SE446406B - NEW COMPOUNDS WITH CARCINOSTATIC AND IMMUNOSTIMULATING EFFECTS PRODUCED BY CULTIVATION OF FUSOBACTERIUM NUCLEATUM TF-031 (FERM 5077, ATCC 31647), PROCEDURE FOR THE PRODUCTION OF SAME AND CARCINOSTATIC - Google Patents

Description

. '446 -406 bacterium nucleatum 1010 (Dental Surgery, go, 322-333 (1974) and gl, 534-539 (1972) (Japanese)). However, no study has yet been conducted on the components obtained from a culture or its excess liquid produced by culturing bacteria belonging to the genus Fusobacterium and the pharmacological å Q activities obtained with these components.

The present inventors have examined the pharmacological activity of a supernatant obtained by culturing bacteria belonging to the genus Fusobacterium and removing the organism from the culture, and have thereby found that a particular component obtained from the supernatant has a carcinostatic effect; that said component has substantially no effect on inhibiting the formation of a colony of cancer cells in a colony foaming assay and does not have a carcinostatic activity by killing cancer cells but, an indirect carcinostatic activity by increasing the indirect host antitumor activity or immunity of the host and the use of assistance of immunity and that said component has very low toxicity and can also be obtained by treatment of the culture. An object of the present invention is to provide a process comprising culturing bacteria belonging to the genus Fusobacterium under anaerobic conditions and recovering the new TF substances with carcinostatic and immunostimulatory activities from the formed cultures or the excess liquid thereof. Another object of the present invention is to produce said TF substances.

A further object of the present invention is to provide a carcinostatic agent containing said TF-2 substances. Other objects and advantages of the present invention will be further elucidated in the following description. 4461406 u 3 As bacteria used in the present invention any TF substance producing bacteria belonging to the genus Fusobacterium can be used, for example Fusobacterium nucleatum is preferred, in particular Fusobacterium nucleatum TF-031 (FERM 5077; ATCC 31647) and the like are used.

The bacteriological properties of Fusobacterium nucleatum TF-031 are as follows: (I) Form (l) Cell shape: spider-shaped (Fig. 1) (2) Cell polymorphism: none (B) Motility: none (4) Spores: none (5) Gram stain: gram-negative (6) Acid resistance: negative (II) Plant conditions in a culture medium (l) TF-agar plate and oblique culture medium Outer shape: a round shape Size: about 1 mm Protuberance: hemispherical shape Structure: dewdrop-like Surface: even Edges: even g Color: milky yellowish-white Y Transparency: opaque (2) TF-a liquid culture medium Degree of growth: lively Grumlignota ooaqulum Precipitation: none Surface growth: none, no growth to a depth of about 5 mm Gas formation: none (III) Physiological properties (l) Formation of hydrogen sulfide: + (2) Reduction of nitrates: - (3) Formation of butyric acid: + (4) Formation of indole: + (5) Ureas: - _ 'ru-ale _ the. 'L »-a» en .m -. V-wwnwwl ..., 446: 406 (6) Catalase: - (7) Starch hydrolysis: - (8) Behavior in relation to oxygen: anaerobic L (9) Formation of ammonia: + § É J '(10) Formation of carbon dioxide: + (11) free-growing area; pH ss, s, temperature 30-4s ° c (12) Formation of gas from saccharides: L-arabinose (-), D-xylose (-), 3-glucose (-), D-mannose fw (-), D -fructose (-), D-galactose (-), malt sugar (-), * «* sucrose (-), trehalose (-), sorbitol (-), mannitol (-), inositol (-), glycerol (-) , starch (-).

The novel TF substances of the present invention are prepared, for example, as follows: Å,: hå 4415106 Culture of TF substance producing bacteria belonging to the genus Fusobacterium I Culture or its excess liquid Hydrophilic k organic solution | Gets Radiating agent 'Soluble fraction -' tion Phosphate buffer & sodium chloride] _ or water I Insoluble. fraction .Separation I | DryingL Water soluble xz $ Yi 'fraction _ _ \ * ~ g Dialysis or ultrafiltration ng o' ïë Drying Q. Internal solution ~ "'“ 3 TF-llo _ _ _ “~ ~ __ Treatment with: ïâzzïäifa fi | Phosphate buffer _ (sodium chloride) Desalination Kšššat Drying TF-l2O TF-130 (TF-1316, l32a, l32b, l33a, l33b, 1323, 136) 1 krrwn T .. \ ._ 446 406 6 Culture or its xl overflow U _ 2 Vattenlosllg x _ fraktion -> _ ff wo 1, f 'Produktion av et, Éf "Ißariumjonerl --9 bariumsalt Y * I Separation o-ål Lösning! l Barium salt Barium removal Separation --- eel Fasš | Solution \ 'Dialysis or ultrafiltration Concentrationl Drying x / TF-140 446 406 _ lW_ »7 Water soluble xz fraction Ä x3 _ L Internal, lDryingr-9 T§; llO solution,> 1 -å Treatment with one, f '* Soluble fraction! quaternary ammonium- K in salt I A quaternary ammonium complex Solution containing a sodium chloride fi Dissolution procedure "'" "'" '] /, "tvâ“ W Washing liquids y Soluble fraction Hydrophilic organic solvent Soluble fraction Separation Precipitation Ä Ü Drying TF-150 446 406 8 The above production process is illustrated as follows: (1) Cultivation of bacteria The cultivation of bacteria belonging to the genus Fusobacterium is carried out by a standard method for culturing anaerobic bacteria.

That is, a culture medium containing a source of nitrogen such as calf brain / heart extract, various peptones, or the like; a source of vitamin such as yeast extract or the like; an inorganic salt such as sodium chloride or the like; a carbon source such as glucose, lactose or the like; a reducing agent, such as L-cystine, sodium sulfite, thioglycollate or the like, its pH is adjusted to 6-8.5, preferably 7.2-8.2, and the bacteria are inoculated into the culture medium, after which a static culture is carried out during anaerobic conditions at 3s-42 ° c, preferably 3e-3s ° c, during ice days, preferably 24-72 hours. It is particularly desirable to use the culture medium described in Table 1 (hereinafter referred to as TF culture medium). The brain / heart infusion which is a calf-brain / heart extract is not always necessary as a carbon source and can be replaced with a heart infusion which is a veal heart extract, a beef extract, a fish extract; a corn steep liquor or the like. Among the various peptones, proteospeptone and phyton peptone are not always necessary and tryptic peptone can be replaced by polypeptone.

When agar is not used, it is desirable to stir the culture.

Table 1 Culture components (g / 1) TF-a TF-b TF-c Tryptic peptone 17 17 17 Phyton peptone 3 3 1,5 Proteospeptone 10 5 5 Brain / heart infusion 35 17,5 - Heart infusion - - 25 Yeast extract 3 3 3 Sodium chloride 7.5 7.5 7.5 Glucose 6 6 6 Lactose 5 5 5 L-cystine 0.25 0.5 0.5 446 406 9 Sodium sulphite 0.1 0.1 0.1 Thioglycolate 0.5 0.5 0.5 Agar O el. 0.7 O el. 0.7 O el. 0.7 (2) Collection of a supernatant from the culture (removal of the organisms) The organisms are removed from the culture obtained according to the above to obtain a supernatant. For removal of the organisms, a common method can be used, for example centrifugation and a filtration method using a filter aid, such as Hyflo Super Cel, and a centrifugation method is particularly preferred with regard to the degree to which the organisms are removed and the yield of the supernatant. .

The organisms are preferably removed in this step but can also be removed in the following step (3). (3) Collection of the carcinostatic substance TF-100 A hydrophilic organic solvent is added to the supernatant or culture obtained as above and the precipitate formed is collected. At this time, the pH of the supernatant or culture is preferably adjusted to 2-7. The hydrophilic organic solvent includes, for example, alcohols such as ethanol, methanol and the like, and ketones such as acetone and the like, although alcohols, especially ethanol, give the best results. The hydrophilic organic solvent is suitably added in concentrations of 30-70% by volume, preferably 50-70% by volume. After the addition of the hydrophilic organic solvent, the resulting mixture is allowed to stand at a low temperature, preferably at about 5 ° C, for several hours to several days to complete the precipitation.

The precipitate thus obtained is separated by conventional methods such as decantation, centrifugation, filtration and the like.

Then water is added in an amount of 10 to 15 times the amount of the precipitate obtained as above (the water can be replaced - «-« - «- 'I% Il -.-! - fw- 1 i _ W, -446' 406. IC of a phosphate buffer or a phosphate buffer containing sodium chloride when it is desired to obtain the substances TF-120 or 130 (1316, 132a, 132b, 133a, 133b, 1323 or 136) as indicated below) and the formed water-insoluble substances are removed with common methods, such as centrifugation, filtration or the like, after which the water-soluble fraction is collected. When the culture is used, the organisms are removed by the above methods. The water-soluble fraction thus obtained is dried by lyophilization or the like to obtain a carcinostatic substance TF-100.

TF-100 has the properties shown in Table 2. (4) Collection of the carcinostatic substance TF-110 The water-soluble fraction obtained under (3) above is subjected to dialysis or ultrafiltration; or alternatively the substance TF-10O is dissolved in water in an amount which is 10-15 times the amount of the substance TF-100 and the resulting solution is subjected to dialysis or ultrafiltration. Low molecular weight substances such as amino acids and inorganic substances are removed by the above treatment. The internal solution obtained by dialysis or ultrafiltration is collected and dried to obtain a carcinostatic substance TF-110.

The substance TF-110 thus obtained has the properties shown in Table 2. (5) Collection of the carcinostatic substance TF-120 The water-soluble fraction obtained according to the method described in (3), or, if necessary, the internal solution the solution obtained by subjecting the water-soluble fraction to dialysis or ultrafiltration (the internal solution obtained according to the method described in (4)), or a solution of a powder of the substance TF-110 in a small amount of water is treated with an ion exchanger. As the ion exchanger a weakly basic ion exchanger is used or preferably a weakly basic ion exchanger with a molecular visibility is used and suitably diethylaminoethyl-Sephadex A-SO (registered trademark 44a> 4osa ~ \ f; ll Å by Pharmacia Co., Ltd.) is used. A column packed with an ion exchanger is balanced, for example, with a phosphate buffer having a pH of about 8, after which the above solution to be treated is passed through the column and the eluate is collected and dried by lyophilization or the like; or alternatively the above buffer and the solution to be treated are placed in a vessel containing an ion exchanger and the contents are stirred and then filtered, after which the filtrate is dried; wherein a carcinostatic substance TF-120 having the properties described below can be obtained. The substance TF-120 can also be obtained by desalting the eluate or filtrate, for example by dialysis using a cellophane tube or the like, by using Sephadex G-25 (registered trademark of Pharmacia Co., Ltd.) or by ultrafiltration and then drying of the same. qß u; The substance TF-120 thus obtained has the properties shown in Table 2. (6) Collection of the carcinostatic substance TF-130 A column containing the adsorbed components from which the substance TF-120 has been removed by treatment by the method described in (5) is treated with a phosphate buffer, preferably a phosphate buffer with a sodium chloride concentration of 0 , 1-0.6 M and a pH of about 8 (hereinafter the phosphate buffer with a sodium chloride concentration of 0.1-0.6 M is indicated by the following designation: a 0.1-0.6 M sodium chloride-phosphate buffer); or alternatively, the water-soluble fraction obtained by the method described under (3) is subjected, if necessary, to dialysis or ultrafiltration and then to treatment with an ion exchanger using a 0,1-0,6M sodium chloride-phosphate buffer to obtain of a fraction which is not eluted with a phosphate buffer free of sodium chloride but eluted with a 0,1-0,6M sodium chloride-phosphate buffer.

This procedure can be performed with either a column system or a batch system. The thus obtained desired fraction eluted with the sodium chloride-phosphate buffer is dried by 446 "lyophilization or the like to obtain a carcinostatic substance TF-130. The eluate containing the desired fraction eluted with said sodium chloride buffered phosphate 3% is desalted, for example by dialysis using a cello-fantub, by molecular sieving using Sephadex G-25, or by ultrafiltration and then drying.

The term "TF-130" as used herein collectively refers to the desired eluate fractions obtained by treating the water-soluble fraction obtained by the method described in (3) or, if necessary, the internal solution 9% 4, the component obtained. by subjecting the water-soluble fraction to dialysis or ultrafiltration with an ion exchanger, which fractions were not eluted with a phosphate buffer free of sodium chloride but eluted with a 0.1-0.6M sodium chloride-phosphate buffer. fractions. (i) A fraction which has not been eluted with a phosphate buffer free of sodium chloride but has been eluted with a 0,6M sodium chloride-phosphate buffer in the above ion exchange treatment l's (the fraction is designated TF ~ 136). fraction which has not been eluted with a 0.1 M sodium chloride phosphate buffer but has been eluted with a 0.2 M sodium chloride phosphate buffer in the above ion exchange treatment (the fraction is designated TF-132a and b; TF-132a is obtained by washing an ion exchanger containing the aforementioned adsorbed component with a 0.1 M sodium chloride phosphate buffer, treating the ion exchanger with a 0.2 M sodium chloride phosphate buffer and collecting the eluted fraction with 0.2 M sodium chloride. -phosphate buffer; and TF-132b is obtained by treating the water-soluble fraction obtained by the method described in (3) or the internal solution component (TF-110) obtained by the method described in (4) with an ion exchanger). (Iii) A fraction which has not been eluted with a 0.2M sodium chloride-phosphate buffer but has been eluted with a 0.3M sodium chloride-phosphate buffer in the above ion exchange treatment (fraction designated TF-133a and b: a and b have the same meaning as described above for substances TF-132a and b). (iv) A fraction which has not been eluted with a 0.1M sodium chloride phosphate buffer but has been eluted with a 0.3M sodium chloride phosphate buffer (v) A fraction which has not been eluted with a 0,5M sodium chloride phosphate buffer but has been eluted with a 0,6M sodium chloride phosphate buffer in the above ionic ion treatment (fraction is designated TF-1323). -136).

Since these substances TF-1316, 132a, 132b, 133a, 133b, 1323 and 136 have common properties in the following respects, the substances having the usual properties are referred to as TF-130.

Ie. the substance TF-130 obtained in the above manner has the following properties: (a) Off-white to light brown powder (b) It prevents the growth of Ehrlich ascites tumor, Ehrlich solid tumor and Sarcom.-180 carcinoma in mice and has a immunostimulatory effect. (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. (d) It has no clear melting point, it begins to decompose at about 110 ° C and decomposes appreciably above 200 ° C. (e) Its infrared absorption spectrum obtained by a KB: tablet method has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 140-1000 and 820 cm -1. (f) The ultraviolet absorption spectrum of its aqueous solution shows a strong absorption at the absorption edge and shows an absorption peak in the vicinity of 256-280 nm.

