BE884864A - NOVEL SUBSTANCES PRODUCED BY FUSOBACTERIUM BACTERIA, PREPARATION METHOD THEREOF AND THERAPEUTIC APPLICATION THEREOF - Google Patents

Description

       

    <EMI ID = 1.1>

  
tion of new substances with carcinostatic and immunostimulatory action, which consists in cultivating under anaerobic conditions bacteria producing TF substances, belonging to the genus Fusobacterium, and collecting the

  
  <EMI ID = 2.1>

  
substances resulting from the culture or its supernatant liquid (said new substance being called

  
  <EMI ID = 3.1>

  
TF substances obtained [they will include TF-100 below,
110, 120, 130 (1316, 132a, 132b, 133a, 133b, 1323, 136),
140 and 150], as well as a carcinostatic agent containing these substances.

  
In recent years, we have come to use extensively, to treat patients with different types of cancer, a treatment based on improving the immunological function of the host and on obtaining a carcinostatic effect with the using immunological function. Among the anti-tumor agents used in this treatment, there are known constituents obtained from organisms of various bacteria, or by culture of various bacteria or polysaccharides obtained from bodies of molds producing spores of Basidiomycetes or from their bodies of cultivated mushrooms. However, these anti-tumor agents are not yet satisfactory by their therapeutic effect, their harmful side effects and their method of production.

  
On the other hand, there are reports on an organism and a supernatant liquid obtained by cultivating bacteria belonging to the genus Fusobacterium, for example

  
  <EMI ID = 4.1>

  
Fusobacterium girans 1012 and Fusobacterium nucleatum 1010 [Dental Surgery, 23, 322-333 (1974), and 21, 534-539 (1972)
(Japanese);. However, no study has yet been done on the constituents obtained from a culture or its supernatant liquid, prepared by cultivating bacteria belonging to the genus Fusobacterium, nor on the pharmacological activities of these constituents.

  
The authors of the present invention studied the pharmacological activity of a supernatant liquid obtained by cultivating bacteria belonging to the genus Fusobacterium and by removing the organisms from the culture, and they discovered that a specific constituent obtained from the supernatant liquid had a carcinostatic action; that said constituent had practically no effect of inhibiting the formation of a colony of cancer cells in a test method for the formation of colonies, and had no carcinostatic action by destruction of the

  
  <EMI ID = 5.1>

  
that said constituent had a very low toxicity and could also be obtained by treating the culture.

  
An object of the present invention is to provide a process consisting in cultivating bacteria belonging to the genus Fusobacterium under anaerobic conditions, and in collecting the resulting culture or its liquid supernatant of new TF substances with carcinostatic and immunostimulatory action.

  
Another object of the invention is to provide said TF substances.

  
Another object of the invention is to provide a carcinostatic agent containing said TF substances.

  
Other objectives and advantages of the invention will emerge from the description which follows.

  
As bacteria used in the present invention, any TF substance-producing bacteria belonging to the genus Fusobacterium can be used, and, for example, Fusobacterium nucleatum is preferably used. Concretely, we use Fusobacterium nucleatum TF-031
(FARM 5077; ATCC 31647).

  
The bacteriological properties of Fusobacterium nucleatum TF-031 are as follows:

  
(I) Form

  

  <EMI ID = 6.1>


  
(II) Conditions for growth in a culture medium

  

  <EMI ID = 7.1>


  
surface growth: none, no growth

  
up! a depth

  
  <EMI ID = 8.1>

  
gas: None

  
(III) Physiological properties
  <EMI ID = 9.1>
 (12) Production of gas from saccharides:

  

  <EMI ID = 10.1>


  
The new TF substances according to the invention are obtained, for example, in the following manner.

  

  <EMI ID = 11.1>
 

  

  <EMI ID = 12.1>
 

  

  <EMI ID = 13.1>
 

  
We will now illustrate the abovementioned obtaining process.

  
(1) Culture of bacteria

  
Culture of bacteria belonging to the genus Fusobacterium is carried out by a usual method of culture of anaerobic bacteria. In other words, a culture medium is adjusted to a pH of 6 to 8.5, preferably 7.2 to 8.2

  
  <EMI ID = 14.1>

  
heart of calf, various peptones or similar products;

  
a source of vitamins, such as a yeast extract or the like; a mineral 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, and the bacteria are inoculated into the culture medium, after which a steady state culture is carried out under anaerobic conditions between 35 and 42 [deg.] C, preferably between 36 and 38 [deg.] C, for 1 to 5 days, preferably for 24 to 72 hours. In particular, it is desirable to use the culture medium described in Table I below (hereinafter called TF culture medium).

   However, an infusion of brainwort which is an extract of the brain-heart of calf is not always necessary as a carbon source and can be replaced by an infusion of heart which is an extract of calf heart, an extract of beef, a fish extract, corn maceration liquor, or a similar product. Among the various peptones, the protease peptone and the phytone peptone are not always necessary, and the trypticase peptone can be replaced by the polypeptone.

  
When agar is not used, it is desirable to proceed with a stirred culture.

TABLE I

  

  <EMI ID = 15.1>


  
(2) Collection of a culture supernatant liquid

  
(removal of organisms)

  
The organisms from the culture obtained above are removed to obtain a supernatant liquid. To collect the organisms, a conventional method can be used, for example centrifugation and a filtration method using a filter aid, such as "Hyflo Super Cel", and in particular a centrifugation method is preferable from the point of view ease of operation, degree of organism removal, and yield of supernatant. The organisms are preferably removed in this step, but they can be removed in the next step (3).

  
(3) Collection of the carcinostatic substance TF-100

  
A hydrophilic organic solvent is added to the supernatant liquid obtained above or to the entire culture, and the precipitate formed is collected. At this point, the supernatant or the culture is preferably adjusted to a pH of 2 to 7. The hydrophilic organic solvent can be, for example, an alcohol such as ethanol or methanol, or a ketone such as acetone, but it is the alcohols, in particular ethanol, which give the best results.

  
The hydrophilic organic solvent should be added so that its concentration is 30 to 70%, preferably 50 to 70%, by volume. After the addition of the hydrophilic organic solvent, the resulting mixture is left to stand at low temperature, preferably at about 5 ° C., for a period of several hours to several days, to complete the formation of the precipitate.

  
The precipitate thus formed is separated by usual methods such as decantation, centrifugation, filtration, etc.

  
Then an amount of water representing 10 to 15 times that of the precipitate obtained is added to the precipitate [the water can be replaced by a phosphate buffer or by a phosphate buffer containing sodium chloride when it is TF- 120 or 130 (1316, 132a, 132b, 133a, 133b,
1323 or 136) which is to be obtained as mentioned below] and the water-insoluble matter which has formed is eliminated by a usual process such as centrifugation, filtration, etc., after which it is collected the water soluble fraction. When the whole culture is used, the organisms are removed by the above-mentioned procedures. The water-soluble fraction thus obtained is dried, by lyophilization and the like, to obtain a carcinostatic substance called TF-100.

  
The TF-100 substance thus obtained has the properties indicated in Table II below.

  
(4) Collection of the carcinostatic substance TF-110

  
The water-soluble fraction obtained in (3) above is subjected to dialysis or ultrafiltration; or else TF-100 is dissolved in an amount of water representing 10 to 15 times that of TF-100, and the resulting solution is subjected to dialysis or ultrafiltration. Low molecular weight substances such as amino acids and minerals are removed by the above process. The internal solution obtained is collected by dialysis or ultrafiltration, then dried to obtain a carcinostatic substance called TF-110.

  
The substance TF-110 thus obtained has the properties indicated in Table II below.

  
  <EMI ID = 16.1>

  
The fraction is treated with an ion exchanger

  
  <EMI ID = 17.1>

  
saire, the internal solution obtained by subjecting the water-soluble fraction to a clause or an ultrafiltration [the internal solution obtained in (4) above], or else a solution of a powder of TF-110 in a small amount of water. As the ion exchanger,

  
  <EMI ID = 18.1>

  
weakly basic ion exchanger having the properties of a molecular sieve and, for example, suitable

  
  <EMI ID = 19.1>

  
ion exchanger, for example using a phosphate buffer having a pH of about 8, after which we do

  
  <EMI ID = 20.1>
-and the eluted solution is collected, then dried by lyophilization or by a similar process .; or else, we <EMI ID = 21.1>

  
  <EMI ID = 22.1>

  
stir the contents of the container, then filter, after which the filtrate is dried. One can thus obtain a carcinostatic substance called TF-120 having the properties described below. TF-120 can also be obtained by demineralizing the eluted solution or the filtrate, for example by dialysis using a

  
'' Cellophane *, or a similar means, using

  
  <EMI ID = 23.1>

  
Ltd.), or by ultrafiltration, then drying.

  
The TF-120 substance thus obtained has the properties indicated in Table II below.

  
(6) Collection of the carcinostatic substance TF-130

  
A column containing the adsorbed constituents is treated, from which TF-120 was removed by the treatment indicated in (5) above with a phosphate buffer, preferably a phosphate buffer having a sodium chloride concentration of 0.1. at 0.6 M and a pH of approximately 8 (below,

  
the phosphate buffer having a chloride concentration

  
  <EMI ID = 24.1> sodium chloride 0.1 - 0.6 M): or the water-soluble fraction obtained in (3) above is subjected, if necessary, to dialysis or ultrafiltration, followed by treatment with an ion exchanger using a 0.1 - 0.6 M sodium chloride-phosphate buffer, to obtain a fraction which is not eluted with a phosphate buffer free of sodium chloride but with 0.1 - 0.6 M sodium phosphate chloride buffer.

  
This procedure can be implemented either continuously in a column or discontinuously. Fraction

  
  <EMI ID = 25.1>

  
to get a substance

  
carcinostat called TF-130. The eluate containing the desired fraction can be demineralized, which is eluted with the aforementioned phosphate-sodium chloride buffer, for example by dialysis 3 using a "Cellophane" tube, by molecular sieving with using Sephadex G-25, or by ultrafiltration, then it is dried.

  
The designation "TF-130" used here collectively designates the desired eluted fractions obtained by treating the water-soluble fraction obtained in (3) above or, if necessary, the internal solution obtained by subjecting to the water-soluble fraction, dialysis or ultrafiltration, using an ion exchanger, fractions which are not eluted with a phosphate buffer devoid of sodium chloride, but eluted with a phosphate buffer -

  
  <EMI ID = 26.1>

  
ment, even example, the following fractions.

  
(i) Fraction which was not eluted with a phosphate buffer devoid of sodium chloride but was eluted with a 0.6 M sodium chloride phosphate buffer in the above-mentioned ion exchange treatment [this fraction is called TF-1316].

  
(ii) Fraction which has not been eluted with a buffer of

  
  <EMI ID = 27.1>

  
0.2 M sodium chloride-phosphate buffer in the above-mentioned ion exchange treatment [this fraction is called TF-132a and b; TF-132a is obtained by washing an ion exchanger containing the above-mentioned adsorbed constituent with a 0.1 M sodium chloride-phosphate buffer, treating the ion exchanger with a 0.2 M sodium chloride phosphate buffer and then collecting the fraction

  
  <EMI ID = 28.1>

  
and TF-132b is obtained by treating the soluble fraction in

  
  <EMI ID = 29.1>

  
(TF-110) obtained in (4) above with an ion exchanger].

  
  <EMI ID = 30.1>

  
0.2 M sodium phosphate chloride but was eluted with 0.3 M sodium chloride phosphate buffer in the above-mentioned ion exchange treatment [this fraction is called TF-133a and b; the symbols a and b have the same meanings as in the case of TF-132a and b, respectively].

  
(iv) Fraction which was not eluted with 0.1 M sodium chloride-phosphate buffer but was eluted with 0.3 M sodium chloride-phosphate buffer in the treatment

  
  <EMI ID = 31.1>

  
called TF-1323].

  
(v) Fraction which was not eluted with 0.5 M sodium chloride-phosphate buffer but was eluted with 0.6 M sodium phosphate-chloride buffer in the above-mentioned exchange exchange treatment ions [this fraction is called TF-136].

