JPS58129977A - Carcinostatic substance tf-300-2, its preparation and carcinostatic agent containing the same - Google Patents

Abstract

PURPOSE:To prepare the titled carcinostatic substance, by culturing specific microbial strain, removing proteins from the cultured liquid, and concentrating the substance by ultrafiltration. CONSTITUTION:Bacterial strain belonging to Fusobacterium genus and capable of producing carcinostatic substance TF-300-2, e.g. Fusobacterium nucleatum TF- 031 (FERM-P No.5077, ATCC-31647), is cultured under anaerobic conditions at about 30-42 deg.C for about 1-5 days, and the microbial cells are removed from the cultured liquid. A hydrophilic organic solvent (e.g. ethanol) is added to the supernatant liquid, the produced precipitate is separated, and the active fraction is collected by adjusting the pH of the solution. The active fraction is treated with an enzyme to effect the removal of proteins, concentrated and desalted by the ultrafiltration (fractionated molecular weight; about 50,000-200,000), and dried to obtain the carcinostatic substance TF-300-2.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention aims at cultivating a novel anticancer substance, more specifically, a bacterium belonging to the genus Hubbacterium, and extracting a fraction having an anticancer effect obtained from the culture solution using a proteolytic enzyme. Anticancer substance TI'-300-2 (Tl'
-! 110-2, TI'-320-2 and TF-3
30-2) and its salts. Furthermore, the present invention relates to a method for producing the anti-cancer substance TF-300-2 and its salt, and an anti-cancer agent containing the same.

BACKGROUND ART In recent years, treatments for various cancer patients that enhance the host's immune function and exert an anticancer effect with the help of the immune function have become popular.

As drugs used in such therapy, components obtained from 1 m cell cultures of various bacteria, or polysaccharides obtained from the fruiting bodies of basidiomycetes or their cultured cells are known. However, there has been little research on components that have excellent anticancer effects obtained from culturing anaerobic bacteria and using the culture solution. Its effects are still unknown.

The present invention was developed by cultivating bacteria belonging to the genus Hubbacterium isolated from the four cavities of humans, and investigating the pharmacological effects of components collected from the supernatant after removing bacterial cells from the culture solution. Certain components obtained by ultrafiltration in conjunction with protein processing have anticancer effects, and can be inhibited indirectly through host-mediated or enhanced host immunity. It was discovered that it has the effect of causing cancerous effects. And this particular ingredient.

The present invention was completed under the heading 1, including the fact that it has favorable properties as an anticancer agent, such as strong anticancer action and few side effects.

The bacterium used in the present invention produces an anticarcinogenic substance TF-300-2 by subjecting a fraction with anticancer activity obtained from the supernatant of culturing the bacterium to an enzymatic treatment with a proteolytic enzyme. Any bacteria belonging to the genus Hubbacterium may be used, and a preferred example is Hubbacterium nucleatum. Specifically, 1 For example, Hubbacterium nucleatum? ? -031 (FliXRM-5077,
ao-51647> and, as is common knowledge in microbiology, strains having these properties, such as natural mutant strains or artificially modified strains, are used.

The mycological properties of Hubbacterium nucleatum TF-031 are as follows.

(1) Morphology (i') Cell shape: spindle-shaped (Figure 1) ■ Presence of cell pleomorphism: None ■ Presence of motility: None ■ Presence of spores: None ■ Durham stain nigerum negative ■ Acid-fastness: Growth status in negative (2) medium ■ TIF-a agar plate and slant medium External shape: Circular Size: Approximately 1M Protuberance: Hemispherical structure: Dewdrop-shaped surface: Smooth Edge: Smooth Color: Milky white Transparency: Opaque ■ Degree of growth of T sea a liquid medium: Strong turbidity; coagulum precipitation: None Surface growth: None, no growth up to about 5M: None 3) Physiological properties ■ Hydrogen sulfide production: + ■ Nitrate Reduction knee ■ Production of butyric acid: + ■ Production of indole: + ■ Urea Zeny 1 ■ Camera Zeny ■ Hydrolysis knee of starch ■ Attitude towards oxygen: Anaerobic ■ Production of ammonia: + [Phase] Production of carbon dioxide: + ■ Growth range: pH 5-8.5 Temperature 30-45°C @ Gas production from sugar L-arabinose (→, D-xylose ←), D-glucose ←), D-mannose ←), D-7 lactose ←
), D- is 2ctose (→, maltose ←), sucrose (→,
Trehalose←), Sorbitol (→, Mannitol (→
, Inosit (→, Chrycerin ←), Starch (→ When examining the above properties in the light of Percy's Manual Ode Determinative Bacteriolozoe 8th edition, this strain is Humbacterium nucleatum (!
Fueobacterium nucleatum)
It closely resembles and belongs to this category.

Next, an example of the method for producing the anticancer substance TIF-300-2 of the present invention will be illustrated and explained as follows.

(1) Separation of a fraction with anticancer activity from culture and culture solution Anticancer substance belonging to the genus Hubbacterium TF-300-2 Culture solution or organic solvent of producing bacteria Separation -- Soluble fraction precipitate PI - Resolution by IK (2) Protein removal treatment and ultrafiltration of the fraction obtained in (1) The typical routes of the above production method are specifically shown below.

(1) Step of culturing and separating a fraction having anticancer activity from the culture solution (!L) Cultivation of bacteria belonging to the genus Hubbacterium is carried out by 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; carbon sources such as glucose and 2-ctose; IL-cystine, sodium sulfite, and thio A medium containing a reducing agent such as glycolate is adjusted to pH 6 to 8.5, preferably 6.5 to 7.5, with sodium hydroxide, and the bacteria are inoculated and heated under anaerobic conditions, preferably at 30 to 42°C. 1-5 days at 32-37℃,
Preferably, the culture is carried out in a static apparatus or with stirring for 1 to 4 days.

In particular, it is preferable to use a medium (hereinafter referred to as τ? medium) listed in the ingredient list below. but.

As a nitrogen source, it is not necessary to use Plain Heart Infusible, which is a cow's brain and heart extract, but Heart Infusyo Y1 Beef Extract, which is a cow's heart extract, can be used as a nitrogen source.
Fish meat extract, corn stazolica extracted from corn, etc. may be substituted, and proteose benoton and phyton pezoton are not necessarily necessary in various pedaton, and polypeptone can be substituted for tritcase penoton. .