Sx fl rorna morn nalcnlrs .anger min f "" WW "" "" "'°"' "d ° k“ m '"" * ° d fnnhonøll uiunhhenngnkoxl, lNlD-kud Bukshw mom kl-HHIH 446 406 14 (g) Den har a positive Molisch reaction, phenol-sulfuric acid reaction, anthron-sulfuric acid reaction, indole-hydrochloric acid reaction and Lowry-Folins reaction and a negative ninhydrin reaction. (h) Elemental analysis values: C: 27.5-39.8% H: 3.2-5.7% N: 2.8-6.3% (i) Its saccharide content measured by a phenol-sulfuric acid method is approx. 19.0 to about 64.0% by weight (expressed as glucose) and its protein content measured by the Lowry-Folin method is in the vicinity of 6.0-28.0% by weight (expressed as calf serum albumin).

The above ion exchange treatment is described in particular in the following. As the ion exchanger used in the following processes, a weakly basic ion exchanger or a weakly basic ion exchanger with molecular visibility is preferred, and suitably, for example, diethylaminoethyl-Sephadex A-50 used in the method described under (5) is used, and the following method can be carried out according to (i) A 0.6M sodium chloride-phosphate buffer having a pH of preferably 8 is passed through a column with an ion exchanger containing the adsorbed component, the solution to be treated has been passed by the method described in (5) and the eluted fraction is collected. , optionally concentrated and desalted by dialysis, ultrafiltration or the like, and then dried by lyophilization or the like to obtain a carcinostatic substance TF-136.

The substance TF-1316 thus obtained has the properties shown in Table 2. (ii) -1) A 0.1M sodium chloride-phosphate buffer with a pH of preferably 8 is allowed to pass through a column with an ion exchanger containing the adsorbed component. through which the solution to be treated has been passed according to the method described under (5), the column is washed, after which a 0.2M sodium chloride phosphate buffer with a pH of preferably about 8 is allowed to pass through the column and the eluted fraction is collected , optionally concentrated and desalted by dialysis, ultrafiltration or the like and then dried by lyophilization or the like to obtain a carcinostatic substance TF-132a.

The substance TF-132a thus obtained has the properties shown in Table 2. (ii) -2) Methods for collecting the fraction which has not been eluted with a 0.1M sodium chloride-phosphate buffer but has been eluted with a 0.2M sodium chloride-phosphate buffer at the ion exchange treatment is carried out in the following manner using the water-soluble fraction obtained by the method described in (3), or the internal solution of the method described in (4) to obtain the substance TF-132b. Ie. the column packed with ion exchanger has been equilibrated with a 0.2-0.3M sodium chloride-phosphate buffer having a pH of preferably about 8, after which the previously mentioned 0.2-0.3M sodium chloride-phosphate buffer of the water-soluble fraction is obtained. by the method described in (3) or the internal solution component, obtained by subjecting the water-soluble fraction to dialysis or ultrafiltration, passing through the column and collecting the eluted solution. If required, the column is washed with the aforementioned 0.2-0.3M sodium chloride-phosphate buffer and the eluted solution and washings are combined. The ion exchange treatment can be performed several times. Thereafter, a solution prepared by diluting the aforementioned eluted solution with a phosphate buffer so that the sodium chloride concentration becomes 0.1 M is passed through a column in which the ion exchanger has been equilibrated with a 0.1 M sodium chloride phosphate buffer having a pH preferably at about 8 and the fraction eluted with the aforementioned 0.1M sodium chloride-phosphate buffer is removed, after which the 0.2M sodium chloride-phosphate buffer is passed through the column to obtain an eluate. In the concrete examples of the above ion exchange treatment, a fraction which has been adsorbed on an ion exchanger equilibrated with the 0.2 M sodium chloride phosphate buffer is collected, adsorbed on an ion exchanger equilibrated with the 0.1 M sodium chloride-phosphate buffer, washed with said 0.1M sodium chloride-phosphate buffer and then eluted with said 0.2M sodium chloride-phosphate buffer, to obtain an eluate. A method can also be used in which, conversely, the fraction which has been adsorbed on an ion exchanger equilibrated with said 0.1M sodium chloride-phosphate buffer is collected and then a process is performed for separating said fraction from a fraction which has been adsorbed on an ion exchanger equilibrated with said 0.2M sodium chloride phosphate buffer and a fraction eluted with said 0.2M sodium chloride phosphate buffer is collected.

The eluate obtained in the above-mentioned manner is optionally concentrated and desalted by dialysis, ultrafiltration or the like and then dried by lyophilization or the like to obtain a carcinostatic substance TF-132b.

The substance TF-132b thus obtained has the properties given in Table 2. (iii) -1) The internal solution component obtained by the method described in (4) is passed through a column with an ion exchanger equilibrated with a phosphate buffer. having a pH preferably at about 8 and a 0.2M sodium chloride-phosphate buffer having a pH preferably at about 8 is passed through said column for washing the column and then a 0.3M sodium chloride-phosphate buffer having a pH of about 8 passes through said column or alternatively, said 0.3M sodium chloride phosphate buffer is allowed to pass through the column which has been treated according to the method described under (ii) -1) and still has a residual adsorbed component.

The eluted fraction is collected, optionally concentrated and desalted by dialysis, ultrafiltration or the like, and then dried by lyophilization or the like to obtain a carcinostatic substance TF-133a.

The substance TF-133a thus obtained has the properties given in Table 2. (iii) -2) The fraction which has not been eluted with a 0.2M sodium chloride-folphate buffer but has been eluted with a 0.3M sodium chloride. riding phosphate buffer in the ion exchange treatment is collected in the following manner, which is the same as described above under (ii) -2) to form TF-133b. Ie. the column packed with ion exchanger and equilibrated with a 0.3M sodium chloride-phosphate buffer having a pH of preferably about 8, then said 0.3M sodium chloride-phosphate buffer of the water-soluble fraction obtained with the sub (3 ) described method, or the internal solution component obtained by subjecting the water-soluble fraction to dialysis or ultrafiltration, is allowed to pass through the column and the eluted solution is collected. If necessary, the column is washed with the 0.3 M sodium chloride phosphate buffer and the eluted solution and washings are combined. The ion exchange treatment can be performed several times. Thereafter, a solution prepared by diluting said eluted solution with a phosphate buffer so that the sodium chloride concentration becomes 0.2 M is passed through a column in which the ion exchanger has been equilibrated with a 0.2 M sodium chloride phosphate buffer having a pH of preferably about 8, and the fraction eluted with said 0.2M sodium chloride phosphate buffer is removed, after which said 0.3M sodium chloride phosphate buffer is passed through the column to obtain an eluate. In the concrete example of the above-mentioned ion exchange treatment, a fraction which has not been adsorbed is collected on an ion exchanger equilibrated with said 0.3M sodium chloride phosphate buffer, adsorbed on an ion exchanger equilibrated with said 0.2 M sodium chloride-phosphate buffer, washed with said 0.2M sodium chloride-phosphate buffer and then eluted with said 0.3M sodium chloride-phosphate buffer to obtain an eluate. A method can also be used in which, conversely, a fraction which has been absorbed on an ion exchanger which has been equilibrated with said 0.2M sodium chloride-phosphate buffer is collected and then a process is carried out for separating said fraction from a fraction which has been adsorbed on a ion exchangers which have been equilibrated with said 0.3M sodium chloride-phosphate buffer and a fraction which has been eluted with said 0.3M sodium chloride-phosphate buffer are collected. The eluate obtained as described above is then optionally concentrated and desalted by dialysis, ultrafiltration or the like and then dried by lyophilization or the like to obtain the desired carcinostatic substance TF-133b.

The substance TF-13b thus obtained has the properties shown in Table 2. (iv) The water-soluble fraction obtained by the method described in (3) or the internal solution component obtained by the method described in (4) is introduced into a .3M sodium chloride-phosphate buffer having a pH of preferably about 8 and the resulting buffer is passed through an ion exchange column previously equilibrated with a .3M sodium chloride-phosphate buffer having a pH of preferably about 8. The eluted solution is adjusted by adding a phosphate buffer so that the sodium chloride concentration becomes 0.1M and then allowed to pass through an ion exchange column which has previously been equilibrated with a 0.1M sodium chloride, phosphate buffer with a pH of preferably about 8 and then said 0, 3M sodium chloride-phosphate buffer pass through said column, after which the eluted fraction is collected, optionally concentrated and desalted by dialysis, ultrafiltration or similar and then dried by lyophilization or the like to obtain a carcinostatic substance TF_13330. The substance TF-1323 thus obtained has the properties shown in Table 2.

(V) The internal solution component obtained by the method described in (4) is passed through a column with an ion exchanger equilibrated with a phosphate buffer having a pH of preferably about 8, and an unsaturated sodium chloride phosphate buffer having a pH of preferably about 8 is allowed to pass through said column for washing the column and then a 0.6M sodium chloride phosphate buffer having a pH of preferably about 8 is passed through said column, or alter- wm-: pwu .vm »; . . .nw-w. 446. 406; Natively, said 0.5M sodium chloride-phosphate buffer is allowed to pass through the column treated as described in the procedure described in (ii) -2) and still has residual absorbed component, after which said 0.6M sodium chloride-phosphate buffer is passed through said column. The eluted fraction is collected, optionally concentrated and desalted by dialysis, ultrafiltration or the like, and then dried by lyophilization or the like to obtain a carcinostatic substance TF-136.

The substance TF-136 thus obtained has the properties shown in Table 2. (7) Collection of the carcinostatic substance TF-14 Barium ions additive culture or its supernatant obtained by the method E fl w fi m fi or to (2) or (2) above the water-soluble fraction, obtained by the method described in (3) above or to the solution obtained by dissolving TF-100 in water, to form a barium salt, after which the precipitate is collected and then subjected to a process for removing barium and the water-soluble fraction is collected and then subjected to dialysis or ultrafiltration to obtain the substance TF-140.

In particular, an aqueous barium hydroxide solution is described, preferably a 0.1-0.5M aqueous barium hydroxide solution is added to the culture or its excess liquid E obtained by the method described under (1) or (2) or to the water-soluble fraction obtained with the under ( 3) described method or to an aqueous solution obtained by dissolving TF-100 in water in an amount of about 10 -15 times the amount of TF-100 and the pH of the resulting mixture is preferably adjusted to 10-13 for formation. of a barium salt. A precipitate is deposited by adding a barium hydroxide solution and the added amount of the barium ions is preferably adjusted so that the concentration of the total barium ions in the final volume of the mixture becomes 0.005-0.1 M. The mixture thus obtained is filtered and a process for separating barium from the obtained 44s-40s-20 barium salt is carried out. Barium is preferably removed by adding barium salt to sodium sulfate, preferably a 5-15% aqueous sodium sulfate solution, the resulting mixture is stirred and then the resulting solution is filtered. The precipitate which has been separated and removed is washed again with an aqueous sodium sulphate solution and the washing liquids are combined with the above-mentioned solution. The resulting solution is subjected to dialysis, ultrafiltration or the like, to remove excess sodium sulfate or low molecular weight substances, and the resulting solution is then dried by lyophilization or the like to obtain a carcinostatic substance TF-140.

The substance TF-140 thus obtained has the properties shown in Table 2. (8) Collection of the carcinostatic substance TF-150 The water-soluble fraction obtained by the method described in (3) above or the internal solution obtained by the method under (3) 4) the method described above is treated with a quaternary ammonium salt and the precipitate formed is collected and subjected to a dissolution process using a solution containing sodium chloride, after which a hydrophilic organic solvent is added to the soluble fraction and the precipitate thus formed is collected and dried to obtain the substance -150. As the quaternary ammonium salt which can be used may be mentioned any complex with a polysaccharide and especially preferred are cetyl-pyridinium chloride, cetyl-trimethylammonium bromide, etc. The treatment with a quaternary ammonium salt is preferably carried out in the presence of a buffer, such as a borate buffer or similar.

The concrete description of the above treatment with a quaternary ammonium salt and the dissociation process using a solution containing sodium chloride are as follows.

For example, an aqueous solution of a quaternary ammonium salt is added dropwise to the water-soluble fraction obtained by the method described under (3) or the internal solution obtained by the lv ~~, ¿; w, ¿~ gnl described in (4). ¥ 1lñv ».,. N,. _ m ,, V,: \., \ »__,. The method, while maintaining a slightly basic pH and the resulting mixture is stirred at 0-50 ° C for 10 minutes to several hours, after which the precipitate is collected. The precipitate is washed several times with a buffer, preferably a 0.1% borate buffer containing 0.1 M sodium chloride and a small amount of a quaternary ammonium salt and then dissociated with a buffer, preferably a 0.1% borate buffer, containing 0 , 5M sodium chloride and a small amount of quaternary ammonium salt to form a soluble fraction and the pH of the soluble fraction is then adjusted to 2-6. A hydrophilic organic solvent, preferably an alcohol, is then added to the soluble fraction so that its concentration finally becomes SO-90% by volume, preferably 70-90% by volume, and the mixture thus obtained is allowed to stand at 0-30 ° C for several hours to 90 hours, after which the precipitate is collected to form a carcinostatic substance TF-150.

The substance TF-150 thus obtained has the properties shown in Table 2. g, ¿J;,; ¿,, * än ._. _1L., ... ".ixïê fl ii 1 iir ~, w¿i I fi alrtl) 446 406 '22 umuw fi muww fl 500 umuwow fi mum .ënomouo fl x .fl wmcwn .coumom .HOS | muw .fi ocmuwš fl m fifi wæ fl ø> oo = w» » uwsm fifl MOQ cmxuw> wwcmn Im al ON fi umøsøëåw cm others mn zoo mmßñ m0; oc f unmu Oman EOoumm: oo UMS fl u »mmm SUW fi ußm .uwñ fl u mmuwumm now al nußm> m øwwxwb fififl n wwmuøc flfi co al uxmnw mother: MM | nw> wwcmuw al SS al umomøšñw cm www Que MMW fi mon uæñøu MWSS al iff so fifi unm> m GwuXw> HH f # W FI MUW flfifi: O fl uxmHm m flfl WA CmMHw> m © GmH0A5E fi umOG5EEw Gm mums: oo WMMS mon umëøv ummu so flfi Iunm: oO Hmšßu mmu al iff Nü fifl n f m> m muXw> Ha fl u wømuøcwc fl o f uxmnu mccwn cmxuw> xmwmoaoxmënmm N HHGDMH Hw> H5m u al SNN umm> m | u fl> WHM wwQmwmuD Amm fi lh fi mmm fl lh fi QNMHIMH mmm fl lmñ w fi m fl nma Q ONHIMH O fifl lmñ OOH | mH W; ||| mmMw @ mm4 vv ~ Hwmmxmcwmm .m f. \ | www-m 44% »40H» 40.6 > wucmu w flfl ë fi umocn fi ëw Gm mums S00 mwmE mon nnE fl u mwuwomm: U fl aunm> m: fi0 UXW> HHH ¥ ÜÜNHUGHS GÖHMXMHM MCGNQ OWHIHB E: WMHIMH ..