  
Since these substances TF-1316, 132a, 132b, 133a,
133b, 1323 and 136 have common properties on the following points, these substances having the common properties are

  
  <EMI ID = 32.1>

  
TF-130 obtained as described above has the following properties:
(a) Grayish-white to light brown powder.
(b) It inhibits the proliferation of Ehrlich ascites tumor in mice, Ehrlich's consistent tumor and Sarcoma-180 carcinoma, and it has an immunostimulatory action.
(c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether.

   (d) It does not have a well-defined melting point, it begins to decompose at around 110 [deg.] C, and it decomposes remarkably above 200 [deg.] C.
(e) Its infrared absorption spectrum obtained by the KBr pellet method has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670- <EMI ID = 33.1>
(f) The ultraviolet absorption spectrum of its aqueous solution exhibits a strong absorption at the absorption edge, and an absorption peak in the vicinity of 256-280 nm.

  
(g) It is positive. to Molisch's reaction, to

  
the phenol-sulfuric acid reaction, the anthrone-sulfuric acid reaction, the indole-hydrochloric acid reaction, and the Lowry-Folin reaction, and negative to the ninhydrin reaction

  
(h) Elementary analysis:

  

  <EMI ID = 34.1>


  
(i) Its saccharide content, measured by the method

  
  <EMI ID = 35.1>

  
by weight (expressed as glucose), and its protein content, measured by the Lowry-Folin method, is in the region of 6.0 to 28.0% by weight (expressed as bovine serum albumin).

  
The aforementioned ion exchange treatment is explained in more detail below. As the ion exchanger used in the following procedures, a weak basic ion exchanger or a weak basic ion exchanger having the properties of a molecular sieve is preferred.

  
  <EMI ID = 36.1>

  
Sephadex A-50 used in (5) above, and the following process can be carried out batchwise.

  
(i) A 0.6 M sodium phosphatechloride buffer is passed through a column of an ion exchanger containing the adsorbed component in which the solution to be treated is passed through (5) above. preferably a pH of about 8, and the eluted fraction is collected, optionally concentrated and demineralized by dialysis, ultrafiltration or a similar process, and then dried by lyophilization or by a similar process to obtain a carcinostatic substance called TF-1316.

  
The substance TF-1316 thus obtained has the properties indicated in Table II below.

  
(ii) -l A phosphate buffer is passed through a column of an ion exchanger containing the adsorbed constituent in which the solution to be treated is passed through (5) above. 0.1 M sodium chloride preferably having a pH of approximately 8, after which a phosphate buffer 0.2 M sodium chloride preferably having a pH of approximately 8 is passed through the column, and collecting the fraction eluted, it is optionally concentrated and demineralized by dialysis, by ultrafiltration or by a similar process, then it is dried by lyophilization or by a similar process, to obtain a carcinostatic substance called TF-132a.

  
. The substance TF-132a thus obtained has the properties indicated in Table II below.

  
(ii) -2 Procedures are used to collect the fraction which has not been eluted with a 0.1 M sodium chloride-phosphate buffer but has been eluted with a buffer. 0.2 M sodium phosphate-chloride in the ion exchange treatment, using the water-soluble fraction obtained in (3) above or the internal solution obtained in (4) above, to obtain the substance TF-132b. In other words, the column packed with the ion exchanger is equilibrated with a 0.2-0.3 M sodium chloride phosphate buffer, preferably having a pH of approximately 8, after which the above buffer is passed through the column. 0.2-0.3 M sodium phosphate-chloride from the water-soluble fraction obtained in (3) above

  
  <EMI ID = 37.1>

  
soluble in water on dialysis or ultrafiltration, and the eluted solution is collected. If necessary, the column is washed with the aforementioned 0.2-0.3 M sodium chloride-chloride buffer, and the eluted solution and the washings are combined. The ion exchange treatment can be repeated several times.

   Then passed through a column in which the ion exchanger has been equilibrated with a 0.1 M sodium chloride-phosphate buffer having, preferably a pH of approximately 8, a solution prepared by diluting the eluted solution. above with a phosphate buffer such that the sodium chloride concentration is 0.1 M, and the fraction which has been eluted is separated with the above 0.1 M sodium phosphate phosphate buffer, after which the above-mentioned 0.2 M 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 is collected which has not been adsorbed by an ion exchanger balanced with the aforementioned 0.2 M sodium chloride-phosphate buffer,

  
  <EMI ID = 38.1>

  
the aforementioned 0.1 M sodium phosphate-chloride buffer, washed with the aforementioned 0.1 M sodium phosphate-chloride buffer, then eluted with the aforementioned 0.2 sodium phosphate-sodium chloride buffer M to obtain an eluate. It is also possible to use a process according to which, conversely, the fraction which has been adsorbed by an ion exchanger balanced with the abovementioned 0.1 M sodium chloride-phosphate buffer is collected, and then a process for separating said fraction from a

  
fraction which has been adsorbed by an ion exchanger balanced with the aforementioned 0.2 M sodium chloride-phosphate buffer, and a fraction which has been eluted with the aforementioned 0.2 M sodium chloride phosphate buffer is collected.

  
The eluate obtained is optionally concentrated as described above and demineralized by dLaly se, by ultrafiltration or by a similar process, then dried by lyophilization or by a similar process, to obtain a carcinostatic substance called TF- 132b.

  
The substance TF-132b thus obtained has the properties indicated in Table II below.

  
(iii) -l) The internal solution obtained in (4) above is passed through a column of a balanced ion exchanger

  
  <EMI ID = 39.1>

  
approximately, and a 0.2 M sodium chloride-phosphate buffer is preferably passed through said column to wash the column, preferably having a pH of approximately 8, then a phosphate buffer is passed through said column. 0.3 M sodium chloride preferably having a pH of about 8, or else passing the aforementioned phosphate-chloride buffer

  
  <EMI ID = 40.1>

  
which still contains the remaining adsorbed component. The eluted fraction is collected and optionally concentrated.

  
  <EMI ID = 41.1>

  
by a similar process, then dried by lyophilization or a similar process to obtain a carcinostatic substance called TF-133a.

  
The substance TF-133a thus obtained has the properties indicated in Table II below.

  
(iii) - 2) We collect in the following manner, which is the

  
  <EMI ID = 42.1>

  
was not eluted with 0.2 M sodium chloride phosphate buffer but was eluted with 0.3 M sodium phosphate phosphate buffer in the ion exchange treatment, to obtain TF-133b. In other words, the column packed with the ion exchanger is balanced with a 0.3 M sodium chloride phosphate buffer, preferably having a pH of approximately 8, after which the abovementioned phosphate buffer is passed through the column. 0.3 M sodium chloride from the water soluble fraction obtained in (3) above or the internal solution obtained by subjecting the water soluble fraction to dialysis or ultrafiltration, and collecting

  
the solution eluted. If necessary, the column is washed with the abovementioned 0.3 M sodium chloride-phosphate buffer, and the eluted solution and the washing waters are combined. The ion exchange treatment can be repeated several times. Next, a solution prepared by diluting the solution is passed through a column in which the ion exchanger has been balanced with a 0.2 M sodium chloride-phosphate buffer having, preferably, a pH of approximately 8. eluted above with a phosphate buffer so that the sodium chloride concentration is equal to

  
0.2 M, and the fraction which has been eluted with the above-mentioned 0.2 M sodium chloride buffer is separated, after which the above 0.3 M sodium phosphate chloride buffer is passed through the column. to get an eluate.

  
In the concrete example of the above-mentioned ion exchange treatment, a fraction is collected which has not been adsorbed by an ion exchanger balanced with the aforementioned 0.3 M sodium chloride-phosphate buffer, it is adsorbed by an ion exchanger balanced with the aforementioned 0.2 M sodium chloride-phosphate buffer, washed with the aforementioned 0.2 M sodium chloride-phosphate buffer, then eluted with the buffer above-mentioned 0.3 M sodium chloride-phosphate to obtain an eluate. It is also possible to use a method according to which, conversely, a fraction which has been adsorbed by an ion exchanger balanced with the abovementioned phosphate chloride chloride is collected.

  
  <EMI ID = 43.1>

  
said fraction from a fraction which has been adsorbed by an ion exchanger balanced with the aforementioned buffer of sodium phosphate 0.3 M, and a fraction which has been eluted with the aforementioned buffer of sodium phosphate 0 is collected. , 3 M.

  
Then, the eluate obtained in the manner described above is optionally concentrated and demineralized by dialysis, by ultrafiltration or by a similar process, then it is dried by lyophilization or by a similar process, to obtain the desired carcinostatic substance, called TF-133b.

  
The substance TF-133b thus obtained has the properties indicated in Table 2 below.

  
(iv) The water-soluble fraction obtained in (3) above or the internal solution obtained in (4) above is placed in the form of a 0.3 M sodium chloride-phosphate buffer. preferably having a pH of approximately 8, and the buffer obtained is passed through an ion exchange column which has been previously equilibrated with a 0.3 M sodium phosphate chloride buffer having preferably a pH around 8.

   The eluted solution is adjusted by adding a phosphate buffer so that the sodium chloride concentration is 0.1 M, then it is passed through an ion exchange column which has been previously balanced with a 0.1 M sodium phosphate-chloride buffer preferably having a pH of approximately 8, and then the said 0.3 M sodium phosphate-chloride buffer is passed through said column, after which it is collected the fraction eluted, it is optionally concentrated and demineralized by dialysis, by ultrafiltration or by a similar process, then it is dried by lyophilization or by a similar process, to obtain a carcinostatic substance called TF-1323.

  
The substance TF-1323 thus obtained has the properties indicated in Table II below.

  
(v) The internal solution obtained in (4) above is passed through a column containing an ion exchanger balanced with a phosphate buffer, preferably having a pH of approximately 8, and it is passed through said column, to washing the column, a 0.5 M sodium chloride-phosphate buffer preferably having a pH of approximately 8, then a 0.6 M sodium phosphate chloride buffer passing preferably through said column pH of about 8, well, the above-mentioned 0.5 M sodium chloride-phosphate buffer which has been

  
  <EMI ID = 44.1>

  
adsorbed remaining, after which the aforementioned 0.6 M sodium chloride-buffer is passed through said column. The eluted fraction is collected, it is optionally concentrated and demineralized by dialysis, by ultrafiltration or by a similar process, then it is dried by lyophilization or by a similar process, to obtain a carcinostatic substance called TF-136.

  
  <EMI ID = 45.1>

  
properties indicated in Table II below.

  
(7) Collection of the carcinostatic substance TF-140

  
  <EMI ID = 46.1>

  
supernatant liquid obtained in (1) or (2) above, or to the water-soluble fraction obtained in (3) above, or to the solution obtained by dissolving TF-100 in water, so as to form a barium salt, after which the precipitate is collected, then the barium is removed, and the water-soluble fraction is collected, then subjected to dialysis or to ultrafiltration to obtain TF -140. To be more precise, an aqueous barium solution is added, preferably a

  
  <EMI ID = 47.1>

  
its supernatant liquid obtained in (1) or (2) above, or to the water-soluble fraction obtained in (3) above, or to an aqueous solution obtained by dissolving TF-100 in a quantity of water representing about 10 to
15 times that of TF-100, and the pH of the resulting mixture is preferably adjusted between 10 and 13 to form a barium salt. A precipitate is deposited under the effect of the addition of a barium solution, and the quantity of barium ions added is preferably adjusted so that the concentration of total barium ions in the final volume of the mixture is 0.005 at 0.1 M. The mixture thus obtained is filtered, and the barium is separated from the barium salt obtained.

   The barium is preferably removed by adding the barium salt to sodium sulfate, preferably in the form of a 5-15% aqueous solution, stirring the resulting mixture, then filtering to obtain a solution. The precipitate which has been separated and removed is washed again with an aqueous solution of sodium sulfate, and the washings are combined with the above-mentioned solution. The solution thus obtained is subjected to dialysis, ultrafiltration or similar treatment to remove excess sodium sulfate or low molecular weight substances, then the resulting solution is dried by lyophilization or by a similar process to obtain a carcinostatic substance

  
  <EMI ID = 48.1>

  
The substance TF-140 thus obtained has the properties indicated in Table II below.