In addition, when not using dowry, it is preferable to perform agitation culture. 3-component table (b) Collection of supernatant from culture solution <Removal of tS bodies) Cells are removed from the culture solution obtained above to obtain a supernatant. Bacterial cells can be removed using conventional methods, such as centrifugation or a transient method using a transit aid such as Hyflo-Purcel, but centrifugation is particularly difficult in terms of operation, degree of bacterial removal, and yield of supernatant. It is preferable.

(e) Add a hydrophilic organic solvent to the supernatant obtained in the separation of the effective fraction having anticancer activity, and collect the resulting precipitate. At this time, the supernatant liquid has a pH of 1.5 to 7, preferably P
Adjust K to around H2 (pH 1.5 to 2.5). As a hydrophilic organic solvent.

Examples include alcohols such as ethanol and methanol, and ketones such as acetone, but alcohols, especially ethanol, give the best results. The concentration of this hydrophilic organic solvent is 60 to 80 (volume ratio), preferably 50
It is preferable to add so that the ratio is -80- (volume ratio). After adding the hydrophilic organic solvent, at a low temperature, preferably about 4
Leave at a temperature of ~5°C for several hours to several days.

Complete the formation of the precipitate.

The precipitate thus formed is separated by conventional procedures such as decantation, centrifugation, and filtration.

Next, generally 5 to 20 times the amount of water is added to this precipitate,
- to obtain an effective fraction. For example, after adjusting to -7.5 to 8 and then removing insoluble parts as desired,
The water-insoluble part (TIF-210) obtained by adjusting the pH to about 3.5 to 4.5 is collected by a conventional method such as centrifugation or filtration. Alternatively, the water-insoluble part (TIF-220) obtained by adjusting the pH to 7.5-8, then removing the insoluble part if desired, and adjusting it to around PF'I6 (p) 15.5-6.5) The water-soluble portion may be separated and collected by a method, and the water-soluble portion may be collected at a pH of around 14 (pH 3.5-4.
The precipitate (TF-230) obtained by adjusting 5) can be separated and collected by a conventional method.
0 and 'I'm' -230) may be subjected to a similar splitting operation with - several times for purification. The fraction having anticancer activity thus obtained is subjected to the next protein removal treatment and target product collection step.

(2) Protein removal treatment and target product collection process The protein removal treatment referred to herein may be any treatment known in the technical field, and when subjected to enzyme treatment such as proteolytic enzyme,
First, the fraction having anticancer activity obtained as described above is treated as water or a slow proteolytic enzyme! These include lonase, papain, trypsin and chymotrigycin.
Preferred examples include zo4nase and trypsin.

For or during enzyme treatment, the aqueous solution is
It is preferable to adjust and maintain it at a value of 7 to 8, so that k is hydroxylated.IJum, potassium hydroxide, soda carbonate, sodium bicarbonate, etc. are used. Furthermore, 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 determined based on the powder (solid) subjected to fermentation treatment.
An amount of about 1 to 2 parts (by weight) is used.

The above enzyme treatment is usually carried out at 30-40°C for 1-72 hours.
Preferably it is carried out for 24 to 48 hours. Also, for example, by adding 1t6 (weight ratio) to the powder (solid), 1 to 2
Enzyme treatment may be performed for 4 hours, and further enzyme treatment may be performed by adding about 11 (weight ratio) enzymes.

After the above protein removal treatment, the anticancer substance TI' is extracted from the water-soluble portion from which insoluble matter has been removed by operations such as centrifugation and filtration, if desired.
-300-2 fraction is collected. From this water-soluble part
To collect the F-300-2 fraction, for example, it is treated by combining one or more of the following methods: - separation method, precipitation fractionation method using a hydrophilic organic solvent, and separation method using an ion exchanger, followed by ultrafiltration. (These operations may be repeated twice). To give a specific example of the collection method, the above-mentioned water-soluble part is preferably adjusted to -2.5 or less, and trichloroacetic acid may be added if desired).The resulting precipitate is removed, and the soluble part is Water-breaking organic solvent 1 For example, alcohol at 30 to 80 g (volume ratio), preferably 60 to 8 g
0% (volume ratio), collect the precipitate of the resulting fraction of the target product, and if desired, then add this precipitate to
For example, Dowex Type 1 (product name), Amberlight I
The non-adsorbed fraction was treated with a strongly basic anion exchange system such as Rmu-400 (trade name) (this operation may be repeated several times), and ultrafiltered (molecular weight cut off at 50,000 yen). ~200,000), concentrated and desalted, and then dried to produce the anticancer substance of the present invention. ? -300-2 is obtained.

Anticancer substance TF-300-2 obtained by adding 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 nodule-type carcinoma, Sarzu FF180 cancer cells and 3-16 meonoma cancer cells in mice, and has an immunostimulatory effect.

ei Dissolves in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, and diethyl ether.

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

(e) The infrared absorption spectrum obtained by the KBr tablet method.

3500~6300.2920.2850.1660~
1620% 1580. ,~1540.1460~14
00.1380~1360°'120.1080~10
It has absorption bands near 20,970 and 820 to 800 cwI-1.

The ultraviolet absorption spectrum of an aqueous solution of (f)-7 shows strong absorption at the absorption end and absorption near 270 to 280 degrees.

(G) Molisch reaction, phenol-sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, and Lowry-Folin reaction are positive, and Ninch F-phosphorus reaction is negative.

(C) Elemental analysis value C: 58 excellent to 47 chi H: 5 regret to 7 regret
-30% (in terms of glucose), and the protein content by the Lowry-Folin method is about 10% or less (in terms of bovine serum albumin). 3011)b 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, magnesium salts, and alkaline earth metal salts such as calcium salts.

In this specification, in the above (1)-C separation of the precipitate obtained by adding a hydrophilic organic solvent to the supernatant liquid, the water-insoluble part obtained by adjusting the precipitate to around pH 4 [ Examples 1-(5) and 4-+4) ), P)' Water-insoluble part obtained by adjusting around I6 [Example 2-+31]
and the water-soluble portion obtained by adjusting the pH to around 6.
Precipitate obtained by adjusting the temperature to around -14 [Example 3-(3
)] were subjected to enzymatic treatment with a proteolytic enzyme, followed by ultrafiltration.

τF-310-2.7F-320-2 and τF, respectively
-330-2.

