water, but aqueous solvents can also be used which contain water-soluble organic solvents, acids, bases or salts. The time required for extraction is a function of the extraction temperature. The extraction is preferably carried out at a temperature of 60 to 100 ° C. for a period of 30 minutes to 10 hours. The extraction is best carried out with constant stirring in a glass vessel or in a vessel with an inner glass lining, in enamelled vessels or in vessels Made of stainless steel

The mycelium extract produced in this way is filtered or centrifuged to remove solids and then cleaned as described below.

In the event that a cultivated culture medium is assumed, the mycelium becomes the culture medium separately, concentrated and then subjected to the cleaning process described below in the same way.

The aqueous extract or the filtered nutrient solution obtained in this way contains, in addition to the polysaccharide, which is the active component of the anti-cancerous substance, a number of other undesirable substances, such as free proteins, nucleic acids or reducing sugars. To remove these substances is a cleaning procedure is required.

With the cleaning method according to the invention, the undesirable substances, especially the Removes extremely harmful components, the free proteins. This cleaning is advantageous through Combination of a number of cleaning methods performed, for example by a combination of one Deproteinization, deionization (which includes neutralization and desalination) and, where appropriate, methods of eliminating other organic substances. To achieve this, you can Processes such as extraction, centrifugation, filtration, acid treatment, salting out, dialysis or Process using ion exchangers can be used.

First of all, however, as the examples show, the polysaccharide is formed from the solids broth precipitated with ammonium sulfate, if necessary after incidents with ethanol and resumption of precipitation in water. Only then do the further cleaning steps follow.

The deproteinization of the redissolved precipitate present in the liquid phase is carried out by adding acids, for example to add sulfuric acid, hydrochloric acid, nitric acid, acetic acid, trichloroacetic acid, picric acid, sulfosalicylic acid, tannic acid and others in an amount that amounts to 0.1 to 3 wt. % corresponds, with the free proteins being precipitated, the proteins thus precipitated being subsequently separated from the liquid phase. The solution freed from the precipitated proteins is then placed on a column with anion exchange resin, which is used in connection with the purification process in the event that a cult ^; ". ated nutrient medium was used as the starting material, is described in more detail. Instead of being placed on the exchange column, the extract can also be subjected to dialysis, whereby for

To get rid of the excess acid, dialysis membranes, such as Cel'ophan foils, can be used. Alternatively, the deprotection can also be carried out directly by ion exchange.

After the end of the dialysis or the ion exchange, the liquid collected in the membrane or in the outlet is concentrated and mixed with 3 to 10 times the volume of an alcohol to produce a Precipitation is added, which is then subjected to freeze-drying or first with alcohol and then with Acetone is washed and then dried. The product obtained in this way is a no hygroscopic, light gray to white powder that is anti-cancerous

Even when using the concentrated culture broth, the polysaccharide is precipitated first, as above specified. To reduce further undesirable substances such as free proteins, free nucleic acids en Various methods are available to remove sugars, various ions or vitamins. So can for example be concentrated, treated with acid, extracted, dialyzed or treated with an ion exchanger. Combinations of these procedures are also possible. To the Inorganic and organic acids are suitable for removing the free proteins, for example trichloroacetic acid. Sulfuric acid, hydrochloric acid, glacial acetic acid, sulfosalicylic acid, picric acid or tannic acid. Referring to the In the entire liquid phase, the concentration of these acids should be about 0.1 to 3%.

Dialysis, which is used to remove lower sugars, free amino acids, various salts or even for Removal of various nucleic acids can be carried out with the aid of a semipermeable membrane, for example with a CeHophane film, a collodion membrane or also with an intestinal membrane. The ion exchange is carried out with anion and cation exchange resins Als Cation exchange resins come, for example, Amberlite IR-120 B, Amberlite 200, Amberlite IR-122, Amberlite IR-

123, Amberlite XE-100, Amberlite IRC-50 and Amberiite IRC-84 (® all Rohm &Haas); Dowex 50W (® from Dow Chemical); Duolite C-3, Duolite C-10, Duolite C-20, Duclite C-25, Duolite C 27, Duolite ES-63, Duolite ES-80 and DuoliteCS-101 (* all from Diamond Alkali) as well as Diaion SK-IA, Diaion SK-IB, Diaion SK-102, Dk- ion SK-103, Diaion SK-104, Diaion S K-106, Diaion SK-110, Diaion SK-112, Diaion SK-116, Diaion SKI, Diaion PK-204, Diaion PK-208, Diaion PK-212, Diaion PK-216 and Diaion PK-228 (® all from Mitsubishi Chemical Industry Co, Ltd.) in question. The following can be used as anion exchangers: Amberlite IRA-400, Amberlite IRA-401, Amberlite IRA-402, Amberlite IRA-405, Amberlite IRA-425, Amberlite IRA-900, Amberlite IRA-904, Amberlite IRA-410, Amberlite IRA- 411, Amberlite IRA-911, Amberlite IRA-910, Amberli- te 1R-45, Amberlite IRA-93, Amberlite IRA-68, and Amberlite IR-4B (Rohm &Haas); Dowex 1, Dowex 2, Dowex 44, Dowex 21K and Dowex 11 (Dow Chemical) as well as Duolite A-2, Duolite A-4, Duolite A-6, Duolite A-7, Duolite ES-15, Duolite A-30T, Duolite A-30B, Duolite A-41, Duolite A-43, Duolite ES-57, Duolite A-40, Duolite A-40LC, Duolite A-42, Duolite A-42LC, Duolite A-40, Duolite A-101D, Duolite A-102, Duolite A-102D and Duolite ES-111 (Diamond Alkali). The free proteins are produced by the procedures already mentioned above removed, the acid treatment being particularly suitable. The removal of the excess acid and the Discoloration is achieved through ion exchange. The exchanger Duolite S-30 or Duolite ES-33 (Diamond Alkali) and the exchangers related to these show above all a particularly favorable decolorization effect. The exchangers Dowex 5OWX2 (Η-type) and Dowex 44 (OH-type) are made for the removal of nucleic acids particularly suitable for the liquid phase.

The end product can also be precipitated by adding strongly polar organic solvents, for example water-soluble alcohols and ketones such as methanol, ethanol, propanol, butanol or acetone. Treatment with lead acetate is understood to mean the addition of lead acetate to the liquid phase, whereby the end product is precipitated as lead salt. The pure end product can come through from this lead salt Removal of the lead can be obtained, for example, by introducing hydrogen sulfide into a Auf Elimination of the precipitate can be effected, whereby the lead is precipitated as lead sulfide.

