JPH0243760B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel anticancer substance, and more particularly to TV-130, an anticancer substance obtained from a culture solution obtained by culturing bacteria belonging to the genus Veillonella. Furthermore, the present invention relates to a method for producing the anticancer substance TV-130 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, there is little research on components that have excellent anticancer effects obtained from the culture solution obtained by culturing anaerobic bacteria, and in particular, there is still little research on the components obtained from the culture solution obtained by culturing anaerobic bacteria belonging to the genus Veilonella and their anticancer activity. unknown. The present inventor cultivated bacteria belonging to the genus Veillonella isolated from the human oral cavity, and investigated the pharmacological effects of the supernatant after removing the bacterial cells from the culture solution. Certain ingredients have strong anticancer effects, and this also means that
In the colony formation suppression method, the effect of inhibiting the formation of cancer cell colonies is extremely small, and instead of an anticancer effect by killing cells, it is an indirect anticancer effect that is mediated by the host or enhances the host's immunity, with the help of the immune system. We discovered that this component has the effect of causing the expression of
The invention has been completed. The bacteria used in the present invention may be any TV-130 producing bacteria belonging to the genus Veillonella, and suitable examples include Veillonella alcalescens and Veillonella parvula. Specifically, for example, Veilonera alcaretscens
TF-V, Veillonella parvula TF-V-1, and strains having these properties as common knowledge in microbiology, such as natural mutant strains or artificially improved strains, are used. Next, Veilonera Alcalescens TF-V
The mycological properties of Veillonella parvula TF-V-1 are as follows. Γ Mycological properties of Veillonella alcalescens TF-V (1) Morphology Cell shape: Spherical (Fig. 1) Presence or absence of cell pleomorphism: None Presence or absence of motility: None Presence or absence of spores: None Gram staining : Gram negative (2) Growth status in medium TF-a agar plate External shape: Round and non-hemolytic Structure: Dewdrop-like Surface: Smooth Color: Milky white TF-a liquid medium Growth level: Normal Turbidity: Cotton fibrous Gas: None (3) Physiological properties Hydrogen sulfide production: - Nitrate reduction: - Propionic acid production: + Indole production: + Urease: - Catalase: + Milk: non-clotting Attitude towards oxygen: anaerobic Lipase: - Lecithinase: - Growth range: Optimum pH around 7 (neutral), optimal temperature 35-40℃ Gas production from sugar L-arabinose (-), D-xylose (-), D. glucose (-), D- Mannose (-), D-fructose (-), D-galactose (-), maltose (-), sucrose (-), trehalose (-), sorbitol (-), mannitol (-), inosit (-), Glycerin (-), starch (-), salicin (-), gelatin (-), esculin (-), cellbiose (-), raffinose (-), rhamnose (-) Γ Veillonella parvula TF-V-1 bacteria Scientific properties (1) Morphology Cell shape: spherical (Figure 2) Presence of cell pleomorphism: None Motility: None Presence of spores: None Gram staining: Gram negative (2) Growth status in medium TF -a agar plate External shape: round and non-hemolytic Structure: dewdrop-like Surface: smooth Color: milky white TF-a liquid medium Growth level: normal Turbidity: cotton fibrous gas: none (3) Physiological properties Production of hydrogen sulfide :- Nitrate reduction: - Propion production: + Indole production: + Urease: - Catalase: - Milk: Does not coagulate Attitude towards oxygen: Anaerobic Lipase: - Lecithinase: - Growth range: Optimum pH around 7 (neutral) ), optimal temperature 35-40℃ Production of gas from sugar L-arabinose (-), D-xylose (-), D-glucose (-), D-mannose (-), D-fructose (-), D-galactose (-), maltose (-), sucrose (-), trehalose (-), sorbitol (-), mannitol (-), inositol (-), glycerin (-), starch (-), salicin ( -), gelatin (-), esculin (-), cellbiose (-), raffinose (-), rhamnose (-)
When considered against the edition, these strains are Veillonella alcaretscens.
alcalescens) and Veillonella parvula, we identified them as belonging to these strains, respectively, and identified these strains as Veillonella alcalescens TF-V and Veillonella parvula TF-1. -V (Veillonella
parvula TF-V-1) and were given accession numbers FERM-P No. 5876 and 5877 to the Institute of Microbial Technology, Agency of Industrial Science and Technology, respectively.
(FERM-P No. 5877). The anticancer substance TV-130 of the present invention is produced, for example, as follows. (a) Cultivation Veillonella alcalescens and Veillonella parvula are cultured by a conventional anaerobic culture method. Namely, nitrogen sources such as various peptones, cow brain, and heart extract; carbon sources such as glucose and lactose; vitamin sources such as yeast extract; reducing agents such as L-cystine, sodium sulfite, and sodium thioglycolate. ; Using a medium containing inorganic salts such as sodium chloride adjusted to pH around 7, particularly preferably PH7, with sodium hydroxide, under anaerobic conditions, preferably at 35 to 40°C.
Culture for 1 to 7 days either by standing still or by stirring. In particular, a medium containing the following components (hereinafter referred to as TF medium):
It is preferable to use However, as a nitrogen source, it is not always necessary to use brain heart intake, which is an extract of cow's brain or heart. Proteose peptone and phitone peptone are not necessarily necessary among various peptones, and polypeptone can be substituted for tryptocase peptone. In addition,
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 by conventional methods such as centrifugation or filtration using a super-assistant such as high-flow super gel, but centrifugation is particularly preferable in terms of operation, degree of bacterial removal, and yield of supernatant. . TV was prepared from the culture solution and supernatant obtained in this way.
The method for collecting -130 is shown schematically as follows.