Next, the anticancer substance of the present invention T? The pharmacological effects of -300-2 are as follows. In addition, in the following pharmacological test, TIF-310-2 was subcutaneously transplanted to the site obtained in Example 4. Next, the test substance was dissolved in a 5% glucose aqueous solution,
The solution 90.2- was simultaneously administered into the tumor and intraperitoneally once a day every 6 days from the 113th day after cancer cell transfer. In addition, to the control group, #-t5% glucose aqueous solution o was administered 2-/once in the same manner.

i! -2.

Margin 3 below: The LD5o value of TF-300-2 in acutely toxic mice (NCR strain, male, 6 weeks old (iv administration)) was greater than ICIP/Kg.

As is clear from the above pharmacological experiments, the anticancer substance TF-300-2 obtained by the method of the present invention is.

It is useful as an anticancer agent, and is expected to be effective in treating various cancer diseases.

The anticancer substance TF-300-2 of the present invention can be used as it is or in the form of a non-toxic salt in a conventional manner in the form of oral, injection, crude drug, etc. dosage forms. Oral preparations may contain one or more 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. In addition, for use as an injection, the above powder TM can be dissolved or suspended in, for example, physiological saline, an aqueous glucose solution, or an aqueous solution containing a local anesthetic.

When the anticancer substance TF-3Q O-2 of the present invention and its non-toxic salt are used in patients, the dosage is appropriately selected depending on the patient's symptoms, but in general, for adults, the dosage is 0.01 to 5.
It is preferable to administer 0*/Kg once to several times a day, and preferred administration methods include oral, subcutaneous, intramuscular, intravenous, or injection into the affected area.

Next, examples and formulation examples of the present invention will be described.
Made by 9'r) K, 1! of tryptocase/distilled water.
Peptone 171I, Heart Infusion 10g,
6g yeast extract.

Eating 7.5g, alcohol 212g lactose 10g,
Sodium sulfate tl17 and thioglycolate
Add TF-e medium 8-e containing 0.59 sodium and sterilize at 120°C for 60 minutes.

The culture solution lacks nitrogen gas after cooling 100++/! /min 1
Ventilate at +iJ'1.

Humbacterium nucleatum TE"-031 (FE[(M
-5077, ATea-61647) in the previous section II solution 11
Conduct under a sterile φ outside. Culture was performed at 67°C with nitrogen gas flow (65 m/'/min) and stirring (30 rpm).
This will be done for the next 6 days. After finishing the meal, add Celite 160 to the medium II solution.
I and cellulose powder80. ! Add i' and stir, filter this under reduced pressure and leave gap 5! I culture sub-supernatant 7.6
You can get 0 (2) m for the samurai lol! Above the nutrient solution ff7.
l: Add IAK Iso hydrochloric acid $171, pH 2.0Ki14
After adjustment, x p / −) L.

11.71 was added to make an ethanol bath solution, and the mixture was heated directly at 4°C for 24 hours. Next, remove the solution part by decantation, and centrifuge at 4°C to collect the precipitate.
x 10' rpm, 5 minutes). This precipitate Vp)4
After sequentially washing with 2.0 60'6 ethanol water bath solution 400-, ethanol 400II+/, acetone 20Qd and diethyl ether 200wK/, drying under reduced pressure yields 6.9 g of powder.

F31 (Suspend the powder obtained in 21 in water 25-,
Add 1N-sodium hydroxide solution, pH 7.5-8
．． After stirring at room temperature for 30 minutes, 1N-salt #k
Adjust the pH to 4.0. Next, after stirring for 2 hours in an ice-cooled F
rpm.

10 minutes) and separate the precipitated psm and supernatant. This precipitate vp) 14.0Kv@@Ly, was washed with 10 m of water, and the precipitate and washing solution were centrifuged (1x 10' rpm, 10
minute). The precipitate was washed with 10% of ethanol and dried under reduced pressure to obtain 1.5 g of the anticancer substance TF-21L.

This tunnel σ has the following properties.

(a) White-gray to pale horizontal powder (b) Mouse σ Noehlrich ascites type cotton, Ehrlich nodular type Kusunoki, Zurcomer 180&#I cyst and B-1
6 It inhibits the proliferation of melanoma cicada cells and has immunostimulatory effects.

(c) Methanol, ethanol, acetone, benzene, chloroform, [? Insoluble in ethyl and diethyl ether.

) without a clear #AI point, 160″C to 265°C
Decompose it with.

The infrared pure absorption spectrum by So I KBrv Yakuden is
6600~6200.2950~2920.1680~
1620.1550-1510.1440.1680.
1240-1220 and 1120-1020 cIn-
There is an absorption force near 1 (Figure 2).

(to) The external radiation absorption spectrum of the water-#-liquid in the Mizuguchi bath fraction in pii7 has strong absorption at the absorption end, and 2
Absorbs at 48-265 nm (Figure 6)
0 (g) Resistance to Molisch reaction, phenol ttmh reaction, anthrone sulfuric acid reaction, indole acid resistance reaction, and Lowry-Folin reaction.

-Elemental analysis value 0: 404-46 excellent H: 5 etc.-7'IN: 9-10
m! (The sugar content of the water-soluble fraction in step 1-7 is about 5 to 25 glucose (glucose equivalent) by the phenol extraction method, and the protein content by the Lowry-Folin method is about 20 g.
Section 50 (bovine serum albumin (calculation 11)).

(1. of the anticancer substance TF-210 obtained in 41 (31).
Suspend 5 g in 15 g of water, add 1N aqueous sodium hydroxide solution to a stirred pot, and adjust to -7.8.

Heat to 67°C, pronase IC (product name manufactured by Kaken Chemical Co., Ltd.:
1.000.000 tyrosine rate/i') 15MQ
After adding the liquid, add several liters of toluene and stir.
-17,8~8. Perform IW# wood treatment for 24 hours at 0.37~40"G. Centrifuge the treatment solution (4000 rpm, 10
), separate the supernatant and precipitate, and add the precipitate to pH 8.0.
Add 6- adjusted water and centrifuge 11g1 (400Q
rpm for 10 minutes) and separate the precipitate and washing solution. Add the top 1' to the washing solution first! Combine the solution and add KIN-hydrochloric acid to P) (2, OK. A precipitate will precipitate. After leaving this solution at 5℃ for 12 hours, centrifuge @(IX10'
rpm, 10 minutes), separated into supernatant and precipitate, added 3IIl/'r of water adjusted to -2.0 to the precipitate, and centrifuged for 1 minute.
111 (IXl 0'rptn, 10 minutes) to separate the precipitate and washing liquid. Clean: Add ethanol to the # solution and the supernatant solution to make a λ80 ethanol solution. After stirring for 2 hours under water cooling, centrifuge (4000 rpm, 10 minutes) to separate the precipitate and supernatant solution. - to do. precipitation eflj'
After washing with 11#A of 180% ethanol water bath solution 51 and ethanol 5-1, it was dried under reduced pressure and immediately disintegrated.
-sto fraction! Get 527 ay.

