JPS6261036B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel anticancer substance, and more particularly to TF-132b, an anticancer substance obtained from a culture solution obtained by culturing bacteria belonging to the genus Fusobacterium. Furthermore, the present invention relates to a method for producing the anticancer substance TF-132b and an anticancer agent containing the same. In recent years, therapeutic methods that enhance the host's immune function and exert anticancer effects with the help of the immune function have become popular in the treatment of various cancer patients. 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, these drugs are still unsatisfactory in terms of efficacy, side effects, and manufacturing methods. On the other hand, bacteria belonging to Fusobacterium, such as Fusobacterium KO31-3B, Fusobacterium fusiphomis W-12, Fusobacterium
There have been reports regarding bacterial cells and supernatant obtained by culturing G. nucleatum 1012 and Fusobacterium nucleatum 1010. [Journal of Stomatology 21, pp. 534-539 (1972),
Journal of Stomatology 23, pp. 322-333 (1974)] However,
The components obtained from the culture solution or supernatant obtained by culturing bacteria belonging to the genus Fusobacterium and their pharmacological effects have not yet been studied. Therefore, the present inventor cultivated bacteria belonging to the genus Fusobacterium and investigated the pharmacological effects of the supernatant liquid from which the bacterial cells were removed. It has a strong anticancer effect, and this means that in the colony formation suppression method, the effect of inhibiting cancer cell colony formation is extremely small, and the anticancer effect is not cell killing, but rather a host-mediated or host immune system enhancement. The present invention was completed based on the discovery that this ingredient has the effect of indirectly exerting anticancer effects with the help of immunity, and that this ingredient has extremely low toxicity. The bacteria used in the present invention may be any TF-132b producing bacteria belonging to the genus Fusobacterium.
Preferred examples include Fusobacterium nucleatum. For example, Fusobacterium nucleatum
Strains such as TF-031 (FARM-5077, ATCC-31647) 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 Outside of TF-a agar plate and slant medium Shape: Circular Size: Approximately 1 mm Elevation: Hemispherical structure: Dewdrop-like surface: Smooth edge Edge: Smooth Color: Milky white Transparency: Opaque TF-a liquid medium growth Degree of turbidity: Agglomeration Sediment: None Surface growth: None, no growth up to about 5 mm Gas: None (3) Physiological properties Hydrogen sulfide production: + Nitrate reduction: - Butyric acid production: + Production of indole: + Urease: - Catalase: - Hydrolysis of starch: - Attitude towards oxygen: Anaerobic Production of ammonia: + Production of carbon dioxide: + Growth range: PH5-8.5 Temperature 30-45℃ From sugar Gas production L-arabinose (-), D-xylose (-), D-glucose (-), D-mannose (-), D-fructose (-), D-galactose (-), maltose (-), Sucrose (-), trehalose (-), sorbitol (-), mannitol (-), inositol (-), glycerin (-), starch (-) Bacteriology No. 8
When examined against the edition, this strain is Fusobacterium nucleatum (Fusobacterium nucleatum).
nucleatum) and belongs to this group. Next, the method for producing the anticancer substance TF-132b of the present invention will be schematically explained as follows.

[Table] The above manufacturing method is specifically shown as follows. (a) Culture Bacteria belonging to the genus Fusobacterium are cultured using a conventional anaerobic culture method. Namely, nitrogen sources such as cow brain, heart extract, and various peptones; vitamin sources such as yeast extract; inorganic salts such as sodium chloride; glucose,
A medium containing a carbon source such as lactose; a reducing agent such as L-cystine, sodium sulfite, and thioglycollate is adjusted to pH 6-8.5, preferably 7.2-8.2 with sodium hydroxide, and the bacteria are inoculated and grown under anaerobic conditions. under conditions of 35-42°C, preferably 36-38°C for 1-5 days.
Preferably, static culture is performed for 24 to 72 hours. especially,
It is preferable to use a medium (hereinafter referred to as TF medium) described in the ingredient table below. However, as a carbon source, brain heart extract of cow brain,
Infusion is not always necessary, and heart infusion (beef heart extract), beef extract, fish meat extract, corn staplerica extracted from corn, etc. may be substituted.
