JPS6141551B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antitumor substance and a method for producing the antitumor substance using Rhizopus oryzae M-24 strain isolated and fixed by the present inventor. BACKGROUND OF THE INVENTION BACKGROUND ART Brewed products have been produced by fermenting various grains with the action of filamentous fungi, namely Rhizopus oryzae. However, the bacteria can be treated with ammonium sulfate, magnesium sulfate, monobasic potassium phosphate, potassium carbonate, calcium carbonate, glucose,
Cultures grown in conventional media made of agar had the drawback of relatively weak enzyme activity, resulting in low saccharification power and low production of organic acids. Therefore, the present inventor conducted intensive research to improve the enzyme activity of this bacterium, cultivated the bacterium using powdered slate extract as a basic culture medium, and pure cultured the spores etc. in sterile sand. As a result of repeating this process, they were able to obtain fixed bacterial cells with strong enzymatic activity, and surprisingly, they discovered that when grains were fermented using this bacterial strain, an excellent antitumor substance was produced. , completed the invention. Therefore, the present invention provides a novel antitumor substance. Furthermore, the present invention provides a method for producing a novel antitumor substance using a new bacterial strain belonging to Rhizopus oryzae. The bacterial strain used in the present invention, fixed by the present inventor, has the following mycological properties. () Morphological properties Colonies start out white and then turn gray on potato dextrose agar medium at 30°C.
The vegetative hyphae are well developed, fast growing, and 23 μm wide.
and forms stolons. The stolons form branched rhizoids when attached to the substrate. Although sporangial stalks can grow directly from hyphae, typical stalks arise from sites where stolons attach to the substrate and form rhizoids. Usually one erect, sometimes two erect. Unbranched, initially colorless and then turning brown. Sporangia, 7.8-14 μm wide, are spherical to sub-spherical, colorless at first, and turn black when mature. It has an apophysis with a diameter of 62 to 150 μm at the lower part of the sporangium. The columnar axis is subspherical to ovoid, and the width is 34 to 110 μm.
It is. After releasing the spores, it collapses into the shape of a closed cup. Spores are olive gray, 6.6-9.3μ
It measures 4.6 to 6.3 μm in size and has a slightly angular subspherical to ovoid or round green pattern on its surface. Chlamydospores are solitary, spherical, oval, or cylindrical, formed in large numbers in vegetative and aerial hyphae. No zygospores are observed. () Physiological properties Growth range Temperature: No growth at 5℃, poor growth at 10℃. 15~40℃
Grow in. Optimum temperature 30℃. PH: 5-7 NaCl salt tolerance 1% Good growth. 3% poor. 5% did not grow. V-P test: - Fermentability of sugars D-glucose (+), D-fructose (+), D-galactose (+), sucrose (+), maltose (+), starch (+), raffinose (-) , inulin (-),
Glycerin (+) Use of sodium citrate: + Use of sodium succinate: + Use of nitrogen sources Ammonium sulfate (+), monosodium glutamate (+), peptone (+), sodium nitrate (-), sodium nitrite (-) Acid production (glucose): + For the above mycological properties, Hesseltine
(1950) and Zycha et al. (1969), it was found that this strain belongs to Rhizopus oryzae, but its enzymatic activity was superior to that of known strains, so it was identified as a new strain, and Rhizopus oryzae Went.・
Eto Prinsen-Gerlings (Rhizopus)
oryzae Went et Prinsen-Geerligs) M-24
(hereinafter referred to as "Rhizopus oryzae M-24"), and was deposited with the Institute of Microbiology, Agency of Industrial Science and Technology as FERMP-7195. The antitumor substance of the present invention is produced by fermenting grains using Rhizopus oryzae M-24 and collecting water-soluble components in the fermentation liquid. Examples of grains used as fermentation raw materials in the method of the present invention include white rice, scrap rice, split rice, barley, millet,
Examples include millet, millet, barley, corn, and corn. These grains are soaked in water to absorb sufficient water, then boiled, and then inoculated with Rhizopus oryzae M-24 for fermentation. Fermentation is preferably carried out for 3 to 5 days, increasing the temperature daily from 30°C at the beginning to around 40°C at the end. Water is added to the fermentation liquid thus obtained, and insoluble matter is removed by filtration, centrifugation, decanting, etc., and an aqueous solution is collected. An antitumor substance can be obtained by drying this aqueous solution by spray drying or the like. The antitumor substance thus obtained has the following physical properties. Elemental analysis values (approx.): C40%, H7%, N1%. Molecular weight: 5000-100000. Melting point: Does not show a clear melting point, but decomposition is observed above 220℃. Specific optical rotation: [α] _D 59.9° (10% water). Ultraviolet absorption spectrum: Maximum absorption near 257 and 263 nm (Figure 1). Infrared absorption spectrum (KBr): 1650,
It has main peaks at 1400 and 1050 cm ^-1 (second
figure). Solubility in solvents: Easily soluble in water. Insoluble in methanol, ethanol, chloroform, ether, acetone, ethyl acetate and n-hexane. Color reaction: Biuretz reaction, xanthoprotein reaction, Miron reaction, Anthrone-sulfuric acid reaction, phenol-sulfuric acid reaction, and cysteine-sulfuric acid reaction are positive. Distinction between basic, acidic and neutral: amphoteric. Material color: white. Sugar content: Sugar content in terms of glucose measured by the phenol sulfuric acid method is approximately 85-90%. Protein content: The bovine serum albumin equivalent protein content measured by the Folin-Lowry method is approximately 1
%. The results of testing the effect of the antitumor substance of the present invention thus obtained are as follows. Test Example 1 Meth-A tumor cells were intraperitoneally inoculated into 6-week-old BALB/c mice with an average weight of 20 g, and after 1 week, the proliferated tumor cells were extracted along with ascites fluid.
10 ⁵ mice were subcutaneously transplanted into the groin region of another 6-week-old BALB/c female mouse, and the powder preparation obtained in Example 2 was dissolved in distilled water for injection, and from 3 days after transplantation to 1 day after transplantation. once
10 mg/mouse was directly administered intratumorally for 7 consecutive days.
Five weeks after tumor implantation, the diameter or weight of the solid tumor was excised and compared with that of a control group in which physiological saline was administered instead of the sample. The obtained results were expressed using the following formula. Tumor inhibition rate (%) = (1 - drug administration group average tumor weight / control group average tumor weight) × 100 Complete cure = number of completely cured mice / total number of mice