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+ + + + + I nwm fi nma .w A + + + + + .. måm fl àa, 2. w .q 3 + + + + + a 221.2. + + + + + 1 37.2. , + + + + + | 27mm. fl + + + + + + oo fl nmu. _ 0 Go fi uxmwu: O fl axmwu v Nco fi uxmwn .v O fl uxmwu fi owuxmwu: O fi uxmwu w å. Nc fi omnä fi son: Ümåoøcn .. om mxcouucm | ow måocwm unom flfi o: .š fl numnc fl z u .: O fi uxmwumumh nwmmxm fi mmm W, 4. Tw fi oä N Swnma. w 6 nu w 6 w 3 3 I r M +. + + + + | om fi nma Y +. + + + + | oï fi ä. + 1. ” + + + + | wm fi um fi%. * ._ Tm fi eyes N Swnm s - «: xJ oå fl mo 13» 05 mo o.om wo .S3 0.2 mo wmTma MW o. ~ H mo flfifl v o.H fi mo om ~ nu H fifl w o.mH nu m ~ m «| ma Y,. v m_- mo H flfl u m.- mo m. «~ mo H fifi v o.mH wo nmm fi ama w. o.æ ~ mv H flfi p o.- wo o_mm mo H flfl »ow ~ mo mmmH | ma o_w ~ nu Hñ fl v m.mH mo mm« mo H flfi u wm ~ wo n ~ m fi | ma W ow ~ mo fififi p c.wH mo o . «W mo H fifi u o_mm mo m ~ mH« ma xn om ~ wo H fifi u o.oH mo o_om mo HHH »o_mm wo w fl m fi lma o.mH mo H fifi u om nu o_m> nu HHM» o_wm mo ow fi ama 1 au o.- nu H fifi u ow ~ fl u o.mw mo H fifi v o_om mu o fifi uma 0. nn om ~ mo H fifl u o_o ~ mv ow «mo fififi u oo« mo ooH | ma zu «NLW 1 ^ wv øo» wE | om m _ o fl uxmum 1 \ 4 SL wOuwñ ÉÃHPHTMHBOA mvmš »uwä Luucwm MQ: uumñ .moxø fi m> m fr.,, ~ GMEDQHGEDHMW> HMM P0. EHOM fl HHWSQCCMCMNMOHN EHOM ... H .HHWÄÜGCHÜHHMMOMW QQMMWCÜWN _. ^ .muuowv N H fl wnma w á .Ill 4426 * 405 35 .MW O.vH m0 HHMM O ~ ß M0 O. ~ m mo Hawa O.mN nu ^ .mvHOmv N AHOQMB 0.3 .B 33 o_o «mo om ~ mo H fifl u om mo Om fi lhñ Ov fi im fi -_. e ...___ 446 406 36 In Table 2, for Sephadex G-50 mä ”dzned“ x ”a 55 mmø x 600 mm column and distilled water were used as eluent; for sephadex G-200 (registered trademark of Pharmacia Co., Ltd.) marked "xx", a 44 mm x 500 mm column was used for TF-110, 120, 32a, 133a, 136 and 140 and a column with 21 mm x 400 mm for TF-1316, 13b, 13b, 1323 and 150, and as eluent a 0.1 M phosphate buffer with a pH of 7 was used for all the compounds and to obtain the HPL chromatogram "xxx". - 2 columns were turned with 7.9 mm x 600 mm below TSK-GEL G30OSW (trademark of Toyo Soda Co., Ltd.) and the elution was carried out at room temperature at a flow rate of 0.8 ml / min. using 0.1M phosphate buffer with a pH of 7 as eluent.

The pharmacological activities of the present carcinostatic substances TF-100, TF-110, TF ~ 120, TF-1316, TF-132a, TF-132b, TF-133a, TF-13b, TF-1323, TF-140 and TF -150 is as follows: (I) Effect of TF substances on cell viability (Colony-forming Assay) Based on the method of Kim. J.H. et al. (Cancer Res. Go, 698 (1965)), HeLa S-3 cells were grown in Eagle's MEM supplemented with 10% calf serum for 3-5 days at 37 ° C, CO 2 incubator, after which the culture was removed and a PBS (-) solution (an aqueous solution containing 8.0 g / l sodium chloride, 0.2 g / l potassium chloride, 1.5 g / l sodium phosphate, monobasic and 0.2 g / l potassium phosphate, dibasic) containing 0,01% by weight ethylenediaminetetraacetic acid and 0.1% by weight of trypsin were added to the glass adhering cells. The cells were withdrawn from the glass surface of the culture flask and dissolved into individual cells by pipetting, and then the cells were counted under a microscope. The cell suspension was diluted with Eagle's MEM supplemented with 20% calf serum so that it could contain 200 cells per ml. After 1 ml of the cell suspension was inoculated into Petri dishes and grown in a CO 2 incubator at 37 ° C for 24 hours, the supernatant obtained according to Example 1 (1) below was added to the cell suspension so ß 4 IL ¿ , .___. x “l 41l6 * '4 {l6 37 that the concentration became 1/16 to l / 128. To each 1 ml portion of the cell suspension cultured at 37 ° C for 24 hours was added each of the test substances and the HeLa cells were cultured for 7 days. After culturing, the culture medium was removed, after which the culture dish was washed with Hanks solution and the cells were fixed with 70% by weight ethanol and then washed with 100% by weight ethanol. After the ethanol was removed, the cells were stained with a "Gimsa" staining solution, after which the colonies were counted and the cell viability was calculated.

The results are shown in Tables 3 and 4. The ceuivšdug figure in the tables was calculated using the following equation. The cell viability of each test substance is an average of 5 experiments.

Number of colonies in a treated Cell viability = group X 190 Number of colonies in an untreated QIUPP: L j, fw oo fi oo fi oo fi HHo »» aox o \ oo wH \ fi ooo ~ m \ H wm m.ß o.om «@ \ ~ «_Ow« _wo o.mß w ~ H \ H uwse fi u om Hmee fi »Nß fl mae fi u fi wo» w: w fi Hm: om> «HHwo 446 '406 ^ HE \ Ha. mn fl sowmmuo m fifl w fl wñ \: muwnm flfl møwmš flfifl wo mm uxwwmw mcmxmuw> wøcmuä fi wuïfw: m0 Uv-Wfm ... ,,. 1 ,,, 39 Taßell 4 Effect of TF substances on cell viability Substance Concentration Cell viability Mig / ml) _ (%) TF-100 1.0 99.4 50 97.4 100 95.9 TF-110 10 94.5 50 88.9 100 '87.3 TF-120 10 98.8 50' 91.2 100 8 83.3 TF-132 10 96.7 5 97.4 100 '95.9' rr-lasa lo \ 96.9 50 95, 4,100 88.8 TF-136 10 95.9 50 96.4 100 95.4 TF-140 10 98.5 50 98.6 100 98.6 200 96.8 500 93.4 .vw 446 406 40 as shown of these experiments, the TF substances of the invention have very low cytoxidal effect on HeLa cells compared to the supernatant. {Üä ~ (II) Immunostimulatory effect _ ag; Four mice of the ICR breed were used for each group. Each m * ü of the test substances was dissolved in 0.2 ml of a physiological saline solution and then administered intraperitoneally to the mice. 24 hours after administration, 0.2 ml of a carbon suspension prepared by mixing 1 ml of Perican Drawing Ink 17 Black (manufactured by Günther-Wagner Co., Ltd.) and 2 ml of a physiological saline solution containing 3% by weight of gelatin were injected intravenously into mouse tail and 1.5, 10 and 15 minutes after the injection, 0.02 ml of blood was collected from the banana using a hematocrit capillary coated with heparin and diluted and hemolyzed immediately with 1.6 ml of a 0.1% by weight aqueous sodium carbonate. was subject to colorimetric night solution. determination at 675 nm and the phagocytotic index, i.e.

The K value, was determined by Halperns et al.

In the mice in the control group, 0.2 ml of a physiological saline solution was administered. log Co - log C K: t-to where Co = the carbon powder content of the blood at time t and C = the carbon powder content of the blood at time t.

The results are shown in Tables 5 to 7. »_- V .k fine, H L ---- il * -> 44sf4o6 41 Table s Dose The average of the K-values Substance (mg / Kg) TF_l0o 10 o.o85l-1 o ¿O1u5 50 'o.o6o5 = o.o3o2 TF_ll0 5 0.o9o1: o.o213 20 o.1o25: o.oo25 TF_l¿o 5 o.o718 1 o.o152 20 o.0559 1 o.o197 dm fl ü ao o .1oo1 1 o.o2u6 ïå TF_l32a 5 o.11o2 1 o.o2o3 Éå 20 o.o8a9: o; o296 L fl w fl n TF_l33a 5_ o.o851 2 0.005? 20 0.o769 1 o.o17u 1222136 5 o.o959 i o.ooa1 20 0.102? r. 0.0l5¿l K ° ntr ° 1l - o.o3oo 5 o.oo2u Table 6% ¿“_ f Substance Dose 'The average of the K-hosts' ams / Ks) 20 o.1o73 s o.oo31 TF_l33b 5 o .o55o = o.oo2o ni 20 o.1o99: 0.oo75 g yi TF_l5O 5 o.1399 = o.oo11 ¿§ ao 9.057? 2 o.oo98 *% TF_l323 5 o.1uoo: c.oo75 “20 o.o685: o.o126 Küntroll _ J”. v .. fw WH ”'"' 'z 4 446 406 42 Table 7 Substance Dose _ The average of the K-values (ms / Ks) l 0.089l: 0.0l56 4 W gg TF-140 5 o.o9u6 x o.o139 6 Ü ia 20 o.o815 1 o.o2oo 1 Q 'à K ° fl tr ° 1l - o.o379 = o.oou8 * M i As can be seen from Tables 5 to 7, the groups to * which each of the TF substances according to the invention, activation of reticuloendothelial macrophage IÄ 4: 1 Vw * had been administered compared to the control group and the cellular immunity of the normal mouse was increased.

(IIl) Carcinostatic activity (a) Antitumor activity against Ehrlich ascites tumor Ehrlich ascites tumor cells were inoculated intraperitoneally into mice of the ICR strain (females, 6 weeks old) in an amount of 1 x 10 5 cells per mouse. Thereafter, each of the test substances was dissolved in 0.2 ml of a physiological saline solution and then administered intraperitoneally to the mice once daily for 7 days from the first day after the inoculation of the tumor cells, or once daily on the first day and from the third day to the seventh day for a total of 6 days. In the case of the test substances in Table 9, each of them was administered once daily on the first day and from the third day to the seventh day for a total of 6 days after the inoculation of the tumor cells. 0.2 ml of a physiological saline solution was administered to the control group in the same manner as above. 14 The results are shown in Tables 8 and 9.

T / C = Number of survival days for mice in a group On which the substance was administered x 100 (%) Number of survival days for mice in the control group l!:! IJ .ßv now in .Of of 43 w \ c oo fl ~ .m H N .> HI m \ m NNHA æ.mw w.mN ^ mx oa H * @ \ 2 @ ~ @ H æuæNÅ © vn @ w \ m = mH ^ ww H m.æN ^ mx fi. m \ o co fi N.w M m.> fl | w \ H mmH ^ æ.æ «mm ~ ^ wx OH fi ß m \ 0 wo fi ~. = M m.wH W xm mmm fi ha w \ H, HWHA æ.> u æ.om ^ wxa: \ o oo fi wm fi æ.> HI Hm = \ N mm fi ^ wm H m.> m ^ mx om om fi nma _ = \ N = mH ^ mm «m .- ^ mx ø fl m \ o oo fi mH H æ.wH IH * m \ m mmH ^ m. ~ ü m. ~ m ^ wx om oHH | @am \ N m = fi ^ o.oH H o. = ~ ^ mx ca Hm Qm \ o oo fi mH M w.æH 1 oo fl nma W om \ m Nwf fw fi fo? N x S. .IJ -Aw-xq »p _. ~ ~ WW», »» ¿«; .-; ~ @ u ~ 44 446 406 mGHwHHwUHmE: u> m cm: OHumvnmHmwQmuv Hwuww nwmmw wuøw: mv mm cwmGHcEß fl wm Nx mGmHHH : mHmmGmu # Hwvum cwmmw w fl mm: mv mm: wmGHCE @ fi mm Hx wwmumuøx uHws um>: oo mwuH0wm cwmcw mums wU: m> wHum> m mHH <x ".¿d4 QH \ o ooH HH H \. ~ H oom ^ o.HH H o. = m ^ N x oH N * \ æ ßvM M _ ~ .®MÅ N un .Å- oH \ z Ü? æóH H = á ~ ^ w. x H m \ o ooH m . ~ H æ.æH IN * m \ N mmH ^ m.oH H æ. = M ^ mx oH nmmH | m & w \ m. WSx H. ~ HM mi? Wxwm \ H æmH ^ m.HH H o. = m ^ mx H m \ o ooH wm w æ.æH | N * m \ z mHm ^ m .: H oo = ^ wx oH nmmH | mB m \ m wwHA æá H o.mm ^ wxmz \ c ooH mH x m .> H | H * = x N å? ~ .HH x mä? Wx øoH wHmTmH.

= \ Q SH NA fl .ämm w x ww w \ o ooH m. ~ H w.> H | w x H w = H ^ m5 fl o.w ~ ^ w x oH w H * w .x m E? wß fl WOT w x m wmTâ. w \ H: m7 ww q. Hñmmx w x H. Tw fi oä w HHwnm ,,.,. A. 446H406 '* 45 QUNHWHÜX UHOS .HNP S00 WÜUHMUWN CQUC fi Ümv flfl ÜÜCM> ÜHHO> Ü NHHÉ un u .å m H fi w fl m fi m \ o oo fi mH fl N.mH I m \ m mæHA o M m.m.m ? f .. M OxNmA w x 2 Q fl nä. m \ m mm? .im fl m6? wxm ma \ o oo fi: .mw m.mH I m \ = æ ~ HA æ. = HH: .æw ^ mx om mn \ m wmH ^> .mn m.Hm ^ mxm oz fi lmß o fi \ = o> fi ^ mw H o.wN ^ mx H m \ N 3.7 m6 fl ma? mx m6 ñümmmw MwWm.mwä mmmä w fl mu mw: m> wH Anv ñmm fl v ummmwm fl m fi fi umm flfi umuum fl c fl ñwm | mmu amucd 1um> m fi mu fl d K o \ B |> wHum> m Mmu \ mEm Mmu \ mEm mm \ m As shown in Tables 8 and 9, the effect on the group administered with each of the TF substances of the invention was prolonged in life, compared to the control group. (b) Antitumor activity against Ehrlich solid tumor (l) Ehrlich tumor cells were transplanted subcutaneously into the armpit of mice of the ICR strain (females, 6 weeks old) in an amount of 4 x 10 6 cells per mouse. Thereafter, each of the test substances was administered to the mice once a day for 7 days from the first day after the transplantation of the tumor cells, or once a day from the first day and from the third day to the seventh day for a total of 6 days.

