JPH02223600A - Anticancer substance TF-300, its manufacturing method, and anticancer agent containing the same - Google Patents

Abstract

NEW MATERIAL:TF-300 and its salt having the following properties. (A) Grayish white to pale brown powder; (B) effective in inhibiting the proliferation of Ehrlich ascites carcinoma, Ehrlich nodular carcinoma, sarcoma 180 cancer cell and B-16 melanoma cancer cell of mouse; (C) soluble in water and insoluble in methanol, acetone, benzene, diethyl ether, etc.; (D) having no definite melting point, starting the decomposition at 180 deg.C and remarkably decomposing at >=195 deg.C; (E) positive to Molish reaction, etc., and negative to ninhydrin reaction; (F) elemental analysis (%), C 38-47, H 5-7, N 1-4, etc. USE:A carcinostatic agent. PREPARATION:A bacterial strain belonging to genus Fusobacterium [e. g. Fusobacterium nucleatum TF-031 (FERM P-5077)] is cultured, a hydrophilic organic solvent is added to the supernatant liquid of the cultured product, a fraction having carcinostatic action is collected from the produced precipitate and the fraction is subjected to enzymatic treatment with a proteinase.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel anti-cancer substance, and more particularly, a protease obtained by culturing a bacterium belonging to the genus Pacterium and obtaining a fraction of a proteolytic enzyme having an anti-cancer effect obtained from the culture solution. The present invention relates to the cancerous substance TF-500 obtained by enzymatic treatment with TF-500 and its salts. Furthermore, the present invention provides the anticancer substance TF.
- A method for producing RoOO and its salts, and an anticancer agent containing the same.

In recent years, methods for treating various cancer patients have become popular, in which the immune function of the host is stimulated and anticancer effects are expressed with the aid of the immune function.

Known drugs used in such therapy include bacterial cells of various bacteria, components obtained from bacterial cell cultures, and polysaccharides obtained from the fruiting bodies of basidiomycetes or their cultured cells. However, there has been little research into the components obtained from the culture solution obtained by culturing anaerobic bacteria and which have excellent anticancer effects.In particular, there is still little research regarding the components obtained from the culture solution obtained by culturing Fusobacterium bacteria and their anticancer activity. unknown.

The present inventor cultivated bacteria belonging to the genus Fusobacterium isolated from the hydrococcal cavity, and investigated the pharmacological effects of the components collected from the supernatant after removing the bacterial cells from the culture solution. It has been discovered that the drug has a strong anticancer effect, and also has the effect of restoring the host-mediated or host immunity and indirectly expressing the anticancer effect with the help of the immune force. The specific component obtained by further subjecting the resulting effective fraction with general anticancer activity to enzymatic treatment with proteolytic enzymes has desirable properties as an anticancer agent, such as strong anticancer activity and few side effects. They discovered this and completed the present invention.

The teeth used in the present invention include Fusobacterium spp., which produces an anticarcinogenic substance TF-300 by subjecting a fraction with anticancer activity obtained from the supernatant of culturing the bacteria to an enzymatic treatment with a protease. Any bacteria belonging to the genus may be used, and a preferred example is Fusobacterium nucleatum. Specifically, for example, Fusobacterium nucleatum TF-'031 (1"KRM-5077, AT
OO-3, 1647) and as a common knowledge in microbiology, strains having such properties, such as natural mutant strains or artificially improved strains, are used.

The mycological properties of Fusobacterium nucleatum TI''-031 are as follows.

(1) Morphology ■ Cell shape: spindle-shaped (Figure 1) ■ Presence or absence of cell pleomorphism: None ■ Presence or absence of motility: None ■ Presence or absence of spores: None ■ Durham stain nigerum negative ■ Acid-fastness: Negative (2 )Growth status in culture medium■ TF-a agar plate and slant medium External shape: bicircular Size: Approximately 1txm Raised bipedal spherical structure: Dewdrop-shaped surface: Smooth Edge: Smooth Color: Milky white Transparency: ° Opaque■ TF-a Degree of growth in liquid medium: Vigorous turbidity: Coagulation #: None Surface growth: None, no growth until about 5 am: None 3) Physiological properties ■ Hydrogen sulfide production: + ■ Nitrate reduction: ■ Production of butyric acid: + ■ Production of indole: ■ Urease: ■ Catalase: ■ Hydrolysis of starch: ■ Attitude towards oxygen: @temperament ■ Production of ammonia: + [phase] Production of carbon dioxide: + ■ Range of growth: pH 5-8.5 Temperature 60-45℃ @ Gas production from sugar L-arabinose (-), D-xylose (-), D-glucose (-), D-mannose (-), D-7 lactose] → , D-galactose (-), maltose (-1, Sho FC-1,) levalose (→, sorbitol (-1, mannitol (-), inosit (-), glycerin (-)
), starch ←) When examining the above properties in the light of the 8th edition of Bird's Manual of Determinative Bacteriology, this strain is Fusobacterium nucleatum (F
It closely resembles and belongs to uSobacterium nucleatum).

Next, an example of the method for producing the anticancer substance TF-500 of the present invention will be schematically explained as follows.

(1) Culture and isolation of a fraction with anticancer activity from the culture solution Culture solution soluble portion of bacteria ← Separation Collection of fraction with anticancer activity Enzyme treatment and collection of target product Fractions separated as above Enzyme treatment (insoluble part ← separation) Collection of soluble part TF-300 The above production method is specifically shown as follows.

(1) Step of culturing and separating a fraction having anticancer activity from the culture solution (a) Cultivation The bacteria belonging to the genus Fusobacterium are cultured by a conventional anaerobic culture method. Namely, nitrogen sources such as cow brain, heart extract, and various inotones; vitamin sources such as yeast extract; inorganic salts such as sodium chloride;
A culture medium containing carbon sources such as glucose and lactose; reducing agents such as L-cystine, sodium sulfite, and thioglycollate is adjusted to pH 6 to 8.5, preferably 6.5 to 7.5, with sodium hydroxide; The bacteria are inoculated and cultured under anaerobic conditions at 30 to 42°C, preferably 52 to 378C, for 1 to 5 days, preferably 1 to 4 days.

Or 1-2 days, 65-42℃, preferably 66-58℃
After culturing at 25°C to 55°C, the cells may be further cultured for 1 to 4 days. In particular, the medium listed in the ingredient list below (hereinafter referred to as TF medium)? It is preferable to use However, as a nitrogen source,
Plain Heart Infusion, which is made from cow brain and heart 11#2 extracts, does not require any strain, and is extracted from heart infusion, which is cow heart extract, beef extract, fish meat extract, and corn. Cornstizorica, etc. may be substituted, and among the various peptones, proteose/peketone and phyton/peptone are not necessarily necessary, and tryptocase, pezotone, and polypeptone may be substituted.

Similarly, when agar is not used, stirring culture 7 is preferably performed.

Below is a table of ingredients in the margin (bl) Collection of supernatant from culture solution (removal of bacterial cells) The supernatant can be obtained by removing the supernatant from the culture solution obtained above.Removal of bacterial cells can be done by conventional methods, such as centrifugation. A filtration method using a filter aid such as , Hyflo-Parcel or the like can be employed, but a centrifugation method is particularly preferable in terms of operation, degree of removal of bacteria, and yield of supernatant liquid.

(A hydrophilic organic solvent is added to the supernatant obtained by separating the effective fraction having C1 anticancer activity Z', and the resulting precipitate is collected. The supernatant at this time preferably has a pH of 1.5 to 7. is pH
Adjust the pH to around 2 (pH 1.5 to 2.5). Examples of hydrophilic organic solvents include alcohols such as ethanol and methanol, and ketones such as acetone, but alcohols, particularly ethanol, give the best results. This hydrophilic organic solvent has a concentration of 50 to 80% (volume ratio),
Preferably, it is added in an amount of 50 to 80 inches (volume ratio). After adding hydrophilic organic solvent, low temperature,
Preferably left at a temperature of about 4 to 5°C for several hours to several days,
Complete the formation of the precipitate.

