JPH021156B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is directed to a novel anticancer substance, more specifically, it is obtained by culturing bacteria belonging to the genus Fusobacterium and subjecting the effective fraction having anticancer activity obtained from the culture solution to enzymatic treatment with a proteolytic enzyme. This article relates to anticancer substance TF-340 and its salt. Furthermore, the present invention relates to a method for producing the anticancer substance TF-340 and its salt, and an anticancer drug containing the same. BACKGROUND ART In recent years, treatments for various cancer patients that enhance the host's immune function and exert anticancer effects with the help of the immune function have become popular. 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 is little research on components that have excellent anticancer effects from the culture fluid obtained by culturing anaerobic bacteria, and in particular, there is still little research on the components obtained from the culture fluid obtained by culturing Fusobacterium bacteria and their anticancer effects. unknown. The present inventor cultivated bacteria belonging to the genus Fusobacterium isolated from the human oral cavity, and investigated the pharmacological effects of the components collected from the supernatant after removing the bacterial cells from the culture solution. This means that the colony formation inhibition method has a strong anticancer effect, and the effect of inhibiting cancer cell colony formation is small, and the anticancer effect is not due to bactericidal vesicles, but rather to host-mediated or host immune system enhancement. It was discovered that the drug indirectly exerts an anticancer effect with the help of immunity. The specific component obtained by further subjecting the resulting effective fraction with anticancer activity to enzymatic treatment with a proteolytic enzyme has favorable properties as an anticancer agent, such as having favorable anticancer activity and few side effects. The present invention was completed based on the discovery that the present invention has the following properties. The bacteria used in the present invention include Fusobacterium, which produces the anticarcinogenic substance TF-340 when the effective fraction with anticancer activity obtained from the supernatant of culturing the bacteria is subjected to 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 (FERM-PNo.5077, ATCC-31647)
As a general knowledge of microbiology, strains having such properties, such as natural mutant strains or artificially improved strains, are used. The mycological properties of Fusobacterium nucleatum TF-031 are as follows. (1) Morphology Cell shape: spindle-shaped (Figure 1) Presence of cell pleomorphism: None Motility: None Presence or absence of spores: None Gram staining: Gram negative Acid-fastness: Negative (2) Growth status in medium TF-a agar plate and slant medium External shape: Circular Size: Approximately 1 mm Protuberance: Hemispherical structure: Dewdrop-like surface: Smooth edges Green: Smooth Color: Milky white Transparency: Opaque TF-a liquid medium Growth level: Vigorous Turbidity: Coagulation precipitation: None Surface growth: None, no growth up to about 5 mm Gas: None (3) Physiological properties Hydrogen sulfide production: + Nitrate reduction: - Butyric acid production: + Indole production: + Urease :- Catalase: - Hydrolysis of starch: - Attitude towards oxygen: Anaerobic Production of ammonia: + Production of carbon dioxide: + Growth range: PH5-8.5 Temperature 30-45℃ Production of gas from sugar L-arabinose ( -), D-xylose (-), D-glucose (-), D-mannose (-), D-fructose (-), D-galactose (-), maltose (-), sucrose (-), trehalose (-), sylvit (-), mannitol (-), inosyte (-), glycerin (-), starch (-)
When considered in light of the edition, this strain is Fusobacterium nucleatum (Fusobacterium nucleatum).
nucleatum) and belongs to this group. Next, an example of the method for producing the anticancer substance TF-340 of the present invention will be schematically explained as follows.

[Table] The above manufacturing method is specifically shown as follows. (1) Steps for culturing and separating effective fractions obtained from the culture solution (a) Cultivation The cultivation of bacteria belonging to the genus Fusobacterium is carried out by the usual culture method for anaerobic bacteria. That is,
Nitrogen sources such as cow brain, heart extract, various peptones; Vitamin sources such as yeast extract;
A medium containing inorganic salts such as sodium chloride; a carbon source such as glucose and lactose; and a reducing agent such as L-cystine, sodium sulfite, and sodium thioglycollate is diluted with sodium hydroxide to pH 6.