[Table] Test Example 2 Mice that have been completely cured by administration of the antitumor substance of the present invention are no longer engrafted and no tumor formation is observed even when newly inoculated with tumor cells. Meth-A tumor cells obtained in the same manner as described in Test Example 1 were first used to form tumors in mice (6 weeks after inoculation) that were completely cured by administering the substance of the present invention to Meth-A tumors. Once the tumor had healed, it was inoculated subcutaneously in the groin on the opposite side to examine engraftment and tumor formation. These results are shown in Table 2.

[Table] Test Example 3 6-week-old mice (ICR strain, female) with an average weight of 22 g
Sarcoma 180 tumor cells were inoculated intraperitoneally, and after one week, the proliferated tumor cells were removed along with ascites fluid.
4 × 10 ⁶ of these cells were transplanted subcutaneously into the groin of another 6-week-old mouse (ICR female), and the material preparation obtained in Example 2 was dissolved in distilled water for injection, and 24 hours after transplantation. The drug was orally administered to the eyes at a dose of 1 g/Kg once a day for 15 consecutive days. Five weeks after tumor implantation, the diameter or weight of the solid tumor was excised and compared with that of a control group in which physiological saline was administered instead of the sample. The interim course was investigated by measuring the diameter of solid tumors. The results are shown in Table 3, and tumor growth was suppressed even when the powder preparation was administered orally at a single dose of 1 g/Kg.