As described above, the purpose of the purification process is to remove undesirable substances from the extract or from the nutrient solution, these undesirable substances being essentially made up Proteins exist, and the isolation of the active polysaccharide from the purified solution thus obtained. The polysaccharide according to the invention, which is obtained by the treatment described above,

it can be characterized as follows: It is a light gray to white powder that cannot be dialysed no sharp melting point, charred on intense heating, is insoluble in common organic Solvents and organic acids, but soluble in water. A 10% aqueous solution of this Substance reacts neutrally. A 0.1% strength aqueous solution shows no characteristic absorptions in the UV range.

If the polysaccharide obtained according to the invention is subjected to a 0.05 molar sodium borate solution Electrophoresis (cellulose acetate membrane, 20 to 25 V / cm, 90 min), so only one is on the anode scite Stain received.

If, however, a 0.1% strength aqueous solution of the polysaccharide is hydrolyzed, it takes so long to dissolve Sulfuric acid is added until the total solution is 1 and that this solution is then 5 hours is heated to 100 ⁰ C, then neutralized by adding barium carbonate and paper chromatographed, glucose can always be detected.

The polysaccharide itself also showed under the most varied of conditions in the paper chromatogram no hike. However, if the 1% strength aqueous solution of the substance is subjected to that described above sulfuric acid hydrolysis and then neutralized with barium carbonate, as the paper chromatography shows gram clearly shows the glucose.

The hydrolysis product of the polysaccharide reacts positively to the following reactions: anisaldehyde reaction, Molisch reaction, anthrone reaction, tryptophane sulfuric acid reaction, chromotropic acid sulfuric acid reaction, carbazole-cysteine-sulfuric acid reaction, aniline hydrochloric acid reaction. Resorcinol hydrochloric acid reaction, ToI-

lens reaction and thioglycolsulfuric acid reaction. The hydrolyzate does not react to ferric chloride and Fehling's solution.

The polysaccharide according to the invention shows no antimicrobial effect against bacteria, fungi and Yeasts such as, for example, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Aspergillus nigel or Candida albicans and thus differs significantly from the antibacterial and anti-carcinogenic agents, obtained from the species belonging to the genus Actinomycetes.

In addition, the polysaccharide does not show any cytotoxicity or other undesirable side effects occur in the known anti-carcinogenic agents, such. B. the decrease in leukocytes, anemia the Liver and other organs, atrophy of the spleen, loss of body weight and loss of appetite. The acute one The toxicity (LD50) of the substance is above 20 g / kg after oral administration and subcutaneous, intramuscular or intravenous injection over 100 mg / kg.

The anti-carcinogenic effect of the polysaccharide according to the invention was verified by the following tests: f

An ICR-JCL series mouse weighing 20 g was intraperitoneally injected with solid cells of the |;

Ehrlich's carcinoma or Sarcoma-180 infected. One week after a sufficient increase in 15, "4 Ascension fluid could be observed in this mouse, the cancer cells it contained were

transferred subcutaneously to other mice at a concentration of 6 million per mouse. These mice became \

Groups of 10 animals each divided, of which the first group was only treated with saline solutions, \

while the other groups with anticancer agents and with the substance according to the invention were treated. The first application was intraperitoneally during a week after the transplant the following treatment was repeated nine times every other day. The ones with the solid cancer cells infected mice were checked for body weight gain five days after the last, the tenth, treatment examined and then examined anatomically to detect side effects.

The results of these tests are compiled in Tables 1 to 12.

The invention is explained in more detail below with the aid of examples.

Example t

2 kg of dried and finely chopped mycelium from Coriolus pubescens (Fr.) Quel. given. In a glass vessel equipped with reflux condenser the mixture to 95 to 100 ⁰ C under stirring was heated for three hours, to thereby effect extraction. The mixture was then cooled to room temperature, filtered through a cloth and the filtrate was concentrated to one liter. After adding 700 g of ammonium sulfate, the filtrate was left to stand in the refrigerator overnight. The precipitate, which had formed overnight, was separated off the next morning by paper filtration

The residue was washed six times with 4 l each time of a saturated ammonium sulfate solution, d. H. so with 24 1 in total, 35 '

washed. The precipitate was then dissolved in about 1 l of water and placed in a Visking cellulose jar, where it was subjected to dialysis for 40 hours in flowing water. After the concentration of the liquid phase in the membrane to 1 l, 100 ml of 10% trichloroacetic acid was added to remove the free proteins. After decolorization with 10 g of activated charcoal, the mixture was decolorized. The filtrate was again subjected to dialysis for 40 hours in running water, after which the liquid phase in the membrane was concentrated to 100 ml. The precipitate which had formed after the addition of 400 ml of ethanol was washed in sequence with SO% ethanol, 99% ethanol and with acetone and _r

then dried. In this way, 60 g of a light gray to white powder was obtained. , .t

The substance obtained in this way is a polysaccharide, as evidenced by the tests mentioned above has been proven. Both this substance and other polysaccharides obtained from the mycelium of other species The Polyporaceae family obtained by the same procedure were tested for their anti-carcinogens Effect investigated. The results are summarized in Table 1

The mycelium used for these experiments was obtained in the following way:

One thousand 11-Erlenmeyer KoIben were washed with 150 ml of the following nutrient solution stated in the ^ ₁

filled: 50;

The flasks were closed with a cotton ball and sterilized for 30 minutes at 120 ° C. The nutrient liquids were then queled in the usual way with Coriolus pubescens (Fr.). inoculated which had been cultivated on an inclined culture soil. After a stationary incubation time of the cultures in the Erlenmayer flasks for 20 days at 23 to 25 ° C., the contents of the flasks were filtered. The mycelium remaining on the filters was washed with 50 liters of water and then dried

1 <~ pi \ suv. 5g Yeast extracts 3g Primary potassium phosphate 03 g Secondary potassium phosphate 03 g Magnesium sulfate heptahydrate 0.3 g Glucose 30 g water 1 liter pH = 6.0

Tabel

family

organism

Efficacy against solid cancer Sarcoma-180

Polyporaceae - ► Corbolus pubescens (Fr.) Quel. Polyporaceae - ► Tyromyces lacteus (Fr.) Murr. Polyporaceae Polyporus confluens (A. et S.) Fr. Polyporaceae - ► Microporus affinis (Blume et NeesJKuntze. Polyporaceae Trametes gibbosa Fr. Polyporaceae Cryptoporus volvatus (PK.) Hubb. Polyporaceae Trametes albida (Fr.) Bourdot et Garzin Polyporaceae - ► Ganoderma lucidum (Fr.) Karst. Polyporaceae - ► Hirschioporusabietinus (Fr.) Donk Polyporaceae - ► Tyromyces incarnatus Imazeki