In the control group, 0.2 ml of a physiological saline solution was administered in the same manner as described above. The M weight of the tumors was determined on the 11th or 14th day after transplantation of the tumor cells. The weight of the tumors was determined by measuring the largest diameter (a) mm and the smallest diameter (b) mm using calipers and was calculated using Å 3 the following equation: 2 ba ä (m9) Tumor weight = The results are shown in Table 10 .-m “@“ nT P 'w mm * + vwwW fl ~ - -44-6' _4106 41 kt \ nÅ ur Åmmrr: RÅ- QQH @om 1 == oø. = w H oH N * Qw ° =. mwxm »Hmwno» HHwmmH »= H nmmH | ma HH @ =. w WHH.

AND. w0.m | w: ~ m .: w H CH N * == @ o. =. m H m »mmaw> wu» aH nNmH | ma Nm Hw.mwx H OCH ==. m 1 H * m = ~ mH w H QH uHmw = o »HHwmmHu = H mHmH | me mh m @. ~ wx N OCH = mm | HN mH.H w H OQH Hcw fl øxnsm Hm mm wm.H w X oo fi uHmwG0uHnwmmHucH oo fi lmü .HN mHf fl WN Om UMÛGmXwNHHÉH Anv Åwv Aummi fl uwnum fl c fl äwm H \ h »H» M » mH »wHcHew <wnmuwnnm OH H fl wßm fi WH- fluïwf mcnm fifl wunwëøv> m cwco fl umucm fi mm fi muu umumw cwmm fi wuwa Cwß mm uäwummm NM mcuw fifi wunwåøu> m mmmww fi mm hm mww fi mm mww fi mm .š i: âa 1 m å o fl m w x 3 m * o: Hw.m m x m vmm fl wawnudH nmmalmm. wm mm.m w x H _ 03. mwd | _ ._ x 3 Sá w x 3 »mocmšmguø fi wm fi .É m .... .. l mm ææ z w x m 1 ... u ä mms w x H 4 6 oOH mm.w I q 4 Nm æm: Ûm w x on u fi mmcOß fl nwmmu flfl H mmm fl lmm. 4 N .. E fi m N .. m F Ümuuowv OH H fi wnm fi x L..l _____, 446-406 f- 49 (2) Ehrlich tumor cells were transplanted subcutaneously into the armpit of mice of the ICR strain (females, 6 weeks old) in a male of 4 x 106 cells per mouse. Thereafter, each of the test substances was administered to the mice divided into groups for intravenous administration, intraperitoneal administration, subcutaneous administration and intramuscular administration, at a dose of 10 mg / kg or 20 mg / kg once a day on the first day. and from the 3rd day to the 7th day for a total of 6 days after the transplantation of the tumor cells. In the control group, 0.2 ml of a physiological saline solution was administered in the same manner as above. The weight of the tumors was determined on the 13th day after the transplantation of the tumor cells.

The results are shown in Table ll. Ü www - »50 oo fi | | m .. ow wm OH ßHmHDxw fl8 MHßGH A: om Hm OH »= m»: xn = w mmm fi lms wm om oz o fi # Hmm = Ou fl HmmMHuGH ww CN mw OH ummGm> muucH oo fl I> = QH ßmmcw> m ~ »= H \ B uxH> Hm © mE | HmE§B ^ wx \ m %% | mmG fl H®HßmHG fl% mw mcmum fl øm ~ H H fi wmmm 51 As shown in Tables 10 and ll, a tumor growth-preventing effect can be observed in the groups On which each of The TF substances of the invention have been administered. (c) Antitumor activity against Sarcom-180 carcinoma Sarcom-180 carcinoma was transplanted intraperitoneally to mice of the ICR strain (females, 6 weeks old) in an amount of 1 x 10 5 cells per mouse. Thereafter, each of the test substances was administered intraperitoneally to the mice at a dose of 1 mg / kg, 5 mg / kg or 10 mg / kg once daily from the first day to the seventh day, repeated for a total of 7 days after transplantation of the tumor cells. or once a day on the first day and from the third day to the seventh day for a total of 6 days. In the control group, 0.2 ml of a physiological saline solution was administered in the same manner as above.

The results are shown in Table 12. 446-406 52 HO, H. »Mama - V = ü H OH \ O OOH m.m H O. = H | v N * O \ O OOOA O H O. ~ = ^ O H O O ~ mH | ma O \ O OOHA O H O. ~ = ^ O H OH A O \ O OOH ~ .OH | O \ N OOHA _O.O ~ ^ O x H H * O \ m OOHA m.> N ^ O H O OOmH | O @ O \ m OOHA ~ .Om ^ O x OH.

O O \ O OOH N.OH | O \ H NHHA OH ~ ^ O x HH * O \ m OOHA O .- ^ O x O OmmH | Oa OO \ O OOHA m.Om ^ O x OH O \ O OOH ~ .OH 1 Hm O \ O HHH mH ~ OHH ONmH = Oe OO \ H OHHA ~ .- ^ O x OO \ N OOHA OO ~ ^ OH OH mwmE w fl mu w fifl m fi m fl fi xv Ammmö Hmmm fl ummc fl uwnuw fl n fi ä fl m H, ummu Hmucê numïw Hmu> wH => HmßHwuw fi uwHmucm N mvïmëv mOQ wumumn fl m V NH HHOQOB ww then 'f446 <4D6 53 QUMHNHDM ßUMÜCWUWHHÜN NWUCNPÜHHÜ> Û ÜÜ HN? NN .S00 .Hun H mšHmHHwuHæE fl u Em cmco fi umucm fi wmcmnß .Hwuum cmmm fl w fl cw mm cmh fl wc fi møwm NK mcHwHHmoHmEøv> m cwaoHumvsmHmmcmuu .Hwuwm mw cwmm. N * m \ H NHHA m. ~ H H w. = ~ ^ O H H OmH «Oa m \ H HHNA 0.0 H 0.0m ^ O H m OH \ O OOH m. ~ H m. = H | H * OH \ H OHNA m.O H m.Om ^ H x H OHmH | ma OH \ m HONA m.HH H 0.0 ~ ^ H x m H \ O OOH O.m H O.mH | N »H \ O OONA Om H ~ m ^ H x HH ~ mH | ma H \ O OONA wO H = m ^> xm mH \ O OOH mm H O. = H 1 OH \ H OONA mO H m.> M ^ O x HN * OH \ OH OONA OH OO = ^ N xm OmmH | Oæ OH \ OH OONA OH 0.0 = ^> K OH * Ü fi f 446 406 54 As can be seen from Table l2, the groups on which each of the TF substances had according to the invention, antitumor activity had been administered, compared to the control group. (d) Antitumor activity against B-16 melanoma (1) B-16 melanoma tumor cells were transplanted subcutaneously into the armpit of mice of the BDFI strain (males, 7 weeks old) in an amount of 1 x 10 6 cells per mouse. Thereafter, the substance TF-133a was administered intraperitoneally to the mice at a dose of 5 mg / kg or 10 mg / kg once a day on the first day and from the third day to the seventh day for a total of 6 days after the transplantation of the tumor cells. In the same manner as above, the control group was treated with 0.2 ml of a physiological saline solution and the 5-FU delivery group was treated with 20 mg / kg 5-FU.

The results are shown in Table 13.

Table 13 Substance Dose (mg / kg x number Tumor mean weight T / C (%) administrations) (g) TF-133 5 x 6 4.07 70 io x 6 2.52 in 43 5-FU 20 x 6 4.21 72 Control - 5.84 100 As shown in Table 13, the groups on which the substance TF-133a had been administered had an obvious tumor growth-preventing activity compared to the control group and the groups on which each of R mg / kg and 10 mg / kg of the substance TF-133a had been administered showed similar and more remarkable tumor growth-preventing activity, compared to the group to which 5-FU had been administered. (2) The transplantation of B-16 melanoma tumor cells and the administration of the substances TF-132a, TF-133a, 5-FU and a physiological saline solution were carried out in the same manner as indicated above under (1). ). On the 21st day after transplantation of the tumor cells, the mice were autopsied and metastases of the melanoma tumor cells were observed on the lungs.

The results are shown in Table 14.

Table 14 Substance Dose (mg / kg x number Mean Prevention of administrations) metastases formation of metastases in percent (%) TF-l32a 5 x 6 l6.0 48 l0 x 6 9.86 69 TF-l33a 5 x 6 9 .3 70 10 x 6 6.7 78 5-FU 20 x 6 24.8 Control - 31.0 0 As shown in Table 14, in the groups on which each of the substances TF-132a and TF-133a had been administered , the metastases of the B-16 melanoma tumor cells had been remarkably prevented compared to the group to which 5-FU had administra- Streraâtå ß (IV) Acute toxicity LDSO for mice is as shown in Table 15. 14 -v Lim '56 Table 15 substance Method of administration LDSO (mg / kg L m TF-100 Intraperitoneal 500 3 TF-110 Intravenous) 25O Intraperitoneal> 100O TF-120 Intravenous) 250 Intraperitoneally> l0O0 TF-l32a Intravenous 300 Intraperitoneally> 500 TF-l33a Intravenous 300 Intraperitoneal Intravenous 300 glntraperitoneally) 5OO TF-132b Intravenous) 100 TF-133 Intravenous) 2OO TF-140 Intravenous 7100 TF-150 Intravenous> 2OO TF-1323 I intravenous> 200 TF-1316 Intravenous> 200 As can be seen from the above pharmacological experiments, the TF substances of the present invention are useful as carcinostatic agents and can be expected to have an effect on various cancers and can also be expected to have a significant effect against particularly solid cancer. . All TF substances according to the present invention have an excellent effect, although the TF substance 130, in particular 132a, 132b, 133a, 133b and 1323, is preferred for various reasons. nas. The TF substances of the invention may be used after administration to various pharmaceutical forms, such as oral drugs, injections, suppositories or the like, although they are preferably used in the form of injections. When used as oral drugs, the oral drugs may contain various excipients, and may be formed into capsules, tablets, powders or granules.

When used in the form of injection solutions, they may be intended for subcutaneous injections, intramuscular injections and intravenous injections, and they are used in the form of a suspension, a solution or a powder, which is dissolved during use. The injection solutions may contain a local anesthetic.

The dosage of the TF substances of the invention is determined according to the patient, although it is generally desirable to administer them in a dose for an adult of 0.01 to 50 mg / kg once a day or several times a day and As for the method of administration, they are preferably administered subcutaneously, intramuscularly or intravenously or in the form of an injection directly into the affected part.

The invention is further described below in the examples and the accompanying drawings, in which Figure 1 shows a microscope photograph of Fusobacterium nucleatum TF-031, used according to the invention, Figure 2 shows an infrared absorption spectrum of the carcinostatic substance TF-100; Figure 3 shows an ultraviolet absorption spectrum of said substance; Figure 4 shows an elution pattern obtained when said substance is subjected to gel filtration using Sephadex G-50; Figure 5 shows an HPL chromatogram; Figure 6 shows an infrared absorption spectrum of the carcinostatic substance TF-110; Figure 7 shows an ultraviolet absorption spectrum of said substance; Figure 8 shows an elution pattern obtained when said substance is subjected to gel filtration using Sephadex G-200; Figure 9 shows an HPL chromatogram of said substance; Figure 10 shows an infrared absorption spectrum of the carcinostatic substance TF-120; Figure 11 shows an ultraviolet absorption spectrum of said substance; Figure 12 shows an elution pattern obtained when said substance is: cast 446 4osf in gel filtration using Sephadex G-200; Figure 13 shows an HPL chromatogram of said substance; Figure 14 shows an infrared absorption spectrum of the carcinostatic substance TF-1316; Figure 15 shows an ultraviolet absorption spectrum of said substance; Figure 16 shows an elution pattern obtained when said substance is subjected to gel filtration using Sephadex G-200; Figure 17 shows an HPL chromatogram of said substance; Figure 18 shows an infrared absorption spectrum of the carcinostatic substance TF-132a; Figure 19 shows an ultraviolet absorption spectrum of said substance; Figure 20 shows an elution pattern obtained when said substance is subjected to gel filtration using Sephadex G-200; Figure 21 shows an HPL chromatogram of said substance; Figure 22 shows an infrared absorption spectrum of the carcinostatic substance TF-132b; Figure 23 shows an ultraviolet absorption spectrum of said substance; Figure 24 shows an elution pattern obtained when said substance is subjected to gel filtration using Sephadex G-200; Figure 25 shows an HPL chromatogram of said substance; Figure 26 shows an infrared absorption spectrum of the carcinostatic substance TF-133a; Figure 27 shows an ultraviolet absorption spectrum of said substance; Figure 28 shows an elution pattern obtained when said substance is subjected to gel filtration using Sephadex G-200; Figure 29 shows an HPL chromatogram of said substance; Figure 30 shows an infrared abortion spectrum of the kareino cheese stinking nucleus TF-13b; Figure 31 shows an ultraviolet absorption spectrum of said substance; Figure 32 shows an elution pattern obtained when said substance is subjected to gel filtration using Sephadex G-200; Figure 33 shows an HPL chromatogram of said substance; Figure 34 shows an infrared absorption spectrum of the carcinostatic substance TF-1323; Figure 35 shows an ultraviolet absorption spectrum of said substance; Figure 36 shows an elution pattern obtained when said substance is subjected to gel filtration using Sephadex G-200; Figure 37 shows an HPL chromatogram of said substance; Figure 38 shows an infrared absorption spectrum of the carcinostatic substance TF-136; Figure 39 shows a ;; aÉ; .wis_à -bvfïä F ”Å ~ training. 446.1, 406 »59 ultraviolet absorption spectrum of said substance; Figure 40 fl shows an elution pattern obtained when said substance is subjected to gel filtration using Sephadex G-200; figure H? 41 shows an HPL chromatogram of said substance; Figure 42 shows an infrared absorption spectrum of the carcinostatic substance TF-140; Figure 43 shows an ultraviolet absorption Figure 44 shows an elution pattern __; 4 spectrum of said substance; obtained when said substance is subjected to gel filtration using Sephadex G-200; and Figure 45 shows an HPL chromatogram of said substance; Figure 46 shows an infrared absorption spectrum of the carcinostatic substance TF-150; Figure 47 shows an ultraviolet absorption spectrum of said substance; Figure 48 shows an elution pattern obtained when said substance is subjected to gel filtration using Sephadex G-200; and Figure 49 shows an HPL chromatogram of said substance.

