JPH0238198B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to Acremonium spp.
The present invention relates to a method for producing polysaccharides exhibiting antitumor activity by culturing polysaccharide-producing bacteria belonging to . In recent years, many methods for producing antitumor substances containing polysaccharides as active ingredients by culturing basidiomycetes have been reported, and some of them are being implemented industrially. On the other hand, it has been reported that some fungi Deuteromycetes produce polysaccharides outside their cells when cultured in a liquid medium, but no fungi that exhibit effective antitumor activity are yet known. Not yet. As a result of investigating Deuteromycetes in soil that have the ability to produce polysaccharides that exhibit effective antitumor activity, the present inventor discovered that Acremonium sp. The present invention was based on the knowledge that a polysaccharide exhibiting effective antitumor activity can be produced by culturing a strain of B. sp . Incidentally, the term "polysaccharide" used in the present invention means a heteropolysaccharide in which different sugars and substances other than sugars are bound. The present invention will be explained in detail below. The present invention involves culturing mycelia or conidia obtained by culturing polysaccharide-producing bacteria belonging to the genus Acremonium in an agar plate medium in a liquid medium, and collecting polysaccharides exhibiting antitumor activity from the culture. It is characterized by The polysaccharide-producing bacterium belonging to the genus Acremonium used in the present invention was isolated from Shiyang (woodland soil, Chichibu City, Saitama Prefecture), and belongs to the Deuteromycotina subphylum (Deuteromycotina). Hypho
Acremonium sp. ( acremonium sp. ) belongs to the Acremonium genus (Acremonium), and is designated as SBT7016 and is designated by the Agency of Industrial Science and Technology Microbial Technology with the number FERMP-6601. It is entrusted to a research institute. The identification of Acremonium sp. SBT7016 is as follows:
W. Gams, “Cephalosporium―artige Shim―
melpilze” p262, G.Fischer: “Stuttgart” 18
(1980) and Seiji Tokumasu: “Bacterial Prevention” vol.8, No..
2, 18 (1980). Below is Acremonium sp . The mycological properties of SBT7016 are shown. (1) Growth Growth on malt agar medium is slow, colonies are fluffy and yellow-brown, turning dark brown due to conidia formation, and the underside and agar become dark brown. Produces brown water droplets on the surface. The growth on potato dextrose agar medium is rapid, fluffy and white, and the color remains white due to conidia formation, and the underside remains white. (2) Morphology Microscopic findings on slide culture on potato dextrose agar medium show that the hyphae are colorless, and the conidiophores grow upright from the side of the aerial hyphae, are colorless, and do not have septa. Conidiophores are mostly unbranched, but contain only a few branches. The phialides are elongated and tapered. The conidia are phialoid, colorless, oval, and unicellular, forming slime balls and clustering at the tip of the conidiophore. (3) Sugar utilization Glucose, mannose, galactose, fructose, sucrose, maltose, lactose, and starch are used, but xylose is not used. In the present invention, Acremonium sp. SBT7016 is first cultured on an agar plate medium to obtain its mycelia or conidia, or a mixture thereof, grown on the surface of the medium. This incubation is usually carried out at 20-30°C, preferably 25-27°C for 10
Do this for ~14 days. In addition, the agar plate medium used here is 0.1 to 0.3% by weight of the potato dextrose agar medium normally used for culturing filamentous fungi, yeast, etc.
Yeast extract is added to adjust the pH to 4-7. Next, in the present invention, the white mycelium or conidia (spores) obtained by culturing as described above, or a mixture thereof, is used as a seed mother, and this is cultured in a liquid medium. When culturing in this liquid medium, static culture or aerated culture should be performed at the initial stage of culture (generally 3 to 6 days), followed by shaking culture or aerated agitation culture for about 4 to 6 days. is preferred. The reason for adopting this culture method is that when the mycelium as a seed mother is inoculated into a liquid culture and immediately subjected to shaking culture or aerated agitation culture without static culture, the mycelium grows vigorously. On the other hand, this is due to a decrease in the production of the target substance, an antitumor active substance whose main component is polysaccharide. The culture temperature in the liquid medium was 20 to 30℃ throughout the two stages mentioned above.