The precipitate thus produced is separated by conventional operations such as decantation, centrifugation, and filtration.

Next, generally 5 to 20 times the amount of water is added to this precipitate,
Divided according to pH to obtain effective fractions. For example, pH 7.5~
After adjusting the pH to 8 and removing the insoluble portion as desired, the water-insoluble portion obtained by adjusting the pH to around 4 (pH 3.5 to 4.5) is collected by a normal method such as centrifugation or filtration. do. Alternatively, after adjusting the pH to 7.5 to 8 and removing the insoluble portion if desired, the water-insoluble portion 7 obtained by adjusting the pH to around 6 (pH 5.5 to 6.5) may be separated and collected by a conventional method. Furthermore, the water-soluble part has a pH of around 4 (P)! 3.5
The precipitate obtained by adjusting the temperature to 4.5) may be separated and collected by a conventional method. The water-insoluble portion or precipitate of the fraction obtained as described above is subjected to a secondary enzyme treatment and a step of collecting the target product.

(2) Enzyme treatment and target product collection step First, fraction 2 having anticancer activity obtained as above.
Dissolve or suspend in water or buffer, add 2 proteolytic enzymes, and perform enzyme treatment according to a conventional method.

Proteolytic enzymes include pronase, panoin, tridisin, chymotrypsin, etc., with pronase and trypsin being preferred. For or during enzyme treatment, adjust the aqueous solution to pH 7-8.
It is preferable to adjust and maintain the temperature at a certain temperature, and for this purpose, sodium hydroxide, potassium hydroxide, soda carbonate, soda bicarbonate, etc. are used. Further, during the enzyme treatment, it is desirable to add a small amount of an organic solvent such as chloroform or toluene to prevent spoilage of the reaction solution. The amount of enzyme used is usually about 1 to 2% of the powder (solid) to be subjected to enzyme treatment.
Amounts around (weight ratio) are used.

The above enzyme treatment is usually carried out at 60 to 40°C for 1 to 72 hours, preferably 24 to 48 hours. Alternatively, for example, you can add about 1% (weight ratio) of enzyme to the powder (solid), perform enzyme treatment for 7 hours for 1 to 24 hours, and then perform enzyme treatment by adding about 1% (weight ratio) of enzyme. good.

After the above-mentioned enzyme treatment, the anticancer substance TF-
Collect 300 fractions. From this water-soluble part, TF-3
To collect the 00 fraction, for example, a pH-based division method,
Precipitation fractionation using a hydrophilic organic solvent, separation method using an ion exchanger, ultrafiltration, etc. may be combined for treatment. Alternatively, one or more of these operations may be repeated twice. To specifically exemplify the collection method, the above-mentioned water-soluble part is preferably adjusted to pH 2.5 or less, and trichloroacetic acid may be added if desired).The resulting precipitate is removed, and the soluble part is 60 to 80% (volume ratio) of a hydrophilic organic solvent, such as alcohol, preferably 60 to 80%
(volume ratio), collect the precipitate of the resulting fraction of the target product, and if desired, convert this into a precipitate, e.g. Treat with a strong basic anion exchanger such as 400 (trade name) (this operation may be repeated several times), collect the non-adsorbed fraction, and concentrate, dry or hydrophilize using a conventional method. Processing such as precipitation using organic solvents Q′]l:
, the anticancer substance TF-300 of the present invention is obtained.

Anticancer substance TI”-500 obtained as above
The properties of are as follows.

(a) White-gray to light brown powder.

(b) It inhibits the proliferation of Ehrlich ascites-type carcinoma, Ehrlich nodular carcinoma, Sarcoma 180 cancer cells, and B-16 melanoma cancer cells in mice, and has an immunostimulatory effect.

(c) Soluble in water, insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, diethyl ether.

) It does not show a clear melting point, and begins to decompose at about 180°C, and significantly decomposes above 195°C.

(e) The infrared absorption spectrum obtained by the KBr tablet method is
3500~3300.2920.2850.1660~
1620.1580-1540.1460-1400.
1380-1360.1120.1080-1020.
It has absorption bands near 970 and 820-800 cm-''.

(f) The ultraviolet absorption spectrum of an aqueous solution at pH 7 has strong absorption at the 1st and 1st ends, and from 246 to 280 nm.
It shows absorption near .

(G) Molisch reaction, phenol-sulfuric acid reaction, acylon sulfuric acid reaction, indole-hydrochloric acid reaction, and Lowry-Follin reaction are positive, and ninhydrin reaction is negative.

(G) Elemental analysis value C: 68% to 47% H: 5% to 7% N: 1% to 4% (S) Sugar content by phenol-sulfuric acid method is approximately 16
% to 60% (in terms of glucose), and the protein content by the Lowry-Follin method is about 10 parts or less (in terms of bovine serum alzomine).

The anticancer substance TF-300 obtained as described above may be converted into a pharmaceutically acceptable non-toxic salt according to a conventional method. Specific examples include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, and the like.

Next, what is the pharmacological effect of the anticancer substance of the present invention? If you show it, you can move on to the next step. In addition, in the following experiments, the above (1) -
In dividing the precipitate obtained by adding hydrophilic organic solution IA to the supernatant liquid of c., the precipitate & the water-insoluble portion obtained by adjusting the pH to around 4 [Example 1-(3))
, water-insoluble part obtained by adjusting the pH to around 6 [Example 2-
(1) ], a precipitate obtained by further adjusting the water-soluble portion obtained by adjusting the pH to around 6 [Example 2
- (2) :) Each of the TFs obtained by using 7'L and subjecting it to enzyme treatment with a proteolytic enzyme
-310, TF-320 and TF-3,150, and the above TF-510L obtained by enzyme treatment
-(4)) was shown as Tr-310 (precious).

(1) Immunostimulation effect Using a group of 6 IOR mice, the test substance was dissolved in physiological saline, and 0.21 nl of the solution was administered intraperitoneally. 24 hours after administration
A carbon suspension of 0.2 rnln prepared by mixing 1 ml of physiological saline containing 6% gelatin was injected into the tail vein of a normal mouse, and at 1, 5, 10, and 15 minutes after injection, the Madocrit capillary was inserted from the orbit into the heparin-coated tube. 0.02 ml of blood was collected using the same method, and immediately diluted with 6 ml of 0.1% sodium carbonate in water, which had a wavelength of 675 nI.
T] to calculate the phagocytosis coefficient
deX): was calculated using the formula of Halpern et al.

In addition, the control group received 0.2 rnl of physiological saline! Bi-administration was administered.

K-1°g Co-log C t-t.

(co=the amount of charcoal in the blood at the time of t, o, C=the amount of the charcoal in the blood at the time) The results are shown in Table-1.

In the target substance administration group, reticuloendothelial macrophages were activated and cell-mediated immunity of normal mice was increased.

(2) Anticancer effect @) Antitumor effect IO in Ehrlich ascites type tumor
R mice (female, 6 weeks old) were intraperitoneally inoculated with 105 Ehrlich ascites-type cancer cells per mouse. °Then, the test substance was dissolved in physiological saline, and the solution Q was 2 m
1i once a day for 7 days from the 1st day after cancer cell inoculation.
The drug was administered intraperitoneally.

In addition, 0.2 ml of physiological saline was administered once to the control group in the same manner.

The results are shown in Table-2.

Margin below As shown in Table 1, compared to the control group, this presentation-2 are shown in Table-6.

Table 6 (b) Antitumor effect on Lucoma 180 cancer 7H0 cells IcR mice (female, 6 weeks old) were subcutaneously transplanted with 107 Lucoma 180 cancer cells per mouse into the axillary region. Next, the test substance was dissolved in physiological saline or 5-glucose aqueous solution, and 0.2 ml of the solution was administered intravenously or intramuscularly once a day for 7 consecutive days, starting from the first day after the transplantation of the cancer cells.