Furthermore, among the various peptones, proteose peptone and phytone peptone are not necessarily necessary, and tryptocase peptone can be substituted with polypeptone. Note that when agar is not used, it is preferable to perform agitation 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 using conventional methods such as centrifugation or filtration using a supernatant such as Hyflo Super Cell, but centrifugation is particularly difficult in terms of operation, degree of bacterial removal, and amount of supernatant obtained. preferable. Furthermore, although it is preferable to remove the bacterial cells at this stage, they may be removed in the next step (c). (c) Collection of anticancer substance TF-132b A hydrophilic organic solvent is added to the supernatant or culture solution obtained above, and the resulting precipitate is collected. At this time, the supernatant liquid or culture solution is preferably adjusted to pH 2-7. 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 30-70%, preferably 50-70%
It is preferable to add it so that After adding the hydrophilic organic solvent, it is left to stand at a low temperature, preferably at a temperature of about 5° C., for several hours to several days to complete the formation of a precipitate. The precipitate thus formed is separated by conventional operations such as decantation, centrifugation, filtration, etc. Next, 10 to 15 times the amount of water or phosphate buffer (salt) is added to this precipitate, and the resulting water-insoluble matter is removed by a conventional method such as centrifugation or sieving. When a culture solution is used, bacterial cells are removed by this treatment. Furthermore, the water-soluble component separated liquid obtained in this way can be directly subjected to ion exchange treatment, but this water-soluble component can be subjected to dialysis or ultrafiltration, and the inner solution can be further processed by lyophilization, etc. The dried powder may be treated with an ion exchanger. The water-soluble components thus obtained or the dialyzed or ultrafiltered internal fluid components are treated with an ion exchanger. As the ion exchanger, it is preferable to use a weakly basic ion exchanger or one having molecular sieving properties; for example, diethylaminoethyl-Sephadex A-50 (trade name, manufactured by Pharmacia) is preferably used. In the ion exchanger treatment, preferably the amount that does not elute with 0.1 molar saline phosphate buffer (in this specification, "molar" means molar concentration) but elutes with 0.2 molar saline phosphate buffer Collect images. A concrete explanation of the ion exchanger treatment method is as follows, for example. That is, a column packed with an ion exchanger is equilibrated with a 0.2 to 0.3 molar saline phosphate buffer, preferably a 0.025 molar phosphate buffer with a pH of 8, and the above precipitate or powder is added to the 0.2 to 0.3 molar saline phosphate buffer. The solution dissolved in water is passed through the tube, and the passing liquid is collected. If necessary, the column is washed with the above 0.2 to 0.3 molar saline phosphate buffer, and the flow through and washing liquid are combined. Further, this ion exchanger treatment may be performed several times. Then the ion exchanger
0.1 molar saline phosphate buffer, preferably PH8
A solution diluted with phosphate buffer so that the saline concentration is 0.1M is passed through a column equilibrated with 0.025M phosphate buffer, and then eluted with the above 0.1M NaCl phosphate buffer. After removing the fraction to be treated, the 0.2M saline phosphate buffer described above is passed through the column to obtain an eluate. The above ion exchanger treatment may be carried out by a batch method. In addition, in the specific example of the above ion exchanger treatment, the fraction that does not adsorb to the ion exchanger equilibrated with 0.2 molar saline phosphate buffer is collected, and the ions equilibrated with 0.1 molar saline phosphate buffer are collected. Ions adsorbed to the exchanger and not eluted with 0.1 molar saline phosphate buffer, eluted with 0.2 molar saline phosphate buffer to obtain the eluate, but on the contrary, equilibrated with 0.1 molar saline phosphate buffer The fraction adsorbed to the exchanger was collected and separated from the fraction adsorbed to the ion exchanger equilibrated with 0.2M saline phosphate buffer.