Example 2

80 100 80 70 60 60 70 70 70 90

45 liters of distilled water were added to 3 kg of dried and powdered mycelium from Grifola gigantea (Fr.) Pilat. In one provided with reflux condenser, glass-lined vessel under stirring the mixture was heated for the purpose of extraction for five hours at 9O ⁰ C. After cooling, it was filtered through a cloth and the filtrate was then concentrated to 51%. The concentrate was then centrifuged for 30 minutes (1500 g), the supernatant solution decanted and mixed with 4250 g ammonium sulfate while stirring and left to stand overnight in the refrigerator. The next morning it was filtered, the residue was collected and dissolved in water and the solution obtained in this way was then applied to a column activated with 1 η hydrochloric acid and containing Amberlite IR-120B NaOH solution added to activated column, which contained Duolite A-7. The effluent was concentrated to one liter by adding 41% ethanol and then filtered. The residue was washed with ethanol and then with acetone, dried and gave a yield of 55 g of a light gray to white powdery substance.

This substance is a polysaccharide as demonstrated by the tests described above became. The substance obtained in this way was combined with the other polysaccharides obtained from the mycelium of others Polyporaceae obtained by the same procedure were examined for their anti-tumor effects. The results are summarized in Table 2

The mycelium used was obtained in the manner described in Example 1. Tabel

family

organism

Efficacy against solid cancer Sarcoma-180

Polyporaceae Trametes cinnabarina Fr. Polyporaceae - Deadaleopsis confragosa (Fr.) Schroet Polyporaceae - * Favolus arcularius (Fr.) Arnes. Polyporaceae Gloeoporus amorphus (Fr.) Clem, et Shear. Polyporaceae Fomitopsis castanea imaz. Polyporaceae Ischnoderma resinosum (Fr.) Karst. Polyporpceae - ► Grifola frondosa (Fr.) S. F. Gray Polyporaceae Amauroderma scopulosum (Berk.) Imaz. Polyporaceae Grifola gigantea (Fr.) Pilat Polyporaceae Tyromyces caesius (Fr.) Karst Polyporaceae Trametes sanguinea (Fr.) Lloyd Polyporaceae Elfvingia applanata (Pers.) Karst

Example 3

90 90 90 60 80 70 80 90 90 70 90 80

35 l of distilled water were added to 1 kg of dried and powdered mycelium from Microporus flabelliformis (Fr.) Kuntze. ^{The mixture was heated to 95 to 98 D} C for 5 hours with stirring for the purpose of extraction in a stainless steel vessel equipped with a reflux condenser and a stirrer. After cooling to room temperature, it was filtered through a cloth. The residue was filtered for the purpose the further extraction with a further 301 distilled water for three hours with stirring at 98 to 100 "C then cooled to room temperature and filtered again through a cloth. Both filtrates were combined and concentrated to 51 under reduced pressure. 4 kg were added to the concentrate with thorough stirring After standing overnight at 5 to 8 ° C., the reaction mixture was centrifuged for 20 minutes at 1300 G the next day

2000 (Amicon Corporation) using a Diaflo UM-2 membrane (Amicon Corporation) at 5 to 10 ° C a pressure of 4.9 bar (5 kg / cm ² ) was achieved. The liquid in the membrane was placed in an Erlenayer flask and, based on the total weight of the mixture, 3% by weight of tannic acid was added. The mixture was stirred for 30 minutes and then centrifuged at 300G for 20 minutes. The supernatant liquid was again ultrafiltered, again using a model 2000 ultrafilter with a Diaflo membrane. The ultrafiltration was carried out for 200 hours at 5 to 8 ° C. and 4.9 bar (5 kg / cm ² ). The liquid in the membrane was mixed with three times the volume merge of ethanol. The mixture was stirred well and the precipitate so formed was then collected on a paper filter. The residue on the filter paper was washed in sequence with 5 l of 75% ethanol, with 3 l of 95% ethanol and finally with one liter of 99% ethanol and finally with one liter of acetone and dried under reduced pressure. In this way, 23 g of a light gray to white powdery substance were obtained.

The substance obtained in this way was found to be a polysaccharide by the tests described above be identified. This substance, along with other polysaccharides, was extracted from the mycelium other Polyporaceae obtained by the method described above for their anticarcinogens Effectiveness examined. The results are summarized in Table 3.

The mycelium used for these experiments was prepared in the manner described in Example 1

Table 3

_ _—

Family organism efficiency against

solid cancer sarcoma-180

Polyporaceae Daedaleopsis styracina (P. Henn.etShir) lmazeki 80 2s

Polyporaceae Gloeophyllum saepiarium (Fr.) Karst. 80

Polyporaceae Fomitopsis officinalis (Fr.) Bond, et Sing. 80

Polyporaceae - »Coriolus consors (Berk) Imaz. 60

Polyporaceae Tyromyces sambuceus (Lloyd.) Imaz. 80

Polyporaceae Polyoporus pes-caprae Fr. 90

Polyporaceae Microporusflabelliformis (Fr.) Kuntze 100

Polyporaceae - »Coriolus flabelliformis (Fr.) Kuntze 70

Polyporaceae— »Fomesfomentarius (Fr.) Kickx 80

Polyporaceae Ganoderma neo-japonicum Imaz. 80

Polyporaceae Trametes dickinsii Berk. 70

Polyporaceae Oxyporus ravidus (Fr.) Bond, et Sing. 60

Polyporaceae— * Hirschioporusfusco-violaceus (Fr.) Donk 60

Polyporaceae Coltrioiaperennis (Fr.) Murrill 80

Polyporaceae Favolusalveolarius (Fr.) Quel. 80

Example 4

To 2.5 kg of dried and powdered mycelium from Coriolus hirsuius (Fr.) Quel were 751 distilled Given water. The mixture was placed in a glass vessel 3 with stirring for the purpose of extraction The customer is heated to 95 to 100 ° C for a long time, occasionally adding a little distilled water in such a way that that the mixture was concentrated to an amount of 25 liters after the Erlvizen. After cooling to room temperature was first filtered through c-ir cloth, then through a filter paper and the filtrate then under reduced pressure to 2.5 liters. After adding 2 kg of ammonium sulfate, the mixture became vigorous stirred, left to stand overnight and filtered through a filter paper the next morning. The residue is taken up The filter paper was washed with a saturated ammonium sulfate solution (101) and then in 24 liters of so dissolved in distilled water. The solution was then put on a column of Duolite C-25-D and then on placed on a column of Amberlite IRA-400 to remove the excess ammonium sulfate. The outlet was concentrated to 500 ml under reduced pressure and applied to a column of Scphadex G-75. These Column was additionally charged with 101 washing water, whereby the fraction with the highest molecular weight was collected and subjected to freeze-drying. After drying, 3.26 g of a light gray to white powdery substance obtained.