Adjust to 8.5 preferably 7.2 to 8.2, inoculate the bacteria,
1 at 35-42℃ preferably 36-38℃ under anaerobic conditions.
Allow to stand or culture with stirring for ~5 days, preferably 1 to 4 days. Alternatively, after culturing at 35-42°C, preferably 36-38°C, for 1-2 days, the culture may be further cultured at 25-35°C for 1-4 days. In particular, it is preferable to use a medium (hereinafter referred to as TF medium) described in the ingredient table below.
However, as a nitrogen source, it is not always necessary to use plain heart infusion, which is made from cow brain and heart extracts. Proteose peptone and phitone peptone are not necessarily necessary among various peptones, and tryptocase peptone can also be substituted with polypeptone. Incidentally, when agar is not used, it is preferable to perform stirring culture.

[Table] (b) Collection of supernatant from culture solution (removal of bacterial cells) Remove bacterial cells from the culture solution obtained above to obtain supernatant. Bacterial cells can be removed by conventional methods, such as centrifugation or filtration using a supernatant such as Hyflo Super Cell, but centrifugation is particularly preferable in terms of operation, degree of bacterial removal, and yield of supernatant. . (c) Collection of anticancer substance TF-240 Add a hydrophilic organic solvent to the supernatant obtained above and collect the resulting precipitate. The supernatant liquid at this time is
Adjust to PH5-7, preferably around PH2 (PH1.5-2.5). 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 is suitably added so that its concentration is 30 to 80% (volume ratio), preferably 50 to 80% (volume ratio). After adding the hydrophilic organic solvent, the mixture is left at a low temperature, preferably about 4° to 5° C., for several hours to several days to complete the formation of a precipitate. The precipitate thus formed is separated by conventional procedures such as decantation, centrifugation, filtration, etc. Then, generally 5 to 20 times the amount of water is added to this precipitate, and the mixture is divided according to pH. For example, PH7.5-8
After adjusting the pH to around 4 (PH 3.5 to 4.5), the water-insoluble part and the water-soluble part are separated by a conventional method such as centrifugation or filtration. The water-soluble portion thus obtained is further adjusted to a pH of around 2 (PH 1.5 to 2.5), and the resulting precipitate is separated from the water-soluble portion by a conventional method such as centrifugation or sieving. If the precipitate is collected, the effective fraction TF-240 can be obtained. The anticancer substance TF-240 obtained as described above has the following properties. (a) White-gray to light brown powder. (b) Mouse Ehrrich ascites-type carcinoma, Ehrlichi's nodule-type carcinoma, Sarcomer 180 cancer cells, and B
-16 It inhibits the proliferation of melanoma cancer cells and has immunostimulatory effects. (c) Insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, and diethyl ether. (d) It does not show a clear melting point and decomposes at 200℃ to 215℃. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3600~3200, 2950~2920, 1680~1620, 1550~
It has absorption bands near 1520, 1410-1360, 1280-1210, 1060, 960 and 820 cm ^-1 (second
figure). (F) The ultraviolet absorption spectrum of an aqueous solution with a pH of 7 has strong absorption at the absorption end and also shows absorption in the vicinity of 250 to 265 nm (Figure 3). (g) Moritsch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, and Lowry-Follin reaction were positive. (h) Elemental analysis values C: 35% to 38% H4% to 5% N: 12% to 14% (li) Sugar content by phenol sulfuric acid method is approximately 15%
~35% (glucose equivalent), and Lowry
The protein content by the phorin method is approximately 20-30%
(in terms of bovine serum albumin). (2) Enzyme treatment and target product collection process First, the fraction with anticancer activity obtained as described above is dissolved or suspended in water or a buffer solution, a protease is added, and the mixture is treated according to a conventional method. Perform enzyme treatment. Examples of the protease include pronase, papain, trypsin, and chymotrypsin, with preference given to pronase and trypsin. It is preferable to adjust and maintain the pH of the aqueous solution at 7 to 8 during the enzyme treatment, and for this purpose, sodium hydroxide, potassium hydroxide, soda carbonate, sodium bicarbonate, etc. are used. Furthermore, during 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% (weight ratio) based on the powder (solid) to be subjected to enzyme treatment. The above enzyme treatment is usually carried out at 30-40℃ for 1-72 hours.