Fractions eluting with 0.2M saline phosphate buffer may be collected. Next, the eluate obtained as described above is concentrated, desalted, and dried by dialysis or ultrafiltration, and the desired anticancer substance TF-132b is obtained. TF-132b thus obtained has the following physical properties. (a) White-gray to light brown powder. (b) It inhibits the proliferation of Ehrlichi's ascites-type carcinoma, Ehrlichi's nodular-type carcinoma, and Sarcoma 180 cancer cells 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 show a clear melting point, and begins to decompose at about 110°C, and significantly decomposes above 200°C. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3600~3200, 2960~2930, 1670~1640, 1550,
It has absorption bands near 1440-1380, 1240, 1140-1000 and 820 cm ^-1 (Figure 2). (f) The ultraviolet absorption spectrum of the aqueous solution shows strong absorption at the absorption end and a shoulder near 265 to 280 nm (Figure 3). (g) Gel filtration (column: 21 mmφ) using Sephadex G-200 (registered trademark of Pharmacia)
× 400 mm, eluent: 0.1M phosphate buffer with pH 7.0), in the ultraviolet absorbance measurement at 260 nm,
It has an absorption band around 150 ml, and in absorbance measurement at 490 nm using the phenol sulfuric acid method, it has an absorption band from around the void volume passage to around 150 ml (Figure 4). (H) High-performance liquid chromatogram using TSK-GEL G3000SW (trade name of Toyo Soda Co., Ltd., column: 7.9 mmφ x 600 mm x 2) (eluent: 0.1 at PH7)
Molar phosphate buffer, flow rate 0.8ml/min, room temperature)
In the ultraviolet absorbance measurement at 220nm, peaks were observed at the front of the solvent, around 36 to 37 minutes, and around 48 to 50 minutes, and in the absorbance measurement at 260 nm, the peaks were at the front of the solvent, around 32 to 39 minutes, and around 45 to 52 minutes. It has a peak around (Fig. 5). (li) Moritsch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-Fillin reaction are positive, and ninhydrin reaction is negative. (nu) Elemental analysis value C: 35.3% to 39.5%, H: 4.5% to 5.6%,
N: 2.8% to 5.4% (le) Sugar content by phenol sulfuric acid method is approx.
The protein content is 23.6% to 45.5% (in terms of glycose) and about 15.5% to 28.0% (in terms of bovine serum albumin) by the Lowry-Follin method. The pharmacological action of the anticancer substance TF-132b of the present invention is as follows. (1) Immunostimulatory effect Using a group of 4 ICR mice, anticancer substances were
Dissolve TF-132b in 0.2ml of physiological saline, add 5 and
20 mg/Kg was administered intraperitoneally. Perikan Drawing Ink 17 24 hours after administration
0.2 ml of carbon suspension prepared by mixing 1 ml of Black (manufactured by Guilnter Wagner) and 2 ml of physiological saline containing 3% gelatin was injected into the tail vein of the mouse, and 1, 5, 10, and 15 minutes after injection, it was inserted into the orbit. Blood 0.02 using heparin-coated hematolith capillary tubes
Collect ml and immediately add 0.1% sodium carbonate aqueous solution.
Dilute hemolyze to 1.6 ml and compare the color at a wavelength of 675 nm to determine the phagocytic 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-t _p (Co=the amount of charcoal in the blood at the time of t _p , C=the amount of charcoal in the blood at the time of t) The results are shown in Table-1.

[Table] As is clear from Table 1, compared to the control group,
In the TF-132b administration group of the present invention, reticuloendothelial macrophages were activated, and cell-mediated immunity of normal mice was increased. (2) Anticancer effect (a) Antitumor effect on Ehrlitsu ascites tumor ICR mice (female, 6 weeks old, 5 mice per group) were intraperitoneally inoculated with 1 x 10 ⁵ Ehrlitsu ascites cancer cells/mouse. did. Then, it was dissolved in 0.2 ml of physiological saline.
TF-132b was administered on day 1 and day 3 to day 7 after cancer cell inoculation.
The drug was administered intraperitoneally once a day until day 1, for a total of 6 days. In addition, the same amount of physiological saline was administered to the control group in the same manner as above. The results are shown in Table-2. T/C = Average survival days of TF-132b administration group / Average survival days of control group × 100 (%)

[Table] As is clear from Table 2, compared to the control group,
A survival effect was observed in the TF-132b administration group, with many mice completely recovering on the 40th day. (b) Antitumor effect on Ehrlichi nodule-type tumor Ehrlichi cancer cells (4×10 ⁶ cells/mouse) were subcutaneously transplanted into ICR mice (female, 6 weeks old, 7 mice per group) in the lower axilla. Then, TF− dissolved in 0.2 ml of physiological saline
132b was administered intravenously once a day from day 1 to day 3 to day 7 after cancer cell transplantation, for a total of 6 days. In addition, the same amount of physiological saline was administered to the control group in the same manner as above. Tumor weight was measured 14 days after cancer cell transplantation. The results are shown in Table-3. The tumor weight was determined by measuring the major axis (a) mm and the minor axis (b) mm of the tumor site using calipers, and using the following formula. Tumor weight = a×b ² /2 (mg)

[Table] As is clear from Table 3, compared to the control group,
Tumor growth suppressive effects were observed in the TF-132b administration group. (c) Antitumor effect against Sarcoma 180 cancer cells Sarcoma 180 was applied to ICR mice (female, 6 weeks old)
1×10 ⁵ cancer cells/mouse were transplanted intraperitoneally. Next, TF-132b dissolved in 0.2 ml of physiological saline was intraperitoneally administered once a day for 7 consecutive days starting from the first day after cancer cell transplantation. In addition, to the control group, the same amount of physiological saline was continuously administered in the same manner as above. The results are shown in Table 4. T/C was determined in the same manner as in (a).