Preferably it is 25-27°C. The medium used for the above-mentioned liquid culture may be a known liquid medium that is generally applied to the culture of microorganisms, for example, it may be a medium that uses glucose as a sugar source and contains peptone and yeast extract. Furthermore, a medium to which inorganic salts, amino acids, vitamins, milk components, etc. are added may also be used. Further, the pH of the liquid medium is suitably in the range of 4 to 7, and it is particularly preferable to use a liquid medium with a pH of 5.5 before sterilization. Although culture in a liquid medium can be carried out successively, as the number of passages increases, the productivity of polysaccharides gradually decreases. Although it should be carried out within 5 times, from the viewpoint of polysaccharide productivity, it is advantageous to perform a new liquid culture using mycelium grown on an agar plate medium as a seed mother. However, as the culture scale increases (e.g. 10
When performing the above liquid culture) Since it is difficult to use the mycelia of the above agar plate culture as a seed mother, it is recommended to use a culture obtained by static culture in a liquid medium as a fungal culture as a seed mother. good.
Alternatively, a first-generation shaken or aerated culture obtained from an agar plate culture may be diluted 3 to 5 times with sterile physiological saline, homogenized, and used as a seed mother. Acremonium sp. SBT7016 used in the present invention
When mycelia (or conidia) are cultured in liquid as described above, a highly viscous substance is produced and the culture itself becomes highly viscous. Extraction techniques can be applied to collect the desired polysaccharide with antitumor activity from this highly viscous culture, but the extraction efficiency is not good if the culture is used as it is because of its high viscosity. Dilute with 3 times as much distilled water, mix thoroughly with a mixer, and apply techniques such as centrifugation and filtration to this mixture to obtain an aqueous fraction. Alternatively, the residue (mycelium) obtained by the above fractionation may be washed two to three times with distilled water, and after adding water again, the mycelium may be crushed with, for example, a Waring blender, and the aqueous solution fraction obtained by centrifugation may be used. Furthermore, this fraction may be combined with the above aqueous solution fraction. Furthermore, it is also possible to use the mycelial suspension as it is. Next, in order to remove impurities such as free proteins, free nucleic acids, and reducing sugars in these aqueous solution fractions,
The fractions are treated with activated carbon, weakly basic ion exchange resin or chromatography. These treatments also decolorize the fraction and remove low-molecular substances. Furthermore, treatments such as salting out with ammonium sulfate, precipitation using lower alcohols or acetone, molecular sieves, and ultrafiltration may be applied alone or in combination. method and enzyme method can also be applied. The polysaccharide collected from the culture as described above is subjected to dialysis against distilled water, concentrated if necessary, and then freeze-dried to obtain a product. Next, the physical properties of the polysaccharide thus obtained (hereinafter referred to as the present substance) will be explained. Appearance: It is white in color and decomposes and carbonizes when heated. Melting point: This substance does not show a clear melting point. Infrared absorption spectrum: The results were measured using the KBr tablet method, as shown in the attached figure. Solubility: Soluble in water and dimethyl sulfoxide, but insoluble in general organic solvents and organic acids. Note that an aqueous solution of this substance does not pass through a semipermeable membrane, the pH of a 2-10% aqueous solution is 6.2-6.4, and no ultraviolet absorption is observed in a 0.1% aqueous solution. Color reaction: Moritsch reaction, Anthrone reaction, and ninhydrin reaction are positive, and Elson-Morgan reaction, Bahaal reaction, and iodostarch reaction are negative. The origin of this substance does not shift even when thin layer chromatography is performed under various conditions. 2~
Hydrolyzed with 4N sulfuric acid, neutralized, and then trimethylsilylated, analyzed by gas chromatography, and glucose and small amounts of mannose and galactose were detected. In addition, this substance