The substance thus obtained was tested using the tests described above as Polysaccharide Identified This substance was identified in common with other polysaccharides derived from the mycelium other Polyporaceae obtained by the method described above for their anticarcinogens Effectiveness examined. The results are shown in Table 4.

The mycelium used for these experiments was obtained in the manner described in Example 1.

Table 4

family

organism

Efficacy against solid cancer Sarcoma-180

Polyporaceae Porodiscuius penduius (Schw.) Murr: !!

Polyporaceae Fomitopsis cytisina (Berk.) Bond, et Sing.

Polyporaceae Polyporus caeruleoprus Peck

Polyporaceae Fomitopsis pinicola (Fr.) Karst.

Polyporaceae Polyporellus brumalis (Fr.) Karst.

Polyporaceae - ► CoriolushirsutusiFr.JQuel.

Polyporaceae Coriolellus heteromorphus (Fr.) Bond, et Sing.

Polyporaceae - * Coriolus conchifer (Schw.) Pat.

Polyporaceae Fomitopsis latissima (Bres.) Imazeki, comb. nov.

Polyporaceae Polyporellus elegans (Fr.) Karst.

Polyporaceae Pclyporus dispansus Lloyd

Polyporaceae Trachyderma tsunodae (Yasuda) Imazeki

Polyporaceae Lenzites betulina Fr.

Example 5

60 60 90 80 90 90 70 70 80 90 60 60 90

A culture of Fomitopsis rhodophaea (Lev.) Imaz. and the nutrient liquid was filtered through a cloth to remove the mycelium. The resulting filtrate (i001) was concentrated to 151 under reduced pressure, and 12.75 kg of ammonium sulfate were added. The added salt was then dissolved with stirring. The resulting solution was left to stand in the refrigerator overnight and centrifuged at 6000 G for one hour the next morning. The supernatant liquid was decanted and the residue was dissolved in 3 l of distilled water. 2550 g of ammonium sulfate were added to this solution and the mixture was again left to stand in the refrigerator overnight. The next morning it was centrifuged at 6000G for one hour. This procedure was repeated five times. The precipitate finally obtained was dissolved in 31% of distilled water. The solution was filtered through No. 2 pore size paper, and the filtrate placed in a cellophane cell was then dialyzed in flowing water for 50 hours. The liquid collected in the cell was concentrated to 11 at room temperature under reduced pressure, and trichloroacetic acid was added so that the trichloroacetic acid concentration was 4% by weight based on the total solution. The solution was left to stand in the refrigerator overnight and then centrifuged at 10,000 G for one hour. The supernatant was again placed in a cellophane cell and dialyzed at 10 ° C for another 150 hours. The liquid collected in the cell was vacuumed at room temperature 11 concentrated and then treated with 31 ethanol. The mixture obtained was left to stand overnight and the next morning the precipitate which had formed was separated off by centrifugation (15 minutes at 1500 G), washed with absolute ethanol, acetone and ether and dried. 19.2 g of a light gray to white, powdery substance were obtained.

In Table 5 are the results of tests on the anticarcinogenic efficacy of the thus obtained Mucopolysaccharide presented along with the results for other mucopolysaccharides included in the similarly obtained from other Polyporaceae.

Table 5

family organism Elementary Yield dose Effect Effect analysis on mg / kg straight against straight against C H sugar solid cancer solid cancer Base Sarcoma-180 from the honest (%) (%) Type(%)

55 Polyporaceae Polyporaceae

Polyporaceae Polyporaceae Polyporaceae

65 Polyporaceae

Fomitopsis rhodophaea (Lev.) Imaz.

Coltricia pusilla Imazeki etYsk-Kobayashi Polyporellus picipes (Fr.) Karst

Daedaleopsis styracina (P. Henn et Shir.) Imazeki Gloeophyllum saepiarium (Fr.) Karst Fomitopsis officinalis (Fr.) Bond, et Sing.

4235 531 0.93

40.02 41.41

5.1! 0.60 634 0.61

4230 631 037 3835 6.88 0.22 42.94 5.16 0.61

300 300 300 300 300 300

80 100 70 80 60 80

70 90 60 90 60 80

Example 6

A cooling liquid from Ganoderma tsugae Murrill prepared in the same manner as in Example 5 had been received. was filtered through a cloth to remove the mycelium. 451 each of the filtrate were used in Concentrated to 4.51 at room temperature under reduced pressure. The insoluble residues were through Centrifugation (20 minutes at 1300 G) removed 3285 g of ammonium sulfate was added to the supernatant liquid, the mixture was stirred well and left to stand in the refrigerator overnight. The next It was centrifuged again at 1500 G for 20 minutes tomorrow. The residue was collected with 31 saturates Ammonium sulfate solution homogenized and centrifuged again for 20 minutes at 1500G The residue was dissolved in one liter of distilled water and applied to an Amberlite IR-120 column activated with hydrochloric acid to remove some of the proteins and some of the ammonium sulfate. The column was then washed with distilled water and the wash water with the outlet combined and then concentrated to 1.5 l at room temperature under reduced pressure. Trichloroacetic acid was added to the concentrated liquid up to a total concentration of 3.5% by weight The batch was left to stand in the refrigerator overnight and the next to separate the proteins Centrifuged at 10,000 G for 40 minutes tomorrow. The supernatant liquid was passed through a column with Duolite A-7, which had been activated with 4% soda solution. This separated the remaining ammonium sulfate and the colored by-products. The column was then distilled with 51% Washed water and the wash water added to the previously obtained run. The combined run was concentrated to 1.5 liters at room temperature under reduced pressure. 4.5 l of ethanol were added to the concentrated liquid and the mixture was left to stand overnight and the next morning Centrifuged at 1400 G for 20 minutes. The precipitate was collected and poured into two liters of 75% Ethanol was taken up and homogenized and centrifuged again for 20 minutes at 1400 G. This treatment was repeated five times and the residue finally obtained was successively washed with absolute ethanol, Acetone and ether washed and then dried. The yield a .. light gray to white powder was 2.5 g.

Table 6 shows the results of the tests for demonstrating the anticarcinogenic effectiveness of the so obtained mucopolysaccharide are combined with the results obtained for other mucopolysaccharides obtained in the same manner from other Polyporaceae.