Preferably it is carried out for 24 to 48 hours. Also, for example, by adding about 1% (weight ratio) of enzyme to the powder (solid),
Enzyme treatment may be performed for 1 to 24 hours, and further enzyme treatment may be performed by adding about 1% (weight ratio) of enzyme. After the above enzyme treatment, a fraction of the anticancer substance TF-340 is collected. To collect the TF-340 fraction, a combination of one or more of, for example, a separation method using PH, a precipitation method using a hydrophilic organic solvent, a separation method using an ion exchanger, etc. may be performed. To give a specific example of the collection method, the above enzyme-treated solution is adjusted to PH2.5 or less (trichloroacetic acid may be added if desired), the resulting precipitate is removed, and the soluble part is added with a hydrophilic organic Add a solvent, e.g. alcohol, to 30-80% (by volume), preferably 60-80% (by volume), collect the resulting precipitate of the desired effective fraction, and if desired, then add this precipitate to For example, Dowex 1-X (product name), Amberlite IRA-
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 hydrophilic by a conventional method. The anticancer substance TF-340 of the present invention can be obtained by treatment such as precipitation using an organic solvent. Anticancer substance TF-340 obtained as above
The properties of are as follows. (a) White-gray to light brown powder. (b) It inhibits the growth of Ehrrich's ascites-type carcinoma in mice and has immunostimulatory effects. (c) Dissolved in water, methanol, ethanol, acetone, benzene, chloroform, ethyl acetate,
Insoluble in diethyl ether. (d) It does not exhibit a clear melting point, and begins to decompose at about 140°C, and significantly decomposes at temperatures above 200°C. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3500~3300, 2920, 2850, 1660~1640, 1580~
1520, 1460~1440, 1410~1340, 1250~1220,
It has absorption bands near 1120-1030, 970 and 835 cm ^-1 . (Fig. 4) (f) The ultraviolet absorption spectrum of an aqueous solution with pH 7 has strong absorption at the absorption end and also shows absorption in the vicinity of 250 to 265 nm. (Figure 5) (g) High performance liquid chromatogram using TSK-GEL G3000SW (trade name of Toyo Soda Co., Ltd., column: 7.5 mmφ x 600 mm x 2) (eluent: 0.11 molar phosphate buffer with PH, flow rate 0.8 ml) /min, room temperature)
In ultraviolet absorbance measurement at 220 nm, there is absorption at the front end of the solvent, around 36 to 62 minutes.
In ultraviolet absorbance measurement at 260 nm, absorption occurs at the front end of the solvent, around 34 to 62 minutes.
(Fig. 6) (H) Moritsch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-Folin reaction are positive, and ninhydrin reaction is negative. (li) Elemental analysis values C: 32% to 34% H: 4% to 6% N: 3% to 5% (nu) The sugar content determined by the phenol sulfuric acid method is approximately
20% to 50% (glucose equivalent), and protein content by Lowry-Follin method is approximately 10%
It is as follows (in terms of bovine serum albumin). (Ru) Although the molecular weight is over 1000, it is extremely difficult to identify. Anticancer substance TF-340 obtained as 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, and alkaline earth metal salts such as magnesium salts and calcium salts. Next, the pharmacological action of the anticancer substance TF-340 of the present invention is as follows. (1) Immunostimulatory effect Using a group of four ICR mice, the test substance was dissolved in physiological saline, and 0.2 ml of the solution was intraperitoneally administered. Perikan Drawing 24 hours after administration
0.2 ml of a carbon suspension prepared by mixing 1 ml of Ink17Black (manufactured by Guilnter Wagner) and 2 ml of physiological saline containing 3% gelatin was injected into the mouse tail vein, and 1, 5, 10, and 15 minutes after injection, the carbon suspension was injected into the mouse via the orbit. Blood was collected using heparin-coated hematocrit capillaries.
Collect 0.02ml, immediately dilute it with 1.6ml of 0.1% sodium carbonate aqueous solution, hemolyze it, compare the color at a wavelength of 675nm, and calculate the phagocytotic index (K value).