Example Gel 1. (1) To each of fifteen 2-liter culture flasks, each equipped with a screw cap, was added 2 liters of a culture medium, containing 34 g of trypticase peptone, 6 g of phyton peptone, 20 g of proteospeptone, 70 g of brain / cardiac infusion, 6 g yeast extract, 15 g sodium chloride, 12 g glucose, 10 g lactose, 0.5 g L-cystine, 0.2 g sodium sulfite, 1.0 g sodium thioglycolate and 1.4 g agar and pH in the culture medium was adjusted to 7. The culture medium was sterilized - changed under 1.2 atm at 120 ° C for 15 minutes, heated in a boiling water bath for 20 minutes and then immediately cooled in water, after which a preculture solution of Fusobacterium nucleatum TF-031, previously prepared by culturing in a culture medium of the same composition as above, inoculated into the above-mentioned water-cooled culture medium under sterile conditions in an amount of 100 ml per culture flask and subjected to a stable culture in an incubator at 37 ° C for 48 hours. After the end of the culture, the culture was centrifuged to remove organisms at 4000 rpm at 5 ° C for 20 minutes. The amount of excess liquid obtained was about 27 liters. 1 446 ms e, 60 (2) To the supernatant obtained under (1) above was added 40 liters of ethanol with stirring at 5 ° C and the resulting mixture was allowed to stand at low temperature until the amorphous precipitate had settled completely. Then the mixture was centrifuged at 6000 rpm at 5 ° C for 15 minutes and the precipitate was collected, washed with ethanol and dried under reduced pressure to obtain about 60 g of the crude powder. (3) In 120 ml of water was dissolved @ 0pg of the crude powder obtained according to (2) above and the water-insoluble materials formed were removed by centrifugation at 7500 rpm for 10 minutes, after which the water-soluble fraction obtained was combined with the washing liquids obtained by washing the water-insoluble material twice with 60 ml portions of water and the resulting solution were dried under reduced pressure to obtain 14 g of off-white to pale brown powder of the substance TF-100.

Example 2.

The solution described in Example 1- (3) and prepared by combining the water-soluble fraction free of water-insoluble materials, with the washing liquids obtained by washing the water-insoluble substances, was subjected to ultrafiltration using a hollow fiber type ultrafiltration system (membrane no. HI-1: prepared by Asahi Kasei Kogyo Kabushiki Kaisha) and the internal solution was subjected to lyophilization to give 10 g of off-white to pale brown powder of the substance TF-110. Example 3.

In a small amount of water, 10 g of the powder obtained according to Example 2 was dissolved and the resulting solution was added to a column packed with diethylaminoethyl-Sephadex A-50 equilibrated with a 0.025M phosphate buffer having a pH of 8 and the eluted solution was collected. Then, 2.5 liters of the same phosphate buffer from above was passed through the column and the eluted solution obtained was combined with the previously collected 61 eluted solution, after which the resulting solution was desalted using a hollow fiber type ultrafiltration system (membrane No. HI -1), was concentrated under reduced pressure and then subjected to lyophilization to give 470 mg of off-white to pale brown powder of the substance TF-120.

Exemgel 4.

In 24 ml of water, 2 g of the powder obtained according to Example 1- (2) were dissolved and the water-insoluble substances were removed by centrifugation at 7500 rpm for 10 minutes. The water-soluble fraction was dialyzed overnight against distilled water at 5 ° C using a cellophane tube and the inner solution was concentrated under reduced pressure.

The concentrated solution was then added to a column packed with 150 ml of diethylaminoethyl-Sephadex A-50, which had previously been equilibrated with a 0.025M phosphate buffer having a pH of 8, and the column was washed by rapidly withdrawing 600 ml of a 0, C25M phosphate ctt on of 8 and then a 0.025M uu an. phosphate buffer with a sodium chloride concentration of 0.6 M and a pH of 8, was passed through the column to obtain an eluate. The eluate was concentrated under reduced pressure to about 100 ml, dialyzed against distilled water at 5 ° C, concentrated again under reduced pressure, desalted using a column of Sephadex G-25 and then subjected to lyophilization to obtain 470 mg of an off-white to slightly white brown substance TF-1316.

Example 5.

The solution described in Example 1- (3) and prepared by combining the water-soluble fraction free of water-insoluble substances and the washing liquids obtained by washing the water-insoluble substances, was dialyzed overnight against distilled water at 5 ° C using a cellophane tube and the inner solution was concentrated under reduced pressure. The concentrated solution was then added to a column packed with 50 g of diethylaminoethyl-Sephadex A-50, which had previously been equilibrated with a 0.025M phosphate buffer having a pH of 8, and 2 liters of a 0.025M phosphate buffer having a pH of 446 cos: e 62 8 and 2.5 liters of a 0.025M phosphate buffer with a sodium clot of 0.1 M and a pH of 8, which successively after which 2.5 liters of a riding concentrate had been passed through the column, 0.025M phosphate buffer with a sodium chloride concentration of 0.2 M and a pH of 8 were passed through the column to elute the active substance adsorbed in the column, and the eluted solution obtained was desalted using a semi-fiber type filtration system (membrane No: HI-1) and lyophilization was then discarded to give 600 mg of an off-white to pale brown powder of the substance TF-132a.

Example 6.

In 1.2 liters of a 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8, 44.2 g of the powder obtained according to Example 1- (2) were dissolved and the suspended insoluble substances were removed by filtration using Hyflo Super Cel, after which the resulting filtrate was added to a 33 cm diameter column packed with 500 ml of diethylaminoethyl-Sephadex A-50, which had previously been equilibrated with E a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8 and the eluted solution was recovered.

The column was washed with a 0.025M phosphate buffer having a sodium chloride concentration of 0.3M and a pH of 8, and the washings were combined with the above-mentioned eluted solution to obtain 1.67 liters of a solution. The solution was again fed to an 8 cm diameter column packed with 400 ml of diethylaminoethyl sephadex A-40, which had previously been equilibrated with a 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8 for 1.64 liters per liter. of an eluted solution. The column was washed with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8 for 6 holes, 4 lw fi ß of 1.64 liters of washing liquids. The eluted solution and the washings were combined and a 0.025 M phosphate buffer having a pH of a was added to obtain 4.98 liters of a 0.025 M phosphate buffer having a sodium chloride concentration of 0.2 M and a pH of 8. The buffer solution thus obtained was added to a column with 8 cm diameter packed with 500 ml of diethylaminoethyl-Sephadex A-50, which had previously been equilibrated with seal J; .hk 1I / Ju (Z) Ch 63 a 0.025M phosphate buffer with a sodium chloride concentration of 0.2M and a pH of 8 and the column was washed with 2 liters of a 0.025M phosphate buffer with a sodium chloride concentration of v a solution prepared 0.2M and a pH of 8. To 7 liters of a by combining the washings and the eluted solution was added a 0.025M phosphate buffer with a pH of 8 for 14 liters of a 0.025M phosphate buffer with a sodium chloride concentration of 0.1M and a pH of The eluted solution obtained was fed to an 8 cm diameter column packed with 500 ml of diethylaminoethyl-Sephadex A-50, which had previously been equilibrated with a 0.025 M phosphate buffer having a sodium chloride concentration of 0.1 M and a pH of 8, and the eluted solution was removed. The column was washed with 2 liters of a 0.025 M phosphate buffer having a sodium chloride concentration of 0.1 M and a pH of 8, after which 2 liters of a 0.025 M phosphate buffer having a sodium chloride concentration of 0.2 M and a pH of 8 were passed through the column and the eluted the solution was collected.

The eluted solution was concentrated and desalted using an ultrafiltration system (filter used: Toyo Ultrafilter UK-10), further desalted using a Sephadex G-25 column, decolorized with activated carbon and then subjected to lyophilization to obtain 880 mg of an off-white to light brown powder of the substance TF-132b.

Example 7.

In a small amount of water, 10 g of the powder of Example 2 was dissolved and the resulting solution was added to a column packed with diethylaminoethyl-Sephadex A-50, which had previously been equilibrated with a 0.025M phosphate buffer having a pH of 8.

Then 5 liters of a 0.025M phosphate buffer with a sodium chloride concentration of 0.2M and a pH of 8 were passed through the column, after which 2.5 liters of a 0.025M phosphate buffer with a sodium chloride concentration of 0.3M and a pH of 8 were passed. through the column for eluting the active substance α 2 which was adsorbed in the column and the resulting eluted solution was concentrated under reduced pressure. The concentrated solution was desalted using a cellophane tube, completely desalted using Sephadex G-25 and then subjected to lyophilization to give 600 mg of an off-white to light brown powder of the substance TF. -l33a.

Example 8.

In 1.2 liters of a 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8 was dissolved 44.2 g of the powder obtained according to Example 1- (2) and the suspended insolubles were removed by filtration with using Hyflo Super Cel, after which the resulting filtrate was added to a 33 cm diameter column packed with 500 ml of diethylaminoethyl-Sephadex A-50, which had previously been equilibrated with a 0.025M phosphate buffer with a sodium chloride concentration of 0.3M and a pH of 8 and the eluted solution was collected. The column * was washed with a 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8, and the washings were combined with the aforementioned eluted solution to obtain 1.67 liters of a solution. The solution was again fed to an 8 cm diameter column packed with 40 ml of diethylaminoethyl-Sephadex A-SO, which had previously been equilibrated with a 0.025M phosphate buffer with a sodium chloride concentration of 0.3M and a pI-I of 8 to obtain 1, 68 liters of an eluted solution. The column was washed with a 0.025M phosphate buffer having a sodium chloride concentration of 0.3M and a pH of 8 to obtain 1.64 liters of washings. The eluted solution and the washings were combined and a 0.025M phosphate buffer with a pH of 8 was added to obtain 4.8 ml of 4.98 liters of a 0.025M phosphate buffer with a sodium chloride concentration * I of 0.2M and a pH of 8. The eluted solution obtained was added to an 8 cm diameter column packed with 500 ml of ethylaminoethyl-Sephadex A-50, which had previously been equilibrated with a 0.025 M phosphate buffer having a sodium chloride concentration of 0.2 M and a pH of 8, and the column was washed with a 0.025M phosphate buffer with a sodium chloride concentration of 0.2M and a pH of 8, after which 2 liters of a 0.025M phosphate buffer with a sodium chloride concentration of 0.3M and a pH of 8 were passed through the column and the eluted solution was collected. The eluted solution was concentrated and desalted using an ultrafiltration system (used filter: Toyo Ultrafilter UK-10), further desalted using a Sephadex G-25 column, decolorized. with activated carbon and then subjected to lyophilization to give 590 mg of an off-white to pale brown powder of the substance TF-13b.

Example 9. (1) In 120 ml of water was dissolved 20 g of the crude powder obtained according to Example 1- (2), and the formed water-insoluble substances were removed by centrifugation at 7500 rpm for 10 minutes, after which the water-soluble fraction obtained and the washing liquids obtained upon washing the water-insoluble substances twice with 60 ml portions of water, combined and then subjected to ultrafiltration using a hollow fiber type ultrafiltration system (membrane No. HI-1) and the inner solution was subjected to lyophilization to obtain 10 g of an off-white to light brown powder. (2) In one liter of a 0.025M phosphate buffer with a sodium chloride concentration of 0.3M and a pH of 8, 25 g of the powder obtained according to (1) above were dissolved and the resulting solution was added to a 33 cm column. diameter packed with 500 ml of diethylaminoethyl-Sephadex A-50, which had previously been equilibrated with a 0.025M phosphate buffer having a sodium chloride concentration of 0.3M and a pH of 8. The column was similarly washed with a 0.025M phosphate buffer having a sodium chloride concentration of 0.3M and a pH of 8 and the eluted solution and the washing liquids were combined in a ratio of 1.6 liters of a mixture. The mixture was added to an 8 cm diameter column packed with 400 ml of diethylaminoethyl-Sephadex A-50, previously equilibrated with a 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8 to give 1.65 liters. of an eluted solution. The eluted solution was combined with 1.7 liters of the washings obtained by washing the column with a 0.025M phosphate buffer with a sodium chloride concentration of 0.3M and a pH of 8, and the resulting mixture was diluted with a 0.025M phosphate buffer with a pH of 8 for the preparation of 10.35 liters of a 0.025M phosphate buffer with a sodium chloride concentration of 0.1M and a pH of 8.

Then, the buffered solution thus obtained was added to an 8 cm diameter column packed with 500 ml of diethylaminoethyl-Sephadex A-50, which had previously been equilibrated with a 0.025 M phosphate buffer having a sodium chloride concentration of 0.1 M and a pH of 8, and the column was washed with 2 liters of a 0.025M phosphate buffer with a sodium chloride concentration of 0.1 and a pH of 8, after which 2 liters of a 0.025M phosphate buffer with a sodium chloride concentration of 0.3M and a pH of 8 were passed through the column and the eluted the solutions were collected. The eluted solution obtained was desalted and concentrated using an ultrafiltration system (filter used: Toyo Ultrafiltei UK-10) and further desalted on Sephadex G-25, decolorized with activated carbon and then subjected to lyophilization to obtain 1.65 g of off-white to light brown powder of the substance TF-1323.

Example 10.

In a small amount of water was dissolved 10 g of the powder obtained according to Example 2, and the resulting solution was added to a column packed with diethylaminoethyl-Sephadex A-50, which had previously been equilibrated with a 0.025M phosphate buffer having a pH of 8. Then 6 liters of a 0.025M phosphate buffer with a sodium chloride concentration of 0.5M and a pH of 8 passed through the column after which 2.5 liters of a 0.025M phosphate buffer with a sodium chloride concentration of 0.6M and a pH of 8 were passed through the column to elute the active the substance adsorbed in the column and the resulting continuous solution was desalted using a semi-fiber type ultrafiltration system (Membrane No. HI-1), concentrated under reduced pressure and then subjected to lyophilization to give 130 mg of off-white to light brown powder. of the substance TF-136.

Exemgel ll.