Table 6

family organism Elementary H Yield dose Effect Effect Type(%) analysis on mg / kg straight against straight against 90 C. sugar solid cancer solid cancer 5.70 Base Sarcoma-180 vom Ehrlich- 90 (%) (%) Polyporaceae Coriolus consors 41.54 6.25 0.77 300 80 100 (Berk.) Imaz. Polyporao?!> E Tyromyces sambuceus 38.06 5.63 0.47 300 90 100 (Lloyd) Imaz. 100 Polyporaceae Coltricia dependens 38.67 6.68 0.25 300 100 (Berk, et Curt) Imaz. 6.87 60 Polyporaceae Polyporus ovinus Fr. 39.07 0.76 300 100 Polyporaceae Fomitopsis insularis 40.96 6.49 0.16 300 100 (Murr.) Imaz. Polyporaceae Ganoderma tsugae Murrill 39.58 0.19 300 60

Example 7

By incubation in 300 ml of a nutrient liquid with the following composition: Composition (I):

Peptones 10g Yeast extracts 2g Malt extracts 2g Primary potassium phosphate 0.1g Secondary potassium phosphate 0.1g Magnesium sulfate heptahydrate 0.1g Glucose 50 g Calcium carbonate 1.5g water 1 liter pH = 6.5

60

after 14 days at 23 to 25 ° C, a mycelium of Tramets gibbosa Fr. obtain. The mycelium was homogenized with 100 ml of an aphysiological saline solution and for inoculation in 23 1

one bottom into a 361 capacity stainless steel fermentation vat.

The nutrient medium, which had the composition given below, had been sterilized ^{at 120 ° C. for 30 minutes:}

S Composition (II):

Peptones 10 g Yeast extract 2g malt extract 2g Primary potassium phosphate 0.1g Secondary potassium phosphate 0.1g Magnesium sulfate heptahydrate 0.1g Glucose 50g Silicone resin 0.05 g Calcium carbonate 13 g water 1 liter pH -6.5

The medium was subjected to aerobic incubation for 10 days at a stirring speed of 110 to 120 rpm and 23 to 25 ° C. and an aeration rate of 0.15 liters / liter / min.

The nutrient medium (151) was filtered through a cloth and the filtrate was concentrated to one liter at room temperature under reduced pressure. 850 g of ammonium sulfate were added to the concentrate, and the mixture was then stirred well and left to stand in the refrigerator overnight. The next morning it was centrifuged at 1600G for 20 minutes. The residue thus obtained was dissolved in one liter of distilled water and the resulting solution was ultrafiltered using an ultrafilter model 2000 with a Diaflo UM-2 membrane (Amicon Corporation, USA). It was filtered for 150 hours under a pressure of 6.87 bar (7 kg / cm ² ). The liquid collected in the filter was placed in a 3 liter beaker and trichloroacetic acid was added to a total concentration of 3% by weight. The mixture was stirred well, left in the refrigerator overnight and centrifuged at 10,000 G for one hour the next morning. The supernatant was applied to a Duolite A-7 column which had been treated with 4% NaOH solution. The column was then washed with 31% of distilled water, the wash water being combined with the previously obtained run-through. The combined runs were concentrated to one liter in vacuo. Acetone was added to the concentrate, and the precipitate thus obtained was washed with pure acetone and with ether and then dried. 3.0 g of a light gray to white powder were obtained.

In Table 7 are the results of the anticarcinogenic efficacy tests of the thus obtained Mucopolysaccharide is given along with the results for the other mucopolysaccharides that obtained from other Polyporaceae by the above process.

40 Table 7 organism Elementary
analysis
C H 6.34 Yield
on
sugar
Base
(%) dose
mg / kg Effect
straight against
solid cancer
Sarcoma-180
(%) Effect
straight against
solid cancer
from the honest
Type(%) 45 family Polyporellus brumalis
(Fr.) Karst
Coriolus pubescens
(Fr.) Qu6l.
Tyromyces lacteus
/ Cr \ Murr 42.65 5.53 0.79 300 80 70 Polyporaceae \ r I. f IVlUI I
Polyporus confluens
(A. et S.) Fr.
Microporus af finis
(Blume et Nees) Kuntze
Trametes gibbosa Fr. 40.55 6.15 0.20 300 70 80 50 Polyporaceae 38.26 6.10
5.59
5.86 0.71 300 90 80 Polyporaceae 39.23
38.20
39.25 Example 8 0.53
0.20
0.25 300
300
300 80
70
80 90
80
90 55 Polyporaceae
Polyporaceae
Polyporaceae 60

220 1 liter incubation bottles were filled with 200 ml each of a malt extract nutrient liquid with the following composition:

Malt extracts (Difco) water

50 g 1 liter

The bottles were closed with cotton plugs 30 minutes sterilized at 120 ⁰ C, allowed to stand for cooling and then with a mycelium of Fomhopsis castanea Imaz. vaccinated that had been grown on a sloping surface. The stationary incubation was carried out for 20 days at 25 to 28 ° C. To remove the mycelium, the fermented nutrient medium was then filtered through a filter cloth kg / cm ² ) was used. The liquid concentrated by ultrafiltration was discharged from the filter and centrifuged at 1500 G for 15 minutes to remove the residues. 2550 g of ammonium sulfate was added to the supernatant liquid. The mixture was left to stand overnight and centrifuged at 1500 G for 15 minutes the next morning residue obtained in this way was taken up in 31% of a saturated ammonium sulfate solution and homogenized and centrifuged again for 15 minutes at 1500 G. This treatment was repeated five times, whereupon the liquid was applied to an Amberlitc IR-200 column which had been activated with 1 η hydrochloric acid was. The column was then washed with one liter of distilled water added to the previously obtained run-through. The pooled runs were left in the refrigerator overnight and centrifuged at 10,000 G for 30 minutes the next morning. The residue obtained in this way was separated off and the supernatant liquid was applied to an activated Dowex 1 column. The run-through was concentrated to one liter at room temperature under reduced pressure, treated with 31% ethanol and the residue obtained after filtration was washed in succession with absolute alcohol, acetone and ether and then dried. 103 g of a light gray to white powder were obtained.

Table 8 shows the results of the tests for demonstrating the anti-carcinogenic effectiveness of the so obtained mucopolysaccharide together with the results of other mucopolysaccharides, obtained from various other Polyporaceae by the method described above had been.