  
(8) Collection of the carcinostatic substance TF-150

  
The water-soluble fraction obtained in (3) above or the internal solution obtained in (4) above is treated with a quaternary ammonium salt, and the precipitate thus formed is collected and then subjected to it. dissociation 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, then dried to obtain TF-150. The quaternary ammonium salt which can be used here generally can be in the form of any complex with a polysaccharide, and particularly preferred examples of this

  
salt include cetyl pyridinium chloride, bromide

  
  <EMI ID = 49.1>

  
Reference to treatment with a quaternary ammonium salt in the presence of a buffer such as a borate buffer or a similar buffer. The concrete description of the above-mentioned treatment with a quaternary ammonium salt and of the dissociation procedure using a solution containing sodium chloride is as follows. For example, an aqueous solution of a quaternary ammonium salt is added dropwise to the water-soluble fraction obtained in (3) above or to the internal solution obtained in (4) above, all keeping the pH low

  
  <EMI ID = 50.1>

  
for a period of 10 minutes to several hours, after which the precipitate which has settled is collected. This precipitate is washed several times with a buffer, preferably a

  
  <EMI ID = 51.1>

  
sodium and a small amount of a quaternary ammonium salt and then dissociate it with a buffer, preferably a 0.1% borate buffer, containing 0.5 M sodium chloride and a small amount of a quaternary ammonium salt, in order to obtain a soluble fraction, then the said soluble fraction is preferably adjusted to pH .2 - 6. Then a hydrophilic organic residue, preferably an alcohol, is added to the soluble fraction so that its concentration

  
  <EMI ID = 52.1>

  
volume, and the mixture thus obtained is allowed to stand in

  
  <EMI ID = 53.1>

  
hours, after which the precipitate which is deposited is collected to obtain a carcinostatic substance called TF-150.

  
The TF-150 substance thus obtained has the properties indicated in Table II below.

  

  <EMI ID = 54.1>


  

  <EMI ID = 55.1>
 

  

  <EMI ID = 56.1>


  

  <EMI ID = 57.1>
 

  

  <EMI ID = 58.1>


  

  <EMI ID = 59.1>
 

  

  <EMI ID = 60.1>


  

  <EMI ID = 61.1>
 

  

  <EMI ID = 62.1>


  

  <EMI ID = 63.1>
 

  

  <EMI ID = 64.1>


  

  <EMI ID = 65.1>
 

  

  <EMI ID = 66.1>


  

  <EMI ID = 67.1>
 

  

  <EMI ID = 68.1>


  

  <EMI ID = 69.1>
 

  

  <EMI ID = 70.1>


  

  <EMI ID = 71.1>
 

  

  <EMI ID = 72.1>


  

  <EMI ID = 73.1>
 

  

  <EMI ID = 74.1>


  

  <EMI ID = 75.1>
 

  

  <EMI ID = 76.1>


  

  <EMI ID = 77.1>
 

  

  <EMI ID = 78.1>


  

  <EMI ID = 79.1>
 

  

  <EMI ID = 80.1>


  

  <EMI ID = 81.1>
 

  
In Table II, for the Sephadex G-50 marked "*", a column 50 mm in diameter and of
600 mm high, and distilled water as eluent. ; for

  
  <EMI ID = 82.1>

  
se a column of 44 mm in diameter and 500 mm high for TF-110, 120, 132a, 133a, 136 and 140, and a column of

  
  <EMI ID = 83.1>

  
133b, 1323 and 150, and as an eluent, a 0.1 M phosphate buffer with a pH of 7 was used for all substances and, to obtain the high performance liquid chromatogram, mark "***" , a column of 7.9 mm in diameter x 600 mm x 2 was used, containing TSK-GEL G300SW (trade name of a product sold by the company Toyo Soda Co., Ltd.), and we proceeded to elution at room temperature at a flow rate of 0.8 ml / min using a phosphate buffer having a pH of 7 as eluent.

  
The pharmacological activities of the present carcinostatic substances TF-100, TF-110, TF-120, TF-1316,

  
  <EMI ID = 84.1>

  
TF-150 are:

  
(1) Effect of TF substances on cell viability

  
(Colon formation test)

  
Based on Kim's method. J.H. et al. [Cancer Res. 25, 698 (.1965)], HeLa 8-3 cells were cultured on MEM Eagle supplemented with serum, [deg.] 10% calf, pendant.

  
3 to 5 days at 37 [deg.] C, in a carbon dioxide incubator, after which the culture is removed, and a solution of PBS (-} (solution) is added to the cells adhering to the glass.

  
  <EMI ID = 85.1>

  
potassium chloride, 1.15 g / l of monobasic sodium phosphate and 0.2 g / l of dibasic potassium phosphate) containing 0.01% by weight of ethylenediaminetetraacetic acid and 0.1% of trypsin. The cells were detached from the glass surface of the culture flask and dissociated into single cells by pipetting, then the number of cells was counted by microscopy. The cell suspension was diluted with MEM Eagle supplemented with serum

  
  <EMI ID = 86.1>

  
per ml. After inoculating 1 ml of the cell suspension in Petri dishes and cultivating it in

  
  <EMI ID = 87.1>

  
added to the cell suspension the supernatant liquid obtained in Example 1 (1), which appears below, so that the concentration changes from 1/16 to 1/128. Then, to each of the 1 ml fractions of the cell suspension cultured at 37 [deg.] C for 24 hours, each of the test substances was added and in each case, the HeLa cells were cultured for 7 days. After this culture, the culture medium was separated, after which the culture dishes were washed with Hank's solution, and the cells were fixed with ethanol at 70% by weight, then washed. with 100% by weight ethanol. After removing the ethanol, the cells were stained with a solution of

  
  <EMI ID = 88.1>

  
calculated the viability of the cells.

  
  <EMI ID = 89.1>

  
IV below. The cell viability was calculated in the Tables from the following equation. Viability

  
  <EMI ID = 90.1>

  
as an average of five identical trials.

  

  <EMI ID = 91.1>
 

  

  <EMI ID = 92.1>


  

  <EMI ID = 93.1>
 

  
TABLE IV: Effect of TF substances on viability

  
cells
  <EMI ID = 94.1>
 As this experiment proves, the TF substances according to the present invention have a very weak cytotoxic effect on HeLa cells, compared to the supernatant.

  
(II) Immunostimulatory activity

  
Four mice of ICR strain were used for each group. Each of the test substances was dissolved in 0.2 ml of physiological saline solution, and then administered intraperitoneally to the mice. Twenty-four hours after administration, 0.2 ml of a carbon suspension prepared by mixing 1 ml of Perikan drawing ink (manufactured by GUnther-Wagner Co., Ltd.) was injected intravenously into the tail of the mice. ) and 2 ml of a physiological saline solution containing 3% by weight

  
  <EMI ID = 95.1>

  
injection, 0.02 ml of blood was collected from the orbit of the mouse eye, using a hepatin coated hematocrit capillary, and the blood was immediately diluted and hemolyzed with 1.6 ml of an aqueous solution at 0.1% by weight of sodium carbonate. This suspension was subjected to colorimetry at 675 nm, and the phagocytotic index, i.e. the K value, was determined from the equation of Halpern et al.

  
To the mice in the control group, 0.2 ml of physiological saline was administered.

  

  <EMI ID = 96.1>


  
  <EMI ID = 97.1>

  
  <EMI ID = 98.1>

  
at time t.

  
The results are shown in Tables V to VII below.

TABLE V

  

  <EMI ID = 99.1>
 

TABLE VI

  

  <EMI ID = 100.1>

TABLE VII

  

  <EMI ID = 101.1>


  
As shown in Tables V to VII, the groups, to which each of the TF substances according to the present invention were administered, showed activation of the reticuloendothelial macrophages, compared to the control group, and the cellular immunity of normal mice was increased.

  
(III) Carcinostatic activity
(a) Anti-tumor activity against Ehrlich's ascites tumor

  
Ehrlich ascites tumor cells were inoculated intraperitoneally with ICR strain mice
(females, 6 weeks old) at a rate of 1 x 105 cells per mouse. Then, each of the test substances was dissolved in 0.2 ml of physiological saline, then administered intraperitoneally to mice once a day for 7 days from the first day after inoculation of the cells. tumors, or once a day on the first day and from the third to the seventh day for a total of 6 days. In the case of the test substances in Table IX, each was administered to mice once a day on the first day and from the third day to the seventh day for a total of 6 days after inoculation of the tumor cells. The control group was administered 0.2 ml of physiological saline,

  
  <EMI ID = 102.1>

  
The results are given in Tables VIII and IX below.

  

  <EMI ID = 103.1>
 

  

  <EMI ID = 104.1>


  

  <EMI ID = 105.1>


  

  <EMI ID = 106.1>
 

  

  <EMI ID = 107.1>


  

  <EMI ID = 108.1>


  

  <EMI ID = 109.1>
 

  

  <EMI ID = 110.1>


  

  <EMI ID = 111.1>


  

  <EMI ID = 112.1>
 

  
As shown in Tables VIII and IX, the group to which each of the TF substances according to the invention was administered benefited from the life-prolonging effect, compared with the control group.

  
  <EMI ID = 113.1> consistent from Ehrlich

  
(1) Ehrlich tumor cells were transplanted subcutaneously in the armpit to ICR strain mice (females, 6 weeks old), at a rate of

  
4 x 106 cells per mouse. Each of the test substances was then 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 on the first day and from the third day to the seventh day for 6 days in total. The control group was administered 0.2 ml of physiological saline, in the same manner as described above. The tumor weights were determined on the 11th or 14th day after the transplantation of the tumor cells. The tumor weights were determined by measuring the large diameter (a) mm and the small diameter (b)

  
  <EMI ID = 114.1>

  
next :

  

  <EMI ID = 115.1>


  
The results are given in Table X below.

  

  <EMI ID = 116.1>


  

  <EMI ID = 117.1>


  

  <EMI ID = 118.1>
 

  

  <EMI ID = 119.1>


  

  <EMI ID = 120.1>


  

  <EMI ID = 121.1>
 

  
(2) Ehrlich tumor cells were transplanted subcutaneously into the armpit of ICR strain mice (females, 6 weeks old) at a rate of

  
  <EMI ID = 122.1>

  
each of the test substances to mice divided into groups for intravenous administration, intraperitoneal administration, subcutaneous administration and intramuscular administration, at a dosage of

  
10 mg / kg or 20 mg / kg once a day on the first day and from the third day to the seventh day, a total of 6 days after transplanting the tumor cells. The control group was administered 0.2 ml of physiological saline 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 given in Table XI below.

  

  <EMI ID = 123.1>
 

  
As shown in Tables X and XI, an effect of inhibiting tumor growth is observed in the groups to which one of the TF substances according to the invention has been administered.

  
(c) Anti-tumor activity against carcinoma

  
Sarcoma 180

  
We transplanted intraperitoneally

  
  <EMI ID = 124.1>

  
(females, 6 weeks old) at a rate of 1 x 10 cells per mouse. Thereafter, each of the test substances was administered intraperiorally to the mice at a dose of mg / kg, 5 mg / kg or 10 mg / kg, once daily from the first day to the seventh day, i.e. for 7 days. total days, after tumor cell transplantation or once a day on the first day and from the third day to the seventh day for a total of 6 days. 0.2 ml of physiological saline was administered to the control group , in the same way as above.

  
  <EMI ID = 125.1>

  
below.

  

  <EMI ID = 126.1>


  

  <EMI ID = 127.1>


  

  <EMI ID = 128.1>
 

  

  <EMI ID = 129.1>


  

  <EMI ID = 130.1>


  

  <EMI ID = 131.1>
 

  
As shown in Table XII, the groups to which one of the TF substances according to the invention was administered had benefited from tumor activity, in comparison with the control group.