It was calculated using the formula of Halpern et al. In addition, 0.2 ml of physiological saline was administered to the control group. K=log Co-log C/t-to (Co=to amount of charcoal in the blood, C=blood charcoal amount at t) The results are shown in Table-1.

[Table] As is clear from Table 1, compared to the control group, TF
In the -340 substance administration group, reticuloendothelial macrophages were activated and cell-mediated immunity in normal mice was increased. (2) Anticancer effect Antitumor effect on Ehrlitsu ascites tumor ICR mice (female, 6 weeks old) were intraperitoneally inoculated with 1×10 ⁵ Ehrlitsu ascites cancer cells per mouse. Next, the test substance was dissolved in physiological saline, and 0.2 ml of the solution was injected from the first day after cancer cell inoculation.
The drug was administered intraperitoneally once a day for 7 consecutive days. In addition, to the control group, 0.2 ml of physiological saline was administered once. The results are shown in Table-2. T/C = Survival days of treatment group/Number of survival days of control group x 100
(%)

[Table] (3) Acute toxicity The LD ₅₀ value in mice (ICR strain, male, 6 weeks old: iv administration) was 200 mg/Kg or higher. As is clear from the above pharmacological experiments, the anticancer substance TF-340 obtained by the method of the present invention is useful as an anticancer agent, and is expected to be effective when used for various cancer diseases. The anticancer substance TF-340 of the present invention can be used as it is or as a non-toxic salt in a conventional manner in the form of oral, injection, suppository, etc. dosage forms. As an oral agent,
It may contain various excipients and can be made into capsules, tablets, powders, and granules. The injection may be subcutaneous, intramuscular, or intravenous, and may be in the form of a suspension, solution, or powder to be dissolved before use. The injection may also contain a local anesthetic. When administering the anticancer substance TF-340 of the present invention and its non-toxic salt to a patient, the dose is appropriately selected depending on the patient's symptoms, but in general, for adults it is 0.01.
It is preferable to administer ~50 mg/Kg in one to several divided doses a day, and 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) Into 10 jar fermenters (manufactured by Marubishi Rika Kenkyusho), 17 g of tryptocase peptone and heart injection were added to 1 portion of distilled water.
10g, yeast extract 3g, salt
TF-e medium 8 containing 7.5 g, glucose 12 g, lactose 10 g, sodium sulfite 0.1 g and sodium thioglycolate 0.5 g
and sterilize at 120°C for 30 minutes. After cooling the culture solution, nitrogen gas is aerated at 100 ml/min for 1 hour. Fusobacterium nucleatum TF-, which was pre-cultured in the TF-e medium mentioned above.
031 (FERM-P No. 5077, ATCC-31647) preculture solution 1 is inoculated under sterile conditions. Culture is 37
This is carried out at ℃ for 3 days under stirring (30 rpm) while flowing nitrogen gas (65 ml/min). After the culture was completed, 160 g of Celite and 80 g of cellulose powder were added to the culture solution and stirred, and the mixture was filtered under reduced pressure to obtain 7.8 g of sterilized culture supernatant. (2) Add 117ml of concentrated hydrochloric acid to 7.8ml of the culture supernatant obtained in (1).
After adding and adjusting the pH to 2.0, ethanol 11.7
Add to make a 60% ethanol solution and incubate at 4℃ for 24 hours.
Leave it for a while. Next, the solution portion was removed by decantation, and the temperature was heated at 4°C to collect the precipitate.
Centrifuge (6 x 10 ³ rpm, 5 minutes). This precipitate was mixed with 400 ml of 60% ethanol aqueous solution with pH 2.0.