In addition, the control group included 5-glucose aqueous solution or physiological saline.
．． 2ml/1l was administered in the same manner. Results (Note) TF
The data regarding llo show the results 7 days after inoculation with cancer cells, and the data regarding TF-510 (semen) and TF-520 show the results 14 days after inoculation with cancer cells.

(c) Antitumor effect IO in Ehrlich's tuberosity tumor
In R mice (female, 6 weeks old), 4 x 10' Ehrlich cancer cells per mouse were transplanted subcutaneously into the lower part of the fluid. Next, the test substance was dissolved in a 5% glucose aqueous solution, and the solution was added to a width of 0.2 ml from 8 days after cell transplantation.
The drug was administered once a day, every other day for 5 days. In addition, to the control group, 0.2 rnl of a 5% glucose aqueous solution was administered once in the same manner. Tumor weight Z was measured 150 days after cancer cell transplantation. The tumor weight was measured using a vernier caliper with the major axis (am) and minor axis (b) of the tumor site, and was calculated using the following formula.

Xb2 Tumor weight = (su) The results are shown in Table-4.

Table 4 Melanoma cancer cells were transplanted subcutaneously into BDFL mice (male, 7 weeks old) at a dose of 106 IX per mouse. Next is the test substance? Dissolve in physiological saline or 5% glucose aqueous solution and administer 2 ml of solution Q intracavitally once a day for 7 days from the first soil after cancer cell transplantation, or
From day 0, it was administered intratumorally once a day for 14 consecutive days, or simultaneously administered intratumorally and intraperitoneally once a day every other day for 6 days. In addition, to the control group, 0.2 ml of physiological saline or 5Lib glucose aqueous solution was administered 11 times in the same manner. The results are shown in Table 7-5.

Table 5 (3) Acutely toxic mice (IOR strain, female, 6 weeks old: 1v administration) The following margin (Note) The data regarding TF-310 shows the results on the 23rd day after cancer cell migration. The data regarding 310 (Sperm) shows the results on the 23rd day after cancer cell migration.

Anticancer substance TF-500 (TF-310, T
F-310 (fine), TF'-320, TF-52
0111), TF-530, TF-330C)
) Both the LD5o and Izu male weighed 10~/kg or more.

As is clear from the above pharmacological experiments, the anticancer substance TF-300 obtained by the method of the present invention is useful as an anticancer agent, and is expected to be effective when used in the treatment of various cancers. .

The anticancer substance TF-500 of the present invention can be used as it is or in the form of a non-toxic salt in the form of oral, injection, crude drug, or other dosage forms in a conventional manner. Oral preparations may contain various excipients 7 and may be in the form of capsules, tablets, powders, or granules. In addition, as an injection, subcutaneous, intramuscular,
It may be an intravenous injection, and may be in the form of a suspension, solution, or powder to be dissolved before use. The injection may also contain salt, glucose, or a local anesthetic.

When using the anticancer substance TF-300 of the present invention and its non-toxic salt-hungry patients, the dosage thereof is appropriately selected depending on the patient's symptoms, but in general, for adults it is 0.005 to 50 m
It is preferable to administer 7 g/kg once to several times a day,
The preferred method of administration is oral, subcutaneous, intramuscular, intravenous, or injection into the affected area.

Next, the present invention will be explained by giving examples and formulation examples.

Example 1 (1) In a 10/10 jar fermenter (manufactured by Marubishi Rika Kenkyusho), 1 g of distilled water, 1 F of Tricytocase Hepton, 10 g of Heart Infusion, 6 g of yeast extract, and salt were added. 7.5I, glucose 12
g, lactose 10 &, sodium sulfite 0.1,!
? and sodium thioglycolate 0.5, li
Add 8e of TFe medium containing ``&'' and sterilize at 120℃ for 60 minutes. After cooling, add 100ml of nitrogen gas to the culture solution.
Vent for 1 hour.

Fusobacterium nucleatum TF-031 (FERM-031) precultured in the above-mentioned TF-e medium
5077, ATOO-61647) under sterile conditions. Cultivation was carried out at 67°C under stirring (60 rpm) while injecting nitrogen gas (65ml for 11 min).
Do it for days. After culturing, add Celite 160.9 to the medium-i solution.
Then, cellulose powder 80F'& was added and stirred, and this was filtered under reduced pressure to obtain 7.8'6v of culture liquid supernatant which was sterilized.

(2) (Add 177ml of concentrated hydrochloric acid to the culture supernatant 7.8e obtained in step 11, adjust the pl to 2.0, then add to ethanol 11, #' to make a 60% ethanol solution,
Leave at 4°C for 24 hours. Then, the solution was removed by decantation, and centrifuged at 4°C (6X 10" rpm, 5 minutes) to collect the precipitate. The precipitate was dissolved in 400 mL of a 60% ethanol aqueous solution of p) 12.0. 400111, washed with 200 ml of acetone and 2 CJO ml of diethyl ether in sequence, and dried under reduced pressure to obtain powder 5.9 F'a?.

(3) Suspend the powder obtained in (2) in 25ml of water and add 1N sodium hydroxide aqueous solution to pH 7.5-8.
．． After stirring at room temperature for 60 minutes, 1N-hydrochloric acid 2
Addition? Adjust to H4.0. Then, after stirring for 2 hours under water cooling, centrifugation (I x 10' rpm, 10 minutes)
Then, separate the precipitate and supernatant. This precipitate? p) 14
．． Wash with 10 ml of water adjusted to zero, and centrifuge the precipitate and washing solution (I x 10' rpm, 10 minutes). The precipitate was washed with 1 Qml of ethanol and then dried under reduced pressure to obtain an anticarcinogenic substance TF-210.

This material has the following properties.

(a) White-gray to pale brown powder (b) It inhibits the proliferation of Ehrlich ascites-type cancer, Ehrlich nodular-type cancer, Zulkoma 180 cancer cells, and B-16 melanoma cancer cells in mice, and has an immunostimulatory effect.

(c) Methanol, ethanol, acetone, benzene,
Insoluble in chloroform, ethyl acetate, diethyl ether.

) It does not have a clear melting point and decomposes at 160°C to 255°C.

(The infrared absorption spectrum by the feather KBr tablet method is 5
600~ro200.2950~2920, 1380~1
620, 1550-1510.1440, 1380.1
240-1220 and 1120-1020cmn+-
There is an absorption band 2 near 1 (Fig. 2).

(f) The ultraviolet absorption spectrum of an aqueous solution of the water-soluble fraction at pH 7 has strong absorption at the absorption end, and 248-26
．． It exhibits absorption near 5 nm (Fig. 6).

(G) Molisch reaction, phenol-sulfuric acid reaction, Anthrone-sulfuric acid reaction, indole-hydrochloric acid reaction, and Lowry-Foulin reaction were positive.

Example Elemental analysis value C: 40% to 46% H: 5% to 7 N: 9 to 10% (Male) The sugar content measured by the phenol-sulfuric acid method of the water-soluble fraction at pH 7 is approximately 5% to 25%. % (in terms of glucose), and the protein content by the Lowry-Follin method is approximately 20
% to 50% (in terms of bovine serum albumin).

(4) (3) Anticancer substance TF'-21
Suspend 151 nlVc of 59 water and add 1N-
Add an aqueous sodium hydroxide solution to adjust the pH to 7.8.