[Table] As is clear from Table 4, compared to the control group,
A survival effect was observed in the TF-132b administration group, with many mice making a complete recovery. (3) Acute toxicity In ICR mice (female, 6 weeks old), TF-
LD ₅₀ value for a single intravenous administration of 132b is >100
It was mg/Kg. As is clear from the results of the above pharmacological experiments, the anticancer substance TF-132b of the present invention is useful as an anticancer agent and is expected to be effective when used for various cancer diseases. It can be expected to have a remarkable effect on type cancer. The TF-132b of the present invention can be administered orally, by injection, or by conventional methods.
It can be used in dosage forms such as suppositories. Oral preparations may contain various excipients and may be in the form of capsules, tablets, powders, or 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. The dosage of TF-132b of the present invention is appropriately selected depending on the patient's symptoms, but generally 0.01
It is preferable to administer up to 50 mg/Kg in one to several divided doses per day, and the preferred method of administration is subcutaneous, intramuscular, intravenous, or injection into the affected area. Next, examples of the present invention will be described. Example 1 (1) Into 15 two-volume culture bottles with screw caps, each bottle contained 34 g of Tryptocase Peptone, 6 g of Phaiton Peptone, 20 g of Proteose Peptone, 70 g of Brain Heart Injection, and 6 g of Yeast Extract. , salt 15g, glucose 12g, lactose 10g, L-cystine 0.5g, sodium sulfite 0.2
Add medium 2, adjusted to pH 7 by adding g, sodium thioglycolate 1.0 g, and agar 1.4 g, autoclave at 120°C for 15 minutes, immediately cool with water, and pre-culture in a medium with the same composition. Fusobacterium nucleatum TF-
Inoculate the pre-culture solution of 031 (Feikoken accession number 5077) at a rate of 100 ml per culture bottle under sterile conditions, and culture it statically for 48 hours in an incubator at 37°C. conduct. After incubation, 4000 r.p. at 5°C.
The culture solution was centrifuged for 20 minutes to remove bacterial cells and a supernatant liquid of about 2.0 m was obtained. (2) Add ethanol to the supernatant obtained in (1) at 5℃ while stirring.
40 and leave it in a cold room until the amorphous precipitate is completely settled. Then, at 5℃, 6000r.
pm, centrifuged for 15 minutes, collected the precipitate, washed with ethanol, and dried under reduced pressure to obtain about 60 g of crude powder. (3) Dissolve 44.2 g of the powder obtained in (2) in 1.2 mol of a 0.025 molar phosphate buffer with a pH of 8 and a salt concentration of 0.3 molar, remove floating insoluble matter using a Hyflo Super Cell, and remove the resulting solution. , salt concentration 0.3
Diethylaminoethyl Sephadex A- equilibrated with 0.025 molar phosphate buffer, pH 8.
50 (trade name, manufactured by Pharmacia) and collect the passing liquid through a 500 ml column (diameter 33 cm). The column was washed with a 0.025 molar phosphate buffer with a pH of 8 and a saline concentration of 0.3 molar, and the washing solution and the above flowthrough were combined.
Obtain a solution of 1.67. Change this again to the salt concentration
Diethylaminoethyl Cephadex A equilibrated with 0.3M PH8 0.025M phosphate buffer
-50 (trade name, manufactured by Pharmacia) and applied to a 400 ml column (diameter 8 cm) to obtain 1.68 mL of the passed-through liquid. The column is washed with 0.025 molar phosphate buffer, pH 8, with a saline concentration of 0.3 molar, yielding a wash solution of 1.64.
Combine the passing liquid and washing liquid and add to this with a pH of 8.
Add 0.025 molar phosphate buffer to make a 0.025 molar phosphate buffer with a pH of 8 and a saline concentration of 0.2 molar at 4.98. This solution was adjusted to a pH of 8 with a salt concentration of 0.2M.
Diethylaminoethyl Sephadex A-50 (product name manufactured by Pharmacia) 500 ml column (diameter 8 cm) equilibrated with 0.025 molar phosphate buffer
Wash the column with 0.025 molar phosphate buffer 2, pH 8, and have a salt concentration of 0.2 molar.