After sequentially washing with 400 ml of ethanol, 200 ml of acetone and 200 ml of diethyl ether, the mixture was dried under reduced pressure to obtain 3.9 g of powder. (3) Suspend the powder obtained in (2) in 25 ml of water, and
Add aqueous sodium hydroxide solution to adjust the pH to 7.5-8.0, stir at room temperature for 30 minutes, then add 1N hydrochloric acid to adjust the pH to 6.0. Next, after stirring for 2 hours under ice cooling, centrifugation (1 x 10 ⁴ rpm, 10 minutes)
and separate the precipitate and supernatant. This precipitate
Wash with 5 ml of water adjusted to pH 6.0, centrifuge the precipitate and washing liquid (1 x 10 ⁴ rpm, 10 minutes), combine the supernatant liquid obtained earlier and the washing liquid, and add 1N-hydrochloric acid to this solution. After adjusting the pH to 4.0 by adding
Leave it for 12 hours below. Then, centrifugation (1
×10 ⁴ rpm, 10 minutes) and separate the precipitate and supernatant. This precipitate was washed with 5 ml of water adjusted to pH 4.0, and the precipitate and washing solution were centrifuged (1×
10 ⁴ rpm for 10 minutes), combine the washing solution and the supernatant obtained earlier, and add 1N-hydrochloric acid to this solution.
After adjusting the pH to 2.0, leave it for 2 hours under cooling with ice water. Then centrifugation (6 x 10 ³ rpm, 10 minutes)
and separate into precipitate and supernatant. This precipitate
Wash with 3 ml of water adjusted to pH 2.0, centrifuge the precipitate and washing solution (6 x 10 ³ rpm, 10 minutes), wash the precipitate with 5 ml of ethanol, dry under reduced pressure, and remove the anticancer substance TF. - Obtain 0.11g of 240. (4) Suspend 0.9 g of the anticancer substance TF-240 obtained as in (1), (2), and (3) above in 10 ml of water, and add 1N aqueous sodium hydroxide solution while stirring. TePH
Adjust to 7.8. Warm to 37℃ and add pronase E.
(Product name manufactured by Kaken Chemical; 1,000,000 tyrosine units/
g) After adding 9 mg, add a few drops of toluene and perform enzyme treatment for 24 hours at pH 7.8-8.0 and 37-40°C with stirring. Centrifuge the treated solution (4000rpm,
10 minutes), separate the supernatant and precipitate, and add to the precipitate.
Add 3 ml of water adjusted to pH 8.0 and centrifuge (4000 rpm, 10 minutes) to separate the precipitate and washing solution. Combine the supernatant solution with the washing solution first, and add to this.
Add 1N hydrochloric acid to adjust the pH to 2.0, and a precipitate will form. After leaving this solution at 5°C for 12 hours,
Centrifuge (1 x 10 ⁴ rpm, 10 minutes) to separate the supernatant and precipitate, add 5 ml of water adjusted to PH2.0 to the precipitate, and centrifuge (1 x 10 ⁴ rpm, 10 minutes).
and separate the precipitate and washing solution. Combine the washing solution with the supernatant, add ethanol to make an 80% ethanol solution, and stir under ice cooling for 2 hours.
Centrifuge (1×10 ⁴ rpm, 10 minutes) to separate the precipitate and supernatant. After sequentially washing the precipitate with 5 ml of 80% ethanol aqueous solution and 5 ml of ethanol, it was dried under reduced pressure to obtain a fraction of the anticancer substance TF-340.
Get 170mg. Dissolve 150 mg of this powder in 5 ml of water, add 1N aqueous sodium hydroxide solution and adjust the pH to 7.0.
In advance with 1N-sodium hydroxide aqueous solution.
A column packed with 25 ml of Amberlite IRA400 (trade name manufactured by Rohm & Haas) adjusted to OH type was applied to the column, and 100 ml of water was passed through the column. Combine all the liquids that passed through the column, and add 1N-hydrochloric acid to adjust the pH.
Adjust to 7.0. This is concentrated under reduced pressure and the pore size
0.3μm Millipore filter (Japan Millipore)
Limited (product name)) and then freeze-dried to obtain 110 freeze-dried products of the anticancer substance TF-340.
Get mg. Example 2 (1) Into 10 jar fermenters (manufactured by Marubishi Rika Kenkyusho), 17 g of tryptocase peptone and heart injection were added to 1 portion of distilled water.
20g, yeast extract 3g, salt
TF-d medium 8 containing 7.5 g, glucose 12 g, lactose 10 g, sodium sulfite 0.1 g and sodium thioglycolate 0.5 g.
and sterilize at 120°C for 30 minutes. After cooling the culture solution, nitrogen gas is aerated at 100 ml/min for 1 hour. Fusobacterium nucleatum TF-, which was pre-cultured in the TF-d medium mentioned above.