A sample hydrolyzed with 4N hydrochloric acid for 14 hours was analyzed using an automatic amino acid analyzer, and galactosamine and a small amount of glucosamine were detected. Furthermore, this substance was made into a suspension of 50 mg/50 ml in water, treated with ultrasonic waves to sufficiently solubilize it, centrifuged, and the resulting supernatant was subjected to gel filtration or ion exchange system. As a result of analysis using column chromatography or a fractionation method that takes advantage of differences in solubility by adjusting the pH of the solution, it was found that neutral sugars whose main constituent is glucose and amino sugars whose main constituent is galactosamine were separated. It was composed of fusions and proteins. The content ratios of these components vary depending on the culture conditions for obtaining this substance and the extraction conditions of the culture, but are generally as follows. Neutral sugars...50-60% by weight (as total hexoses by the phenol-sulfuric acid method) Amino sugars...25-35% by weight (as hexosamines by the indole-hydrochloric acid method) Proteins...5-15% by weight (as proteins by the low-fluorin method) Next, the pharmacological properties of the polysaccharide obtained by the present invention will be explained. (1) Acute toxicity Mice are ICR-JCL strain, 4-5 weeks old, weight 20~
This substance was administered orally and intraperitoneally to 25 rats of the Wistar strain, 4 to 5 weeks old, and weighing 110 to 140 g in each test group, and the acute toxicity test was conducted as follows. This was done by After administration of this substance, each test animal was observed for general symptoms, body weight changes, and deaths for one week, and then sacrificed and autopsied. The results are shown in the table below, and it can be seen that the LD ₅₀ value is extremely high.

[Table] (2) Antitumor activity The antitumor activity of this substance was tested using an in vivo test method in mice inoculated subcutaneously with Sarcoma 180 solid tumors and in mice intraperitoneally implanted with Ehrlish tumor cells. (b) Antitumor activity against Sarcoma180 solid tumor: Test animals were ICR-JCL, 6-week-old female mice (body weight
Sarcoma180 tumor cells were cultured intraperitoneally in mice in the ascites form and were passaged every week. For the test, cells from ascites were removed one week after inoculation, and 0.1 ml of physiological saline containing approximately 4 million cells was subcutaneously transplanted into the lower right flank of test mice. 24 hours after transplantation, dissolve this substance in physiological saline to a concentration of 50 mg/10 ml.
0.25 ml of the solution sterilized at 120°C for 15 minutes was intraperitoneally administered to mice, and the administration was continued for 10 consecutive days. Control mice received 0.25 ml of sterile physiological saline in the same manner. Thirty days after tumor implantation, the mice were dissected, and the grown solid tumors were excised and weighed for comparison with a control group. Each group consisted of 10 mice. As a result, this substance exhibited strong antitumor activity with a tumor suppression rate of 87.3%, and the tumors in 6 out of 10 animals in the treatment group completely disappeared. Here, the tumor suppression rate for Sarcoma180 solid tumors is a value calculated using the following formula. Tumor inhibition rate = _Cs - _Ts / _Cs × 100 _Cs : Average tumor weight of the control group _Ts : Test group The effective dose of this substance against Sarcoma180 solid tumors is 5 to 15 mg/Kg per day. (body weight) and can be formulated and applied by conventional methods. (b) Antitumor activity against Ehrlish ascites tumor: Test animals were ICR-JCL, 8-week-old female mice (body weight
25g ± 3g), 1 x 10 ⁶ Ehrlish tumor cells were transplanted into each of 4 groups of 11 mice, and 24 hours later, this substance was administered to each of 3 groups other than the control group.
25 mg/Kg (body weight)/day, 10 mg/Kg (body weight)/day, and 2.5 mg/Kg (body weight)/day were administered intraperitoneally for 10 days to observe the survival effect and healing effect in each group. In addition, only physiological saline was administered to the control group. The results were as follows.