  
(d) Antitumor activity against melanoma B-16
(1) B-16 melanoma tumor cells were transplanted subcutaneously into the armpit of BDF1 strain males (males, 7 weeks old) at a rate of 1 × 10 6 cells per mouse. Subsequently, the TF- substance was administered intraperitoneally to the mice.
133a 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 tumor cells. In the same manner as above, the control group was administered 0.2 ml of physiological saline solution and 20 mg / kg of substance called "5-FU" at

  
another group formed for this purpose-

  
The results are given in Table XIII below.

TABLE XIII

  

  <EMI ID = 132.1>


  
As shown in Table XIII, the groups administered TF-133a benefited from apparent tumor growth inhibiting activity compared to the control group, and the groups administered either 5 mg / kg, or 10 ng / kg of TF-133a, benefited from an activity of inhibition of tumor growth equal and more remarkable respectively, in comparison with, the group to which the substance 5-FU was administered.

  
(2) We transplanted

  
  <EMI ID = 133.1>

  
TF-132a, TF-133a, 5-FU and physiological saline

  
  <EMI ID = 134.1>

  
day after tumor cell transplant, mice were dissected, and metastasis of melanoma tumor cells was observed in the lungs.

  
The results are given in Table XIV below.

  
  <EMI ID = 135.1>

  

  <EMI ID = 136.1>


  
As shown in Table XIV, in the groups administered either TF-132a or TF-133a, metastasis of B-16 melanoma tumor cells was remarkably inhibited compared to the group administered administered the

  
  <EMI ID = 137.1>

  
(IV) Acute toxicity

  
  <EMI ID = 138.1>

  
in Table XV below.

TABLE XV

  

  <EMI ID = 139.1>
 

  
As demonstrated by the above pharmacological experiments, the TF substances according to the invention are useful as carcinostatic agents, it can be expected to have activities against various cancer diseases, and it can be expected that they have remarkable activities against particularly recalcitrant cancers. All the TF substances according to the invention have excellent effects, but from a certain number of points of view, it is the TF-130 substance which is preferred, and in particular the substances 132a, 132b, 133a, 133b and
1323.

  
The TF substances according to the invention can be used after having put them in various pharmaceutical forms

  
  <EMI ID = 140.1>

  
suppositories, etc., but they are preferably used in the pharmaceutical form of an injection. When used in the form of oral medications, these medications can contain a variety of excipients, and can be taken as capsules, tablets, powders or granules. When used in the form of injections, the latter may be subcutaneous, intramuscular or intravenous injections, and they are used in the form of a suspension, solution or

  
  <EMI ID = 141.1>

  
injectables may contain local anesthetic-

  
The dosage of TF substances according to the invention is chosen appropriately depending on the condition of the patient, but in general, it is desirable to administer them, for an adult, at a dose of 0.01 to 50 mg / kg once a day or several times a day, and as for the mode of administration, they are preferably administered by subcutaneous, intramuscular or intravenous injections, or else by injections in the diseased part.

  
The invention will be better explained below with reference to Examples and the accompanying drawings, in which Figure 1 shows a microscopic photograph showing the form of Fusobacterium nucleatum TF-031 used in this 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 diagram obtained when said substance was subjected to gel filtration using Sephadex G-50; Figure 5 shows a high performance liquid 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 diagram obtained when said substance was subjected to gel filtration using Sephadex G-200; Figure 9 shows a high performance liquid 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 diagram obtained when said substance was subjected to gel filtration using Sephadex G-2; Figure 13 shows a high performance liquid 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 diagram obtained when said substance was subjected to gel filtration using Sephadex G-200; Figure 17 shows a high performance liquid 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 <EMI ID = 142.1>

  
obtained when said substance 3 was subjected to gel filtration using Sephadex G-200; Figure 21 shows a high performance liquid 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 diagram obtained when said substance was subjected to gel filtration using Sephadex G-200; Figure 25 shows a high performance liquid 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 a diagram <EMI ID = 143.1>

  
gel filtration using Sephadex G-200; Figure 29 shows a high performance liquid chromatogram of said substance; Figure 30 shows an infrared absorption spectrum of the carcinostatic substance TF-133b; Figure 31 shows an ultraviolet absorption spectrum of said substance; Figure 32 shows an elution diagram obtained when said substance was subjected to gel filtration using Sephadex G-200; Figure 33 shows a high performance liquid 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 diagram obtained when said substance was subjected to gel filtration using Sephadex G-200; Figure 37 shows a high performance liquid chromatogram of said substance; Figure 3R shows an infrared absorption spectrum of the carcinostatic substance TF-136; Figure 39 shows an ultraviolet absorption spectra of said substance; Figure 40 shows an elution diagram obtained when said substance was subjected to gel filtration using Sephadex G-200; the figure
41 represents a high performance liquid chromatogram of said substance; Figure 42 shows an infrared absorption spectrum of the carcinostatic substance TF-14 # ; FIG. 43 represents an ultraviolet absorption spectrum of said substance:

   Figure 44 shows an elution diagram obtained when said substance was subjected to gel filtration using Sephadex G-200; and Figure 45 shows a high performance liquid 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 diagram obtained when said substance was subjected to gel filtration using Sephadex G-200; and Figure 49 shows a high performance liquid chromatogram of said substance.

Example 1

  
(1) In each of 15 2-liter culture bottles each fitted with a threaded stopper, two liters of culture medium containing 34 g of try.pticase peptone, 6 g of phytone peptone were placed; 20 g of proteose peptone,
70 g of a brain and heart infusion, 6 g of yeast extract, 15 q of sodium chloride, 12 g of glucose,

  
10 g lactose, 0.5 g L-cystine, 0.2 g sodium sulfite, 1.0 g sodium thioglycollate and 1.4 g agar, and the culture medium was adjusted to pH of 7.

  
  <EMI ID = 144.1>

  
for 15 minutes, it was warmed in a boiling water bath for 20 minutes and immediately after

  
cooled with water, after which a solution of Fusobacterium preculture was inoculated into the above water-cooled culture medium under sterile conditions

  
  <EMI ID = 145.1>

  
medium having the same composition as above, in an amount of 100 ml per culture bottle, and it was subjected to a steady state culture in an incubator at 37 [deg.] C for 48 hours. After completion of the culture, the culture was centrifuged, to remove organisms, at 4000 rpm at 5 [deg.] C for 20 minutes. The volume of the supernatant obtained was approximately 27 liters.

  
(2) To the supernatant liquid obtained in (1) above, 40 liters of ethanol were added, with stirring, at 5 [deg.] C, and the resulting mixture was allowed to stand in a room at low temperature until the amorphous precipitate is completely deposited. Then, the mixture was centrifuged at 6000 rpm at 5 [deg.] C for 15 minutes, and collected

  
  <EMI ID = 146.1>

  
under reduced pressure to obtain approximately 60 g of bcute powder.

  
(3) In 120 ml of water, 20 g of the raw powder obtained in (2) above were dissolved, and the water-insoluble matter formed at that time was separated by centrifugation.

  
  <EMI ID = 147.1>

  
the water-soluble fraction thus obtained in the wash waters obtained by washing the water-insoluble materials twice with 60 ml water fractions, and the resulting solution was dried under reduced pressure to obtain 14 g of '' a greyish-white to light brown powder of TF-100.

Example 2

  
The solution described in Example 1- was subjected to

  
(3), which had been prepared by combining the water-soluble fraction devoid of materials insoluble in

  
  <EMI ID = 148.1>

  
insoluble in water, ultrafiltration using a hollow fiber type ultrafiltration device
(membrane N [deg.] HI-1: manufactured by Asahi Kasei Kogyo Kabushiki Kaisha), and the internal solution was subjected to lyophilization to obtain 10 g of a grayish white to light brown powder of TF-110

Example 3

  
In a small amount of water, 10 g of

  
  <EMI ID = 149.1>

  
resulting solution in a column packed with diethylamino-

  
  <EMI ID = 150.1>

  
0.025 M having a pH of 8, and the solution was collected

  
  <EMI ID = 151.1>

  
  <EMI ID = 152.1>

  
eluted solution obtained with the eluted solution previously collected, after which the resulting solution was demineralized using a hollow fiber ultrafiltration device (Membrane N [deg.] HI-1), it was concentrated under reduced pressure, then lyophilized to obtain 470 mg of a greyish-white to light brown powder

  
from TF-120.

Example 4

  
In 24 ml of water, 2 g of the powder obtained in Example 1- (2) were dissolved, and the water-insoluble matters were separated by centrifugation at 7500 rpm for
10 minutes. The water-soluble fraction was dialyzed overnight by exchange with distilled water at 5 ° C. using a "Cellophane" tube and the internal solution was concentrated under reduced pressure. Then the concentrated solution was passed through a packed column

  
  <EMI ID = 153.1>

  
previously equilibrated with a 0.025 M phosphate buffer having a pH of 8, and the column was washed with 600 ml of a 0.025 M phosphate buffer having a pH of 8, after which it was passed through the column a 0.025 M phosphate buffer having a sodium chloride concentration of 0.6 M and a pH of 8, to obtain an eluate. This eluate was concentrated under reduced pressure to approximately 100 ml, it was dialyzed by exchange with distilled water at 5 [deg.] C, it was again concentrated under reduced pressure, it was demineralized using a Sephadex G-25 column, and then freeze-dried to get
470 mg of a greyish-white to light brown powder of TF-1316.

Example 5

  
The solution described in Example 1- (3), which had been prepared by combining the water-soluble fraction devoid of water-insoluble matter, was dialyzed overnight to the washing waters obtained by washing the water insoluble matter, by exchange with distilled water at 5 [deg.] C using a "Cellophane" tube, and the internal solution was concentrated under reduced pressure.

   The concentrated solution was then passed through a column packed with 50 g of diethylaminoethyl-Sephadex A-50 which had been previously equilibrated with a 0.025 M phosphate buffer having a pH of 8, and it was passed successively through the column 2 liters of a 0.025 M phosphate buffer having a pH of 8 and 2.5 liters of a 0.025 M phosphate buffer having a sodium chloride concentration of 0.1 M and a pH of 8, after which 2.5 liters of a 0.025 M phosphate buffer having a sodium chloride concentration of 0.2 M and a pH of 8 was passed through the column in order to elute the active substance adsorbed in the column, and the eluted solution obtained was demineralized using a hollow fiber ultrafiltration device (Membrane N [deg.] HI-1),

   then it was freeze-dried to obtain 600 mg of a grayish-white to light brown powder of TF-132a.

Example 6

  
In 1.2 liters of 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of

  
  <EMI ID = 154.1>

  
and the insoluble matters in suspension were separated by filtration using "Hyflo Super Cel", after which the filtrate obtained was passed through a 33 cm column.

  
  <EMI ID = 155.1>

  
which had been previously equilibrated with a 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8, and the eluted solution was collected. The column was washed with 0.025 M phosphate buffer having a sodium chloride concentration of

  
0.3 M and pH 8, and the washings were combined with the above-mentioned eluted solution to obtain 1.67 liters of a solution. This solution was again passed through an 8 cm diameter column packed with 400 ml of diethylamino-

  
  <EMI ID = 156.1>

  
with 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8, to obtain 1.68 liters of an eluted solution. The column was washed with 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8, to obtain

  
  <EMI ID = 157.1>

  
and the washings, and to the assembly was added a 0.025 M phosphate buffer having a pH of 8 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 passed through a B cm diameter column packed with 500 ml of diethylaminoethyl-Sephadex A-50 which had been previously equilibrated with a 0.025 M phosphate buffer. having a 0.2 M sodium chloride concentration and a pH of 8, and the column was washed with 2 liters of 0.025 M phosphate buffer having a 0.2 M sodium chloride concentration and a pH of 8.-To 7 liters of a solution prepared by combining the washings with the eluted solution, was added

  
a 0.025 M phosphate buffer having a pH of 8, to obtain 14 liters of a 0.025 M phosphate buffer having a sodium chloride concentration of 0.1 M and a pH of 8.