Dissolve this powder 627~ in water 5-, add a 1N sodium hydroxide water bath solution to adjust LIK, adjust it to OH type with an IN-sodium hydroxide water source 1[K], and add 1N sodium hydroxide. Pour 200 g of water into a column filled with IR Mu4oo (trade name, manufactured by Rohm & Haas) 45sd, combine all the liquids that passed through the column, and add IN-hydrochloric acid to adjust H7 and OK. Shrink this to # under reduced pressure and use Seien's Millipore filter with a pore diameter of 0.3.
(trade name manufactured by Nippon Milivore Ltd.) and then freeze-dried to obtain anti-cancer substance F-310 freeze-dried product 1
-21019 obtained.

This material has the following properties.

(a) White-gray to pale brown powder (b) It inhibits the proliferation of Ehrlich's ascites-type cancer, Ehrlich's nodule 11@, Sarcoma 180 cancer cells and B-16 melanoma I cancer cells and has an immunostimulatory effect.

Dissolved in water, methanol, ethanol, acetone, benzene, etaform% Vinegar 11-! -f-le,
'Insoluble in diethyl ether.

(b) Suiseki has a melting point of @14ii, starts to decompose at about 180°C, and decomposes at #L<< above 195°C.

(e) KBr - Infrared absorption vector according to the Penal Code.

6500~3600.2920.2850.1660~
1620.1580-1540.1460-1400.
1380-1360.1120.1080-1020.
970 and 820-80 [1 (has an absorption band near W-' (4th and 1).

(f) The ultraviolet absorption spectrum of the water bath liquid of P117 has strong absorption at the absorption end and also shows absorption in the vicinity of 246 to 259 mwa (Fuki 5shi 1).

() I TSK -GEL G 3 Q Q I S
W (Toyo Iita Co., Ltd. f1 product name, columnar, 5m
mg x 600 mm
y/min, room temperature) is 220 nm when measuring the ultraviolet edge absorption 1 at the tip of the medium, 28 to 62 minutes, and 46 to 6 minutes.
Absorption '%: 4F! 260 nm
In the o'r ultraviolet absorbance measuring instrument, the base end of the bath medium, 42
Absorption occurs around ~60 minutes (Figure 6).

(e) Molisch reaction, phenol spear a! kW reaction, Anthrone sulfur reaction, indole cyanide reaction, and Lowry-Folin reaction were positive, and Ninh P11 reaction was negative.

(Eleven element analysis value 0: 38.0 width ~ 42 division H: 5.0 winter ~ 7I6
N: 1 to 4 m (N) The sugar content by the phenol-sulfuric acid method is approximately 20
The protein content according to Reference-604 (glucose converted IN) and the Lowry-Folin method is about 10L6 or less (in terms of bovine serum albumin).

(5) Anticancer substance TIF-310140 obtained from upper sweetfish
IIQ will be doubled and S salary will be given. This number of pairs was filtered through a Millipore filter (trade name manufactured by Nippon Millihoari S.A.F.) with a pore size of 0.2 μm, and then freeze-dried.
1o-2 lyophilized product 66~ served.

Did you get it like this? 1tll transgenic substance TF'-3
10-2 had the properties described in the claims, and had a sugar content (calculated by 1 glucose) of about 16 to 60.

Example 2 (11 5.9 g of powder obtained by the method of Example 1-121)
was suspended in water 2511/2, added with 1N sodium hydroxide aqueous solution to give pi-17.5 to 8.0, and diluted with 6.
After stirring for 0 minutes, adjust the temperature to 6.0 by adding 1N-acidic acid. Then, after stirring with water cooling for 2 hours, centrifuge # (I
x 10' rpm for 10 minutes) and separate the precipitate and supernatant. This precipitate was washed with water 51 adjusted to p) 16.0, and the precipitate and washing solution were centrifuged*(l×10'rpm).
, 10 minutes). The washing solution is combined with the previously diluted 4% supernatant and subjected to the treatment described in Example 6-(11).
After washing the precipitate with ethanol for 5 seconds, the pressure was reduced to 0 to obtain anticancer substance TF-220V 1.06.9.

This σ has the following property V.

(a) White-gray color f! A brown powder (b) inhibits the proliferation of Ehrlich ascites type, Ehrs-Nohi nodule type, Zulkoma 180 static cells and B-16 melanoma lytic cells in mice, and has an immunostimulatory effect.

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

) Clear l (dot not shown, decomposes at 160°C to 240”C.

The infrared absorption spectrum obtained by the M KBr tablet method is 3
600-6200.2950-2920.1680-1
620.1550-1510.1440.1680.1
240-1220 and 1120-1020cIs-1
It has an absorption band near (Fig. 7).

(To) Water soluble fraction water at pH 7
Absorbs near 8 at 66 nm (Silk Figure 8).

(T1 Molisch reaction, phenol salmon Ii reaction, Anthrone sulfur eII reaction, indole editing acid reaction, and Lowry-Folin reaction are positive. 74 N = 7 ~ 9 (Ill p) The sugar content of the water OT #4 fraction at 17 by the phenol-sulfuric acid method is approximately 5 ~ 204 (y glucose equivalent), by the Lowry-Folin method. The protein content is approximately 104% or less (beef protein content is about 10% or less).

(1 of the anticancer substance TI'-220 obtained in 21(II)
g) was suspended in 10 Il of water, and while stirring, 1N aqueous sodium hydroxide solution was added to adjust the solution to p) 17.8.

Heat to 67°C, pronase B (product name manufactured by Kaken Chemical Co., Ltd.:
1.000.000 tyrosine units/g) 15■V#71
Add a few drops of toluene to the D bond, stir, and bring to a pH of 7.8.
～B, Enzyme treatment for 24 hours at 0.67-40'O.