031 (FERM-P No. 5077, ATCC-31647) preculture solution 1 is inoculated under sterile conditions. Culture is 37
This is carried out at ℃ for 3 days under stirring (30 rpm) while flowing nitrogen gas (65 ml/min). After completion of the culture, 160 g of Celite and 80 g of cellulose powder were added to the culture solution and stirred, and the mixture was filtered under reduced pressure to obtain 7.8 ml of culture supernatant. (2) Add 117ml of concentrated hydrochloric acid to 7.8ml of the culture supernatant obtained in (1).
After adding and adjusting the pH to 2.0, ethanol 11.7
Add to make a 60% ethanol solution and incubate at 4℃ for 24 hours.
Leave it for a while. Next, the solution portion was removed by decantation, and the temperature was heated at 4°C to collect the precipitate.
Centrifuge (6 x 10 ³ rpm, 5 minutes). This precipitate was mixed with 400 ml of 60% ethanol aqueous solution with pH 2.0.
After sequentially washing with 400 ml of ethanol, 200 ml of acetone and 200 ml of diethyl ether, the mixture was dried under reduced pressure to obtain 4.5 g of powder. (3) Suspend the powder obtained in (2) in 45 ml of water and
Add aqueous sodium hydroxide solution to adjust the pH to 7.5-8.0, stir at room temperature for 30 minutes, then add 1N hydrochloric acid to adjust the pH to 4.0. Then centrifugation (1x
10 ⁴ rpm, 10 minutes) and separate the precipitate and supernatant. This precipitate was washed with 5 ml of water adjusted to pH 4.0, and the precipitate and washing solution were centrifuged (1×
10 ⁴ rpm for 10 minutes), combine the washing solution and the supernatant obtained earlier, and add 1N-hydrochloric acid to this solution.
After adjusting the pH to 2.0, leave it for 2 hours under ice water cooling. Then centrifugation (6 x 10 ³ rpm, 10 minutes),
Separate the precipitate and supernatant. This precipitate has a pH of
Wash with 3 ml of water adjusted to 2.0, centrifuge the precipitate and washing solution (6 x 10 ³ rpm, 10 minutes), wash the precipitate with 5 ml of ethanol, dry under reduced pressure, and remove the anticancer substance TF-240. Obtain 0.12g of. (4) 0.9 g of the anticancer substance TF-240 obtained as in (1), (2), and (3) above was treated in the same manner as in Example 1-(4) to Lyophilized product of substance TF-340
Get 115mg. Example 3 The anticancer substance TF-240 obtained in Example 1-(3)
0.9g was treated with an enzyme using 9mg of trypsin (manufactured by Difco) instead of pronase E, and when collecting the precipitate from the water-soluble part adjusted to pH 2 after the enzyme treatment, an 80% ethanol solution was used. 60 instead
A freeze-dried product of the anticancer substance TF-340 was prepared in the same manner as in Example 1-(4) except for making a % ethanol solution.
Get 105mg. Example 4 After enzymatically treating the anticancer substance TF-240 obtained in Example 1-(3) in the same manner as in Example 1-(4), the precipitate was collected from the water-soluble portion adjusted to pH 2. At this time, a 60% ethanol solution was used instead of an 80% ethanol solution, and the same procedure as in that example was carried out to obtain 101 mg of a freeze-dried product of the anticancer substance TF-340. Example 5 Anticancer substance TF-240 obtained in Example 1-(3)
was treated with an enzyme in the same manner as in Example 1-(4), and then PH2
Instead of collecting the precipitate from the water-soluble part adjusted to
Add 40% trichloroacetic acid to make the concentration of trichloroacetic acid 10%, adjust the water-soluble part to 80% ethanol solution, collect the precipitate, and then perform the same procedure as in that example to determine anticancer properties. Obtain 90 mg of lyophilized substance TF-340. Drug Example: 1 mg of lyophilized anticancer substance TF-340 was filled into a vial. When used, it is dissolved in sterile physiological saline or a solution containing 0.5% lidocaine and used as an injection solution.