[Table] The substances obtained by the present invention are
The same antitumor activity as above was also shown against Sarcoma180 ascites tumor. EXAMPLES The present invention will be explained in more detail with reference to Examples below. Example 1 Medium composition: Polypeptone 10g Yeast extract 5.0g Glucose 30g Water 1 A liquid medium consisting of a ratio of PH = 5.5 was dispensed into 50 500ml Erlenmeyer flasks of 200ml each, and after attaching cotton plugs, 120ml
Acremonium sp. SBT7016, which had been sterilized at ℃ for 15 minutes and separately cultured on a potato dextrose agar medium supplemented with 0.3% yeast extract, was inoculated into the above liquid medium, and statically cultured at 23 to 27℃ for 3 days. Summer. This was continued for 5 days at 23-27℃.
Rotary shaking culture was performed at 180 rpm to obtain highly viscous culture 10. To this was added 10% hot water, and after stirring and mixing with a mixer, the mixture was centrifuged at 10,000 g for 20 minutes to remove mycelium, and a viscous transparent liquid was obtained. In this solution, a mixture of activated carbon and celite (1:1), which had been treated with concentrated hydrochloric acid, thoroughly washed with water, then washed with ethanol and acetone, and dried, was added in a weight ratio of 4.
% and stirred at room temperature for 16 hours. The activated carbon-celite mixture was removed by filtration, and the resulting solution was dialyzed against distilled water at 5° C. for 48 hours, and then freeze-dried to obtain 27.5 g of an off-white powder. The substance obtained in this way is a polysaccharide consisting of polymers of neutral sugars whose main component is glucose and amino sugars whose main component is galactosamine, and proteins.As a result of testing for its antitumor effect, ICR-
JCL, 10mg/intraperitoneal dose in 6 week old mice
The inhibition rate against Sarcoma180 solid tumor in Kg is
It was 87.3%. Example 2 Medium composition: Polypeptone 5g Yeast extract 3g Glucose 30g Monopotassium phosphate 0.5g Dipotassium phosphate 0.5g Magnesium chloride 0.3g Manganese chloride 0.05g Water 1 A liquid medium consisting of a ratio of PH = 5.5 was prepared in 500ml triangular 100ml portions. Dispense into flasks, attach cotton stoppers, and incubate at 120℃ for 15 minutes.
Acremonium sp. SBT7016, which had been separately cultured on an agar slant medium, was inoculated into the sterilized medium by a conventional method, and the mixture was left to stand at 25°C for 4 days. On the other hand, 20
Add a liquid medium with the above composition to a jar fermenter.
10, sterilized and cooled at 120℃ for 30 minutes, homogenized the above culture with a Waring blender, transplanted it, and cultured it statically for 2 days at 25℃, followed by an aeration rate of 0.4vvm. Aerated agitation culture was carried out for 5 days at a stirring speed of 250 rpm. The obtained culture was diluted twice, and the mycelium was separated and removed using a cloth. The transparent viscous liquid thus obtained has a volume ratio of 10
Add 2% Amberlite XAD-2 at room temperature.
Mix and stir for hours. After separating the Amberlite resin, an equal amount of ethanol was added to the supernatant, and the resulting white precipitate was vacuum-dried, resulting in a dark gray powder.
24.5g was obtained. This powder is a polysaccharide consisting of various polymers of neutral sugars whose main component is glucose and amino sugars whose main component is galactosamine, and proteins.As a result of testing for its antitumor activity, it was found to be ICR-JCL in 6-week-old mice. With an intraperitoneal dose of 10 mg/Kg in
The inhibition rate against Sarecoma 180 solid tumor was 83.5%.

[Brief explanation of drawings]

The attached diagram illustrates an infrared absorption spectrum measured by the KBr tablet method of an antitumor substance composed of a polysaccharide obtained according to the present invention.

Claims (1)

[Scope of Claims] 1. Mycelia or conidia obtained by culturing polysaccharide-producing bacteria belonging to the genus Acremonium in an agar plate medium, or a mixture thereof, in a liquid medium, and A method for producing an antitumor polysaccharide, which comprises collecting a polysaccharide exhibiting antitumor activity. 2.Culture in a liquid medium is carried out by standing or aerating culture at 20 to 30°C for 3 to 5 days, and then culturing with shaking or aeration with stirring at 25 to 27°C for 4 to 6 days. The manufacturing method described in paragraph 1.