Add 0.025 molar phosphate buffer to make a 0.025 molar phosphate buffer 14 with a pH of 8 and a salt concentration of 0.1 molar. This solution was adjusted to a pH of 8 with a salt concentration of 0.1M.
Diethylaminoethyl Sephadex A-50 (product name manufactured by Pharmacia) 500 ml column (diameter 8 cm) equilibrated with 0.025 molar phosphate buffer
After removing the flow-through, the column was adjusted to the salt concentration.
After washing with 0.025 molar phosphate buffer 2 of 0.1 molar pH 8, 0.025 molar phosphate buffer 2 of PH8 with a saline concentration of 0.2 molar is applied, and the eluate is collected. This eluate was concentrated and desalted using an ultrafiltration system (filter used: Toyo Ultra Filter UK-10), further desalted using a Sephadex G-25 column, and decolorized using activated carbon. Freeze-dry to obtain 880 mg of white-gray to light brown powder of FT-132b. Example 2 1 mg of the powder obtained in Example 1 was filled into a vial. This is used as an injection solution dissolved in sterile physiological saline or a solution containing 0.5% lidocaine.

[Brief explanation of the drawing]

Figure 1 shows Fusobacterium nucleatum TF.
Fig. 2 is an infrared absorption spectrum of the anticancer substance TF-132b of the present invention, Fig. 3 is an ultraviolet absorption spectrum of the same substance, and Fig. 4 is a photomicrograph showing the form of the anticancer substance TF-132b of the present invention. Figure 5 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-132b having the following properties obtained from the culture solution obtained by culturing 132b-producing bacteria. (a) White-gray to light brown powder. (b) It inhibits the proliferation of Ehrlichi's ascites-type carcinoma, Ehrlichi's nodular-type carcinoma, and Sarcoma 180 cancer cells 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 show a clear melting point, and begins to decompose at about 110°C, and significantly decomposes above 200°C. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3600~3200, 2960~2930, 1670~1640, 1550,
It has absorption bands near 1440-1380, 1240, 1140-1000 and 820 cm ^-1 . (f) The ultraviolet absorption spectrum of the aqueous solution shows strong absorption at the absorption end and a shoulder near 265 to 280 nm. (g) Gel filtration (column: 21 mmφ) using Sephadex G-200 (registered trademark of Pharmacia)
x 400 mm, eluent 0.1 molar phosphate buffer with pH 7.0), in the ultraviolet absorbance measurement at 260 nm, the
It has an absorption band around 150 ml, and in absorbance measurement at 490 nm using the phenol sulfuric acid method, it has an absorption band from around the void volume passage to around 150 ml. (H) High-performance liquid chromatogram using TSK-GEL G3000SW (trade name of Toyo Soda Co., Ltd., column: 7.9 mmφ x 600 mm x 2) (eluent: 0.1 at PH7)
Molar phosphate buffer, flow rate 0.8ml/min, room temperature)
In the ultraviolet absorbance measurement at 220nm, peaks were observed at the front of the solvent, around 36 to 37 minutes, and around 48 to 50 minutes, and in the absorbance measurement at 260 nm, the peaks were at the front of the solvent, around 32 to 39 minutes, and around 45 to 52 minutes. It has a peak around (li) Moritsch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-Foulin reaction are positive, and ninhydrin reaction is negative. (nu) Elemental analysis value C: 35.3% to 39.5%, H: 4.5% to 5.6%,
N: 2.8% to 5.4% (le) Sugar content by phenol sulfuric acid method is approx.