[Brief explanation of drawings]

Figure 1 is a micrograph showing the morphology of Fusobacterium nucleatum TF-031 used in the present invention, Figure 2 is the infrared absorption spectrum of the anticancer substance TF-240, Figure 3 is the ultraviolet absorption spectrum of the same substance, and Figure 4 is the ultraviolet absorption spectrum of the same substance. The figure shows the infrared absorption spectrum of the anticancer substance TF-340, Figure 5 shows the ultraviolet absorption spectrum of the same substance, and Figure 6 shows the high performance liquid chromatogram of the same substance.

Claims (1)

[Claims] 1. Anticancer substance TF belonging to the genus Fusobacterium
An anticancer substance TF-340 and its salts having the following properties obtained by culturing the 340-producing bacteria and subjecting the fraction with anticancer activity obtained from the culture solution to enzymatic treatment with a proteolytic enzyme. . (a) White-gray to light brown powder. (b) It inhibits the growth of Ehrrich's ascites-type carcinoma in mice and has immunostimulatory effects. (c) Dissolved in water, methanol, ethanol, acetone, benzene, chloroform, ethyl acetate,
Insoluble in diethyl ether. (d) It does not exhibit a clear melting point, and begins to decompose at about 140°C, and significantly decomposes at temperatures above 200°C. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3500~3300, 2920, 2850, 1660~1640, 1580~
1520, 1460~1440, 1410~1340, 1250~1220,
It has absorption bands near 1120-1030, 970 and 835 cm ^-1 . (F) The ultraviolet absorption spectrum of an aqueous solution with a pH of 7 has strong absorption at the absorption end and also shows absorption in the vicinity of 250 to 265 nm. (G) High performance liquid chromatogram (eluent: PH7
0.1M phosphate buffer, flow rate 0.8ml/min, room temperature)
When measuring ultraviolet absorbance at 220 nm, it shows absorption at the front end of the solvent, around 36 to 62 minutes.
In ultraviolet absorbance measurement at 260 nm, absorption occurs at the front end of the solvent, around 34 to 62 minutes. (H) Moritsch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-Follin reaction are positive, ninhydrin reaction is negative (l) Elemental analysis values C: 32% to 34% H: 4% to 6% N : 3% to 5% (nu) The sugar content determined by the phenol sulfuric acid method is:
Approximately 20% to 50% (glucose equivalent), and protein content by Lowry-Follin method is approximately 10%
% or less (bovine serum albumin equivalent). 2 Anticancer substance TF belonging to the Fusobacterium genus
-340 It is obtained by adding a hydrophilic organic solvent to the supernatant liquid obtained by culturing the producing bacteria, and enzymatically treating the fraction with anticancer activity of the precipitate with proteolytic enzymes. The anticancer substance TF-340 and its salt according to claim 1. 3 Anticancer substance TF belonging to the Fusobacterium genus
-340 The precipitate produced by adding a hydrophilic organic solvent to the supernatant obtained by culturing the producing bacteria is divided by pH.
Claim 1, which is obtained by subjecting a precipitate having an anticancer effect that occurs between pH 4 and pH 2 to enzymatic treatment with a proteolytic enzyme, and then collecting a fraction of the anticancer substance TF-340; or The anticancer substance TF-340 and its salt according to item 2. 4. Claim 1, wherein the enzymatic treatment with a proteolytic enzyme is pronase or trypsin treatment.
The anticancer substance TF-340 and its salt according to any one of items 3 to 3. 5 Anticancer substance TF belonging to the Fusobacterium genus
The anticancer substance TF-340 and its salt according to any one of claims 1 to 4, wherein the -340 producing bacterium is Fusobacterium nucleatum. 6 Anticancer substance TF belonging to the Fusobacterium genus
-340 Anticancer activity characterized by subjecting the fraction having anticancer activity of the precipitate produced by adding a hydrophilic organic solvent to the supernatant obtained by culturing the producing bacteria to enzymatic treatment with a protease. Method for producing substance TF-340 and its salt. 7 Anticancer substance TF belonging to the Fusobacterium genus
-340 The precipitate produced by adding a hydrophilic organic solvent to the supernatant obtained by culturing the producing bacteria is separated by pH.