In 200 ml of water, 15 g of the crude powder obtained according to Example 1- (2) were dissolved and the water-insoluble substances were removed 67 by centrifugation at 7500 rpm for 10 minutes, after which the pH of the water-soluble fraction thus obtained was adjusted to 10 g. dropwise addition of 0.2M aqueous barium hydroxide solution. White precipitate precipitated upon addition of the aqueous barium hydroxide solution. Barium ions were added so that the concentration of total barium ions in the final volume was 0.0 μM, after which the solution was stirred for 30 minutes and the precipitate was collected by filtration. The precipitate of a barium salt obtained was suspended in 10 ml of a 10% strength by weight aqueous sodium sulfate solution, stirred sufficiently and then filtered, after which the filtrate was collected. The precipitate was resuspended, stirred and filtered in the same manner as above once with 10 ml of a 10% strength by weight aqueous sodium sulfate solution and twice with 5 ml portions of said solution and each filtrate was collected. Thereafter, the remaining precipitate was washed twice with 10 ml portions of water and then filtered to obtain washing liquids. The said collected filtrate and the washing liquids were combined, dialyzed against distilled water and concentrated under reduced pressure, and then subjected to lyophilization to obtain 0.45 g of an off-white to pale brown powder of the substance TF-140.

Example l2.

In 450 ml of water, 32 g of the powder obtained according to Example 1- (2) were dissolved and the suspended insolubles were removed by filtration using Hyflo Super Cel, after which the resulting filtrate was dialyzed overnight against running water using a cellophane tube. To the dialyzed solution was added dropwise 80 ml of a 20% by weight aqueous cetylpyridinyl chloride solution with stirring, and at the same time, 200 ml of a 10% by weight borate buffer (pH 9.0) was added, and the resulting mixture was stirred at room temperature for 30 minutes. were collected by filtration. The precipitate was suspended in 150 ml of a 0.1% by weight borate buffer (pH 9.0) containing 0.1 M sodium chloride and 0.2% by weight of cetylpyridinium chloride, and the resulting suspension was stirred for 30 minutes. Then the precipitate was collected _,., L; 68 by filtration. The collected precipitate was suspended in 150 ml of a 0.1% by weight borate buffer (pH 9.0) containing 0.5M sodium chloride and 0.2% cetylpyridinium chloride, and the resulting suspension was stirred and dissociated for 30 minutes. Thereafter, the precipitate was separated from the suspension by filtration to obtain a soluble fraction, after which the precipitate separated by filtration was again subjected to the same dissociation procedure as above to obtain a soluble fraction. The two soluble fractions were combined and in the resulting soluble fraction the pH was adjusted to 3-4 with 10% hydrochloric acid, after which ethanol was added so that its concentration finally became 80% by volume.

The resulting solution was allowed to stand overnight at 5 ° C and the precipitate formed was collected by filtration to obtain 5.6 g of an off-white to light brown powder of the substance TF-150. greparation example l.

Each vial was packaged with 1 mg or 5 mg of each of the powders of the substances TF-100, 110 and 120 obtained according to Examples 1, 2 and 3. The powders are dissolved in a sterilized physiological saline solution, a solution containing 0.5% lidocaine or similar when to be used and then used for injections.

Preparation Example 2.

Each vial was packaged with 1 mg or 5 mg of each of the powders of the substances TF-1316, 13a and 132b obtained according to Examples 4, 5 and 6. The powders are dissolved in a sterilized physiological saline solution, a solution containing 0.5%. lidocaine or similar when to be used and then used for injections.

Preparation Example 3.

One vial was packaged with 1 mg or 5 mg of the powder from the substance TF-133a obtained according to Example 7. The powder is dissolved in a sterilized physiological saline solution, a solution containing 0.5% lidocaine or the like when it is to be administered; sf; li J .. 446 -406 69 is inverted and then used for an injection.

Preparation Example 4.

A vial was packaged with 1 mg or 5 mg of the powder from the substance TF-133b obtained according to Example 5. The powder is dissolved in a sterilized physiological saline solution, a solution containing 0.5% lidocaine or the like when to be used and then used for a injection.

Preparation Example 5.

One vial was packaged with 1 mg or 5 mg of the powder from the substance TF-1323 obtained according to Example 9. The powder is dissolved in a sterilized physiological saline solution, a solution in WW containing 0.5% lidocaine or the like when to be used and then used. for an injection. »Www fi Preparation Example 6.

Each vial was packed with 1 mg or 5 mg of each of the powders of substances TF-136, 140 and 150 obtained according to Examples 10, 11 and 12. The powders are dissolved in a sterilized physiological saline solution, a solution containing _ Vfß fi 0.5% lidocaine or similar when to be used and then used for injections.

Claims (31)