Table 8

family organism Elementary Yield dose Effect effect analysis on mg / kg degree against degree against C H sugar solid cancer solid cancer Base Sarcoma-180 vom Ehrlich- (%) (%) Type(%)

Polyporaceae Polyporaceae

Polyporaceae Polyporaceae

Favolus arcularius (Fr.) Arnes

Gloeoporus amorphus (Fr.) Clem et Shear Fomitopsis castanea Imaz. Ischnoderma resinosum (Fr.) Karst.

39.48 6.21 0.50 38.31 5.81 0.%

39.69 41.99

5.15
5.76

0.72 0.79

300
300

300
300

90 90

90 70

100 90

100
80

Example 9

With constant addition of water, 40 g of corn flour were boiled for three hours at 100 ° in one liter of water. After cooling, the slurry was filtered and 10 g of cane sugar was added to the filtrate. 813 1 l incubation bottles were each filled with 180 ml of this grain extract nutrient medium. The bottles were sealed with cotton balls and sterilized at 120 ° C. for 30 minutes. After cooling to room temperature, they were treated with previously cultivated mycelium from Rigidoporus ulmarius (Fr.) Imaz. vaccinated. The stationary incubation was carried out at 23 to 27 ° C for 20 days. The mycelium was then separated from the fermented nutrient medium. The filtrate was concentrated to 101 under reduced pressure at room temperature, and 8.5 kg of ammonium sulfate were added. After stirring vigorously, the liquid was left to stand in the refrigerator overnight and filtered the next morning. The residue remaining on the filter paper was washed with a saturated ammonium sulfate solution, dissolved in 2 l of distilled water and then dialyzed in flowing water for 60 hours using a cellophane cell. The solution in the cellophane cell was then concentrated to one liter and trichloroacetic acid was added to a concentration of 3.5% by weight. After standing again overnight in the refrigerator, centrifugation was carried out at 10,000 G for 30 minutes the next morning. The residue was separated off and the supernatant liquid was subjected to ultrafiltration, using a Diaflo UM-2 membrane with a diameter of 150 mm at a pressure of 6.87 bar (7 kg / cm ² ). Filtration and washing were carried out with 160 liters of distilled water. The ultrafiltration achieved a concentration to 600 ml. 1.8 l of ethanol were added to the concentrated liquid and the mixture was then left to stand in the refrigerator overnight. The next morning it was centrifuged at 1600 G for 20 minutes. The residue was taken up in 3 l of distilled water and the excess ethanol was removed under reduced pressure with constant replenishment of the water. Freeze-drying this liquid gave 29.1 g of a light gray to white powder.

In Table 9 are the results of the anticarcinogenic efficacy tests of the thus obtained Mucopolysaccharide is given along with the results for other mucopolysaccharides obtained from various other Polyporaceae were obtained by the same procedure.

25 30 35 40

50

55

60

65

Table 9 organism Element door Yield dose Effect Effect family analysis on mg / kg straight against straight against C H sugar solid cancer solid cancer Base Sarcoma-180 from the honest (%) (%) Type(%)

Polyporaceae PolypoFjjCeae Polyporaceae Polyporaceae Polyporaceae Polyporaceae

Grifola frondosa (Fr.) S. F. Gray Hirschioporus versetiiis (Berk.) Imaz. Laetiporus versisporus (Lloyd) Imazeki Rigidoporus durus (Jungh.) Imaz. Polyporellus aquamosus (Fr.) Karst Rigidoporus ulmarius (Fr.) Imaz.

39.06 6.77 032 300 100 90

42.65 537 0.25 300 70 70

39.01 5.47 0.43 300 90 100

42.71 6.74 0.62 300 100 100

40.48 5.25 0.17 300 80 90

42.01 5.21 0.97 300 70 80

Example 9a

The above example was reworked with the strains of the Polyporaceae family given in Table 9a below, the same results being obtained within the error limits as in Example 9. For the strain Coriolus versicolor (Fr.) the degree of effectiveness against solid cancer Sarcoma was 180 investigated and showed an efficiency of 100 according to the method given here.

Table 9a

Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae

Trametes trogii Berkeley Fomitopsis rhodophaea (Lev.) Imaz. Coltricia pusilla Imazeki et Ysk. Kobayashi Polyporeilus pioipes (Fr.) Karst. Coriolus pargamenus (Fr.) Pat Tyromyces guttulatus (Pk.) Murr. Polyporus tuberaster Fr. Daedaleopsis nippenica Imazeki Coltrica dependens (Berk, et Curt.) Imaz. Polyporus ovinus Fr. Fomitopsis insularis (Murr.) Imaz. Ganoderma tsugae Murrill Grifola albicans Imazeki Coriolus versicolor (Fr.) Qu6l. Grifola umbellata (Fr.) Pilät Piptoporus betulinus (Fr.) Karst. Daedaleopsis tricolor (Fr.) Bond, et Sing. Ganoderma boninense Pat. Trametes orientalis (Yas.) Irnaz. Favolus emerici (Cooke) Imazeki Fomitopsis pubertatis (Lloyd) Imaz. Fomitopsis vinosa (Berk.) Imaz. Porodisculus pendulus (Schw.) Murrill Fomitopsis cytisina (Berk.) Bond, et Sing. Polyporus caeruleoprus Peck Fomitopsis pinicola (Fr.) Karst. Polyporus dispansus Lloyd Trachyderma tsunodae (Yasuda) Imaz. Lenzites betulina Fr. Fomitopsis rosea (Fr.) Karst. Oxyporus ravidus (Fr.) Bond, et Sing. Hirschioporus fuscoviolaces (Fr.) Donk Coltricia perennis (Fr.) Murr. Favolus aiveolarius (Fr.) Quel. Grifola gigantea (Fr.) Pilät Tyromyces caesius (Fr.) Karst. Trametes sanguinea (Fr.) Lloyd Elfvingia applanata (Pers.) Karst.

12th

Continuation of table 9a

Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae

Hirschiopcrus abietinus (Fr.) Donk Tyromyces incarnatus Imazcki Amauroderma scopulosum (Berk.) Lmaz. Polyporus pes-caprae Fr. Microporusflabelliforrnis (Fr.) Kuntze Coriolus biformis (Klotz.) Pat. Fomes fomentarius (Fr.) Kickx Ganoderma neo-japonicum lmaz.