【table】

[Table] Drying

Claims (1)

[Claims] 1. Anticancer substance belonging to the genus Veillonella TV-
TV-130 is an anticancer substance obtained by culturing 130-producing bacteria and having the following properties from the culture solution. (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 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
3500~3150, 2970~2870, 1690~1620, 1560~
It has absorption bands near 1520, 1490-1320, 1280-1200, 1120-1000 and 980-970 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 247 to 270 nm. (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: 31% to 42%, H: 3% to 7%, N: 2%
~12% 2 Anticancer substance belonging to the genus Veillonella TV-
130 A hydrophilic organic solvent is added to the culture solution or supernatant obtained by culturing the producing bacteria, the resulting precipitate is collected, water is added to this to remove water-insoluble materials, and then this is subjected to dialysis or ultrafiltration. The anticancer substance TV-130 according to claim 1, which is obtained by filtering and further treating the same with an ion exchanger. 3 Anticancer substance TV belonging to the genus Veillonella
130. The anticancer substance TV-130 according to claim 1 or 2, wherein the 130-producing bacterium is Veillonella alkalezcens. 4 Anticancer substance belonging to the genus Veillonella TV-
130 The anticancer substance according to claim 1 or 2, wherein the producing bacterium is Veillonella parvula.
TV-130. 5 Anticancer substance belonging to the genus Veillonella TV-
130 A hydrophilic organic solvent is added to the culture solution or supernatant obtained by culturing the producing bacteria, the resulting precipitate is collected, water is added to this to remove water-insoluble materials, and then this is subjected to dialysis or ultrafiltration. The following properties are obtained by filtration and further treatment with an ion exchanger: (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 110°C, and significantly decomposes at temperatures above 200°C. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3500~3150, 2970~2870, 1690~1620, 1560~
It has absorption bands near 1520, 1490-1320, 1280-1200, 1120-1000 and 980-970 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 247 to 270 nm. (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: 31% to 42%, H: 3% to 7%, N: 2%
A method for producing the anticancer substance TV-130 having ~12%. 6. The method for producing the anticancer substance TV-130 according to claim 5, wherein the hydrophilic organic solvent is alcohol. 7. Anticancer properties according to claim 5 or 6, characterized in that a hydrophilic organic solvent is added to the culture solution or supernatant at a concentration of 30 to 80% (volume ratio). Method for producing substance TV-130. 8. The anticancer substance TV- according to any one of claims 5 to 7, characterized in that it is eluted with a phosphate buffer containing sodium chloride during ion exchanger treatment.
130 manufacturing methods. 9. The method according to any one of claims 5 to 7, characterized in that the ion exchanger treatment is performed with an ion exchanger equilibrated with a phosphate buffer containing 0.1 molar salt, and the non-adsorbed fraction is collected. A method for producing the anticancer substance TV-130 described in Section 1. 10. Claims 5 to 10 are characterized in that in the ion exchanger treatment, a fraction is collected that does not elute with a phosphate buffer containing 0.1 molar salt, but elutes with a phosphate buffer containing 0.3 molar salt. A method for producing the anticancer substance TV-130 according to any one of Section 8. 11. Claims 5 to 11 are characterized in that in the ion exchanger treatment, a fraction is collected that does not elute with a phosphate buffer containing 0.3 molar salt, but elutes with a phosphate buffer containing 0.4 molar salt. A method for producing the anticancer substance TV-130 according to any one of Section 8. 12. Claims 5 to 12 are characterized in that, in the ion exchanger treatment, a fraction that does not elute with a phosphate buffer containing 0.4 molar sodium chloride but elutes with a phosphate buffer containing 0.6 molar sodium chloride is collected. A method for producing the anticancer substance TV-130 according to any one of Section 8. 13 Anticancer substance belonging to the genus Veillonella TV-
130 Claim 9, 10, or 12, wherein the producing bacterium is Veillonella alkalezcens
A method for producing the anticancer substance TV-130 described in Section 1. 14 Anticancer substance belonging to the genus Veillonella TV-
The anticancer substance TV-130 according to claim 11, wherein the 130-producing bacterium is Veillonella parvula.
manufacturing method. 15 Anticancer substance belonging to the genus Veillonella TV-
An anticancer agent characterized by containing TV-130, an anticancer substance obtained by culturing 130-producing bacteria and having the following properties obtained from the culture solution. (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 110°C, and significantly decomposes at temperatures above 200°C. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3500~3150, 2970~2870, 1690~1620, 1560~
It has absorption bands near 1520, 1490-1320, 1280-1200, 1120-1000 and 980-970 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 247 to 270 nm. (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: 31% to 42%, H: 3% to 7%, N: 2%
~12%