1. 70 4446, MTG ~ PATENTKRAV l. Process for the preparation of a TF substance with carcinostatic and immunostimulatory activity, designated TF-100, TF-1010, TF-120, TF-130 (TF-1316, TF-132a, TF-132b, TF-132a , TF-13b, TF-1323, TF-136), TF-140 or TF-150, characterized by culturing TF substance-producing bacteria belonging to Fusobacterium nucleatum TF-031 (FERM 5077; ATCC 31647) in a culture medium containing nitrogen sources, carbon sources, vitamin sources, reducing agents and inorganic salts with or without agar under anaerobic conditions, adding a hydrophilic organic solvent to the culture or its excess liquid, collecting the precipitate thus formed and then the water-soluble fraction of the precipitate, or if desired by the following benenaiingef (ii, (zí, (3) or (4): (l) subjecting the water-soluble fraction to dialysis or ultrafiltration, then collecting a desired fraction from the internal solution thereof or subject to so If desired, the fraction in question is treated with an ion exchanger and collects an adsorbed fraction or a non-adsorbed fraction, or adds a quaternary ammonium salt to the fraction collected from the inner solution, collects the resulting precipitate and then undergoes this dissociation treatment and collects being fraction from the treated liquid; (2) the water-soluble fraction is subjected to ion exchange treatment and the adsorbed fraction or the non-adsorbed fraction is collected; 3) add barium ions to the water-soluble fraction (the culture or its supernatant can be used instead of the water-soluble fraction), collect the obtained barium salt, remove barium from the salt, »V w l fl nnu fi, 71” 446-'406 then subject the water-soluble the fraction dialysis or ultrafiltration and collects a desired fraction A, ¿ßâ. from its inner solution; (4) a quaternary ammonium salt is added to the water-soluble fraction, the precipitate obtained is collected, subjected to this dissociation treatment and Å is then collected a desired fraction from the treated liquid. A process according to claim 1, characterized in that the water-soluble fraction of the precipitate is separated from the precipitate and then dried. 'E g 3. A method according to claim 1, characterized in that the water-soluble fraction of the precipitate is subjected to dialysis or ultrafiltration and that the substances with carcinostatic and immunostimulatory activity are recovered from the inner solution. 4. A process according to claim 1, characterized in that the water-soluble fraction of the precipitate is, if desired, subjected to dialysis or ultrafiltration and then treated with an ion exchanger and that the non-adsorbed fraction is collected. r w, 5. A process according to claim 1, characterized in that the water-soluble fraction of the precipitate is, if desired, subjected to dialysis or ultrafiltration and then treated with an ion exchanger and that the substances with carcinostatic and immunostimulatory activity are collected from the adsorbed fraction. 6. A process according to claim 5, characterized in that the adsorbed fraction is treated with a 0.6M sodium chloride-phosphate buffer and that the eluted fraction is collected. \ §, 72 446 406 '_ 7. A process according to claim 5, characterized in that a fraction which has been adsorbed with a 0.1 M sodium chloride-phosphate buffer, but has been eluted with a 0.2M sodium chloride-phosphate buffer, is collected from the adsorbed fraction. 8. A process according to claim 5, characterized in that a fraction which has been adsorbed with a 0.2M sodium chloride-phosphate buffer, but has been eluted with a 0.3M sodium chloride-phosphate buffer, is collected. 9. A process according to claim 5, characterized in that a fraction which has been adsorbed with a 0.1 M sodium chloride-phosphate buffer, but has been eluted with a 0.3M sodium chloride-phosphate buffer, is collected. 10. A process according to claim 5, characterized in that a fraction which has been adsorbed with a 0.5M sodium chloride-phosphate buffer, but has been eluted with a 0.6M sodium chloride-phosphate buffer, is collected. 11. ll. Process according to Claim 1, characterized in that the water-soluble fraction of the precipitate is subjected to dialysis or ultrafiltration, in that a 0.2-0.3M sodium chloride-phosphate buffer of the water-soluble fraction of the precipitate or in a 0.2-0 The 3M sodium chloride-phosphate buffer of the internal solution component obtained by dialysis or ultrafiltration is prepared and then treated with an ion exchanger, equilibrated with a 0.2-03M sodium chloride-phosphate buffer, to collect a fraction which has passed through the ion exchanger. this eluted solution is then adjusted so that its sodium chloride concentration is 0.1M and then treated with an ion exchanger, equilibrated with a 0.1M sodium chloride-phosphate buffer, to be adsorbed thereon, and then a fraction which has been adsorbed is collected with a 0.1 M sodium chloride-phosphate buffer but has been eluted with a 0.2 or 0.3M sodium chloride-phosphate buffer. e W. gl 73 A process according to claim 1, characterized in that the water-soluble fraction of the precipitate is subjected to dialysis or ultrafiltration, that a 0.3M sodium chloride-phosphate buffer of the water-soluble fraction of the precipitate or a 0.3M sodium chloride-phosphate buffer of the inner solution component obtained by dialysis or ultrafiltration is prepared and then treated with an ion exchanger, equivalent to a 0.3 M sodium chloride phosphate buffer, to collect a fraction which has passed through the ion exchanger, that the sodium chloride concentration in this eluted solution is then adjusted to 0. , 2M and that the solution is then treated with an ion exchanger, equilibrated with a 0.2M sodium chloride-phosphate buffer to be adsorbed therefrom, and that a fraction is subsequently collected which has been adsorbed with a 0.2M sodium chloride-phosphate buffer but has been eluted with a 0.3M sodium chloride phosphate buffer. 13. A process according to claim 1, characterized in that barium ions are added to the water-soluble fraction of the precipitate, that the barium salt formed is collected and barium is removed from the salt and that the water-soluble fraction is collected and subjected to dialysis or ultrafiltration. \ 14. A process according to claim 1, characterized in that the water-soluble fraction of the precipitate is treated with a quaternary ammonium salt, that the precipitate formed is collected and then subjected to a dissociation procedure using a solution containing sodium chloride, that a hydrophilic organic solvent is added to the resulting soluble fraction to form a precipitate and that the precipitate thus formed is collected. A method according to any one of claims 1 to 14, characterized in that the culture medium used for culturing the bacteria contains: tryptican peptone, zytan peptone, proteospeptone, a brain-heart infusion (or cardiac infusion), 7% "446 - 4136 yeast extracts, sodium chloride, glucose, lactose, L-cystine, sodium sulphite and thioglycolate or together with agar. Process according to any one of claims 1 to 14, characterized in that the cultivation is carried out at 35 "to 42 ° C" Q for 1 to 5 days in a culture medium whose pH has been adjusted to 6.0 to 8 , 5. 17. A method according to any one of claims 1-14, characterized in that the cultivation is carried out at 36 ° to ¿§ ° C for 1 to 3 days in a culture medium whose pH has been adjusted to 7.2 to 8.2. 18. A process according to any one of claims 1 to 14, characterized in that the hydrophilic organic solvent added to the culture or its supernatant is an alcohol. 19. A process according to any one of claims 1 to 14, characterized in that the hydrophilic organic solvent is added to the culture or the supernatant in a solvent concentration of 30 to 70% by volume. 20. A method according to any one of claims 5-12, characterized in that the ion exchanger is a weakly basic ion exchanger or a weakly basic ion exchanger with molecular visibility. IN 21. The method of claim 5, wherein a 0.1-0.6M sodium chloride phosphate buffer is used to elute the fraction adsorbed on the ion exchanger. 22. A process according to any one of claims 5-12 or 21, characterized in that the pH of the sodium chloride-containing phosphate buffer used to treat the adsorbed fraction is about B. 75 t. 'N61 405 23. A process according to any one of claims 6 to 12 or 21, characterized in that the fraction which has been subjected to ion exchange treatment is concentrated, desalted and dried. 24. A process according to claim 14, characterized in that the quaternary ammonium salt is cetylpyridinium chloride. 25. A method according to claim 14, characterized in that the treatment with the quaternary ammonium salt is carried out in the presence of a borate buffer. 26. A process according to claim 14, characterized in that the solution containing sodium chloride is a borate buffer containing sodium chloride and a quaternary ammonium salt. 27. A method according to claim 1, characterized in that Fusobacterium nucleatum TF-031 (FERM 5077; ATCC 31647) is inoculated into a culture medium, the pH of which is adjusted to 7.2 to 8.2, containing trypticase peptone, phyton peptone, proteospeptone, a brain-heart infusion (or a cardiac infusion), yeast extract, sodium chloride, glucose, lactose, L-cystine, sodium sulfite and thioglycolate, or inoculated into a culture medium containing these and agar, and then cultured under anaerobic conditions at 36 ° to 38 ° C for 1 to 3 days; an alcohol is added to the culture or its excess liquid in a concentration of 50 to 70% by volume; the precipitate formed is collected; the water-soluble fraction of the precipitate is subjected, if desired, to dialysis or ultrafiltration and then treated with a weakly basic ion exchanger or a weakly basic ion exchanger with a molecular visibility to remove the non-adsorbed fraction; a fraction which has been eluted with a 0.1 or 0.2M sodium chloride phosphate buffer is removed from the adsorbed fraction; and then a fraction which has been eluted with a 0.3M sodium chloride-phosphate buffer is collected, concentrated, desalted and dried. The method of claim 1, characterized in that Fusobacterium nucleatum TF-031 (FERM 5077; ATCC 31647) is inoculated into a culture medium adjusted to pH 7.2 to 8.2, containing trypticase peptone, phyton peptone, proteospeptone, a brain cardiac infusion (or a cardiac infusion), yeast extract, sodium chloride, glucose, lactose, L-cystine, sodium sulphite and thioglycolate, or a culture medium containing these and agar, and then cultured under anaerobic conditions at 36 to 38 ° C for 1 to 3 days ; an alcohol is added to the culture or its excess liquid in a concentration of 50 to 70% by volume; the precipitate formed is collected; the water-soluble fraction of the precipitate is optionally subjected to dialysis or ultrafiltration and a 0.2-0.3M sodium chloride phosphate buffer of the water-soluble fraction of the precipitate or a 0.2 ~ 0.3M sodium chloride-phosphate buffer of the inner solution component, obtained by dialysis or ultrafiltration , then, treated with a weakly basic ion exchanger or weakly basic ion exchanger with a molecular visibility that has been equilibrated with a 0.2-0.3M sodium chloride-phosphate buffer; the fraction which has been passed through the ion exchanger is collected; this eluted solution is adjusted to a sodium chloride concentration of 0.1 to 0.2M and then treated with a weakly basic ion exchanger or a weakly basic ion exchanger with a molecular visibility which has been equilibrated with a 0.1-1,2M sodium chloride phosphate buffer to be adsorbed thereon. ; and a fraction eluted with a 0.2-0.3M sodium chloride phosphate buffer is collected from the adsorbed fraction, I. 4, J 'f. Å - - _ 'lgh J is concentrated, desalted and dried. Carcinostatic TF substance selected from TF-100, TF-1010, TF-120, TF-130 (TF-1316, TF-1332, TF-132b, TF-133a, TF-133b, TF -136), TF-140 or TF-150, which are prepared by culturing Fusobacterium nucleatum TF-031 (FERM 5077; ATCC 31647) under anaerobic conditions and collecting the carotenoid substances from the culture or its supernatant. 4 * 06 liquid, (A) wherein said TF-100 has the following properties: (a) Off-white to light brown powder. (b) It prevents the growth of Ehrlich ascites tumor and Ehrlich solid tumor in mice and has an immunostimulatory activity. (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. (d) It has no clear melting point, begins to decompose at about 110 ° C and decomposes significantly above 200 ° C. U (e) Its infrared absorption spectrum, obtained by a gm KBr tablet method, has absorption bands in the vicinity of V 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, l 1240, ll40-1000 and 820 cm . (f) The ultraviolet absorption spectrum of its aqueous solution shows strong absorption at the absorption edge and shows an absorption peak in the vicinity of 256-260 nm. (g) When fractionated by gel filtration (column: 50 mm diameter x 600 mm, eluent: distilled water) using Sephadex G-50 (registered trademark of Pharmacia Co., Ltd.), it has absorption bands at elution volumes close to the void volume - 400, 430-530-700-800 and 840-870 ml at the ultraviolet absorbance measurement at 260 nm; and at elution volumes in the vicinity of the void volume - 390 and 410-430 ml at the absorbance measurement at 620 nm by an anthron-sulfuric acid method. (h) Its high-resolution liquid chromatogram (eluent: 0.1 M phosphate buffer, pH 7, flow rate: 0.8 ml / min, at room temperature), obtained using μm; fi? of "TSK-GEL G300 SW" (trade name of Toyo Soda Co., Ltd., column: 7.9 mm diameter x 600 mm x 2) has. peaks near the solvent front, 38-60, äf and 65 min. at the ultraviolet absorbance measurement at I 220 nm and 260 nm. L-V __... w! 76 44t- 406 i _. (i) It reacts positively in the Molisch reaction, the phenolic-sulfuric acid reaction, the anthron-sulfuric acid reaction, the indole-hydrochloric acid reaction, the Lowry-Folin reaction and the ninhydrin reaction. (j) Elemental analysis values: C: 10.6-35.7%, H: 4.2-5.2%, N: 4.2-5.2%. (k) Its saccharide content. measured by a phenol-sulfuric acid method, is about 40.0 to about 46.0% by weight, expressed as glucose, and its protein content, measured I by the Lowry-Folin method, is about 20.0 to about ,,,,, 23 .0% by weight, expressed as calf serum albumin; (B) wherein said TF-110 has the following properties: (a) Off-white to light brown powder. (b) It prevents the growth of Ehrlich ascites tumor in * WW mice and has an immunostimulatory effect. vw (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. (d) It has no clear melting point but begins to decompose at about 110 ° C and decomposes well above 200 ° C. (C) Its infrared absorption spectrum, obtained by a KBr tablet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, (1240, 1140-iooe een sin em '1.' (f) The ultraviolet absorption spectrum of its aqueous solution shows strong absorption at the absorption edge and shows an absorption peak near 256-260 nm. (G) When fractionated by gel filtration (column: 44 mm diameter x 500 mm, eluent: 0.1M phosphate buffer, pH 7) using Sephadex G-200 (registered trademark of Pharmacia Co., * Ltd.), it has absorption band at the elution volumes é in the vicinity of the empty volume - 380 and 600-920 ml at the ultraviolet absorbance measurement at 260 nm, at elution volumes close to the void volume - san, 420-520 and 6: 0-760 ml at absorbnnnmlnninunn what i * ..._ '- * ï (h) 79? 44GfÅ06 490 nm by a phenolic-sulfuric acid method, and at elution volumes close to the void volume - 380, 420-520 and 620-760 ml at absorbance the measurement at 620 nm with an anthron-sulfuric acid method. Its high-resolution liquid chromatogram (eluent: 0.1M phosphate buffer, pH 7, flow rate: 0.8 ml / min at room temperature), obtained using "TSK-GEL G3000 SW" (trade name of Toyo Soda Co., Ltd., column : 7.9 mm diameter x 600 mm x 23) has peaks in N near the solvent front, 38-60, and 65 min. (i) (j) (k) in the ultraviolet absorbance measurement at 220 nm and 260 nm. It reacts positively in the Molisch reaction, the phenolic-sulfuric acid reaction, the anthron-sulfuric acid reaction, the indole-hydrochloric acid reaction and the Lo / ry-Folin reaction and negatively in the ninhydrin reaction. Elemental analysis values: C: 35.9-41.0%, H: 4.5-5.2%; N: 4.2-5.2%. Its saccharide content, measured by a phenol-sulfuric acid method, is about 30.0 to about 69.0 weight percent, expressed as glucose, and its protein content, measured by the Lowry-Folin method, is about 18.0 to about 22.0 weight percent. , expressed as calf serum albumin; (C) wherein said TF-120 has the following properties: (a) (b) (c) (d) (e) Off-white to light brown powder. It prevents the growth of Ehrlich ascites tumor in mice and has an immunostimulatory effect. It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. It has no clear melting point but begins to decompose at about 110 ° C and decomposes considerably above 200 ° C. Its infrared absorption spectrum, obtained by a KBr tablet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550, 1380-1360, 1140-looo aan szo cm'l. fm iÉkÉ _ ... min _x ,, _ Juan-Li - 80 446 406- i i. (f) (9) (i) (j) (k) The ultraviolet absorption spectrum of its aqueous solution shows strong absorption at the absorption edge and shows an absorption peak in the vicinity of 268-272 nm. When fractionated by gel filtration (column: 44 mm diameter x 500 mm, eluent: 0.1 M phosphate buffer, pH 7) using Sephadex G-200, it has absorption band at elution volume near the void volume - 325 and 775 875 ml at the ultraviolet absorbance measurement at 260 nm, at elution volumes close to the void volume - 360, 360-480 and 510-760 ml at the absorbance measurement at 490 nm using a phenolic-sulfuric acid method, and at elution volumes near the void volume - 380, 420-520 and 620-760 ml in the absorbance measurement at 620 nm with an anthron-sulfuric acid method. Its high-resolution liquid chromatogram (eluent: 0.1M phosphate buffer, pH 7, flow rate: 0.8 ml / min., At room temperature), obtained using "TSK-GEL G3000 SW" (column: 7.9 mm diameter x 600 mm x 2) has peaks near the solvent front, 38-60 min. at the ultraviolet absorbance measurement at 220 nm and 260 nm. It reacts positively in the Molisch reaction, phenolic-sulfuric acid reaction, anthron-sulfuric acid reaction, indole-hydrochloric acid reaction and the Lowry-Folin reaction and negatively in the ninhydrin reaction. Elemental analysis values C: 35.1-40.2%, H: 4.5-5.5%, N: 2.0-3.1%. Its saccharide content, measured by a phenol-sulfuric acid method, is about 56.0 to about 73.0 weight percent, expressed as glucose, and its protein content, measured by the Lowry-Folin method, is about 9.0 to about 1.3 weight percent. , expressed as calf serum albumin; (D) wherein said TF-130 has the following properties: (a) (b) Off-white to light brown powder. It prevents the growth of Ehrlich ascites tumor, (C) (d) (e) (f) (9) (h) (i) s: 44ó.406le Ehrlich solid tumor and sarcoma-180 carcinoma in mice and has an immunostimulatory effect. It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. It has no clear melting point, begins to decompose at about 110 ° C and decomposes considerably above 200 ° C. Its infrared absorption spectrum, obtained by a KBr tablet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-120 and 820 cm -1. The ultraviolet absorption spectrum of its aqueous solution shows strong absorption at the absorption edge and shows an absorption peak in the vicinity of 256-280 nm. It reacts positively in the Molisch reaction, the phenolic-sulfuric acid reaction, the anthron-sulfuric acid reaction, the indole-hydrochloric acid reaction and the Lowry-Folin reaction and negatively in the ninhydrin reaction. Elemental analysis values C: 27.5-39.8%, H: 3.2-5.7%, N: 2.8-6.3%. Its saccharide content measured by a phenol-sulfuric acid method is about 19.0 to about 64.0 weight percent, expressed as glucose, and its protein content, measured by the Lowry-Folin method, is about 6.0 to about 28.0 weight percent, expressed as as calf serum albumin; (E) wherein said TF-1316 has the following properties: (a) (b) Off-white to light brown powder. It prevents the growth of Ehrlich ascites tumor, Ehrlich solid tumor and sarcoma-180 carcinoma in mice and has an immunostimulatory effect. It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. It has no clear melting point, begins to decompose at about 110 ° C and decomposes considerably above 200 ° C. Its infrared absorption spectrum, obtained by a KBr tablet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, .