Heat to 67°C and use pronase E (product name manufactured by Kaken Chemical Co., Ltd.).
000,000 tyrosine units/g) After adding 15In9y, add a few drops of toluene and adjust the pH to 7.8 while stirring.
8. Perform enzyme treatment between 0.37 and 40°C. Centrifuge the treated solution (4000 rpm, 10 minutes), separate the supernatant and precipitate, and adjust the pH of the precipitate to 8.0. Add 6 ml of water and centrifuge (4000 rpm, 10
minutes) and separate the precipitate from the washing solution. A precipitate is deposited by adding 1N hydrochloric acid to the washing solution and adjusting the pH to 2.0. This solution was left at 5°C for 12 hours and then centrifuged (j x 10' rprn, 10
The supernatant liquid and precipitate were separated, and the precipitate contained PI (2,
Add 5 rnl of water adjusted to zero and centrifuge 1ζIn(1
x10' rpnn, 10 minutes) and separate the precipitate and wash Rw. Add 7 parts of ethanol to the washing solution to obtain an 80% ethanol solution, stir under water cooling for 2 hours, and then centrifuge (4000 rpm, 10 minutes) to separate the precipitate and supernatant. Precipitate 80% ethanol aqueous solution 5
ml and 5 ml of ethanol, and dried under reduced pressure to obtain the fraction 'fx527m' of the anticancer substance TF-310.
Get 9.

Dissolve 627 m of this powder in 5 ml of water, add 1N aqueous sodium hydroxide solution and adjust to PI-17.0.
A column packed with 45 ml of Amberlite RA400 (trade name, manufactured by Rohm & As, Ltd.), which had been adjusted to an OH type with a 1N aqueous sodium hydroxide solution, was applied to the column, and 200 Ill of water was passed through the column. Combine all the ingredients and adjust the pH to 7.0 by adding 1N hydrochloric acid. This is concentrated under reduced pressure, filtered using a Millipore filter (trade name, manufactured by Nippon Millipore Ltd.) with a pore size of 0.3 μm, and then lyophilized to obtain a lyophilized product of the anticancer substance TF-510.

This material has the following properties.

(a) White-gray to light brown powder (b) Ehrlich ascites-type carcinoma of mice, has Ehrlich activity.

(c) Soluble in water, insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, diethyl ether.

) It does not show a clear melting point, and begins to decompose at about 180°C, and significantly decomposes above 195°C.

(e) The infrared absorption spectrum obtained by the KBr tablet method is
3500~3300.2920.2850.1660~
1620.1580-1540.1460-1400.
1380-1360.1120.1080-1020.
It has absorption bands near 970 and 820 to 800 cm-'' (Fig. 4).

(f) The ultraviolet absorption spectrum of an aqueous solution at pH 7 has strong absorption at the absorption end and absorption near 246 to 259 (see Figure 5).

()) TSK-GEL G 3 Q OQ SW (
Toyo Soda Co., Ltd. product name, column: 7.5 mm9
6X 60 Qmm near,
It has an absorption of 2 around 46 to 64 minutes, and in the ultraviolet absorbance measurement at 260 nm, the solvent front part, 42 to 60 minutes
There is an absorption near the minute (Fig. 6).

(H) Positive for Moritzunyu reaction, phenol-sulfuric acid reaction, anthrone-sulfuric acid reaction, indole-hydrochloric acid reaction, and Lowry-Foulin reaction, and negative for ninhydrin reaction.

Concave Elemental analysis value C: 38.0%~42% I(: 5.0%~7%
N: 1% to 4% 0) Sugar content by phenol-sulfuric acid method is approximately 20%
~60% (in terms of glucose) and the protein content by the Lowry-Follin method is about 10% or less (in terms of bovine serum albumin).

(5) Anticancer substance TF-310140 obtained above
Dissolve 50ml/vc of 1% water and use Ultraf Ilter UK-50 (Ultrafiltration membrane product name manufactured by Toyo Okishi Co., Ltd.:
The mixture is ultrafiltered using a molecular weight fraction (molecular weight cutoff: 5×' 104 ) and concentrated 100 times. Millipore filter with a pore diameter of 0.2 μm (product name manufactured by Nippon Millipore Limited)
The anticancer substance TF'-3 is filtered and then freeze-dried.
10 purified lyophilized products 881n9 are available. The thus obtained purified product of the anticancer substance TF-310 had the properties described in the claims, and the sugar content (calcose conversion X) was about 16 to 60.

Example 2 (1) Powder 3.9 obtained by the method of Example 1-(2+)
& 7 Suspended in 251 nl of water, added 2 1N aqueous sodium hydroxide solution to adjust pH to -7.5 to 8.0, and stirred at room temperature.
After stirring for 0 minutes, 1N hydrochloric acid was added to adjust the pH to 6.0. Next, after stirring for 2 hours under water cooling, centrifugation (1
x 10' rpm for 10 minutes) and separate the precipitate and supernatant. This precipitate was mixed with 5 m of water adjusted to pH 6.0.
The precipitate and washing solution were centrifuged (I x 10
'rpm, 10 minutes). The washing solution is combined with the previously obtained supernatant and subjected to the treatment described in Example 6-(). On the other hand, the precipitate was washed with 5 ml of ethanol and dried under reduced pressure to obtain the anticancer substance TF-220'(<,061
Get 1.

This material has the following properties.

(a) White gray to light brown powder (b) Mouse Ehrlich ascites-type carcinoma, Ehrlich nodule-type carcinoma, Delcoma-180 carcinoma 5fB cells and B-1
6 It inhibits the proliferation of melanoma cancer cells and has immunostimulatory effects.

() → Insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, diethyl ether.

(-1 No clear melting point 7, decomposes at 160°C to 240°C.

(e) The infrared absorption spectrum obtained by the KBr tablet method is
6600~5200.2950~2920, 1380~
1620.1550-1510.1440, 1380.
124.0~1220 and 1120~1020cm-
It has an absorption band near 1 (Fig. 7).

f) The ultraviolet absorption spectrum of an aqueous solution of the water-soluble fraction at pH 7 has strong absorption at the absorption end, and 248-266
It shows absorption in the vicinity of nm (Fig. 8).

(G) Molisch reaction, phenol-sulfuric acid reaction, Anthrone-sulfuric acid reaction, indole-hydrochloric acid reaction, and Lowry-Foulin reaction were positive.

(Example: Elemental analysis value C: 40% to 42% H: 5% to 7% Nia% to 9% (IJI The sugar content of the water-soluble surface by the phenol-sulfuric acid method at pH 7 is approximately 5% to 20%) H (glucose equivalent)
, and the protein content determined by the Lowry-Follin method is approximately 10% or less (calculated as bovine serum albumin).

<2) Anticancer substance TF-220 obtained in (1)
The suspension was suspended in 10 ml of 11x water, and the pH was adjusted to 7.8 by adding a 1N aqueous thorium hydroxide solution while stirring.

Heat to 57°C, pronase E (trade name manufactured by convenience chemical company;
1,000.000 tyrosine units/g) 15In9
'(&After addition, add a few drops of toluene, under stirring, p
H7, 8-8. Perform enzyme treatment at 67-40°C for 24 hours. The treated solution was centrifuged (4000 rpm, 10 minutes), the supernatant liquid and precipitate were separated, 5 rnl of water adjusted to pH 8.0 was added to the precipitate, and centrifuged (4000 rpm,
10 minutes) and separate the precipitate and washing solution. Combine the washing solution with the supernatant liquid and add 1N hydrochloric acid to adjust the pH to 2.
If adjusted to 0, a precipitate will separate out. This solution was heated to 12°C at 5°C.
After standing for an hour, centrifugation (I x 10' rpm,
10 minutes), separate the supernatant liquid and precipitate, and add pH 2 to the precipitate.
, add 5 ml of water adjusted to 0, and centrifuge (1 x 1
0' rpm for 10 minutes) and separate the precipitate and washing solution. Combine the washing solution with the supernatant solution and add ethano/L/Y 8
After making 0% ethanol solution and stirring under water cooling for 2 hours,
Centrifuge (4000 rpm 10 minutes) and separate the precipitate and supernatant.
g! Separate L. Add the precipitate to 80% ethanol aqueous solution 5
ml and 5 ml of ethanol, and dried under reduced pressure to obtain fraction 1 of the Nr'i-controlling substance TF-320.
Obtain 66m9.