Claim 6, characterized in that a precipitate having an anticancer effect generated at around PH4 to around PH2 is subjected to enzymatic treatment with a proteolytic enzyme, and then a fraction of the anticancer substance TF-340 is collected. A method for producing the anticancer substance TF-340 and its salts as described in 2. 8 After the enzymatic treatment, the operation of collecting the fraction of the anticancer substance TF-340 is performed by combining one or more of a PH separation method, a precipitation fractionation method using a hydrophilic organic solvent, or an ion exchanger separation method, Anticancer substance TF
7. The method for producing the anticancer substance TF-340 and its salts according to claim 7, which is an operation of collecting a fraction of TF-340. 9 The operation of collecting a fraction of the anticancer substance TF-340 after enzymatic treatment is to divide the water-soluble portion after enzymatic treatment with a proteolytic enzyme by pH and adjust the pH to 2.5 or less. The cancer-fighting method according to claim 7 or 8, which is an operation of adding a hydrophilic organic solvent to a fraction of 10% of the total amount, dividing the resulting precipitate with an ion exchanger, and collecting the non-adsorbed fraction. sexual substance TF
-340 and its salt production method. 10 The anticancer property according to any one of claims 6 to 9, wherein the operation of adding a hydrophilic organic solvent to the supernatant liquid is an operation of adjusting the supernatant liquid to around PH2 and then adding the hydrophilic organic solvent. Method for producing substance TF-340 and its salt. 11. The method for producing the anticancer substance TF-340 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 with a concentration of 30% to 80%
12. The method for producing the anticancer substance TF-340 and its salts according to any one of claims 6 to 11, characterized in that the anticancer substance TF-340 and its salts are added to the supernatant liquid so as to have a volume ratio of (volume ratio). 13. The anticancer substance TF- according to any one of claims 6 to 12, wherein the enzymatic treatment with a proteolytic enzyme is pronase or trypsin treatment.
340 and its salt manufacturing method. 14 Anticancer substances belonging to the genus Fusobacterium
An anticancer substance TF-340 or its salt having the following properties obtained by culturing TF-340-producing bacteria and subjecting the fraction with anticancer activity obtained from the culture solution to enzymatic treatment with a protease. An anticancer agent characterized by containing. (a) White-gray to light brown powder. (b) It inhibits the growth of Ehrrich's ascites-type carcinoma in mice and has immunostimulatory effects. (c) Dissolved in water, methanol, ethanol, acetone, benzene, chloroform, ethyl acetate,
Insoluble in diethyl ether. (d) It does not exhibit a clear melting point, and begins to decompose at about 140°C, and significantly decomposes at temperatures above 200°C. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3500~3300, 2920, 2850, 1660~1640, 1580~
1520, 1460~1440, 1410~1340, 1250~1220,
It has absorption bands near 1120-1030, 970 and 835 cm ^-1 . (f) The ultraviolet absorption spectrum of an aqueous solution with a pH of 7 has strong absorption at the absorption end and also shows absorption in the vicinity of 250 to 265 nm. (G) High-performance liquid chromatogram using TSK-GEL G3000SW (trade name of Toyo Soda Co., Ltd., column: 7.5 mmφ x 600 mm x 2) (eluent: 0.1 at PH7)
Molar phosphate buffer, flow rate 0.8 ml/min, room temperature)
In ultraviolet absorbance measurement at 220 nm, absorption occurs at the front end of the solvent, around 36 to 62 minutes.
In ultraviolet absorbance measurement at 260 nm, absorption occurs at the front end of the solvent, around 34 to 62 minutes. (H) Moritsch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-Follin reaction are positive, ninhydrin reaction is negative (l) Elemental analysis values C: 32% to 34% H: 4% to 6% N : 3% to 5% (nu) The sugar content by the phenol sulfuric acid method is approx.
20% to 50% (glucose equivalent), and protein content by Lowry-Follin method is approximately 10%
It is as follows (in terms of bovine serum albumin).