Example 10

1324 1-1 incubation bottles were filled with 200 ml of a nutrient liquid each, which had the following composition:

Glucose 40 g Corn steep liquor 20 g Sodium nitrite 3g Primary potassium phosphate 0.5 g Calcium carbonate 3g water 1 liter

The filled nutrient liquid was sterilized by heating for 30 minutes at 120 ° C. and then cooled to room temperature. The fluid was mixed with a mycelium from Ganoderma boninense Pat inoculated, which had previously been obtained after the stationary incubation (21 days at 20 to 23 ° C). To the Removal of the mycelium, the nutrient fluid was filtered after the incubation and the filtrate was concentrated to 101 at room temperature under reduced pressure. To the concentrate, 301 ethanol was added, and gradually Standing overnight in the refrigerator was filtered through filter paper the next morning. The residue was taken up the filter paper was washed with absolute ethanol and then with pure acetone and ether and then dried The dried residue was dissolved in one liter of distilled water and used for Removal of insoluble residue, filtered through No. 5C filter paper. The filtrate was mixed with 850 g of ammonium sulfate and stirred well, left to stand in the refrigerator overnight and the next morning centrifuged at 1500G. The residue was collected in one liter of saturated ammonium sulfate solution taken up and homogenized and centrifuged again at 1500G for 15 minutes. This treatment was repeated six times, and the residue finally obtained was dissolved in one liter of distilled water and filtered through No. 2 filter paper. The filtrate was successively on an activated Duolite C-27 and applied to an Amberlite IR-401 column, washed with 2 L of distilled water, the wash water was again added to the run-throughs, the run-throughs thus combined then being concentrated to 830 ml under reduced pressure and room temperature. The concentrate became 2490 ml pure Acetone was added, whereupon the mixture was left to stand overnight in the refrigerator and the next morning 15 Centrifuged at 1500G for minutes. The residue was taken up with 31% 75% acetone washed and then dissolved in 31% distilled water with constant water supply, the acetone stripped off under reduced pressure and the resulting solution was subjected to freeze-drying. It 36.7 g of a light gray to white powdery substance were obtained.

Table 10 shows the results of the tests for demonstrating the anti-carcinogenic effectiveness of the so obtained mucopolysaccharide, with the results for other mucopolysaccharides as well obtained from other species of Polyporaceae by the same procedure was.

Table 10 organism Elementary-
analysis
C H Yield
on
sugar
Base
(%) dose
mg / kg Effect effect
degree against degree against
solid cancer solid cancer
Sarcoma-180 vom Ehrlich-
(%) Type(%) 90
100
80 family Daedaleopsis tricolor
(Fr.) Bond, et Sing.
Ganoderma boninense Pat
Coriolus hirsutus
(Fr.) Source. 39 ^ 9 6.16
4137 6.13
42.48 5.68
Example U 030
0.51
0.17 300
300
300 90
100
90 Polyporaceae
Polyporaceae
Polyporaceae - ►

to

20 25 30 35 40 45 50 55 60 65

220 1-1 incubation bottles were each precipitated with 220 ml of a nutrient liquid that had the following composition:

13th

Peptone Glucose Water

10g 40g 1 liter

After filling of this medium, the so-called Sabouraud medium, the bottles were sealed with cotton plugs and heated for sterilization for 30 minutes at 12O ⁰ C. After cooling to room temperature, the nutrient fluid was inoculated with mycelium from Ganodenna iucidum (Fr.) Karst. The inoculation mycelium was previously prepared by stationary incubation (18 days at 24 to 26 ° C). To remove the My / cls, the incubated nutrient fluids were filtered through a cloth, at room temperature under reduced pressure

Concentrated to pressure to 3.31 and centrifuged at 1300G to remove remaining residues. After this Add 2460 g of ammonium sulfate to the supernatant liquid and then stir well the liquid was left to stand in the refrigerator overnight and at the next morning for 20 minutes Centrifuged 1500 G. The remaining residue was taken up, distributed homogeneously in 3.3 l of saturated ammonium sulfate solution and centrifuged at 1500 G for 20 minutes. After repeating this procedure eight times Initially, the residue finally obtained was dissolved in 31% of distilled water. The solution was to a Dowex 50W and placed on an Amberlite IR-401 ion exchange column, which were then washed with 3 l of distilled water. The wash water was added to the previously obtained runs. the pooled runs were then ultrafiltered. After washing with 301 distilled water it was the ultrafiltration carried out until the total volume of the liquid was only 1.1 l. The so obtained liquid was applied to a Duolite S-30 column, which with 4% hydrochloric acid and with 4% Soda solution had been activated. The column was washed with 31% of distilled water, with the wash water being added back to the run-through. It was then concentrated to 1.11 under reduced pressure at room temperature. After adding 3.31 ethanol, the concentrate was left to stand in the refrigerator overnight and centrifuged at 1500G for 15 minutes the next morning. The received

Precipitate was dissolved in 31% of distilled water and treated under reduced pressure at room temperature to remove the ethanol while constantly compensating for the loss of water. After freeze-drying the remaining liquid, 2.1 g of a light gray to white powder were obtained.

Table 11 shows the results of the tests for demonstrating the anti-carcinogenic effectiveness of the so obtained mucopolysaccharide, including the results for the mucopolysaccharides which have been obtained from other species of Polyporaceae by the same procedure.

Table 11

family organism Elementary H Yield dose Effect Effect analysis on mg / kg straight against straight against C. sugar solid cancer solid cancer 6.11 Base Sarcoma-180 from the honest (%) (%) Type(%) 40 Polyporaceae Coriolellus heteromorphus 41.85 5.42 0.26 300 80 70 (Fr.) Bond, et Sing. Polyporaceae - Coriolus conchifer 39.48 6.43 0.55 300 80 90 (Schw.) PaL Polyporaceae Fomiiopsis latissima 38.53 5.73 0.59 300 70 70 45 (Bros.) Imazeki, comb, nov. 6.45 Poiypöraceae Polyporeilus oiegans
(Fr.) KarsL
Cryptoporus volvatus 4033 0.56 300 60 60 Polyporaceae (PK.) Hubb. 38.14 6.16 0.35 300 80 90 Trametes albida 50 Polyporaceae (Fr.) BourdotetGalzin 39.94 5.21 0.64 300 60 70 Ganoderrna! Ueidum Poiyporaceac (Fr.) Karst. 42 _: 85 Example 12 0.16 300 80 70 55

205 1-1 incubation bottles were each filled with 180 ml of the so-called Mayer culture medium with the following composition:

Cane sugar Ammonium nitrite Primary potassium phosphate Magnesium sulfate heptahydrate Calcium phosphate

water

50g 10g 5g

1 liter

After sterilization under pressure (30 minutes at 120 ⁰ C) and cooling to room temperature, the medium with mycelia of Coriolus pargamenus (Fr.) PaL vaccinated was previously by a stationary incubation

14th

(At 25-27 "C for 20 days).