Where f! Centrifuge the lI solution (4000 rl) m, 10 minutes)
Then, separate the supernatant and precipitate, add A4-conditioned water 6- to the precipitate at p) 18.0, and centrifuge * (40 (J Or
Elm, 10 minutes) and separate the sediment and washing solution. Combine the washing solution with the supernatant solution and mix with KIM-hydrochloric acid. p) 12. [If adjusted to 1, a precipitate will separate out. This solution was packed for 12 hours at 5-9 and centrifuged (1
Separate the supernatant and precipitate, add 5 d of purified water, and centrifuge (1 x 10' rpm, 10 minutes).
minutes) and separate the precipitate and liquid. The supernatant was combined with the washing solution, ethanol was added to make an 80% ethanol solution, stirred for 2 hours under water cooling, and centrifuged at Hi (400%).
Dr. rpm 10 minutes) and separate the precipitate and supernatant liquid Vf+.

The precipitate was sequentially washed with 5 sl of 80% ethanol water bath and 511% ethanol, and then dried under pressure to obtain 166% of the anticancer substance TF-320.

This powder was dissolved in 150 to 5 s of V water, adjusted to -7.0 by adding a 1N aqueous sodium hydroxide solution, and adjusted to an OH type in advance with a 1N sodium hydroxide water bath. Apply to a column packed with 25- (trade name) manufactured by AND) AS Co., Ltd.,
Combine all the liquids that passed through the column when flowing 1N-
Add Hatosuke and make Ichihime -7.0. This was filtered under reduced pressure using a Millipore filter (manufactured by Nippon Millipore Ltd.) with a pore size of 0.6 μm, and then freeze-dried to obtain a freeze-dried product v110 of anticancer-1TF-320.

This σ) has the following properties.

Mark: White-gray to light brown powder.

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

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

←1 Clear one; No points are shown; decomposition starts at about 180°C and gradually decomposes at temperatures above 195'O.

The infrared absorption spectrum according to the KBr Sea Bream Law and Criminal Law is
3500~6600.2920, 2850.1660~
1620.1580~1540.1460~1400,
1380-1660.1120.1080-1020.
It has absorption bands near 970 and 820 to 8QQm-1 (Fig. 9).

The ultraviolet absorption spectrum of an aqueous solution of (f)-7 shows strong absorption at the absorption end and absorption near 250 to 270 nm (10 thickness I).

(To + Tl3K -GIIJ a 3000 SW
(Product name of Toyo*Tatsu Co., Ltd., Columnia, 5111
High performance liquid chromatography using φX60 (llX2) (
Bathing*: A7 0.1M phosphate buffer, flow rate 0.8m
//min, room temperature) in the ultraviolet absorbance measurement at 220 nm, the solvent front part, around 28 to 62 minutes, 43 to 64
In ultraviolet absorbance measurement at 10.260 nm, absorption occurs at K over 65 to 56 minutes at the front end of the solvent (Figure 11).

(a) Molisch reaction, phenol-acid reaction, anthrone sulfuric acid reaction, indole salt 61 Bt reaction, and Lowry 7-4-117 Shio reaction were positive, and Ninhypurine reaction 1 was negative.

(January original wood value C: 684 ~ 42 H: 5 ~ 7 N: 1
% ~ 4 Excellent) Sugar content by phenol sulfuric acid method is approximately 20%
~60% (in terms of f-lucose) and the protein content by the Lowry-Giolin method is about 10% or less (in terms of bovine aldemin).

(31 upper Me te 1% rat 1. ta III m property W
'['F'-320,100all-dissolved in water 50s/Ultrafilter UK-5[1 (Toyo P Paper Co., Ltd. ultrafiltration barrel Product name: Fractional bond≠molecular weight 5 x 104
．． , ) and concentrated to 100@. This concentrated solution was filtered through a Millipore filter with a pore size of 0.2 μm (manufactured by Millipore, manufactured by Millipore), and then lyophilized to form an anticancer substance TI"-320-20 stretched dried product 72.
get ~. The anti-cancer drug 'iiT' served in this way
-320-2, had the properties described in the claims, and had a sugar content (in terms of glucose) of about 16 to 60.

Kyo-? 11J-salt rH was added to the combined solution of the washings and synovial fluid obtained in 11 Example 2-tl to adjust the temperature to -4.0, and then the solution was incubated at below 5°C for 12 hours. . Centrifuge (1 x 10'rpm, IQ minutes) under dry conditions to separate the precipitate and supernatant synovial fluid. This precipitate was washed with 5 WIt of adjusted water, and the precipitate and washing solution were centrifuged at H (1x
10'rpm, 10 minutes). Pour the precipitate into ethanol for 5s
/ After washing with
vO, 489m.

This material has the following properties.

(b) White-gray to light brown powder.

(b) It inhibits the proliferation of Ehrlich ascites-type carcinoma, Ehrlich nodule-type carcinoma, Zulkoma-18 (LEA) and B-16 melanoma cancer cells in mice, and has an immunostimulatory effect.

(/Sai) Insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, and diethyl ether.

2) Decomposes at 185°C to 225°C without showing obvious m-pickling.

(e) The infrared absorption spectrum according to the KBr Detergent Law and Criminal Law is
6600~3200, 2950~2920.1680~
1620.1550~1510°1440.1680.
1240-1220 and 1120-1020 (to) -
It has an absorption band near 1 (#L12 is 1).

(f) The ultraviolet absorption spectrum of an aqueous solution of pl-17 is l
& There is strong absorption at the end, and from 249 to 264 nm
(131g)).

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

- Redundancy analysis value C: 42% ~ 451 EI: 5e6 ~ 7% N: 10 ~ 114 (The M content of sugar by the 111 phenol sulfuric acid method is approximately 5% ~
The protein content according to the Lowry Fist 7 Orin method is approximately 60 to 60 units (1) in terms of serum albumin.

(2+ +11 Anticancer substance Tl230
] 0.48 g was suspended in 5 s of water, and an aqueous solution containing 1N sodium hydroxide was added under stirring to altE to -7,8.