__-_ _., Sz 446 4066 1240, 1140-iooo and azo cm'1. (f) The ultraviolet absorption spectrum of its aqueous solution shows strong absorption at the absorption edge and shows an absorption peak in the vicinity of 256-260 nm. (g) When fractionated by gel filtration (column: 21 mm diameter x 400 mm, eluent: 0.1M phosphate buffer, pH 7) using Sephadex G-200, it has an absorption band at elution volumes near the empty volume - 160 ml in the ultraviolet absorbance measurement at 260 nm, and an absorption band at elution volumes close to the empty volume - 160 ml in the absorbance measurement at 490 nm using a phenolic sulfuric acid method. (h) Its high-resolution liquid chromatogram (eluent: 0.1M phosphate buffer, pH 7, flow rate: 0.8 ml / min, at room temperature) obtained using "TSK-GEL G3000 SW" (column: 7.9 mm diameter x 600 mm x 2) has peaks near the solvent front and 40-60 min. at the ultraviolet absorbance measurement at 260 nm. (i) It reacts positively in the Molisch reaction, the phenolic-sulfuric acid reaction, the anthron-sulfuric acid reaction, the indole-hydrochloric acid reaction and the Lowry-Folin reaction and the negative reaction in the ninhydrin reaction. (j) Elemental analysis values C: 30.0-34.0%, H: 3.8-4.4%, N: 4.9-5.7%. (k) Its saccharide content, measured by a phenol-sulfuric acid method, is about 35.0 to about 50.0% by weight, expressed as glucose, and its protein content, measured by the Lowry-Foin method, is about 10.0 to about 23% by weight. .0% by weight, expressed as calf serum albumin; (F) wherein said TF-132a has the following properties: (a) Off-white to light brown powder. (b) It prevents the growth of Ehrlich ascites tumor, Ehrlich solid tumor and sarcoma-180 carcinoma in mice and has an immunostimulatory effect. 1 få as 446ï40ón. (c) It is soluble in water and insoluble in methanol, ethanol, N acetone, benzene, chloroform, ethyl acetate and diethyl ether. Å 0 ,, (d) It has no clear melting point, begins to decompose 'Å à at about 110 ° C and decomposes considerably above 200 ° C. (e) Its infrared absorption spectrum, obtained by a KBr tablet method, has absorption bands in the vicinity of hf, 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, hf - 1240, 1140-1000 and 820 and others . (f) The ultraviolet absorption spectrum of its aqueous solution shows strong absorption at the absorption edge and shows an absorption peak in the vicinity of 270-280 nm. (g) When fractionated by gel filtration (column: 44 mm diameter x 500 mm, eluent: 0.1 M phosphate buffer, pH 7) using Sephadex G-200, V has the absorption band at elution volumes close to Ü ¿* Lie the empty volume - 340, 600-700 and 720-880 ml at the k k ultraviolet absorbance measurement at 260 nm; at few elution volumes in the vicinity of the empty volume - 340, 340-580 and 720-900 ml in the absorbance measurement at 490 now with a phenol-sulfuric acid method, “and at elution volumes near the empty volume - 340 and -f 340 -580 ml at the ahsorbane measurement at 620 nm by an anthron-sulfuric acid method. I (h) Its high-resolution liquid chromatogram (eluent: 0.1M phosphate buffer pH 7, flow rate: 0.8 ml / min, at room temperature) obtained using "TSK-GEL G3000 SW" (column: 7.9 mm diameter x 600 mm x 2) has peaks in the vicinity of the solvent Å 1 front, 30, 38-60 and 65 min. at the ultraviolet absorbance measurement at 220 nm, and in the vicinity of the solvent front, 62 and 65 minutes. at the ultra- - féšäâä violet absorbance measurement at 260 nm. (i) It reacts positively in the Molisch reaction, the phenolic-sulfuric acid reaction, the anthron-sulfuric acid reaction, the indole-hydrochloric acid reaction and the Lowry-Folin reaction and the t 1 (b): J (f) (9) (h) 446 "406 (j) Elemental analysis values C: 34.8-39.8%, H: 4.5-5.7%, N: 2.8 (K) Its saccharide content, measured by a phenolic-sulfuric acid method, is about 55.0 to about 64.0% by weight, expressed as glucose, and its protein content, measured by the Lowry-Folin method, is about 18.0 to about 28.0% by weight, expressed as calf serum albumin, the TE-132 having the following properties: (a) Off-white to light brown powder, preventing the growth of Ehrlich ascites tumor, Ehrlich solid tumor and sarcoma-180 carcinoma in mice and It has an immunostimulatory effect. It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. It has no clear melting point, begins to decompose at about 110 ° C and decomposes considerably above 200 ° C. absorption spectrum, obtained by a KBr tablet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-iooo and azo cm-1. The sorption spectrum of its aqueous solution shows strong absorption at the absorption edge and has an inflection point in the vicinity of 265-280 nm. When fractionated by gel filtration (column: 21 mm diameter x 400 mm, eluent; 0.1 M phosphate buffer, pH 7) using Sephadex G-200, it has a weak absorption band at elution volumes close to the empty volume - 150 ml at the ultraviolet absorbance measurement at 260 nm, and an absorption band at elution volumes close to the empty volume - 150 ml at the absorbance measurement at 490 nm using a phenolic sulfuric acid method. Its high-resolution liquid chromatogram (eluent: 0.1M phosphate buffer, pH 7, flow rate: 0.8 ml / min., At room temperature) obtained using TSK-GEL G3000 SW "(column: 7.9 mm diameter x 600 mm (i) (j) (K) 446 406 x 2) has peaks near the solvent front, 36-37 and 48-50 minutes at the ultraviolet absorbance measurement at 220 nm, and has very low peaks in near the solvent front, 32-39 and 45-52 minutes at the ultraviolet absorbance measurement at 260 nm. It reacts positively in the Molisch reaction, phenolic-sulfuric acid reaction, anthron-sulfuric acid reaction, indole-hydrochloric acid reaction and the Lowry-Folin reaction and negatively in Elemental analysis values C: 35.3-39.5%, H: 4.5-5.6%, N: 2.8-5.4% Its saccharide content, measured by a phenol-sulfuric acid method, is approx. 23.4 to about 45.5% by weight, expressed as glucose, and its protein content, measured by the Lowry-Folin method, is about 15.5 to about 28.0% by weight, expressed as cal. vserumalbumin; (H) wherein said TF-133a has the following properties: (a) (b) (c) (d) (e) (f) (9) Off-white to light brown powder. It prevents the growth of Ehrlich ascites tumor, Ehrlich solid tumor, sarcoma-180 carcinoma in mice and has an immunostimulatory effect. It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. It has no clear melting point, begins to decompose at about 110 ° C and decomposes considerably above 200 ° C. Its infrared absorption spectrum, obtained by a KBr tablet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 124o, 1140-iooo and szo cm-1. The ultraviolet absorption spectrum of its aqueous solution shows strong absorption at the absorption edge and shows an absorption peak in the vicinity of 258-262 nm. When fractionated by gel filtration (column: 44 mm diameter x 500 mm, eluent: 0.1 M phosphate buffer, pH 7) using Sephadex G-200, it has absorption bands at elution volumes close to * t (h) (in ) (j) (K) (I) wherein said TF-133 has the following properties: (a) (b) (0) (d) 446 406 the void volume - 300 and 630-940 ml in the ultraviolet absorbance measurement at 260 nm, an absorption band at elution volumes near the void volume - 420 ml at the absorbance measurement at 490 nm with a phenolic sulfuric acid method, and an absorption band at elution volumes near the void volume - 420 ml at the absorbance measurement at 620 nm with a anthron sulfuric acid method. Its high-resolution liquid chromatogram (eluent: 0.1M phosphate buffer, pH 7, flow rate: 0.8 ml / min, at room temperature) obtained using "TSK-GEL G3000 SW" 7.9 mm diameter x 600 mm x 2) has peaks in the vicinity of solvent 38 and 50 min. at the ultraviolet absorbance (column: front, measurement at 220 nm, and near the solvent front, 50-60 and 62 minutes at the ultraviolet absorbance measurement at 260 nm. It reacts positively in the Molisch reaction, phenolic-sulfuric acid reaction, anthron-sulfuric acid reaction, indole-hydrochloric acid reaction and Lowry-Folin reaction and negative in ninhydrin reaction Elemental analysis values C: 28.0-36.6%, H: 3.5-5.1%, H: 4.5-6.3% Its saccharide content, measured by a phenolic-sulfuric acid method, is about 26.0 to about 35.0% by weight, expressed as glucose, and its protein content, measured by the Lowry-Folin method, is about 22.0 to about 28.0. weight percent, expressed as calf serum albumin; Off-white to light brown powder.It prevents the growth of Ehrlich ascites tumor, mhrllch aunt tumor and uarkøm-lßè carcinoma hol men and has an immunostimulatory effect.It is soluble in water and insoluble in methanol, ethanol, acetone, benzene , chloroform, ethyl acetate and diethyl ether, it has no clear melting point, begins to decompose a (e) (f) tg) (h) (i) (j) (k) 87 446 406 at about 110 ° C and decomposes considerably above 200 ° C. Its infrared absorption spectrum, obtained by a KBr tablet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-iooo and azo am'1. The ultraviolet absorption spectrum of its aqueous solution shows strong absorption at the absorption edge and has an inflection point near 270-280 nm. When fractionated by gel filtration (column: 21 mm diameter x 400 mm, eluent: 0.1 M phosphate buffer, pH 7) using Sephadex G-200, it has an absorption band at elution volumes close to the void volume - 170 ml at the ultraviolet absorbance measurement at 260 nm, and an absorption band at elution volumes close to the empty volume - 150 ml at the absorbance measurement at 490 nm with a phenolic sulfuric acid method. Its high-resolution liquid chromatogram (eluent: 0.1M phosphate buffer, pH 7, flow rate: 0.8 ml / min, at room temperature) obtained using "TSK-GEL G3000 SW" (column: 7.9 mm diameter x 600 mm x 2), has peaks near the solvent front, 49-50 min. at the ultraviolet absorbance measurement at 220 nm, and in the vicinity of the solvent front, 38-39 and 52 min. at the ultraviolet absorbance measurement at 260 nm. It reacts positively in the Molisch reaction, the phenolic-sulfuric acid reaction, the anthron-sulfuric acid reaction, the indole-hydrochloric acid reaction and the Lowry-Folin reaction and negatively in the ninhydrin reaction. Elemental analysis values C: 31.1-38.5%, H: 3.9-5.2%, N: 3.4-4.7%. Its saccharide content, measured by a phenolic-sulfuric acid method, is about 19.0 to about 24.5% by weight, expressed as glucose, and its protein content, measured by the Lowry-Folin method, is about 12.9 to about 22.9% by weight. , expressed as calf serum albumin; G; 1, - w '111%),. \ -_ A__ i; »W y v 2 *: so (K) wherein the TF-1323 has the following properties: (a) Off-white to light brown powder. (b) It prevents the growth of Ehrlich ascites tumor, Ehrlich solid tumor and sarcoma-180 carcinoma in mice and has an immunostimulatory effect. (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. (d) It has no clear melting point, begins to decompose at about 110 ° C and decomposes significantly above 200 ° C. (e) Its infrared absorption spectrum, obtained by a KBr tablet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cnfl. VM (f) The ultraviolet absorption spectrum of its aqueous solution shows starch absorption at the absorption edge and has an inflection point near 265-280 nm. (G) The needle is fractionated by gel filtration (column: 21 mm x 400 mm , eluent: 0.1M phosphate buffer, pH 7) using Sephadex G-200, it has an absorption band at elution volumes close to the empty volume -, 160 ml at the ultraviolet absorbance measurement at 260 nm, and an absorption band at elution volumes near the empty volume - 150 ml at the absorbance measurement at 490 nm by a phenol-sulfuric acid method. (h) Its high-resolution liquid chromatogram (eluent: 0.1 m phosphate buffer, pH 7, flow rate: 0.8 ml / min at room temperature) obtained using "TSK-GEL G3000 SW" (column: 7.9 mm diameter x 600 mm x 2) has peaks near the solvent front, 36 and 48-50 minutes in the ultraviolet absorbance measurement at 220 nm, and peaks in the vicinity of the solvent front, 36 and 50 ml n. at the ultraviolet absorbance measurement at 260 nm. (i) It reacts positively in the Molisch reaction, phenolic-sulfuric acid reaction, anthron-sulfuric acid reaction, indole-hydrochloric acid reaction and the Lowry-Folin reaction and v. vi f = (L) 446 406. negative in the ninhydrin reaction. (j) Elemental analysis values C: 29.9-39.4%, H: 3.9-5.6%, N: 2.8-5.4%. (k) Its saccharide content, measured by a phenol-sulfuric acid method, is about 19.0 to about 25.0 weight percent, expressed as glucose, and its protein content, measured by the Lowry-Folin method, is about 11.0 to about 17%. .0% by weight, expressed as calf serum albumin; wherein the TF-136 has the following properties: (a) Off-white to light brown powder. (b) It prevents the growth of Ehrlich ascites tumor, Ehrlich solid tumor and sarcoma-180 carcinoma in mice and has an immunostimulatory effect. (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. (d) It has no clear melting point, begins to decompose at about 110 ° C and decomposes significantly above 200 ° C. (e) Its infrared absorption spectrum, obtained by a KBr tablet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cm -1. (f) The ultraviolet absorption spectrum of its aqueous solution shows strong absorption at the absorption edge and shows an absorption peak in the vicinity of 256-260 nm. (g) When fractionated by gel filtration (column: 44 mm diameter x 500 mm, eluent: 0.1 M phosphate buffer, pH 7) using Sephadex G-200, it has an absorption band at elution volumes close to 540-930 ml at the ultraviolet absorbance measurement at 260 nm, and all fractions have a weak absorption band at the absorbance measurement at 490 nm with a phenol-sulfuric acid method and at the absorbance measurement at 620 nm with an anthron-sulfur kidney method. (h) Its high-resolution liquid chromatogram (eluent: 0.1M phosphate buffer, pH 7, flow rate: 0.8 ml / min., at room temperature * obtained using ma., -Wßg fifi w in arg-sr ».w fi ff fl vïk- raw riff »Ü 4 ,. 1, rr _.:. «~ w». «~ .-- WÖ * K. 90 446 406 (i) (j) (k) av" TSK-GEL G3000 SW "(column: 7 , 9 mm diameter x 600 mm x 2) has peaks near the solvent front, 28-40, and 42-60 min at the ultraviolet absorbance measurement at 220 nm, and peaks near the solvent front and 42-60 min at the ultraviolet absorbance measurement at 260 nm. It reacts positively in Molisch reaction, phenolic-sulfuric acid reaction, anthron-sulfuric acid reaction, indole-hydrochloric acid reaction and Lowry-Folin reaction and negatively in ninhydrin reaction. Elemental analysis values C: 27.5-32.6%, H: 3.2-4.0%, N: 5.0-6.1%, its saccharide content, measured by a phenol-sulfuric acid method, is about 19.0 to about 30.0% by weight, expressed as glucose, and its protein content, measured by the Lowry-Folin method, is approx 6.0 to about 12.0 weight percent, expressed as calf serum albumin; (M) wherein said TF-140 has the following properties:. (b) (C) (d) (e) (f) (ä) _Gray white to light brown powder. It prevents the growth of Ehrlich ascites tumor in mice and has an immunostimulatory effect. It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. It has no clear melting point, begins to decompose at about 210 ° C and decomposes considerably above 280 ° C. Its infrared absorption spectrum, obtained by a KBr tablet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, l670-1640, l550, 1440-1380, 1240, 1140-iooo and szo cm'1. The ultraviolet absorption spectrum of its aqueous solution shows strong absorption at the absorption edge and shows an absorption peak in the vicinity of 255-260 nm. When fractionated by gel filtration (column: 44 mm diameter x 500 mm, eluent: 0.1 M phosphate buffer, pH 7) using Sephadex G-200, it has absorption bands at elution volumes close to ,, rs ešalvškjf f, rpsiMsrW -i », 9: 446 406 É 'the empty volume - 300, 500-900 and 900-1000 ml at the Ä ultraviolet absorbance measurement at 260 nm, and at ÄV elution volumes in the vicinity of the empty volume - fiffgi soo, aso-eso and sso-100 m1 in the absorbance measurement% \ M at 490 nm by a phenol-sulfuric acid method. (h) Its high-resolution liquid chromatogram (eluent: 0.1M phosphate buffer, pH 7, flow rate: 0.8 ml / min., at room temperature) obtained using TSK-GEL G3000 SWÜ (column: 7.9 mm diameter x 600 mm x'2) has peaks near the solvent front, J and 40-60 min. at the ultraviolet absorbance measurement at 220 nm and 260 nm. , (i) It reacts positively in the Molisch reaction, the phenolic-sulfuric acid reaction, the anthron-sulfuric acid reaction, in the indole-hydrochloric acid reaction and the Lowry-Folin reaction and in the negative reaction in the ninhydrin reaction. (j) Elemental analysis values C: 22.0-28.0%, H: 3.0-3.5%, N: 5.0-6.5%. (k) Its saccharide content, measured by a phenol-sulfuric acid method, is about 5.0 to about 15.0% by weight, expressed as glucose, and its protein content, measured by the Lowry-Folin method, is about 23.0 to about 32% by weight. .0% by weight, expressed as calf serum albumin; and (N) wherein said TF-150 has the following properties: (a) Off-white to light brown powder. ¿? F (b) It prevents the growth of Ehrlich ascites tumor in 1 Å mice and has an immunostimulatory effect. (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. (d) It has no clear melting point, begins to decompose at about 110 ° C and decomposes significantly above 200 ° C. (e) The ultzaviolet absorption spectrum of its aqueous solution shows strong absorption at the absorption edge and shows an absorption peak in the vicinity of 255-260 nm. M (f) Its infrared absorption spectrum, obtained with an íf fi niw s f? in? ._ hrs; q Å '3 32 446 406 1 KBr tablet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380 / 1240, 1140-1ooo and szo cm'1. (g) When fractionated by gel filtration (column 21 mm diameter x 400 mm, eluent; 0.1M phosphate buffer, pH 7) using Sephadex G-200, it has absorption bands at elution volumes close to the void volume - 100 and 100-160 ml in the ultraviolet absorbance measurement at 260 nm and an absorption band at elution volumes close to the empty volume - 150 ml in the absorbance measurement at 490 nm by a phenolic sulfuric acid method. (h) Its high-resolution liquid chromatogram (eluent: 0.1M phosphate buffer, pH 7, flow rate 0.8 ml / min., at room temperature) obtained using TsK-GEL G3000 SW (Column: 7.9 mm diameter x 600 mm'n 21 has peaks in the vicinity of the solvent front, 32, and 48-50 minutes at the ultraviolet absorbance measurement at 220 nm, and in the vicinity of the solvent front, 32, and 48-50 minutes at the ultraviolet absorbance measurement at 260 nm. (i) It ° reacts positively in the Molisch reaction, phenolic-sulfuric acid reaction, anthron-sulfuric acid reaction, indole-hydrochloric acid reaction and the Lowry-Folin reaction and negatively in the ninhydrin reaction. (j) Elemental analysis values C: 31.0- 34.0%, H: 4.0-4.4%, N: 2.8-3.2% (k) Its saccharide content, measured by a phenol-sulfuric acid method, is about 40.0 to about 55 , 0% by weight, expressed as glucose, and its protein content, measured by the Lowry-Folin method, is about 7.0 to about 14% by weight, expressed as calf serum albumin. A carcinostatic agent, characterized in that it comprises a carcinostatic substance TF-100, 110, 120, 130 (1316, 132a, 132b, 133a, 133b, 1323, 136), 140 or 150 according to claim 29 and a pharmaceutical aid. .m ~ »», - vm ~ .¿a-,: “- \ I> -w \ -w11cg; \, v q -. -vmmxfrfäbraßar / * ßr "» * «~ '* f'. ^ '" f' “* '.1 446 4os_ Carcinostatic agent according to claim 30, characterized in that the carcinostatic substance is TF-132a, 132b, 133a, 133b or 1323 according to claim 29.