The protein content is approximately 23.6% to 45.5% (in terms of glucose) and approximately 15.5% to 28.0% (in terms of bovine serum albumin) according to the Lowry-Follin method. 2 Anticancer substance TF belonging to the Fusobacterium genus
- Collect the precipitate produced by adding a hydrophilic organic solvent to the culture solution or supernatant obtained by culturing the 132b-producing bacteria, and if desired, after dialysis or ultrafiltration of the water-soluble components of the precipitate. , and then treated with an ion exchanger and collecting a fraction that does not elute with 0.1 molar saline phosphate buffer but elutes with 0.2 molar saline phosphate buffer, Claim 1
The anticancer substance TF-132b described in Section 1. 3 Anticancer substance TF belonging to the Fusobacterium genus
The anticancer substance TF-132b according to claim 1 or 2, wherein the -132b-producing bacterium is Fusobacterium nucleatum. 4 Anticancer substance TF belonging to the Fusobacterium genus
- Collect the precipitate produced by adding a hydrophilic organic solvent to the culture solution or supernatant obtained by culturing the 132b-producing bacteria, and if desired, after dialysis or ultrafiltration of the water-soluble components of the precipitate. A method for producing an anticancer substance TF-132b, which is then treated with an ion exchanger. 5 Anticancer substance TF belonging to the Fusobacterium genus
- Collect the precipitate produced by adding a hydrophilic organic solvent to the culture solution or supernatant obtained by culturing the 132b-producing bacteria, and if desired, after dialysis or ultrafiltration of the water-soluble components of the precipitate. , and then treated with an ion exchanger to collect a fraction that does not elute with 0.1 molar saline phosphate buffer but elutes with 0.2 molar saline phosphate buffer. Method for producing anticancer substance TF-132b. 6 Anticancer substance TF belonging to the Fusobacterium genus
A hydrophilic organic solvent is added to the culture solution or supernatant obtained by culturing -132b-producing bacteria, the resulting precipitate is collected, and the precipitate is dissolved in 0.2 to 0.3 molar saline phosphate buffer to remove water-insoluble matter. After removal, this is treated with an ion exchanger equilibrated with 0.2 to 0.3 molar saline phosphate buffer to collect the flow-through solution, and this flow-through solution is adjusted to reduce the salt concentration.
Adjusted to 0.1M, treated with an ion exchanger equilibrated with 0.1M NaCl phosphate buffer, eluted with 0.1M NaCl phosphate buffer, not eluted with 0.2M NaCl phosphate buffer. 5. A method for producing the anticancer substance TF-132b according to claim 4, which comprises collecting a fraction of the anticancer substance TF-132b. 7. The anticancer substance according to any one of claims 4 to 6, wherein the hydrophilic organic solvent is alcohol.
Manufacturing method of TF-132b. 8. The anticancer substance TF- according to claim 7, characterized in that a hydrophilic organic solvent is added to the culture solution or supernatant at a concentration of 30 to 70%.
132b manufacturing method. 9 Claim 4 in which the ion exchanger is a weakly basic ion exchanger or one that also has molecular sieving properties
-Anticancer substance TF-132b described in any of Items 6 to 6
manufacturing method. 10 Anticancer substance belonging to the genus Fusobacterium
The following properties obtained from the culture solution by culturing TF-132b-producing bacteria: (a) White-gray to light brown powder. (b) It inhibits the proliferation of Ehrlichi's ascites-type carcinoma, Ehrlichi's nodular-type carcinoma, and Sarcoma 180 cancer cells 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 show a clear melting point, and begins to decompose at about 110°C, and significantly decomposes above 200°C. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3600~3200, 2960~2930, 1670~1640, 1550,
It has absorption bands near 1440-1380, 1240, 1140-1000 and 820 cm ^-1 . (f) The ultraviolet absorption spectrum of the aqueous solution shows strong absorption at the absorption end and a shoulder near 265 to 280 nm. (g) Gel filtration (column: 21 mmφ) using Sephadex G-200 (registered trademark of Pharmacia)
x 400 mm, eluent 0.1 molar phosphate buffer with pH 7.0), in the ultraviolet absorbance measurement at 260 nm, the
It has an absorption band around 150 ml, and in absorbance measurement at 490 nm using the phenol sulfuric acid method, it has an absorption band from around the void volume passage to around 150 ml. (H) High-performance liquid chromatogram using TSK-GEL G3000SW (trade name of Toyo Soda Co., Ltd., column: 7.9 mmφ x 600 mm x 2) (eluent: 0.1 at PH7)
Molar phosphate buffer, flow rate 0.8ml/min, room temperature)
In the ultraviolet absorbance measurement at 220nm, peaks were observed at the front of the solvent, around 36 to 37 minutes, and around 48 to 50 minutes, and in the absorbance measurement at 260 nm, the peaks were at the front of the solvent, around 32 to 39 minutes, and around 45 to 52 minutes. It has a peak around (li) Moritsch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-Folin reaction are positive, and ninhydrin reaction is negative. (nu) Elemental analysis value C: 35.3% to 39.5%, H: 4.5% to 5.6%,
N: 2.8% to 5.4% (le) Sugar content by phenol sulfuric acid method is approx.
The protein content is approximately 23.6% to 45.5% (in terms of glucose) and approximately 15.5% to 28.0% (in terms of bovine serum albumin) according to the Lowry-Follin method. 1. An anticancer agent comprising the anticancer substance TF-132b.