Heat to 37°C, pronase E (product name manufactured by Kaken Chemical Co., Ltd.:
After adding 1.000.000 tyrosine units #) 5.0■, add a few drops of toluene and under stirring, p) 17.8~
8.0. 37-40°C-C-24alt [perform elementary treatment. Centrifuge the treated solution (4000 rpm, 10 minutes),
Separate the supernatant liquid and precipitate, and remove the precipitate KpH8,0Kg1l
Add 3wLl of water and centrifuge (4000
rpm, 10 minutes) and separate the precipitate and washing solution. If the washing solution is combined with the previous ±f# solution and KIN-hydrochloric acid is added to the solution and adjusted to -2, OK, a precipitate will be deposited. This solution v5
After standing at ℃ for 12 hours, centrifugation (I
'rpm, 10 minutes) to separate the supernatant synovial fluid and precipitate, add water 6II / adjusted to pH 2.0 to the precipitate, perform ischemic separation (IX10'rpm, 10 minutes), and separate the precipitate and washing solution. Separate. Add 710 to 80 ethanol to the cleaning solution, add 710 to 80 ethanol, stir under water cooling for 2 hours, and add Liaoxin 11i (4000 rpm,
10 minutes) and separate the precipitate and supernatant synovial fluid.

The precipitate was washed with an aqueous solution of 804 ethanol and ethanol, and then dried under reduced pressure to remove the anticancer substance TF.
Get 100 ■ from 330.

Dissolve 100μ of this powder in 5W of water, add 1N hydroxide solution, adjust to -7, OK vI4,
Amberlite RA400 (Rohm Ann F
・Column K filled with 4 products manufactured by Haas Co., Ltd. (product name) 15-
When using 60wdvfl of water, combine all the liquid that passed through J Kara 1,
Add 1N hydrochloric acid to adjust the temperature to 17.0 Km. This is concentrated under reduced pressure, filtered through a Millipore filter with a pore size of 0.3 μm (Japan Millipore Ltd. Urako product name), and then freeze-dried to obtain a freeze-dried product of the anticancer substance TF-330.

This material has the following properties.

(b) White-gray to light brown powder book.

It inhibits the N-sensitivity of Ehrlich ascites type mice and has an immunostimulating effect.

(c) methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, dissolved in water.

Insoluble in diethyl ether.

2) It does not reach a clear melting point of 1, but begins to decompose at about 180°C, and decomposes noticeably above 195°C.

(e) The infrared absorption spectrum obtained by the ICBr tablet method.

3500~3300.2920.2850.1660~
1620.1580-1540.1460-1400.
1680~1360°1120.1080~1020.
Absorption band V near 970 and 820~8QQcM-1
The ultraviolet absorption spectrum of an aqueous solution at pH 7 shows strong absorption at the absorption end and absorption near 250 to 260 nm (Saba 15).

()l To T8-Gi G3 Q 008W (Toyo Soda Co., Ltd.'s product name, Columnia, 5 Kamuda x 600
High performance liquid chromatography column (#output: P) using 2)
(0.1 molar phosphate buffer solution in 7, speed u, s mti min, room temperature) has an absorption at the front part of the solvent over K of around 36 to 64 minutes in the 220 nmfN external absorption measurement. 260 nm ultraviolet 11ii! In absorbance measurement, absorption occurs at the tip of # medium over a period of about 35 to 62 minutes (Fig. #116).

-Morisch reaction, phenol-sulfuric acid reaction, Anthro 7
The oxalic acid reaction, indole-hydrochloric acid reaction, and Lowry-Funa 11 reaction were positive, and the ninhydrin reaction was negative.

(su) Elemental analysis value 0: 381~424 H: 5th to 7th ratio N: 14~
4) The sugar content by the phenol-sulfuric acid method is approximately 20
The protein content according to the Ouli-Forin method and the protein content is about 1011 or less (in terms of glucose) and less than 1011 (in terms of glucose).

(3) Anticancer substance TF-33070 obtained above
Dissolve 50wIIK in ~Y water, filter with Millipore filter (Japanese 5l JTIe Arimite Sodori brand name), and then dry with OIR#. Obtain 49~.

Anticancer @'N TP-330- obtained in this way
2. The properties described in the claims are VN, and the sugar content (in terms of gelcose) is generally about 16 to 3 degrees. It was the beginning of Part 1 of the 18th.

Example 4 (1) A 90-6 jar fermenter (MeJ-U type manufactured by Marubishi Rika Kenkyusho) was mixed with 70 σ of distilled water.

Tryptocase Peptone 1190.9, Heart Infusion 1050g, Yeast Extract 2
10g, salt 525. u, ff lucose 840.9. 700 g lactose, 7 I sodium stanmite, 659 sodium thioglycolate and 1 potassium phosphate dibasic
Contains 759, 1 f6T1'-f culture mY7JD, 1
flIt and rIi for 15 minutes with 18-U ◎Into the culture medium,
Cooled nitrogen main gas is passed through at 2501/min for 1 hour.

Humbacterium nucleatum TP-031 (vwhv-5
077, ATOO-31647) pre-culturing solution approximately 900I
II/v Inoculate the above culture solution. Culture was carried out at approximately 36°C with stirring (9 tR1/min) and nitrogen gas injection (250 tR1/min).
0r, p, m) for 4 days. After culturing, add the serum to the culture solution) 10? /Carved and cellulose powder 5g/
! The supernatant of the culture solution was added and stirred, and the culture solution was treated with P under reduced pressure to remove exposure.

(7.5-eK m
Endure 113w? After adjusting the temperature to -260, add 11,4% ethanol to the solution and leave at 4°C for 24 hours. Then, remove the solution by decantation, and centrifuge at 4° C. (4×10′ rpm, 10 minutes) to collect the precipitate. The precipitate was diluted twice with 100 g of 60% ethanol water bath solution of pi-12.0, and ethanol 15 (J+m/, meta/
After washing for a long time (with 2 times OwKc and 150 g of acetone), air dry the powder to 6.0.9 vM.

+31 (suspended in powdered water 60- obtained in step 21,
Add 4N-sodium hydroxide aqueous Th solution, -7,5 to 8
．． After stirring at room temperature for 15 minutes, 4N hydrochloric acid was added to adjust the pH to 4. Then, after standing for 2 hours under water cooling, centrifugation (I x 10'rpm) was performed.

