JPS6241215B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing an anticancer substance by treating cells of microorganisms belonging to the genus Bacteroides, Megasphira, and Pasteurella. Conventionally, substances prepared by various methods from the cell walls of bacteria belonging to the genus Propionibacterium, Streptococcus, and Bifidobacterium have been known as substances that immunologically suppress the proliferation of cancer cells. . This type of substance aims to exert anticancer effects while compensating for the weakened immune system of patients undergoing treatment, and therefore can be expected to have a high therapeutic effect when used in combination with existing anticancer drugs. . It is said that many of these substances contain polysaccharides as a component, but their chemical structures are largely unknown, and currently nothing that can be put to practical use has been obtained. It is hoped that a substance with the following properties will appear. As a result of screening various bacteria for their anticancer properties, the present inventors found that substances with high anticancer effects were present in the cell wall fractions of strains belonging to the genera Bacteroides, Megaspherella, and Pasteurella. The present invention involves the production of an anticancer substance by treating the cell walls of microorganisms belonging to the genus Bacteroides, Megasphira, and Pasteurella with a microbial cell lytic enzyme, and then dialysis in water and freeze-drying the resulting dialysis fluid. It is the law. These bacteria can be cultured using commonly used methods, such as using meat extract, peptone, or nitrogen as a nitrogen source.
A medium containing yeast extract, corn staple liquor, etc., glucose, sucrose, maltose, lactose, etc. as a carbon source, and further supplemented with amino acids, inorganic components, etc. is used. Cultivation conditions are appropriate to the characteristics of each bacterium, with aerobic conditions being best for Pasteurella and anaerobic conditions being best for Bacteroides and Megasphere. The culture temperature is
Perform at around 37℃ for 24 to 72 hours. After stopping the culture, the bacterial cells are collected by centrifugation and washed with physiological saline to thoroughly remove adhering culture components. Next, heat sterilize. For heat treatment, suspend bacteria in saline solution and heat at 60 to 75℃ for 15 to 30 minutes.
If heated for 30 minutes, it can be sterilized while preserving its anticancer activity. Although the thus obtained killed bacteria already have anticancer activity, the present inventors conducted research to obtain even higher anticancer activity. It was found that the obtained method had an excellent effect. When carrying out cell wall lytic enzyme treatment, freeze-drying the killed cells as a pretreatment and degreasing them with an organic solvent such as chloroform-methanol is advantageous for the next enzyme treatment, and further reduces toxicity and anticancer activity. often gives more favorable results. For example, the cell wall lytic enzyme treatment removes the dead bacteria described above.
Uniformly suspend in 1/50 molar phosphate buffer with pH 6.5, and add enzyme crystals to this at a ratio of 0.1 to 0.1 to the dry weight of the bacterial cells.
Add 3.0% and react at 35-38°C for 1-5 hours. Dialysis is performed using a cellophane membrane, molecular sieve membrane, collodion membrane, etc. to remove low molecular weight substances such as inorganic ions. The obtained dialyzed fluid is dehydrated and dried by a method such as freeze-drying to obtain a powdery solid substance having anticancer activity, which is the object of the present invention. Examples will be shown below, but the present invention is not limited to the following examples. Example 1 Peptone 1%, yeast extract 0.5%, meat extract 0.3
%, soluble starch 0.05%, glucose 0.15%,
Pacteroides fragilis SS vulgatus in medium 5 consisting of 0.02% L-cysteine, 0.5% L-cysteine and 0.4% disodium phosphate.
ATCC−8482 (Bacteroides fragils SS.
vulgatus) 50c.c. ( ¹⁰⁹ pieces/cc) and simultaneously inoculated with 0.34% L-ascorbic acid and 0.2% L-cysteine.
% mixed solution is added and incubated for 72 hours at 37°C. The resulting culture solution was centrifuged at 15,000 G for 50 minutes to collect bacterial cells, which were washed three times with physiological saline to obtain 11 g of wet bacterial cells. The wet bacterial cells were suspended in physiological saline and incubated at 60°C for 30
Heat treatment was performed for a minute. Next, this product was freeze-dried to obtain 2.3 g of freeze-dried bacterial cells in the form of white powder. 1 g of this powder was placed in 20 ml of a chloroform-methanol (3:1) mixed solvent, stirred for 5 hours while cooling with ice water, and the solution was filtered. Repeat this operation three times, remove the liquid under reduced pressure, and remove the defatted bacterial cells.
Obtain 0.9g. The cells were suspended in 50 ml of 1/50 molar phosphate buffer at pH 6.5, and 0.018 g of lysozyme (manufactured by P-L Biochemicals Inc.) was added.
React for 4 hours at 37°C. After the reaction, transfer the reaction solution to a cellophane bag (cellulose tube for dialysis 20/3
2.Put it in a cup (manufactured by Union Carbide Corp.) and rinse it with running water for 24 hours.
Time dialysis. The contents are then freeze-dried to obtain 0.5 g of a white powdery anticancer substance. Example 2 Megafiller was added to the medium 5 used in Example 1.
Elsday ATCC−25940
(Megasphaeraelsdenii) and statically cultured at 37°C for 72 hours. The obtained culture solution was centrifuged and washed according to the method of Example 1 to obtain 10 wet bacterial cells.
g, followed by heat treatment and freeze drying,
2.2 g of freeze-dried bacterial cells in the form of black powder were obtained. Further, 1 g of this powder was subjected to defatting, enzyme treatment, dialysis, centrifugation, and freeze-drying according to the method of Example 1 to obtain 0.5 g of a grayish-white powdered anticancer substance. Example 3 Heart infusion broth medium (Eiken Co., Ltd.)
Pasteurella multocida NCTC-5
3195 (Pasteurella multcida) 50 c.c. (10 ⁹ pieces/cc)
inoculated and incubated statically at 37°C for 48 hours. The obtained culture solution was centrifuged and washed according to the method of Example 1 to obtain 5 g of wet bacterial cells. The wet cells were heat-treated and freeze-dried according to the method of Example 1 to produce freeze-dried cells in the form of white powder.
1.1g was obtained. Furthermore, 0.5 g of this powder is defatted to obtain 0.4 g of defatted bacterial cells. The defatted bacterial cells were allowed to exist in the buffer solution of Example 1, and 0.008 g of lysozyme was added and reacted, followed by dialysis, centrifugation, and freeze-drying to obtain 0.2 g of the anticancer substance in the form of a white powder. Ta. The substances obtained in Examples 1, 2 and 3 are all positive (+) in the Morritsch reaction and Fehring reaction and negative (-) in the ninhydrin reaction. They also show no solubility in methanol, ethanol, chloroform or water. These materials, when subjected to elemental analysis, exhibit the following carbon, hydrogen and nitrogen contents:

[Table] Genus)
Example 3 (Pasteurella genus) 45.30 6.31 12.66
Moreover, these have infrared absorption spectra as shown in FIG. 1, FIG. 2, and FIG. 3, respectively. In view of these characteristic absorptions in the infrared spectrum, it is considered that the products obtained by the method of the present invention are probably composites having a basically common structure. Next, the physiological activity of the anticancer substance obtained according to the present invention is shown below. A fixed amount of the substance obtained according to the method of Examples 1 to 3 was suspended in physiological saline to prepare a sample. As a control, the freeze-dried bacterial cells in each Example were suspended in physiological saline and used as a sample. Groups of 10 ICR mice were inoculated intraperitoneally with 10 ⁶ Ehritz tumor cells.
At the same time, the sample prepared by the above method is intraperitoneally administered to mice, and this administration is then repeated every 3 days for a total of 6 times. The doses of the anticarcinogenic substance and freeze-dried bacterial cells per mouse were in accordance with the doses in the table. The mortality status of the mice treated as described above was observed for 50 days after tumor cell inoculation, and the survival rate on the 50th day was expressed as %.

[Table] Next, the acute toxicity of the anticancer substance of the present invention in mice is as follows.

[Table] As is clear from the above sample results, the anticancer substance obtained by the method of the present invention has an excellent anticancer effect, and it shows that the anticancer substance obtained by the method of the present invention has an excellent anticancer effect, and the blood division and leukocyte reduction that are often observed when such substances are administered. No side effects such as weight loss, loss of appetite, or loss of appetite were observed. The anticancer substance according to the method of the present invention is prepared by dispersing it in sterile physiological saline.
It is particularly useful in preventing cancer in domestic animals when administered intravenously or locally in doses of 5 to 40 mg/Kg per day.

[Brief explanation of the drawing]

In the accompanying drawings, Figures 1, 2 and 3
The figures show infrared absorption spectra of anticancer substances obtained by the method of the present invention from the bacterial cell walls of microorganisms of the genus Bacteroides, genus Megasphirella, and genus Pasteurella, respectively.

Claims (1)

[Claims] 1. After treating the bacterial cell walls of microorganisms belonging to the genus Bacteroides, Megasphaera, and Pasteurella with a microbial cell wall lytic enzyme, the dialyzed fluid obtained by dialysis in water was used. A method for producing an anticancer substance, characterized by dehydration and drying.