  
The eluted solution thus obtained was passed through a column 8 cm in diameter, packed with 500 ml of diethyl-

  
  <EMI ID = 158.1>

  
liberated with a 0.025 M phosphate buffer having a sodium chloride concentration of 0.1 .1 and a pH of 8,

  
and the eluted solution was separated. The column was washed with 2 liters of 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 buffer were passed through the column. 0.025 M phosphate having a sodium chloride concentration of 0.2 M and a pH of 8, and the eluted solution was collected. The eluted solution was concentrated and demineralized using an ultrafiltration device (filter used: Toyo Ultrafilter UK-10), it was demineralized again using a column packed with Sephadex G-25, was discolored using activated charcoal and then lyophilized to obtain 880 mg of a grayish-white to light brown powder of TF-132b.

Example 7

  
In a small amount of water, 10 g of the powder obtained in Example 2 were dissolved, and the resulting solution was passed through a column packed with diethylaminoethyl-Sephadex A-50, which had previously been balanced with 0.025 M phosphate buffer having a pH of 8. Then, 5 liters of a 0.025 M phosphate buffer having a sodium chloride concentration of 0.2 M and a pH of 8 was passed through the column, after which 2.5 liters were passed through the column. of a 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8, in order to elute the active substance adsorbed in the column, and the eluted solution obtained was concentrated under reduced pressure.

   The concentrated solution was demineralized using a "Cellophane" tube, it was completely demineralized using Sephadex G-25, then it was freeze-dried to obtain 600 mg of a powder

  
  <EMI ID = 159.1>

Example 8

  
  <EMI ID = 160.1>

  
a sodium chloride concentration of 0.3 M and a pH of 8, 44.2 g of the powder obtained in Example 1- (2) were dissolved, and the insoluble materials in suspension were separated

  
  <EMI ID = 161.1>

  
A-50 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, and the eluted solution was collected. The column was washed with 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 above-mentioned eluted solution to obtain 1.67 liters of 'a solution

  
  <EMI ID = 162.1>

  
with 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8, to obtain 1.68 liters of an eluted solution. The column was washed with 0.025 M phosphate buffer having a concentration of

  
  <EMI ID = 163.1>

  
1.64 liters of washing water. The eluted solution and the washings were combined and a 0.025 M phosphate buffer having a pH of 8 was added to the mixture to obtain 4.98 liters of a 0.025 M phosphate buffer having a concentration of 0.2 M sodium chloride and a pH of 8. The eluted solution thus obtained was passed through a column 8 cm in diameter packed with 500 ml of diethylaminoethyl-Sephadex A-50, which had previously been equilibrated with 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 0.025 M phosphate buffer having a sodium chloride concentration of 0.2 M and a pH of 8, after which 2 liters of a 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8 are passed through the column.

   and the eluted solution was collected. The eluted solution was concentrated and demineralized using an ultrafiltration device (Filter

  
  <EMI ID = 164.1>

  
made using a column packed with Sephadex G-25, it was discolored using activated charcoal, then it was freeze-dried to obtain 590 mg of a grayish-white to brown powder clear from TF-133b.

Example 9

  
(1) In 120 ml of water, 20 g of the crude powder obtained in Example 1- (2) were dissolved, and the water-insoluble matter formed at that time was separated by centrifugation at 7500 rpm for 10 min, after which we reunited

  
  <EMI ID = 165.1>

  
washing obtained by washing water-insoluble matter twice with 60 ml fractions of water, then it was subjected to ultrafiltration using a hollow fiber ultrafiltration device (Membrane N [deg.] HI- 1), and the internal solution was subjected to lyophilization to obtain 10 g of a grayish-white to light brown powder.

  
(2) In 1 liter of 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8, 25 g of the powder obtained by the treatment indicated in (1) ci were dissolved above and the resulting solution was passed through a 33 cm diameter column packed with

  
  <EMI ID = 166.1>

  
previously balanced with 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8. The column was likewise washed with a 0.025 M phosphate buffer having a sodium chloride concentration of 0 , 3M and a pH of 8, and the eluted solution and the washings were combined to obtain 1.6 liters of a mixture. This mixture was passed through a column of

  
  <EMI ID = 167.1> Sephadex A-50, which was likewise balanced with a 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8, to obtain 1, 65 liters of an eluted solution. The solution chosen was combined to 1.7 liters of washing water obtained by washing the column with a 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8, and was diluted. the resulting mixture with a 0.025 M phosphate buffer having a pH of 8, to obtain 10.35 liters of a 0.025 M phosphate buffer having a sodium chloride concentration of 0.1 M and a pH of 8.

  
The buffer solution thus obtained was then passed through 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 one

  
  <EMI ID = 168.1>

  
0.1 M sodium chloride and pH 8, after which 2 liters of 0.025 M phosphate buffer having 0.3 M sodium chloride concentration and pH 8 are passed through the column , and the eluted solution was collected. The eluted solution obtained was demineralized and concentrated, using an ultrafiltration device (Filter used:

  
  <EMI ID = 169.1>

  
on Sephadex G-25, it was discolored using activated charcoal, then it was freeze-dried to

  
  <EMI ID = 170.1>

  
TF-1323.

Example 10

  
In a small amount of water, 10 g of the powder obtained in Example 2 were dissolved, and the resulting solution was passed through a column packed with diethylaminoethyl-Sephadex A-50 which had previously been balanced. with a 0.025 M phosphate buffer having a pH of 8. Then 6 liters of a 0.025 M phosphate buffer having a chloride concentration were passed through the column.

  
  <EMI ID = 171.1>

  
the column 2.5 liters of a 0.025 M phosphate buffer having a sodium chloride concentration of 0.6 M and a pH of 8, in order to elute the active substance adsorbed in the column, and the solution was demineralized intermediate obtained using a hollow fiber ultrafiltration device (Membrane? HI-1), it was concentrated under reduced pressure, then it was subjected to lyophilization.

  
  <EMI ID = 172.1>

  
clear of TF-136.

Example II

  
15 g of crude powder were dissolved in 200 ml of water

  
  <EMI ID = 173.1>

  
insoluble in water by centrifugation at 7500 rpm for 10 minutes, after which the water-soluble fraction thus obtained is adjusted to pH 10, by adding thereto a 0.2 M aqueous baryte solution dropwise. A white precipitate was deposited under the effect of the addition of the aqueous baryte solution. Barium ions were added so that the concentration of total barium ions in the final volume was 0.01 M, after which the solution was stirred for 30 minutes, and the precipitate was collected by filtration. The barium salt precipitate thus obtained was suspended in 10 ml of a 10% by weight aqueous solution of sodium sulphate, stirred sufficiently, then filtered and the filtrate was collected. The precipitate was again suspended, stirred and then filtered in the same manner as above,

  
once with 10 ml of a 10% by weight aqueous solution of sodium sulfate, then twice with frac-

  
  <EMI ID = 174.1>

  
filtrate. In addition, the remaining precipitate was washed twice with 10 ml portions of water, then filtered to obtain washings. The above-mentioned collected filtrate and the washing waters were combined, the whole was dialyzed by exchange with distilled water, concentrated under reduced pressure, then lyophilization was carried out to obtain 0.45 g of a greyish-white to light brown powder of TF-140.

Example 12

  
In 450 ml of water, 32 g of the powder obtained in Example 1- (2) were dissolved, and the insoluble matters in suspension were separated by filtration using "Hyflo Super Cel", after which the filtrate obtained was dialyzed overnight by exchange with running water using a "Cellophane" tube. To the dialyzed solution was added drop &#65533; drop 80 ml of a solution

  
  <EMI ID = 175.1>

  
with stirring, while adding 200 ml of a 10% by weight borate buffer (pH 9.0) to it, and the resulting mixture was stirred at room temperature for 30 minutes, after which the precipitate was collected by filtration . The precipitate was suspended in 150 ml of a buffer.

  
  <EMI ID = 176.1>

  
sodium and 0.2% by weight cetyl pyridinium chloride, and the resulting suspension was stirred for 30 minutes. The precipitate of the suspension was then collected by

  
filtration, and it was again suspended in
150 ml of a borate buffer (pH 9.0) having the composition

  
  <EMI ID = 177.1>

  
sion thus obtained, after which the precipitate was collected by filtration. The collected precipitate was suspended in 150 ml of 0.1% by weight borate buffer

  
  <EMI ID = 178.1>

  
cetyl pyridinium chloride, and the resulting suspension was stirred and dissociated for 30 minutes. The precipitate was then separated from the suspension by filtration to obtain

  
  <EMI ID = 179.1>

  
precipitate separated by filtration the same dissociation as above to obtain a soluble fraction. The two soluble fractions were combined, and the resulting soluble fraction was adjusted to a pH of 3 to 4 using hydrochloric acid

  
at 10%, after which ethanol was added to it so that

  
its concentration is finally 80% by volume. We have

  
  <EMI ID = 180.1>

  
  <EMI ID = 181.1>

  
to obtain 5.5 g of a grayish-white to light brown powder of TF-150.

  
Preparation example 1

  
Each vial was filled with 1 mg or 5 mg of each of the powders of TF-100, 110 and 120 obtained in Examples 1, 2 and 3. These powders are dissolved in a sterilized physiological saline solution, a solution containing 0.5 % lidocaine, or a similar solution, at the time of use, then they are used as injections.

  
Preparation example 2

  
Each flask was filled with 1 mg or 5 mg of each of the powders of TF-1316, 132a and 132b obtained in Examples 4, 5 and 6. These powders are dissolved in a sterilized physiological saline solution, a solution containing 0.5 % lidocaine, or a similar solution, at the time of use, then they are used as injections.

  
Preparation example 3

  
A 1 mg or 5 mg vial of the TF-133a powder obtained in Example 7 was filled. This powder is dissolved in a sterilized physiological saline solution, a solution containing 0.5% of lidocaine, or a solution similar, at the time of use, then it is used in the form of injection.

  
Preparation example 4

  
A 1 mg or 5 mg vial of the TF-133b powder obtained in Example 8 was filled. This powder is dissolved in a sterilized physiological saline solution, a solution containing 0.5% of lidocaine, or a solution similar, at the time of use, then it is used in the form of injection.

  
Preparation example 5

  
A 1 mg or 5 mg vial of the TF-1323 powder obtained in Example 9 was filled. This powder is dissolved in a sterilized physiological saline solution, a solution containing 0.5% of lidocaine, or a solution similar, at the time of use, and then it is used in the form of an injection.

  
Preparation example 6

  
Each flask was filled with 1 mg or 5 mg of each of the powders TF-136, 140 and 150 obtained in Examples 10, II and 12. These powders are dissolved in a sterilized physiological saline solution, a solution containing 0.5% lidocaine, or a similar solution,

  
at the time of use, then they are used in the form of injections.

CLAIMS

  
1. Process for the preparation of substances with carcinostatic and immunostimulatory action, characterized in that it consists in cultivating, under anaerobic conditions, bacteria producing the TF substance, belonging to the genus Fusobacterium, and in collecting said substances from the culture or from its supernatant.