10 minutes) and separate the supernatant and precipitate. This precipitate K
IISv: Pure f > 1: Water 6dv'JJDLW&lI
Solution a)-fx was added with 4N aqueous sodium hydroxide solution to pH 7.5-8. OK - Adjust. Then, stir at room temperature for 15 minutes, then add 4N-salt #k to rJ(4,OK-
Let stand on ice for 2 hours. Then centrifugation (1 x 10
' rpm, 10 minutes) Collect the L precipitate. This precipitate v44.0 was placed on a fc gauze with water 411/ml arranged next to it, centrifuged for 10 minutes at I ).

111I#Ih material TF-2 obtained by pressing in this way
10 has the same properties as y=4 obtained in Example 1.
T' (humidity 1 meeting) water 6mKJ11? Cloudy and stirred FK saturated ammonium carbonate water W4 armpit [1] and adjust to -7,8. This was heated to Y67℃, and pronase E (product name manufactured by Kaken Chemical Co., Ltd.: 1,000,000 tyrosine units/9
) After adding 7J[l of 21~V, add a few drops of toluene V and perform enzyme treatment under shaking at pl-17, 8~8.0, 37~40℃ for 2 hours, then add 4N-\JA acid to the treated solution. to -1,0 to precipitate. After allowing this suspension to stand still under water cooling for 2 hours, it was centrifuged # (I x 10'ri
) m, 10 minutes) C, separate the supernatant liquid. This supernatant liquid Kx
Add 29 liters of water and rinse in a 60-degree ethanol water bath to precipitate. This was left to stand for 2 hours under water cooling, and then centrifuged (6x 103 rpm).

10 minutes) and collect the precipitate. The precipitate was sequentially washed with a 60% ethanol water bath, 10ml of ethanol, and 10ml of acetone, and then air-dried to remove the anticancer substance TF.
-5105154 is obtained. %: Dissolved in 60 d of water and aqueous with 4N water) Pi'18 in IIum water age solution
．． OK aJd bottle filled with DOWEX+×4＠-H# (part name manufactured by Dow Chemical Company) CI 30〆 filled with 5K, water 100sl/11' when swimming 5 times''
1i11 so that all l & becomes -6,5 to 80
Do E. Apply this to ultra filter σ -50 (Toyo p
Ultra p filtration membrane manufactured by Paper Co., Ltd., molecular weight fraction 5X1
0') K and carry out an extreme pass and S- to 100 times. This concentrated solution was filtered through a Millipore filter with a pore size of 0.3 μm.
This millibore product (part name manufactured by Risted) is then lyophilized to produce a lyophilized product of 1111#h substance TIP-610-2 (230 yen). I obtained in this way
l+ lump substance Ty-310-2 is patented i11'#l'5
1? It has the properties described in the range of , and generally its grain content (l
(in terms of lucose) is approximately 16 to 604↑, and the infrared absorption spectrum, ultraviolet absorption spectrum, and fast liquid chromatogram of representative books are as shown in Figure 19, Figure 20, and Silk Figure 21, respectively. .

Formulation Example 1 Inhibition #IJ member TI'-61[J-2'? 1' Fill a vial with a water bath solution of 7.5 to 0.5 or 1 to 0.5 or 1. When using this, sterile physiological saline, lidocaine 0.5%
The solution is dissolved isobarically in a bath solution containing 5-glucose or a 5-glucose solution and used as an injection solution.

Formulation Example 2 -7.0 to 7.5 containing abrasive material end Tl'-320-2
Fill a vial with an aqueous solution of 0.5 or 1 unit to obtain a dry product of 1l111 thermal substance '1TF-320-2yA. When used, sterile saline, lidocaine 0.
The solution containing 5% is used as an injection solution by dissolving it in 5% dextrose water #liquid.

Formulation Example 6 -7,0 to 7.
Fill a vial with the water bath solution of 0.5 or 1 #1 unit σJ%tll cancerous g1 quality T? -350-2 lyophilized product is obtained.

When used, the dough is dissolved in sterile physiological saline, 0.5% lidocaine solution, or 5% glucose water and used as an injection solution.

[Brief explanation of the drawing]

#! Figure 1 shows Funpacterium panuku lz used in the present invention.
7 Tom TF-031 (F) type 1at' micro-photograph showing
Figure 2 shows the infrared acupuncture absorption spectrum of the anticancer substance TF-210, and the ultraviolet absorption spectrum of the same substance 61. Figure 4 shows the infrared absorption spectrum of the anticancer substance TF-310σ, and Figure 5 shows the ultraviolet absorption spectrum of the same substance.
Figure 6 is a fast liquid chromatography ram of the same substance, and the seventh piece is the infrared absorption vector of the anticancer substance TF-220, #18
Doctors are concerned with the ultraviolet absorption spectrum of 11 substances, and Figure 9 shows
Infrared and 1 absorption spectra of the control substance TF-320, Figure 10 is the ultraviolet absorption spectrum of the same substance, Figure 11 is the t
I! The liquid chromatogram of the RI substance, Figure 12, is the anticancer substance T? -230 infrared absorption spectrum, l11
3h is 1) The ultraviolet absorption spectrum of the substance, Figure 14 is the infrared absorption spectrum of the anticancer substance TF-330, and Figure 15 is the ultraviolet absorption spectrum of the same substance. 16
Figure 17 shows the infrared absorption spectrum of the II+1+ TF-630-2 of the present invention. 19-1 is the anticancer substance TF-310- of the present invention.
2 extra spider absorption spectrum, 13120 section 1 is the ultraviolet absorption spectrum of the liquid, and the 21st is the same high performance liquid chromatography column. 1st page: 10μ Continued from page 1 0 Inventor Yuri Sugita 2-2 Shimoakae Municipal Housing, Toyama City 3 204 0 Inventor Yoshiko Yamamoto 970-6 Takatsuka, Namekawa City 0 Inventor Minamimukai Matsuwaka-cho, Toyama City 9-1 0 Inventor Takako Hori Prefectural Housing 4-02 542- 1383 Mukaishinjo, Toyama City

Claims (9)