[Table] Test Example 4 As a result of acute toxicity tests using mice and rats, the LD ₅₀ was 5 g/Kg or more for oral administration and 2 g/Kg or more for intraperitoneal administration. As is clear from the above test results, the antitumor substance of the present invention has low toxicity and has an excellent suppressive effect on various tumors, especially solid tumors that are considered difficult to treat. In order to administer the antitumor substance of the present invention to humans or animals, general methods can be adopted, such as oral administration, subcutaneous, intramuscular, intravenous or intratumoral injection, intrarectal administration, and external administration. , can be administered intravenously. The dosage varies depending on the type of tumor, age, symptoms, etc., but it is recommended to administer approximately 0.2 to 2000 mg/Kg/day, especially 3 to 500 mg/Kg/day divided into 1 to 6 times a day. preferable. In addition, an immune enhancer can also be used in combination with the antitumor substance of the present invention. Next, an example will be given and explained. Example 1 (i) Soak 60 g of soybeans in water for 12 hours to absorb water, grind, add 1 portion of water, and boil for 1 hour. 7 g of calcium chloride is added to this to coagulate it, insoluble protein is removed, and 1 part of water is added to the solution to obtain a solution A. On the other hand, 200 g of potatoes were soaked in 0.1% water for 1 hour, washed with water, peeled, and boiled for 1 hour with 1 portion of water added thereto. After this, water is added to the liquid to make 1 and use it as liquid B. The above solutions A and B are mixed at a ratio of 6:4 to obtain a basic culture solution. (ii) Grinding 50g of slate (less than 70-80 mesh)
Place in 500 ml of basic culture medium and incubate for 30 minutes on a boiling water bath.
Heat for a minute. The slate extract thus obtained was
Add to the basic culture solution to a concentration of ~10% and use it as a culture solution. (iii) The sand, which contains almost no organic matter, is thoroughly washed with distilled water and roasted for 30 minutes in a dry heat sterilizer at 160°C. (iv) Pour the culture solution into a Petriciale, inoculate it with filamentous fungi (Rhizopus sp.) isolated from Chinese noodles, and culture at 25-30°C for 5-10 days until the bacterial cells have sufficiently grown mycelia and spores. Select, 50 sand obtained in (iii)
g and stir thoroughly in a sterile box to mix. After putting this in a shear dish and storing it in a freezer at -4℃ to 10℃ for 5 days, the spores were separated and one platinum loop was inoculated into a test tube medium (Meyer's modified artificial medium), and the spores were kept at 25 to 30℃. It was stored in a thermostat for 5-10 days. This cold and constant temperature storage was repeated 10 times to obtain the fixed M-24 strain. Example 2 1 kg of polished rice was thoroughly washed, soaked in water for 12 hours to absorb sufficient water, and then boiled. Add 400 ml of sterilized water to this white rice, mix well, and inoculate it with the M-24 strain fixed in Example 1. At 30°C for 24 hours, at 35°C for 24 hours, and at 40°C for 24 hours. Fermentation was carried out. 1 to 1.5 parts of water was added to this fermentation liquid, heated to 45°C, and filtered after 24 hours. Spray dryer (hot air inlet temperature 250-300℃, outlet temperature 80-300℃)
170℃) for a residence time of 5 to 80 seconds,
A white powder antitumor substance was obtained.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of the antitumor substance of the present invention, and FIG. 2 shows the infrared absorption spectrum of the same substance.

Claims (1)

[Scope of Claims] 1. An antitumor substance having the following physical properties. Elemental analysis values (approx.): C40%, H7%, N1%. Molecular weight: 5000-100000. Melting point: Does not show a clear melting point, but decomposition is observed above 220℃. Specific optical rotation: [α] _D 59.9° (10% water). Ultraviolet absorption spectrum: Maximum absorption near 257 and 263 nm. Infrared absorption spectrum (KBr): 1650,
It has major peaks at 1400 and 1050 cm ^-1 . Solubility in solvents: Easily soluble in water. Insoluble in methanol, ethanol, chloroform, ether, acetone, ethyl acetate and n-hexane. Color reaction: Biuretz reaction, xanthoprotein reaction, Miron reaction, Anthrone-sulfuric acid reaction, phenol-sulfuric acid reaction, and cysteine-sulfuric acid reaction are positive. Distinction between basic, acidic and neutral: amphoteric Color of substance: white. Sugar content: Sugar content in terms of glucose measured by the phenol sulfuric acid method is approximately 85-90%. Protein content: The bovine serum albumin equivalent protein content measured by the Folin-Lowry method is approximately 1
%. 2. A method for producing an antitumor substance, which comprises fermenting grains using Rhizopus oryzae M-24 strain and collecting water-soluble components in the fermentation liquid.