    

Claims (1)

2. Method according to claim I, characterized in that the bacteria producing substance are cultivated TF belonging to the genus Fusobacterium, under anaerobic conditions, a hydrophilic organic solvent is added to the culture or to its supernatant, after which the substances with carcinostatic action are separated from the precipitate formed <EMI ID = 182.1> 3. Method according to claim 2, characterized in that the water-soluble fraction of the precipitate is separated from the precipitate and then dried.  <EMI ID = 183.1> what the precipitate formed is collected, the fraction of the precipitate which is soluble in water is subjected to dialysis or ultrafiltration, and the substances with carcinostatic and immunostimulatory action are collected from the internal solution. 5. Method according to claim 2, characterized in that the precipitate formed is collected, subjected, if necessary, to the fraction of the precipitate which is soluble in water, dialysis or ultrafiltration, then it is treated with an ion exchanger, and the non-adsorbed fraction is collected. 6. Method according to claim 2, characterized in that the precipitate formed is collected, subjecting, if necessary, to the fraction of the precipitate which is soluble in water, dialysis or ultrafiltration, then it is treated with an ion exchanger, and the fraction is collected  <EMI ID = 184.1> stimulating. 7. Method according to claim 6, characterized in that the fraction adsorbed is treated with a 0.6 M sodium chloride-phosphate buffer, and the eluted fraction is collected. 8. Method according to claim 6, characterized in that one collects from the adsorbed fraction a fraction which has not been eluted with a 0.1 M sodium chloride-phosphate buffer but has been eluted with a phosphate chloride chloride 0.2 M sodium 9. Method according to claim 6, characterized in that a fraction is collected which has not been eluted with a 0.2 M sodium phosphate-chloride buffer, but has been eluted with a phosphate-chloride buffer. sodium 0.3 M. 10. Method according to claim 6, characterized in that a fraction is collected which has not been eluted with a 0.1 M sodium phosphate-chloride buffer but has been eluted with a sodium phosphate-chloride buffer 0.3 M. 11. Method according to claim 6, characterized in that a fraction is collected which has not been eluted with a 0.5 M sodium phosphate-chloride buffer but has been eluted with a sodium phosphate-chloride buffer 0.6 M. 12. Method according to claim 2, characterized in that the precipitate formed is collected; if necessary, the fraction of the precipitate which is soluble in water is subjected to dialysis or ultrafiltration; a 0.2-0.3 M sodium phosphate-chloride buffer of the precipitate fraction which is soluble in water is prepared, or a 0.2-0.3 M sodium phosphate-chloride buffer is prepared. the internal solution obtained by dialysis or ultrafiltration, then the buffer is treated with an ion exchanger balanced with a 0.2-0.3 M sodium chloride-phosphate buffer; a fraction is collected which has passed through the ion exchanger;  this eluted solution is then adjusted so that its sodium chloride concentration is 0.1 M, and it is then treated by an ion exchanger balanced with a 0.1 M sodium chloride-phosphate buffer to be adsorbed by the ion exchanger; and then a fraction is collected which has not been eluted with a 0.1 M sodium chloride-phosphate buffer but has been eluted with a 0.2 or 0.3 M sodium chloride-phosphate buffer. 13. Method according to claim 2, characterized in that the precipitate formed is collected; if necessary, the fraction of the precipitate which is soluble in water is subjected to dialysis or ultrafiltration, a 0.3 M sodium chloride-phosphate buffer is prepared from the fraction of the precipitate which is soluble in water or a 0.3 M sodium phosphate-chloride buffer of the internal solution obtained by dialysis or ultrafiltration, then this buffer is treated with an ion exchanger balanced with a phosphate chloride chloride 0.3 M sodium; a fraction is collected which has passed through the ion exchanger; the sodium chloride concentration of this eluted solution is then adjusted to 0.2 M and the solution is then treated by an ion exchanger balanced with a phosphate buffer.  <EMI ID = 185.1> the ion exchanger; and then a fraction is collected which has not been eluted with a 0.2 M sodium chloride-phosphate buffer but has been eluted with a 0.3 M sodium phosphate chloride buffer. 14. Method according to claim 1, characterized in  <EMI ID = 186.1> supernatant to form a barium salt, or a hydrophilic organic solvent is added to the culture or its supernatant, and the precipitate formed is collected, then barium ions are added to the water-soluble fraction of the precipitate as well collected, or else barium ions are added to a solution of a powder obtained by drying said fraction of the precipitate which is soluble in water, so as to form a barium salt; the barium salt thus formed is collected and then the barium is removed therefrom; the water-soluble fraction is collected and subjected to dialysis or ultrafiltration. 15. Method according to claim 1, characterized in that a hydrophilic organic solvent is added to the culture or to its supernatant liquid; the precipitate formed is collected; the fraction of the precipitate which is soluble in water is treated with a quaternary ammonium salt, or the internal solution obtained by subjecting the water-soluble fraction to dialysis or to ultrafiltration; the precipitate formed is collected and then subjected to dissociation using a solution containing sodium chloride; a hydrophilic organic solvent is added to the soluble fraction obtained, to form a precipitate: and the precipitate thus formed is collected. 16. Method according to any one of claims 1 to 15, characterized in that the culture medium used to cultivate the bacteria contains sources of nitrogen, sources of carbon, sources of vitamins, agents.  <EMI ID = 187.1> agar. 17. Method according to any one of claims 1 to 15, characterized in that the culture medium used to cultivate the bacteria comprises trypticase peptone, phytone peptone, protease peptone, a brain infusion and heart or an infusion of heart, a yeast extract, sodium chloride, glucose, lactose, L-cystine, sodium sulfite and sodium thioglycollate, possibly associated with agar.  <EMI ID = 188.1> 1 to 15, characterized in that the cultivation takes place between 35 [deg.] And 42 [deg.] C for 1 to 5 days in a culture medium adjusted to a pH of 6.0 to 8.5. 19. Method according to any one of claims 1 to 15, characterized in that one proceeds to the culture between  <EMI ID = 189.1> adjusted to a pH of 7.2 to 8.2. 20. Method according to any one of claims 1 to 15, characterized in that the bacteria belonging to the  <EMI ID = 190.1> nucleatum. 21. Method according to any one of claims 2 to 15, characterized in that the hydrophilic organic solvent which is added to the culture or to its supernatant liquid is an alcohol. 22. Method according to any one of claims 2 to 15, characterized in that the hydrophilic organic solvent is added to the culture or to its supernatant liquid with  <EMI ID = 191.1> 23. Method according to any one of claims 6 to 13, characterized in that the ion exchanger is a weakly basic ion exchanger or a weakly basic ion exchanger having the properties d 'a molecular sieve. 24. The method of claim 6, characterized in that a 0.1-0.6 M sodium chloride-phosphate buffer is used to elute the fraction adsorbed by the ion exchanger. 25. Method according to any one of claims 7 to 13 or according to claim 24, characterized in that the pH of the phosphate buffer containing sodium chloride which is used to treat the adsorbed fraction is 8 approx. 26. Method according to any one of claims  <EMI ID = 192.1> centration, demineralization and drying to the fraction to which the ion exchange treatment was subjected. 27. Method according to claim 15, characterized in  <EMI ID = 193.1> cetyl pyridinium. 28. The method of claim 15, characterized in that the treatment is carried out with the quaternary ammonium salt in the presence of a borate buffer. 29. Method according to claim 15, characterized in that the solution containing sodium chloride is a borate buffer containing sodium chloride and a quaternary ammonium salt. 30. Method according to claim 6, characterized in  <EMI ID = 194.1> a culture medium adjusted to a pH of 7.2 to 8.2 comprising trypticase peptone, phytone peptone, proteose peptone, an infusion of brain and heart or an infusion of heart, yeast extract, sodium chloride, glucose, lactose, Lcystine, sodium sulfite and sodium thioglycollate, or to a culture medium comprising the same components and agar, then the culture is carried out in  <EMI ID = 195.1> an alcohol is added to the culture or to its supernatant, at a concentration of 50 to 70% by volume; the precipitate formed is collected: if necessary, the fraction of the precipitate which is soluble in water is subjected to dialysis or ultrafiltration, then it is treated by a weakly basic ion exchanger or by a weakly basic ion exchanger having the properties of a molecular sieve, for separating the non-adsorbed fraction; separating from the adsorbed fraction a fraction which has been eluted with a 0.1 or 0.2 M sodium chloride-phosphate buffer; and then collected, concentrated, demineralized and then dried a fraction which has been eluted with a 0.3 M sodium chloride-phosphate buffer. 31. Method according to claim 6, characterized in that bacteria of Fusobacterium nucleatum are inoculated a culture medium adjusted to a pH of 7.2 to 8.2, comprising trypticase peptone, phytone peptone, proteose peptone, a brain and heart infusion or a heart infusion, yeast extract, sodium chloride, glucose, lactose, Lcystine, sodium sulfite and sodium thioglycollate, or to a culture medium comprising the same constituents and agar, then culture under anaerobic conditions between 36 [deg.] and 38 [deg.] C for 1 to 3 days; an alcohol is added to the culture or to its supernatant liquid, at a concentration of 50 to 70% by volume; the precipitate formed is collected; if necessary, the fraction of the precipitate which is soluble in water is subjected to dialysis or ultrafiltration. then treating a 0.2-0.3 M sodium phosphate-chloride buffer from the precipitate fraction which is soluble in water, or a  <EMI ID = 196.1> internal solution obtained by dialysis or ultrafiltration, by a weakly basic ion exchanger or by a weakly basic ion exchanger having the properties of a molecular sieve, which is previously balanced with a phosphate chloride chloride buffer. sodium 0.2-0.3 M; the fraction which has passed through the ion exchanger is collected; or_ adjust this eluted solution so that it has a sodium chloride concentration of 0.1 to 0.2 M, then it is treated with a weakly basic ion exchanger or with a weakly basic ion exchanger having the properties of a molecular sieve, which has been previously balanced with a phosphate buffer-  <EMI ID = 197.1> the ion exchanger; and a fraction which has been eluted with a 0.2-0.3 M sodium phosphate buffer is collected from the adsorbed fraction, concentrated, demineralized and then dried. 32. Method according to claim 20, 30 or 31, characterized in that the bacteria of Fusobacterium nucleatum originate from a strain of TF-031. 33. Carcinostatic substance called TF-100, characterized by the following properties: (a) It is in the form of a grayish-white to light brown powder. (b) It inhibits the proliferation of ascites tumor and consistent Ehrlich tumor in mice and <EMI ID = 198.1> (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. (d) It does not have a well-defined melting point, it <EMI ID = 199.1> is clearly above 200 [deg.] C. (e) Its infrared absorption spectrum, obtained by the KBr pellet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550, <EMI ID = 200.1> (f) The ultraviolet absorption spectrum of its aqueous solution exhibits a strong absorption at the absorption edge and an absorption peak in the vicinity of 256-260 nm. (g) When fractionated by gel filtration (column: 50 mm of 0 x 600 mm, eluent: distilled water) in, of Pharmacia Co., Ltd. using Sephadex G-50 (registered trademark!), it has absorption bands with elution volumes in the vicinity of the empty volume - 400, 430 - 530, 700 - 800, and 840 - 870 ml in the measurement of ultraviolet absorption at 260 nm; and the elution volumes in the vicinity of the empty volume - 390, and 410 - 430 ml in the absorption measurement at 620 nm, using the anthrone-sulfuric acid method. (h) Its high performance liquid chromatogram (eluent: 0.1 M phosphate buffer with a pH of 7, flow rate: 0.8 ml / min, at room temperature), obtained using "TSK-GEL G300 SW "(trade name of a product sold by Toyo Soda Co., Ltd., column: 7.9 mm 0 x 600 mm x 2) has peaks near the front of the solvent, at 38 - 60, and at 65 min in the measurement of ultraviolet absorption at 220 nm and 260 nm.  <EMI ID = 201.1> phenol-sulfuric acid reaction, anthroneacid sulfuric reaction, indole-hydrochloric acid reaction,  <EMI ID = 202.1> Ninhydrin. (j) His elementary analysis gives the following results:  <EMI ID = 203.1> (k) Its saccharide content, measured by the phenol-sulfuric acid method, is from about 40.0 to about 46.0% by weight, expressed in glucose, and its protein content, measured by the Lowry- method Folin, is around 20.0 at approximately 23.0% by weight, expressed as serum albumin of calf. 34. Carcinostatic substance called TF-110, characterized by the following properties: (a) It is in the form of a grayish white to light brown powder. (b) It inhibits the proliferation of Ehrlich's ascites tumor in mice, and it has an action <EMI ID = 204.1> (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. (d) It has no well-defined melting point, it <EMI ID = 205.1> clearly above 200 [deg.] C. (e) Its infrared absorption spectrum, obtained by the KBr pellet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670- <EMI ID = 206.1> (f) The ultraviolet absorption spectrum of its aqueous solution exhibits a strong absorption at the absorption edge, and an absorption peak in the vicinity of 256-260 nm. (g) When fractionated by gel filtration (column: 44 mm x 0 500 mm, eluent: phosphate buffer  <EMI ID = 207.1>  <EMI ID = 208.1> (registered trademark), it has absorption bands at the  <EMI ID = 209.1> at 600 - 920 ml in the measurement of ultraviolet absorption at 260 nm, at the elution volumes in the vicinity of the empty volume - 380, 420 - 520, and 630 - 760 ml in the absorption measurement at 490 nm, by the phenol-sulfuric acid method; and to the elution volumes in the vicinity of the empty volume - 380, 420 -520, and 620 - 760 ml in the measurement  <EMI ID = 210.1> sulfuric. (h) Its high performance liquid chromatogram (eluent: 0.1 M phosphate buffer with a pH of 7, flow rate: 0.8 ml / min, at room temperature), obtained using "TSK-GEL G300 SW (trade name of a product sold by Toyo Soda Co .. Ltd. column: 7.9 mm 0 x 600 mm x 2) has peaks in the vicinity of the solvent front, at 38 - 60 and 65 min in measurement of ultraviolet absorption at 220 nm and 260 nm. (i) It is positive to the Molish reaction, to the phenol-sulfuric acid reaction, to the anthroneacidic sulfuric reaction, to the indole-hydrochloric acid reaction, and to the Lowry-Folin reaction, and it is negative to the  <EMI ID = 211.1> (j) His elementary analysis gives the following results:  <EMI ID = 212.1> (k) its saccharide content, measured by the phenol-sulfuric acid method, is approximately 30.0 to approximately 69.0% by weight, expressed as glucose, and its protein content, measured by the Lowry-Folin method, is from approximately 18.0 to approximately 22.0% by weight, expressed as  <EMI ID = 213.1> 35. Carcinostatic substance called TF-120, characterized by its following properties: (a) It is in the form of a grayish white to light brown powder. (b) It inhibits the proliferation of Ehrlich's ascites tumor in mice, and it has an immunostimulatory action. (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, <EMI ID = 214.1> (d) It does not have a well-defined melting point, it begins to decompose at around 110 [deg.] C, and it decomposes clearly above 200 [deg.] C. (e) Its infrared absorption spectrum, obtained by the KBr pellet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550, <EMI ID = 215.1> (f) The ultraviolet absorption spectrum of its aqueous solution shows a strong absorption at the absorption edge, and an absorption peak in the vicinity of 268 - 272 nm. (g) When fractionated by gel filtration (column: 44 mm of 0 x 500 mm, eluent: 0.1 M phosphate buffer having a pH of 7) using Sephadex G-200, it presents absorption bands at the elution volumes in the vicinity of the volume vacuum - 325, and at 775 - 875 ml in the measurement of ultraviolet absorption at 260 nm; to the elution volumes in the vicinity of the empty volume - 360, to 360 - 480, and at 510 - 760 ml in the absorption measurement at 490 nm by the phenol-sulfuric acid method; and the elution volumes in the vicinity of the empty volume - 380, to 420 -  <EMI ID = 216.1> by the anthrone-sulfuric acid method. (h) Its high performance liquid chromatogram (eluent: 0.1 M phosphate buffer with a pH of 7, flow rate: 0.8 ml / min, at room temperature), obtained using "TSK-GEL G300 SW "(column: 7.9 mm 0 x 600 mm x 2), has peaks in the vicinity of the solvent front and at 38 - 60 min in the measurement of ultraviolet absorption at 220 nm and 260 nm. (i) It is positive to the Molisch reaction, to the phenol-sulfuric acid reaction, to the anthroneacid sulfuric reaction, to the indole-hydrochloric acid reaction and to the Lowry-Folin reaction, and it is negative to the reaction to Ninhydrin. (j) His elementary analysis gives the following results:  <EMI ID = 217.1> (k) Its saccharide content, measured by the phenol-sulfuric acid method, is approximately 56.0 to approximately 73.0% by weight, expressed in glucose, and its protein content, measured by the Lowry- method Folin, is approximately 9.0 to approximately 13.0% by weight, expressed as calf serum albumin. 36. Carcinostatic substance called TF-130, characterized by the following properties: (a) It is in the form of a grayish white to light brown powder. (b) It inhibits the proliferation of the ascites tumor. <EMI ID = 218.1> Sarcoma 180 carcinoma in mice and it has an immunostimulatory action. (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. (d) It does not have a well-defined melting point, it begins to decompose at around 110 [deg.] C, and it decomposes clearly above 200 [deg.] C. (e) Its infrared absorption spectrum, obtained by the BKr pellet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 in-1. (f) The ultraviolet absorption spectrum of its aqueous solution shows a strong absorption at the absorption edge, and an absorption peak in the vicinity of 256 - 280 nm. (g) It is positive to the Molisch reaction, to the phenol-sulfuric acid reaction, to the anthroneacid sulfuric reaction, to the indole-hydrochloric acid reaction, and to the Lowry-Folin reaction, and it is negative to the reaction. Ninhydrin. (h) His elementary analysis gives the following results:  <EMI ID = 219.1> (i) Its saccharide content, measured by the phenol-sulfuric acid method, is approximately 19.0 # about 64.0% by weight, expressed as glucose, and its protein content, measured by the Lowry-Folin method; is about 6.0 to about 28.0% by weight, expressed as calf serum albumin. 37. Carcinostatic substance called TF-1316, characterized by the burning properties: (a) It is in the form of a grayish white to light brown powder. (b) It inhibits the proliferation of Ehrlich's ascites tumor, Ehrlich's consistent tumor and Sarcoma-180 carcinoma in mice, and it has an immunostimulatory action. (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, <EMI ID = 220.1> (d) It has no well-defined melting point, it begins to decompose at around 110 [deg.] C, and it decomposes <EMI ID = 221.1> (e) Its infrared absorption spectrum, obtained by the KBr pellet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550,  <EMI ID = 222.1> (f) The ultraviolet absorption spectrum of its aqueous solution shows a strong absorption at the absorption edge, <EMI ID = 223.1> (g) When it is fractionated by gel filtration (column: 21 mm of 0 x 400 mm, eluent: 0.1 M phosphate buffer having a pH of 7) using Sephadex G-200,  <EMI ID = 224.1> in the vicinity of the empty volume - 160 ml in the measurement of ultraviolet absorption at 260 nm; and an absorption band at the elution volumes in the vicinity of the empty volume - 160 ml in the absorption measurement at 490 nm, by the phenol-sulfuric acid method. (h) Its high performance liquid chromatogram (eluent: 0.1 M phosphate buffer with a pH of 7, flow rate: 0.8 ml / min, at room temperature), obtained using "TSK-GEL G300 SW "(column: 7.9 mm x 0 600 mm x 2), has peaks in the vicinity of the solvent front and at 40 - 60 min in the measurement of ultraviolet absorption at 220 nm, and in the vicinity of the solvent front, and at 40 - 60 min in the measurement d absorption at 260 nm. (i) It is positive &#65533; the Molisch reaction, the phenol-sulfuric acid reaction, the anthroneacid-sulfuric reaction ,. to the indole-hydrochloric acid reaction, and to the Lowry-Folin reaction, and it is negative to the reaction with Ninhydrin. (j) His elementary analysis gives the following results:  <EMI ID = 225.1> (k) Its saccharide content, measured by the phenol-sulfuric acid method, is from about 35.0 to about 50.0%  <EMI ID = 226.1> measured by the Lowry-Folin method, is approximately 10.0 to approximately 23.0% by weight, expressed as bovine serum albumin. 38. Carcinostatic substance called TF-132a, characterized by the following properties: (a) It is in the form of a grayish-white to light brown powder. (b) It inhibits the proliferation of Ehrlich's ascites tumor, Ehrlich's consistent tumor and Sarcoma-180 carcinoma in mice, and it has an <EMI ID = 227.1> (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. (d) It does not have a well-defined melting point, it begins to decompose at around 110 [deg.] C, and it decomposes clearly above 200 [deg.] C. (e) Its infrared absorption spectrum, obtained by the KBr pellet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670- <EMI ID = 228.1> (f) The ultraviolet absorption spectrum of its aqueous solution exhibits a strong absorption at the absorption edge,  and an absorption peak in the vicinity of 270 - 280 nm. (g) When fractionated by gel filtration (column: 44 mm of 0 x 500 mm, eluent: 0.1 M phosphate buffer having a pH of 7) using Sephadex G-200, it presents absorption bands at the elution volumes in the vicinity of empty volume - 340, 600 - 700, and  <EMI ID = 229.1> 260 nm; the elution volumes in the vicinity of the empty volume - 340, 340 - 580, and 720 - 900 ml in the measurement of absorption at 490 nm, by the phenolacidic sulfuric method; and to the elution volumes in the vicinity of the empty volume - 340, and 340 - 580 ml in the measurement of absorption at 620 nm by the anthroneacid sulfuric method. (h) Its high performance liquid chromatogram (eluent: 0.1 M phosphate buffer having a pH of 7, flow rate. 0.8 ml / min, at room temperature), obtained using "TSK-GEL G300 SW "(column: 7.9 mm x 0 600 mm x 2), has peaks in the vicinity of the solvent front, at 30, at 38 - 60 and at 65 min in the measurement of ultraviolet absorption at 220 nm; and in the vicinity of the front of the solvent, at 62 and 65 min in the measurement of ultraviolet absorption at 2 60 nm. (i) It is positive to Molisch's reaction, to the  <EMI ID = 230.1> sulfuric acid, to the indole-hydrochloric acid reaction, and to the Lowry-Folin reaction, and it is negative to the reaction with Ninhydrin. (j) His elementary analysis gives the following results:  <EMI ID = 231.1> (k) Its saccharide content, measured by the phenol-sulfuric acid method, is approximately 55.0 to approximately 64.0% by weight, expressed as glucose, and its protein content, measured by the Lowry-Folin method, is from approximately 18.0 to approximately 28.0% by weight, expressed as bovine serum albumin. 39. Carcinostatic substance called TF-132b, characterized by the following properties: (a) It takes the form of a grayish-white to light brown powder. (b) It inhibits the proliferation of Ehrlich's ascites tumor, Ehrlich's consistent tumor and Sarcoma-180 carcinoma in mice, and it has an immunostimulatory action. (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, <EMI ID = 232.1> (d) It does not have a well-defined melting point, it begins to decompose at around 110 [deg.] C and it decomposes clearly above 200 [deg.] C. (e) Its infrared absorption spectrum, obtained by the KBr pellet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670- <EMI ID = 233.1> (f) The ultraviolet absorption spectrum of its aqueous solution exhibits a strong absorption at the absorption edge,  and a shoulder in the vicinity of 265 - 280 nm. (g) When fractionated by gel filtration (column: 21 mm of 0 x 400 mm, eluent: 0.1 M phosphate buffer having a pH of 7), using Sephadex G-200, "it has a low absorption band at the elution volumes in the vicinity of the empty volume - 150 ml in the measurement of ultraviolet absorption at 260 nm; and a band <EMI ID = 234.1> vacuum - 150 ml in the absorption measurement at 490 nm by the phenol-sulfuric acid method. (h) Its high performance liquid chromatogram (eluent: 0.1 M phosphate buffer having a pH of 7, flow rate: 0.8 ml / min, at room temperature), obtained  <EMI ID = 235.1> x 600 mm x 2), has peaks near the front of the solvent, at 36 - 37 and at 48 - 50 min, in the measurement of ultraviolet absorption at 220 nm, and very low peaks near the front of the solvent , at 32 - 39, and at 45 - 52 min, in the measurement of ultraviolet absorption at 26 0 nm. (i) It is positive to the Molisch reaction, to the phenol-sulfuric acid reaction, to the anthroneacid sulfuric reaction, to the indole-hydrochloric acid reaction, to the Lowry-Folin reaction, and it is negative to the reaction to Ninhydrin. (j) His elementary analysis gives the following results:  <EMI ID = 236.1> (k). Its saccharide content, measured by the phenol-sulfuric acid method, is approximately 23.6 to approximately 45.5% by weight, expressed as glucose, and its protein content, measured by the Iowry-Folin method, is from about 15.5 to about 28.0% by weight, expressed as calf serum albumin. 