[Claims] (1) Anticancer substance TF-30 having the properties shown below
0-2 and its salt. It 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
3500~3600.2920.2850°1660~
1620.1580-1540.1460-1400.
1!180~1360°1120.1080~1020
．． It has absorption bands near 970 and 820-800 cII-1. (f) The ultraviolet absorption spectrum of the aqueous solution of -7 has strong absorption at the absorption end and absorption near 270 to 280 degrees. ()) Molisch reaction, phenol-sulfuric acid reaction, anthrone-sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-Folin reaction are positive, and ninhydrin reaction is negative. (C) Elemental analysis value C: 38-47cm H: 5-7cm N: 1-4cm (W) Sugar content by phenol-sulfuric acid method is about 16s
The protein content according to the Lowry-Folin method is approximately 10 or less (in terms of bovine serum albumin). (2) Collecting the anticancer substance TIF-300-2 or its salt from the culture solution or its supernatant obtained by culturing the anticancer substance TF-300-2 producing bacteria belonging to the genus Hubbacterium KJII. TF-300, an anticancer substance characterized by
-2 and a method for producing its salt. (3) Anticancer substance TF belonging to the genus Hubbacterium
-300-2 Producing bacteria are cultured, and a fraction having anticancer activity obtained from the culture solution or its supernatant is subjected to protein removal treatment and then subjected to ultrafiltration. The manufacturing method described in paragraph 2. (4) Anticancer substance TF belonging to the genus Hubbacterium
-300-2 A precipitate produced by adding a hydrophilic organic solvent to the supernatant obtained by culturing the producing bacteria -- or after subjecting the fraction with anticancer activity obtained from the precipitate to protein removal treatment. 3. The manufacturing method according to claim 3, which comprises performing ultrafiltration. (5) The production method according to claim 3 or 4, wherein the protein removal treatment is an enzyme treatment using a protease. (6) Is an anticancer substance of the Hubacterium genus? ? -300-2 The fraction with anticancer activity obtained from the precipitate obtained by adding a hydrophilic organic solvent to the supernatant obtained by culturing the producing bacteria was subjected to enzymatic treatment with a proteolytic enzyme. 5. The manufacturing method according to claim 4, which comprises filtration. (7) A hydrophilic organic solvent is added to the supernatant obtained by culturing bacteria belonging to the genus Hubbacterium, and the resulting precipitate is divided by -. 7. The production method according to claim 6, which comprises subjecting the product to enzyme treatment and then collecting the anticancer substance fraction. (8) The fraction with anticancer activity obtained by adding a hydrophilic organic solvent to the supernatant and dividing the resulting precipitate into - into -
8. The production method according to claim 7, wherein the fraction is 3.5 to 4.5 and is insoluble in water. (9) The fraction with anticancer activity obtained by dividing the precipitate produced by adding a hydrophilic organic solvent to the supernatant liquid by -3.5 to 4.5 by adding water to the precipitate 9. The manufacturing method according to claim 8, wherein the water-insoluble portion is obtained by adjusting the water-insoluble portion. OI Precipitate produced by adding a hydrophilic organic solvent to the supernatant #
9. The production method according to claim 8, wherein the fraction having an anticancer effect obtained by dividing one substance based on pHk is a water-insoluble fraction having a p) of 15.5 to 6.5. The fraction with anticancer activity obtained by dividing the precipitate produced by adding a hydrophilic organic solvent to the al supernatant is
Soluble in water at pH 5.5-6.5 p) 13.5-4.5
9. The production method according to claim 8, which is a water-insoluble fraction. 12. The production method according to any one of claims 5 to 11, wherein the enzymatic treatment with α3 protease is f-lonase or trypsin treatment. 03 After the enzymatic treatment, the anticancer substance fraction is collected by a combination of at least one of a fractionation method using -, a fractional precipitation method using a hydrophilic organic solvent, or a separation method using an ion exchanger, followed by ultrafiltration. 13. The production method according to any one of claims 5 to 12, which is an operation of collecting the anticancer substance fraction. (+41 The operation of collecting the anticarcinogenic substance fraction after enzymatic treatment is to divide the water-soluble part after enzymatic treatment with a proteolytic enzyme by - and adjust the pH to 2.5 or less. The production method according to claim 1.3, which is an operation of adding a hydrophilic organic solvent to the fraction, treating the resulting precipitate with an ion exchanger, and collecting the non-adsorbed fraction.09 Enzyme treatment The operation for collecting the post-carcinogenic substance fraction is to divide the water-soluble part after enzymatic treatment with a proteolytic enzyme into multiple fractions and adjust the pi to 42.5 or less to obtain an effective fraction of the water-soluble part. The manufacturing method according to claim 13, which comprises adding a hydrophilic organic solvent to the Oe fraction, treating the resulting precipitate with an ion exchanger, and subjecting the non-adsorbed fraction to ultra-P. Claims 3 to 15, which involve ultrafiltration using a filtration membrane having a molecular weight of 50,000 to 200,000.
The manufacturing method described in any of the paragraphs. An operation of adding a hydrophilic organic solvent to the supernatant liquid is -1,5
17. The production method according to any one of claims 2 to 16, which comprises adding a hydrophilic organic solvent to the supernatant liquid adjusted to a concentration of 2.5 to 2.5. The manufacturing method according to any one of claims 2 to 17, wherein the hydrophilic organic solvent added to the supernatant liquid is alcohol. θ field The method of manufacturing according to any one of claims 2 to 18, characterized in that a hydrophilic organic solvent is added to the supernatant liquid so that its concentration is 30 to 80 N (volume ratio). (a) The production method according to any one of claims 2 to 19, wherein the anticancer substance T C 300-2-producing bacterium belonging to the genus Humbacterium is Fumbacterium nucleatum. (c) Anticancer substance T belonging to the genus Hubbacterium? −
TIF-300-2, an anti-carcinogenic substance having the following properties, obtained by culturing the 300-2 production plant and subjecting the fraction with anti-carcinogenic activity obtained from the culture solution to protein removal treatment, or a salt thereof An anticancer agent characterized by containing. It has a calming and immunostimulating 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℃,
Decomposes significantly above 195°C. (e) The infrared absorption spectrum obtained by the KBr tablet method. 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-1. (f) The ultraviolet absorption spectrum of an aqueous solution at pH 7 has strong absorption at the absorption end, and is 270 to 280n! 110
It shows absorption in the vicinity. (g) Molisch reaction, phenol-sulfuric acid reaction. Anthrone sulfuric acid reaction, indole hydrochloric acid reaction. The Lowry-Folin reaction was positive, and the ninhydrin reaction was negative. (c) Elemental analysis value C: 38-4716 H: 5 excellent ~ 7% N: 1 arrogant ~ 4
Unfortunately, the sugar content by the phenol-sulfuric acid method is approximately 1
The protein content according to the Lowry-Folin method is approximately 10 parts or less (in terms of bovine serum alzomine).