Dissolve 150 Ing of this powder in 5 ml of water and
Add sodium hydroxide aqueous solution to adjust the pH to 7.0,
1N-hydroxide in advance) OH with IJum aqueous solution
Add 25 ml of Amberlite RA400 (trade name, manufactured by Rohm & Haas Co., Ltd.) adjusted to a mold and the liquid that passed through the column when 100 ml of water was poured into the packed column, and add 1N-hydrochloric acid to pH 7.0. Adjust to.

This was concentrated under reduced pressure and filtered through a Millipore filter (trade name manufactured by Nippon Millipore Ltd.) with a pore size of 0.6 μm.
Then, the anticancer substance TF is freeze-dried to obtain 20 freeze-dried products t110In9.

This material has the following property 7.

(b) White-gray to light brown powder.

(b) It inhibits the growth of Ehrlich ascites-type carcinoma in mice and has immunostimulatory effects.

(/1 Soluble in water, insoluble in methanol, ethanol, aptane, benzene, chloroborum, ethyl acetate, diethyl ether.

2) It does not have a clear melting point, but begins to decompose at about 18°C and significantly decomposes above 195°C.

(Infrared absorption spectrum by J-I KBr tablet method is 3500-3500.2920,2850.16
60~1620.158o~154o, 1460~14
00.1380~1560.1120.1080~10
It has absorption band 2 near 20.970 and 820 to 800 CIn-1 (Fig. 9).

(f) The ultraviolet absorption spectrum of an aqueous solution of Pl-17 has strong absorption at the absorption end, and 250 to 270 nm.
Absorption near m? (Figure 10).

(g) TSK-GKT, G5 Q QQ SW
(Product name of Toyo Soda Co., Ltd., column: 7.5mm0
High performance liquid chromatogram (
Eluent: 0.1 molar phosphate buffer, pH 7, flow rate 0.
8 ml 7 minutes, room temperature) is 22 On, in the ultraviolet absorbance measurement of n, the solvent front part, around 28 to 62 minutes, 4
It has absorption 2 in the vicinity of 5 to 64 minutes, and when measuring ultraviolet absorbance at 260 nm, it has absorption in the solvent front region and in the vicinity of 35 to 56 minutes (Figure 11).

(g) Molisch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, Inzole hydrochloric acid reaction, Lowry-Folin reaction were positive, and ninhydrin reaction was negative.

(Male Elemental analysis value C: 68% to 42% H: 5% to 7% N: 1% to 4q6 (nu) The sugar content by the phenol-sulfuric acid method is approximately 20%.
% to 60% (in terms of glucose) and the protein content according to the Lowry-Follin method is about 10% or less (in terms of bovine serum albumin).

(3) Anticancer substance TF-520,10 obtained above
Dissolve 0m9 in 50ml of water and use Ultra Filter UK
-50 (Ultrafiltration membrane manufactured by Toyo Okishi Co., Ltd., trade name: Sekku Te Ga Mokenkei 5 x 10') and concentrated 100 times. This concentrated solution is filtered through a Millipore filter (trade name, manufactured by Nippon Millipore Limited) with a hole diameter of 0.2 μm, and then freeze-dried to obtain 72 drops of a purified freeze-dried product of the anticancer substance TF-620. Does the purified product of the anticancer substance TF-320 thus obtained have the properties described in the claims? The sugar content (in terms of glucose) is approximately 16-5
It was 0%.

Example 6 (1) A solution of the washing solution obtained in Example 2-(1) and the supernatant liquid was adjusted to pH 4.0 by adding 7 IN-hydrochloric acid, and then left at 56C or lower for 12 hours. Then centrifugation (
lx10'rpm for 10 minutes) and separate the precipitate and supernatant liquid x. This precipitate was mixed with 5 m of water adjusted to pH 4.0.
Wash the precipitate with washing solution and centrifuge (1X 1Q'
rpm, 10 minutes). Precipitate ethanol 5 ml
After washing with
Obtain 0.48 g.

This material has the following property 7.

(b) White-gray to light brown powder.

(b) Mouse Ehrlich ascites-type carcinoma, Ehrlich nodule-type carcinoma, Solucomer 180 cancer cells, and B-16
It inhibits the proliferation of melanoma cancer cells and has immunostimulatory effects.

←→ Methanol, ethanol, acetone, benzene,
Insoluble in chloroform, ethyl acetate, diethyl ether.

) It does not have a clear melting point 2 and decomposes between 1856C and 225C.

(e) The infrared absorption spectrum obtained by the KBr tablet method is
6600~6200.2950~2920, 1380~
1620.1550-1510.1440.1580.
1240~1220 and 1120~'l 020cm
It has an absorption band near -1 (Fig. 12).

(f) The ultraviolet absorption spectrum of an aqueous solution at pH 7 has strong absorption at the absorption end, and also shows absorption χ in the vicinity of 249 to 264 nm (Figure 16).

(G) Molisch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, in-1-l-hydrochloric acid reaction, and Lowry-Follin reaction were positive.

(Example: Elemental analysis value C: 42% to 45% H2 5% to
7 qbN: 10% ~ 11th grade The content of Kinuta by the phenol-sulfuric acid method is approximately 5% ~ 25
% (in terms of glucose) and the protein content determined by the Lowry-Follin method is about 60% to 60% (in terms of bovine serum albumin).

f2) Anticancer substance TF-230 obtained in (1)
cv O, 48! Suspend in 5 ml of water and adjust the pH to 7.8 by adding 7 ml of 1N aqueous sodium hydroxide solution while stirring.

Heat to 67°C, Zolonase E (trade name manufactured by convenience chemical company;
1,000,000 tyrosine units/&) 5. OIψ
After adding the Pl-
17,8~8. [), wait for enzyme treatment at 37-40°C for 24 hours. Processed liquid centrifugation (4000 rpm, 10 minutes)
Then, separate the supernatant liquid and precipitate, and turn it into a precipitate! 3) 18.0
Add 5 ml of water adjusted to
00 rpm for 10 minutes) and separate the precipitate and washing solution.

A precipitate is precipitated by adding 1N hydrochloric acid to the washing solution and the supernatants Wi and Y to adjust the pH to 2.0. This solution? After standing at 5°C for 12 hours, centrifugation (I
10' rpm, 1° min), separate the supernatant and precipitate, and settle. Add 5 ra12 of water adjusted to P112.0 to the grains, centrifuge (1X 10' rpm, 10 min), and precipitate. Separate laundry and cleaning fluids. Combine the washing solution with the previous supernatant, add 7 mL of ethanol to make an 80% ethanol solution, stir for 2 hours under water cooling, centrifuge, and VI (4 [) DO rpm).
, 10 minutes) and separate the precipitate and supernatant.

The precipitate was sequentially washed with 5 ml of an aqueous ethanol solution and 5 ml of ethanol, and then dried under reduced pressure to obtain a fraction of the anticancer substance TF-360.

Dissolve this powder in 5 rnl of 100FIQ water and 1N
-Adjust the pFI to 7.0 by adding an aqueous sodium hydroxide solution, and OH in advance with a 1N aqueous sodium hydroxide solution.
Amber Light E RA400 (trade name manufactured by Rohm & Haas) i5mA adjusted to the mold! Column passing liquid 7 when 60 rnl of water was passed through the packed column
Add 7 portions of 1N hydrochloric acid to adjust the pH to 7.0. This is concentrated under reduced pressure, filtered through a Millipore filter (trade name manufactured by Nippon Millipore Ltd.) with a pore size of 0.6 μm, and then freeze-dried to obtain the anti-cancer substance T-550. 1! Get 19.

This material has the following property 7.