After filtering the nutrient liquid through a cloth, it was reduced at room temperature under reduced pressure Pressure reduced to 2.5 l. The concentrate was mixed with 2 kg of ammonium sulfate and then good touched. After standing overnight, centrifugation was carried out at 1500 G for 15 minutes the next morning. The residue was collected and dissolved in 3 liters of distilled water. The solution was activated on an Amberlite IR-200 column. The column was washed with one liter of distilled water, the wash water added to the run-through, and the combined run-throughs were imbibed after standing overnight Filtered through a filter paper in the refrigerator. The filtrate was applied to a Duolite A-6 column, with the The column was washed with 31% distilled water and the wash water was again added to the run-through became. The combined runs were reduced to one liter at room temperature under reduced pressure constricted. 20 g of lead acetate were added to the concentrated liquid with stirring. After the addition was stirred for another hour. After standing in the refrigerator overnight, she turned 30 the next morning Centrifuged at 3000 G for minutes. The residue was taken up and distilled in one liter Homogenized water. This treatment was then repeated for 30 minutes with 3000 G zeinrifiJ "i": i was repeated five times and the residue finally obtained was suspended in one liter of water Precipitate obtained after introducing hydrogen sulfide into the suspension for 30 minutes, was filtered off. The filtrate was placed on a sand bath in an evaporation dish and kept under constant pressure Addition of the evaporated water then evaporated to dryness after 10 hours. The dry residue was taken up in one liter of distilled water and filtered through No. 5C filter paper. That Filtrate was sequentially passed through a filtered Duolite C-25D, an Amberlite IRA-410 and onto a Duolite S-30 column. The columns were washed with one liter of distilled water and the wash water added to the runs. After concentration at room temperature under reduced pressure to a 3 liters of methanol were added to liters and the mixture was left to stand in the refrigerator overnight. The residue, which was separated by centrifugation (15 minutes at 1500 G), was then poured into one liter dissolved in distilled water. The methanol was removed under reduced pressure at room temperature and deducted constant water supply to the solution. The remaining liquid was subjected to freeze-drying. The yield was 8.0 g of a light gray to white powdery substance.

Table 12 shows the results of these tests demonstrating the anticarcinogenic efficacy of the so obtained mucopolysaccharide, the results for other mucopolysaccharides also being shown in The table was recorded using the same procedure as described above other species of Polyporaceae had been preserved.

Table 12 organism Elementary H Yield dose Effect Effect Type(%) family analysis on mg / kg straight against straight against 90 C. sugar solid cancer s solid cancer 5.99 Base Sarcoma-180 vom Ehrlich- 100 (%) (%) 60 Daedaleopsis nipponica 40.80 6.76 0.73 300 100 Polyporaceae Imazcki 5.04 90 Trametes dickinsii Berk 42.00 0.90 300 100 60 Polyporaceae - Coriolus pargamenus 39.46 6.79 0.18 300 60 Polyporaceae - (Fr.) Pat. 6.05 90 Tyromyces guttulatus
(Pk.) Murr.
Cantharellus floccosus 38.60 0.20 300 100 Polyporaceae Schw. 40% 5.59 0.46 300 60 Polyporaceae Polyporus tuberaster Fr. 39.19 0.78 300 80 Polyporaceae

Claims (5)

Patent claims: 1. Polysaccharide with anti-tumor activity, which under normal conditions is solid, amorphous, water-soluble, is not hygroscopic and not dialyzable and only leaves a stain on the anode cord during electrophoresis with a cellulose acetate membrane at 20-25 V / cm for 90 min in sodium borate solution, always shows a positive glucose reaction after hydrolysis with 1 η sulfuric acid, whereby the hydrolyzate of the polysaccharide gives neither the phenol reaction with iron (III) chloride nor the Fehling's reaction to reducing sugars, but positive for the anisaldehyde reaction, dk Molisch reaction, the anthrone reaction, the tryptophan-sulfuric acid reaction, the anilia reaction. Hydrochloric Acid Re- action, the resorcinol-hydrochloric acid reaction. the carbazole-cysteine-sulfuric acid reaction, the Tollens reaction and the thioglycolsulphuric acid reaction reacts, and which can be obtained from a liquid mycelium extract of a fungus of the Basiodiomycetes class or from a culture broth of this fungus, that a Basidiomycetes fungus from the Polyporaceae family is used, the polysaccharide rois the liquid extract obtained by extracting the mycelium with water or syr ^ ij aqueous solvents below 120 ⁰ C has been obtained, or precipitates from the concentrated culture broth by adding ammonium sulfate to the resulting precipitate redissolving in water. The aqueous solution is further purified in a manner known per se, using a combination of at least one dialysis and at least one Acid treatment or ultrafiltration or at least one ultrafiltration and / or at least one ion exchange subjects the solution thus obtained to isolate the polysaccharide. centered and the residue dries or the polysaccharide precipitates from the aqueous solution with a highly polar organic solvent and dries the precipitate formed 2. Polysaccharide according to claim 1 from a liquid extract obtained by extracting the mycelium of the fungus with hot water or a hot aqueous solvent at a temperature between 60 and 100 ° C has been received 3. A method for obtaining a polysaccharide with antitumor activity according to claim 1 from a liquid mycelial extract of a fungus of the Basidiomycetes class or from a culture broth of this fungus by precipitation and purification, characterized in that the Basidiomycetes fungus used is one from the Polyporaceae family, the polysaccharide from the liquid extract obtained by extracting the mycelium with water or an aqueous solvent has been obtained below 120 ⁰ C, or from the concentrated culture broth with ammonium sulphate precipitates, the resulting precipitate redissolves in water, the aqueous solution is further purified in a manner known per se, whereby it is a combination of at least one dialysis and at least one acidification or ultrafiltration or at least one ultrafiltration and / or at least one lonenau ¹ · <u "<. ch subjects, concentrating the thus obtained solution for the isolation of the polysaccharide and the Rücksiaru dries or the polysaccharide from the aqueous Solution with a strongly polar organic solvent is precipitated and the precipitate formed is dry 4. The method according to claim 3, characterized in that one uses a liquid extract by extraction of the mycelia of the fungus is obtained with hot water or a hot aqueous solvent at a temperature between 60 and 100 ⁰ C. 5. The method according to claim 3 or 4, characterized in that a pre-precipitation with ethanol is carried out before the precipitation with ammonium sulfate and the precipitate is dissolved in water and then falls with ammonium sulfate. 6. Use of the polysaccharide according to claim i as an active ingredient in Ar'.itumormittel.