40. Carcinostatic substance called TF-133a, characterized by the following properties: (a) It is in the form of a grayish white to light brown powder. (b) It inhibits the proliferation of Ehrlich's ascites tumor, Ehrlich's consistent tumor, Sarcoma-180 carcinoma in mice, and it has an immunostimulatory action. (c) It is soluble in water, and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. (d) It does not have a well-defined melting point, it begins to decompose at around 110 [deg.] C and decomposes clearly above 200 [deg.] C. (e) Its infrared absorption spectrum, obtained by the KBr pellet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670- <EMI ID = 237.1> (f) The ultraviolet absorption spectrum of its aqueous solution exhibits a strong absorption at the absorption edge,  and an absorption peak in the vicinity of 258 - 262 nm. (g) When fractionated by gel filtration (column: 44 mm of 0 x 500 mm, eluent: phosphate buffer 0, <1> M having a pH of 7) using Sephadex G-200,  <EMI ID = 238.1> in the vicinity of the empty volume - 300, and 630 - 940 ml in the measurement of ultraviolet absorption at 260 nm; an absorption band at the elution volumes in the vicinity of the empty volume - 420 ml as far as absorption at 490 nm by the phenol-sulfuric acid method; and an absorption band at the elution volumes in the vicinity of the empty volume - 420 ml in the absorption measurement at 620 nm by the anthrone-sulfuric acid method. (h) Its high performance liquid chromatogram (eluent: 0.1 M phosphate buffer having a pH of 7, flow rate: 0.8 ml / min, at room temperature), obtained  <EMI ID = 239.1> 600 mm x 2) has peaks in the vicinity of the front of the solvent, at 38 and 50 min in the measurement of ultraviolet absorption at 220 nm; and in the vicinity of the solvent front, at 50 - 60, and at 62 min in the measurement of ultraviolet absorption at 260 nm. (i) It is positive to the Molisch reaction, to the phenol-sulfuric acid reaction, to the anthroneacid sulfuric reaction, to the indole-hydrochloric acid reaction, and to the Lowry-Folin reaction, and it is negative to the reaction Ninhydrin. (j) His elementary analysis gives the following results:  <EMI ID = 240.1>  (k) Its saccharide content, measured by the phenol-sulfuric acid method, is approximately 26.0 to approximately 35.0% by weight, expressed as glucose, and its protein content, measured by the Lowry-Folin method, is from approximately 22.0 to approximately 28.0% by weight, expressed as bovine serum albumin. 41. Carcinostatic substance called TF-133b, characterized by the following properties: (a) It is in the form of a grayish white to light brown powder. (b) It inhibits the proliferation of Ehrlich's ascites tumor, Ehrlich's consistent tumor and <EMI ID = 241.1> stimulating. (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, and diethyl ether. (d) It does not have a well-defined melting point, begins to decompose at around 110 [deg.] C, and decomposes clearly above 200 [deg.] C. (e) Its infrared absorption spectrum, obtained by the KBr pellet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670- <EMI ID = 242.1> (f) The ultraviolet absorption spectrum of its aqueous solution has a strong absorption at the absorption edge, and a shoulder in the vicinity of 270 - 280 nm. (g) When fractionated by gel filtration (column: 21 mm of 0 x 400 mm, eluent: 0.1 M phosphate buffer having a pH of 7) using Sephadex G-200, it presents an absorption band at the elution volumes in the vicinity of the volume vacuum - 170 ml in the measurement of ultraviolet absorption at 260 nm; and an absorption band at the elution volumes near the empty volume - 150 ml in the absorption measurement at 490 nm by the phenol-sulfuric acid method. (h) Its high performance liquid chromatogram (eluent: 0.1 M phosphate buffer with a pH of 7, flow rate: 0.8 ml / min, at room temperature), obtained using "TSK-GEL G300 SW "(column: 7.9 mm x 0 600 mm x 2) has peaks near the front of the solvent, and at 49 - 50 min in the measurement of ultraviolet absorption at 220 nm; and in the vicinity of the front of the solvent, at 38 - 39 and at 52 min in the measurement of ultraviolet absorption at 260 nm. (i) It is positive for the Molisch reaction, the phenol-sulfuric acid reaction, the anthroneacid sulfuric reaction, the indole-hydrochloric acid reaction,  <EMI ID = 243.1> reaction to Ninhydrin. (j) His elementary analysis gives the following results:  <EMI ID = 244.1> (k) Its saccharide content, measured by the phenol-sulfuric acid method, is approximately 19.0 to approximately 24.5% by weight, expressed as glucose, and its protein content,  <EMI ID = 245.1> approximately 22.9% by weight, expressed as albumin of calf serum. 42. Carcinostatic substance called TF-1323, characterized by the following properties: (a) It is in the form of a grayish white to light brown powder. (b) It inhibits the proliferation of Ehrlich's ascites tumor, Ehrlich's consistent tumor and Sarcoma-180 carcinoma in mice, and it has an immunostimulatory action. (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. (d) It has no well-defined melting point, begins to decompose at around 110 ° C, and decomposes clearly above 220 [deg.] C. (e) Its infrared absorption spectrum, obtained by the KBr pellet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670- <EMI ID = 246.1> (f) The spectrum of ultraviolet absorption of its aqueous solution has a strong absorption at the absorption edge,  <EMI ID = 247.1> (a) When it is fractionated by gel filtration (column. 21 mm 0 × 400 mm, eluent: 0.1 M phosphate buffer having a pH of 7) using Sephadex G-200, it presents an absorption band at the elution volumes in the vicinity of the volume vacuum - 160 ml in the measurement of ultraviolet absorption at 260 mm; and an absorption band at the elution volumes in the vicinity of the empty volume - 150 ml in the absorption measurement at 490 nm by the phenol-sulfuric acid method. (h) Its high performance liquid chromatogram (eluent: 0.1 M phosphate buffer having a pH of 7, flow rate: 0.8 ml / min, at room temperature), obtained using "TSK-GEL G300 SW "(column: 7.9 mm x 0 600 mm x 2) has peaks in the vicinity of the front of the solvent, at 36 and 48 - 50 min in the measurement of ultraviolet absorption at 220 nm; and peaks in the vicinity of the solvent front, it 36 and at 50 min in the measurement of ultraviolet absorption at 260 nm. (i) It is positive to the Molisch reaction, to the phenol-sulfuric acid reaction, to the anthroneacid sulfuric reaction, to the indole-hydrochloric acid reaction, and to the Lowry-Folin reaction, and it is negative to the reaction Ninhydrin. (j) Its elementary analysis gives the following results;  <EMI ID = 248.1> (k) Its saccharide content, measured by the phenol-sulfuric acid method, is from about 19.0 to about 25.0% by weight, expressed in glucose, and its protein content, measured by the Lowry- method Folin, is approximately 11.0 to  <EMI ID = 249.1> veal. 43. Carcinostatic substance called TF-136, characterized by the following properties: (a) It is in the form of a grayish-white to light brown powder. (b) It inhibits the proliferation of Ehrlich's ascites tumor, Ehrlich's consistent tumor, and Sarcoma-180 carcinoma in mice, and it has an immunostimulatory action. (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. (d) It does not have a well-defined melting point, begins to decompose at around 110 [deg.] C and decomposes clearly above 200 [deg.] C. (e) Its infrared absorption spectrum, obtained by the KBr pellet method, has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670- <EMI ID = 250.1> (f) The ultraviolet absorption spectrum of its aqueous solution exhibits a strong absorption at the absorption edge,  and an absorption peak in the vicinity of 256-260 nm. (g) When fractionated by gel filtration  <EMI ID = 251.1> 0.1 M having a pH of 7) using Sephadex G-200, it presents absorption bands with elution volumes in the vicinity of 540 - 930 ml in the measurement of ultraviolet absorption at 260 nm, and all fractions have a slight absorption band in the absorption measurement at 490 nm by the phenol-sulfuric acid method and in the absorption measurement at 620 nm by the anthrone-sulfuric acid method. (h) Its high performance liquid phase chromatogram  <EMI ID = 252.1> flow rate: 0.8 ml / min, at room temperature), obtained using "TSK-GEL G300 SW" (column: 7.9 mm x 0 600 mm x 2), has peaks near the front of the solvent, at 28 - 40, and at 42 - 60 min in the measurement of ultraviolet absorption at 220 nm; and peaks near the solvent front and at 42 - 60 min in the absorption measurement  <EMI ID = 253.1>  <EMI ID = 254.1> phenol-sulfuric acid reaction, anthrone-sulfuric acid reaction, indole-hydrochloric acid reaction, and Lowry-Folin reaction, and negative reaction to Ninhydrin reaction. (j) His elementary analysis gives the following results:  <EMI ID = 255.1> (k) Its saccharide content, measured by the phenol-sulfuric acid method, is from about 19.0 to about 30.0% by weight, expressed in glucose, and its protein content, measured by the Lowry- method Folin, is approximately 6.0 to approximately 12.0% by weight, expressed as bovine serum albumin. 44. Carcinostatic substance called TF-140, characterized by the following properties: (a) It is in the form of a grayish white to light brown powder. (b) It inhibits the proliferation of Ehrlich's ascites tumor in mice, and it has an immunostimulatory action. (c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, and diethyl ether. (d) It has no well-defined melting point, <EMI ID = 256.1> significantly above 280 [deg.] C. (e) Its infrared absorption spectrum, obtained by the KBr pellet method, has absorption bands- <EMI ID = 257.1>  <EMI ID = 258.1> (f) The ultraviolet absorption spectrum of its aqueous solution shows a strong absorption at the absorption edge, and an absorption peak in the vicinity of 255 - 260 nm. (g) When fractionated by gel filtration (column: 44 mm of 0 x 500 mm, eluent: 0.1 M phosphate buffer having a pH of 7), using Sephadex G-200, it presents absorption bands at the elution volumes in the vicinity of empty volume - 300, 500 - 900, and 900 -  <EMI ID = 259.1> and to the elution volumes in the vicinity of the empty volume - 500, 650 - 850, and 850 - 1000 ml in the absorption measurement at 490 nm by the phenol-sulfuric acid method. (h) Its high performance liquid chromatogram (eluent: 0.1 M phosphate buffer with a pH of 7, flow rate: 0.8 ml / min, at room temperature), obtained using "TSK-GEL G300 SW "(column: 7.9 mm 0 x 600 mm x 2) has peaks near the front of the solvent, and 40 - 60 min as far as absorb-  <EMI ID = 260.1> (i) It is positive to the Molisch reaction, to the phenol-sulfuric acid reaction, to the anthroneacid sulfuric reaction, to the indole-hydrochloric acid reaction, and to the Lowry-Folin reaction, and negative to the reaction to the Ninhydrin. (j) Its elementary analysis gives the following results  <EMI ID = 261.1> (k) Its saccharide content, measured by the phenol-sulfuric acid method, is from about 5.0 to about 15.0% by weight, expressed in glucose, and its protein content, measured by the Lowry- method. Folin, is approximately 23.0% to approximately 32.0% by weight, expressed as calf serum albumin. 45. Carcinostatic substance called TF-150, characterized by the following properties: (a) It comes in the form of a white powder - <EMI ID = 262.1> (b) It inhibits the proliferation of Ehrlich's ascites tumor in mice, and it has an action <EMI ID = 263.1> (c) It is soluble in water and insoluble in <EMI ID = 264.1> ethyl acetate and diethyl ether. (d) It has no well-defined melting point, begins to decompose at around 110 [deg.] C, and decomposes clearly above 200 [deg.] C. (e) The ultraviolet absorption spectrum of its solution <EMI ID = 265.1> 47. Carcinostatic agent according to claim 46, characterized in that said carcinostatic substance empty volume - 150 ml in the absorption measurement at  <EMI ID = 266.1>  <EMI ID = 267.1>  <EMI ID = 268.1>  <EMI ID = 269.1>  <EMI ID = 270.1>  <EMI ID = 271.1>  <EMI ID = 272.1>  <EMI ID = 273.1>  <EMI ID = 274.1> vea ;:, 46. Carcinostatic agent characterized in that it  <EMI ID = 275.1> 130, 1316, 132a, 132b, 133a, 133b, 1323, 136, 140 or 150 according to claim 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45, and a pharmaceutically acceptable adjuvant. is TF-132a, 132b, 133a, 133b or 1323 according to claim 38, 39, 40, 41 or 42.