(b) White-gray to light brown powder.

(b) It inhibits the growth of Ehrlich ascites-type carcinoma in mice and has an immunostimulatory effect.

(c) Soluble in water, insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, diethyl ether.

) It does not have a clear melting point χ, and begins to decompose at about 180°C, and significantly decomposes above 195°C.

(e) Infrared absorption spectrum by 1cBr tablet method is 3500-3300, 2920.2850.1660
~1620.1580~1540.1460~1400
, 1380~1360°1120.1080~1020
．． It has an absorption band 7 near 970 and 820 to 8 DD cm (FIG. 14).

F) The ultraviolet absorption spectrum of an aqueous solution at pH 7 has a strong absorption at the absorption end and an absorption 7 in the vicinity of 250 to 260 nm (Figure 15).

(TOI TEIK-GEL G 3 Q Q O8
W (Product name of Toyo Soda Co., Ltd., Column: 7.5
High performance liquid chromatogram (mm fl
In the ultraviolet absorbance measurement of m, the solvent front part, 66
It has an absorption in the vicinity of ~64 minutes, and in the ultraviolet absorbance measurement at 260 nm, it has an absorption in the solvent front region, in the vicinity of 65 to 62 minutes (Figure 16).

(Positive for Timorish reaction, phenol-sulfuric acid reaction, anthrone-sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-fluorin reaction, negative for ninhydrin reaction.

(Male elemental analysis value C: 68%-42% H: 5%-7% N: 1
%~4% (nu) The sugar content by the phenol-sulfuric acid method is approximately 20%.
% to 60% (in terms of glucose) and the protein content according to the Lowry-Follin method is about 10% or less (in terms of bovine serum albumin).

(3) The anticancer substance TFi obtained above was dissolved in 50 ml of 5070η hot water and filtered using Ultra Filter UK-5.
0 (Ultra-Oki filtration membrane trade name, manufactured by Toyo Okishi Co., Ltd.: Benyu Molecular Weight Cutoff 5X10') and concentrated 100 times. This concentrated solution was passed through a Millipore filter with a pore size of 0.2 μm (
The product is filtered using Nippon Millipore Limited (trade name) and then freeze-dried to obtain purified freeze-dried product 49m97 of the anticancer substance TF-660. The purified product of the anticancer substance TF-330 obtained in this way has the property 7 described in the claims, and generally has a sugar content (in terms of glucose) of about 16 to
60%, and the typical infrared absorption spectrum and ultraviolet absorption spectrum were as shown in FIGS. 17 and 18.

Example 4 (1) 10d Jar Fermenter (Marubishi Rika JLF
1 g of distilled water (manufactured by Kyusho), 17 g of tryptocase peptone, 20 I of Heart Infusion, 7.5 g of Yeast Extract 6!11 salt, 12 g of glucose, 10 I of lactose, 0.0 g of sodium sulfite. ,9
81 g of TF-d medium containing 0.5 F and sodium thioglycolate was added and incubated at 120° C. for 60 minutes.

After cooling the culture solution, it was aerated with nitrogen gas for 1 hour at 2100 ml for 11 minutes. Fusobacterium nucleatum TF-051 (precultured in the above-mentioned TF-d medium)
FiM-5077, ATCC-61647) Wi, 1e7all - inoculated under sterile conditions. Culture is 6
The mixture was stirred (30 rpm) for 5 days at 7°C while nitrogen gas was introduced (65 ml for 7 minutes). After culturing, add 160 g of celite and 80 g of cellulose powder to the culture solution.
9'a was added and stirred, and this was filtered under reduced pressure to obtain 7.86 ml of sterilized culture supernatant.

(2) Add 117 ml of 7.81 K concentrated hydrochloric acid to the culture supernatant obtained in (1) to adjust the pH to 2.0, then add 1.7 d' of ethanol to make a 60% ethanol solution,
Leave at 4°C for 24 hours. Then, a portion of the solution was removed by decantation, and the solution was centrifuged at 4° C. for 5 minutes (6×103 rpnn, 5 minutes) to collect the precipitate. This precipitate was mixed with 400ml of 60% ethanol aqueous solution at pH 2.0.
After sequentially washing with 400 ml of ethanol, 200 ml of acetone and 20 CJml of diethyl ether, it was dried under reduced pressure to form a powder with a weight of 4.5 cm. 7 Y get.

Ru.

The powder obtained in (31(2)) was prepared in Example 1-(
31, Example 2-(1) and Example 3-(1) to produce anticancer substances TF-210, TF'-
220 and TF-250'&n respectively.

(4) Anticancer substance TF-2,10 obtained in (3)
, TF-220 and TF-250Y respectively Example 1-(4), Example 2-(2) and Example 3-(2)
), and the anticancer substance TF-510, TF
Lyophilized products of 620 and TF-350 are obtained in amounts of 220 da, 120 In9 and 75 m9, respectively.

Example 5 The anticancer substance TF-210 obtained in Example 1-(3) was treated with enzymes using trypsin (manufactured by Difco) 15#ll' instead of 5.9' and pronase E. and when collecting the precipitate from the water-soluble part adjusted to pH 2 after enzyme treatment, a 60% ethanol solution was used instead of an 80% ethanol solution. Manipulated, freeze-dried product of anti-cancer substance TF-510 20
Get 0In9.

Similarly, the following anticancer substances Ti''-320 and T
A freeze-dried product of F-350 was obtained.

Actual sister example 6 Example 1-(31, Example 2-(11 or Example 3-
The anticancer substance TF-210 obtained in (1),
TF-220 or TF-230 Bi, Example 1-(4)
After enzyme treatment in the same manner as in Example 2-(2) or Example 5-(2), when collecting the precipitate from the water-soluble part adjusted to pH 2, it was added to an 80% ethanol solution. The anticancer substances TF610 and TF were adjusted to a 60% ethanol solution, and then the same procedure as in Example 7 was carried out for /l.
190 m9.981n9 or 66 da of lyophilized product of TF'-320 or TF'-500 was obtained.

Example 7 Example 1-(3), Example 2-(1) or Example 5-(
1) Obtained anticancer substance TF −
210, TF-220 or TF-2,50 were treated with enzymes in the same manner as in Example 1-(4), Example 2-(2) or Example 6-(2), and then adjusted to pH 2. Instead of collecting the precipitate from the water-soluble part, add a 40% aqueous solution of trichloroacetic acid to adjust the water-soluble part to an 80% ethanol solution when the concentration of trichloroacetic acid is YID%, and collect the precipitate. After that, perform 7 operations similar to those in the above example, and use the anticancer substance TF-310, TF-620 or T.
E'-550 freeze-dried product heat 192ηV, 102+11
& or obtain 65In9.

Example 8 (1) 901 Jar Fermenter (Marubishi Rika Institute H: MSJ"2 m) K-1701 (1') To distilled water, tryptocase pezotone 1190 g, heart injection 1050 g, yeast・Extract 210g, food fM 525g, glucose 840g
, TF-f medium containing 700 g of lactose, 7 I of sodium sulfite, 55.9 g of sodium thioglycolate and 175 g of potassium dibasic acid was added,
Sterilize for 15 minutes at °C. Add nitrogen gas v2 to the culture solution after cooling.
Aerate at 50 ml/min for 1 hour.

Fusobacterium nucleatum TF-031 (FERM"), which had been precultured in the above-mentioned TF-f medium,
5077, ATC! O-31647) preculture solution approx. 9
001nlχ Inoculate the above-mentioned culture solution.

Cultivation is carried out at about 66° C. for 4 days with stirring (90 r, p, m) and nitrogen gas flow (C2C25D/min).

After culturing, 10 ft/l of cellulose powder and 5 g/l'i of cellulose powder were added to the culture solution, stirred, and the supernatant of the culture solution was sterilized by filtration under reduced pressure of about 65 d't.

(Add 7.51 VCa of the aliquoted culture supernatant obtained in 21(1) and 115 ml of hydrochloric acid to adjust the pH to 2.0+c, then add ethanol 1.46 to make a 60% ethanol solution, and prepare at 4°C.
Leave it for 24 hours. Next, remove the solution part by decantation, and centrifuge at 4°C to collect the precipitate (
4X 10” rpm, 10 minutes).
p82.0 60% ethanol aqueous solution 111 D Oml
So twice, ethanol 1501i/! , methanol 150
After washing twice with ml and 150 ml of acetone, air dry to obtain a powder of 6.0 ml.

(3) Suspend the powder obtained in (2) in 50 ml of water and 4N-hydroxide) Add IJum aqueous solution to pH 7.5.
After stirring at room temperature for 15 minutes, the pH was adjusted to 4.0 by adding 4N hydrochloric acid.
After standing for an hour, centrifugation (I x 10' rpm,
10 minutes) and separate the supernatant and precipitate. For washing, 6 ml of water was added to this precipitate to form a suspension, and 4N sodium hydroxide aqueous solution 7 was added to adjust the pH to 7.5-8.0.Then, the mixture was stirred at room temperature for 15 minutes, and then 4N - Add hydrochloric acid to adjust the pli to 4.o, and let it stand for 2 hours under water cooling. Then, centrifuge 'i''jfQ (I X104rpn
1, 10 minutes) and sink; collect the dead food.

The precipitate was washed with 4 ml of water adjusted to pH 4.0 and centrifuged (I x 10' rpm, 10 minutes) to obtain a precipitate (TF-210) with a wet weight of 6.2 J.

Thus obtained n-type 4+'t'i substance TF-
210 had the same properties 2 as those obtained in Example 1.

(T-F-2106,2,9 (fresh weight) obtained in 41(3) was suspended in 6 rnl of VC water, and the pH was adjusted to 7.8 by adding saturated ammonium carbonate aqueous solution while stirring. Heat to 67°C and add pronase E (trade name, manufactured by convenience chemical company).
, 000.000 tyrosine units/g): 21lIr
After adding 9'&, add a few drops of toluene and shake, P
Enzyme treatment is performed for 2 hours at I-17,8-8.0.67-4o0c. Next, 4N-hydrochloric acid was added to the treatment solution to pH 1,
0 and allow precipitation to occur. This suspension #! χ After standing for 2 hours under water cooling, centrifugation (I X I Q' rpm, 1
0 minutes) and separate the supernatant MY. Add 1/- to this supernatant.
Add 29ml 1"f of water, add 60ml of ethanol aqueous solution,
Let the precipitate precipitate out. After leaving it for 2 hours under cooling water, centrifuge it (6X 103rpo+, 10 minutes) to form a precipitate. Collect. Add the precipitate to 5 ml of 60% ethanol aqueous solution.
, ethanol iQmJ and acetone 10rnl, and then air-dried to remove the anticancer substance TF-610315I.
Get n9 yen.

This was dissolved in 73 Qrnl of water, adjusted to P) 18.0 using a 4N-sodium hydroxide aqueous solution, and filled with 53 ml of DOWEX I The total amount of liquid passing through the column when 100ml of water was poured into the column was PI-16, 5 to 7.
．． Adjust so that it becomes 0. Ultra filter U
K-50 (product name of ultra-ν filtration membrane manufactured by Toyo P Paper Co., Ltd.; molecular weight cut off 5 x 10') + 2 ultrafiltration steps 10
Reduce a to 0 times. This concentrated solution was filtered through a Millipore filter (trade name manufactured by Nippon Millipore Limited) with a pore diameter of 0.3 μm, and then freeze-dried to obtain the anticancer substance TF-311.
I would like 230 mg of purified lyophilized product. The purified product of the anticancer substance TF-410 obtained as in step 7 has the properties described in the claims, and generally has a sugar content (in terms of glucose) of about 16 to 60, and a representative The infrared absorption spectrum, ultraviolet absorption spectrum, and high performance liquid chromatogram of the sample were as shown in Figures 19, 20, and 21 of 2''L.

Formulation Example 1 A lyophilized product of the anticancer substance T17'ilO was filled into Toda7 vials. When used, this solution is dissolved in sterile physiological saline or a solution containing 0.5% lidocaine and used as an injection solution.

Formulation Example 2 Anticancer substance TF-,! The lyophilized product of +20 was filled into a 1-inch vial. When used, this solution is dissolved in sterile physiological saline or a solution containing 0.5% lidocaine and used as an injection solution.

Formulation Example 6 A lyophilized product of anti-cancer substance TF-350 was filled into a 1η vial. At the time of opening, the drug is dissolved in sterile physiological saline or a solution containing 0.5% lidocaine and used as an injection.

Formulation Example 4 Anticancer substance TF-z + 10, pH 7, 0-7.5 aqueous solution M'< Filled in a vial, 0.5 1 unit of anticancer substance TF-310 freeze-dried product I can beg. This is
When used, it is used as an injection by dissolving it in sterile physiological saline, a solution containing 0.5% lidocaine, or a 5% aqueous glucose solution.

Formulation Example 5 Anticancer substance TF-310 (edge)? Contains pH 7,0-7
．． Fill the aqueous solution of No. 5 into a vial bottle to obtain 0.5 or 1 to 1 unit of anticancer substance TF-510 (sugi) freeze-dried product 2. When using this drug, add sterile physiological saline and lidocaine 0.5%.
It is used as an injection solution by dissolving it in a solution containing it or 5% Zodou Fishing Water Solution.

Formulation Example 6 Fill a vial with an aqueous solution containing anticancer substance TF-520 at pH 7.0 to 7.5 to obtain a lyophilized product of anticancer substance 1, T2 = g 20 per 0.5 or 1 m9 units. . When using this, use sterile physiological saline, lidocaine 0.5%,
- ・ ・ ・ Use as an injection solution by dissolving it in a 5% aqueous glucose solution or a 5% aqueous solution.

Formulation Example 7 Containing anticancer substance TF-550 P) 17.0 to 7.5
The anti-cancer substance TF-550 can be lyophilized in 0.5 or 1 mg units by filling an aqueous solution into a vial. When used, this drug is dissolved in sterile physiological saline, a solution containing 0.5% lidocaine, or a 5% aqueous glucose solution and used as an injection solution.

[Brief explanation of the drawing]

Figure 1 is a micrograph showing the form 7 of Fusobacterium nucleatum TF-051 used in the present invention, Figure 2 is the infrared absorption spectrum of the anticancer substance TF-210, and Figure 6 is the ultraviolet absorption spectrum of the same substance. Figure 4 shows the infrared absorption spectrum of the anticancer substance TF-310 of the present invention.
The figure shows the ultraviolet absorption spectrum of the same substance, Figure 6 shows the high-performance liquid chromatogram of the same substance, and Figure 7 shows the anticancer substance TF-
220, FIG. 8 is the ultraviolet absorption spectrum of the same substance, and FIG. 9 is the anticancer substance TF6 of the present invention.
Figure 10 is the ultraviolet absorption spectrum of the same substance, Figure 11 is the high performance liquid chromatogram of the same substance, the 12th factor is the infrared absorption spectrum of the anticancer substance TF-230, Figure 15 is the same substance. The ultraviolet absorption spectrum of the substance, Fig. 14 is the infrared absorption spectrum of the anticancer substance TF-'3ROO of the present invention, Fig. 15 is the ultraviolet absorption spectrum of the same substance, and Fig. 16 (2) is the high-speed liquid of the same substance. Chromatogram, Figure 17 shows the anticancer substance TF-3 of the present invention.
18 is the ultraviolet absorption spectrum of the same substance, FIG. 19 is the infrared absorption spectrum of the anticancer substance TF-310 (precious) of the present invention,
Figure 0 shows the ultraviolet absorption spectrum of the same substance, and Figure 21 shows the controlled liquid chromatogram 7 of the same substance. Figure 3 Figure 5 Figure 10 Figure 13 Figure 15 Figure 18 Oρ Usumi

Claims (17)

[Claims] (1) An anticarcinogenic substance TF-, which has the following properties, is obtained by culturing bacteria belonging to the genus Fusobacterium and subjecting the fraction with anticancer activity obtained from the culture solution to enzymatic treatment with a proteolytic enzyme. 300 and its salt. (a) White-gray to light brown powder. (b) It inhibits the proliferation of Ehrlichi's ascitic carcinoma, Ehrlichi's nodal carcinoma, Sarcoma 180 cancer cells, and B-16 melanoma cancer cells and has an immunostimulatory effect. (c) Soluble in water, insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, diethyl ether. (d) It does not show a clear melting point, and begins to decompose at about 180°C and significantly decomposes at 195°C or higher. (e) The infrared absorption spectrum obtained by the KBr tablet method is 3
500-3300, 2920, 2850, 1660-1
620, 1580-1540, 1460-1400, 1
380-1360, 1120, 1080-1020, 9
It has absorption bands near 70 and 820 to 800 cm^-^1. (f) The ultraviolet absorption spectrum of an aqueous solution at pH 7 has strong absorption at the absorption end and also shows absorption in the vicinity of 246 to 280 nm. (G) Moritsch reaction, phenol-sulfuric acid reaction, Anthrone-sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-Follin reaction are positive, and ninhydrin reaction is negative. (h) Elemental analysis value C: 38% to 47% H: 5% to 7% N: 1% to 4% (li) Sugar content determined by the phenol-sulfuric acid method is approximately 16%
~60% (in terms of glucose) and the protein content by the Lowry-Follin method is about 10% or less (in terms of bovine serum albumin). (2) A fraction with anticancer activity of the precipitate produced by adding a hydrophilic organic solvent to the supernatant liquid obtained by culturing bacteria belonging to the genus Fusobacterium is obtained by subjecting it to enzymatic treatment with a proteolytic enzyme. The anticancer substance TF-300 and its salts according to claim 1. (3) Add a hydrophilic organic solvent to the supernatant obtained by culturing Fusobacterium genus, divide the resulting precipitate based on pH, and enzymatically treat the fraction with anticancer activity with a proteolytic enzyme. The anticancer substance TF-30
The anticancer substance TF-300 and its salt according to claim 1 or 2, which is obtained by collecting a fraction of 0. (4) The anticancer substance TF-300 and its salt according to any one of claims 1 to 3, wherein the enzymatic treatment with a proteolytic enzyme is pronase or trypsin treatment. (5) The anticancer substance TF-300 and its salt according to any one of claims 1 to 4, wherein the bacterium belonging to the genus Fusobacterium is Fusobacterium nucleatum. (6) The method is characterized in that a fraction having an anticancer effect of a precipitate produced by adding a hydrophilic organic solvent to a supernatant obtained by culturing bacteria belonging to the genus Fusobacterium is subjected to enzymatic treatment with a protease. A method for producing anticancer substance TF-300 and its salt. (7) Add a hydrophilic organic solvent to the supernatant obtained by culturing Fusobacterium genus, divide the resulting precipitate based on pH, and enzymatically treat the fraction with anticancer activity with a proteolytic enzyme. and then the anticancer substance TF-3
7. The method for producing the anticancer substance TF-300 and its salts according to claim 6, characterized in that the 00 fraction is collected. (8) The operation for collecting a fraction of the anticancer substance TF-300 after enzyme treatment is at least one of pH-based separation method, precipitation fractionation method using hydrophilic organic solvent, ion exchanger or ultrafiltration method. The anticancer substance T
The method for producing the anticancer substance TF-300 and its salts according to claim 7, which is an operation of collecting a fraction of F-300. (9) The operation of collecting a fraction of the anticancer substance TF-300 after enzyme treatment is the water obtained by dividing the water-soluble portion after enzyme treatment with a proteolytic enzyme according to the pH and adjusting the pH to 2.5 or less. Claim 7, which is an operation in which a hydrophilic organic solvent is added to the effective fraction of the soluble portion, the resulting precipitate is treated with an ion exchanger, and the non-adsorbed fraction is collected by ultrafiltration. or the method for producing the anticancer substance TF-300 and its salts according to item 8. (10) The control according to any one of claims 6 to 9, wherein the operation of adding the hydrophilic organic solvent to the supernatant liquid is an operation of adjusting the supernatant liquid to around pH 2 and then adding the hydrophilic organic solvent. A method for producing cancerous substance TF-300 and its salt. (11) The method for producing anticancer substance TF-300 and its salts according to any one of claims 6 to 10, wherein the hydrophilic organic solvent added to the supernatant liquid is alcohol. (12) Hydrophilic organic solvent at a concentration of 30% to 80% (
12. A method for producing anticancer substance TF-300 and its salts according to any one of claims 6 to 11, characterized in that the anticancer substance TF-300 and its salts are added to the supernatant liquid so as to have a volume ratio of TF-300 to TF-300. (13) The operation of adding a hydrophilic organic solvent to the supernatant liquid and dividing the precipitate obtained by pH is the same as adding water to the precipitate and dividing it by pH.
13. The method for producing the anticancer substance TF-300 and its salts according to any one of claims 7 to 12, which is an operation of adjusting the water-insoluble portion to around 4 and collecting the obtained water-insoluble portion. (14) The operation of dividing the precipitate obtained by adding a hydrophilic organic solvent to the supernatant liquid by adding water to the precipitate and p
14. The method for producing the anticancer substance TF-300 and its salts according to any one of claims 7 to 13, which comprises an operation of adjusting the temperature around H6 and collecting the water-insoluble portion. (15) The operation of dividing the precipitate obtained by adding a hydrophilic organic solvent to the supernatant liquid by adding water to the precipitate and p
The anticancer substance T according to any one of claims 7 to 14 is an operation in which the water-soluble portion obtained by adjusting the pH to around pH 6 is further adjusted to around pH 4, and the resulting precipitate is collected.
Method for producing F-300 and its salt. (16) The method for producing anticancer substance TF-300 and its salts according to any one of claims 6 to 15, wherein the enzymatic treatment with a proteolytic enzyme is pronase or trypsin treatment. (17) Anticancer substance TF-, which has the following properties, is obtained by culturing bacteria belonging to the genus Fusobacterium and subjecting the fraction with anticancer activity obtained from the culture solution to enzymatic treatment with a proteolytic enzyme. 300 or a salt thereof. (a) White-gray to light brown powder. (b) It inhibits the proliferation of Ehrlichi's ascitic carcinoma, Ehrlichi's nodal carcinoma, Sarcoma 180 cancer cells, and B-16 melanoma cancer cells and has an immunostimulatory effect. (c) Soluble in water, insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, diethyl ether. (d) It does not show a clear melting point, and begins to decompose at about 180°C and significantly decomposes at 195°C or higher. (e) The infrared absorption spectrum obtained by the KBr tablet method is 3
500-3300, 2920, 2850, 1660-1
620, 1580-1540, 1460-1400, 1
680-1360, 1120, 1080-1020, 9
It has absorption bands near 70 and 820 to 800 cm^-^1. (f) The ultraviolet absorption spectrum of an aqueous solution at pH 7 has strong absorption at the absorption end and also shows absorption in the vicinity of 246 to 280 nm. (g) Moritsch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-Follin reaction are positive, and ninhydrin reaction is negative. (h) Elemental analysis value C: 38% to 47% H: 5% to 7% N: 1% to 4% (li) Sugar content determined by phenol-sulfuric acid method is approximately 16%
~60% (in terms of glucose) and the protein content by the Lowry-Follin method is about 10% or less (in terms of bovine serum albumin).