JPS6043387A - Antitumor agent and its preparation - Google Patents

Abstract

PURPOSE:To obtain a novel antitumor agent, by fermenting grain by Rhizopus oryzae M-24 strain, collecting a water-soluble component in the fermented solution. CONSTITUTION:Grain such as polished rice, waste rice, etc. is fermented by Rhizopus oryzae M-24 strain (FERM-P 7195) by koji process. The koji is extracted with water to give a water-soluble substance, which is subjected to spray drying, to give an antitumor agent. This antitumor agent has the following physical properties. Elemental analysis value (about): C 40%, H 7%, and N 1%. Molecular weight: 5,000-100,000. Melting point: decomposing at >=220 deg.C. Solubility in solvent: easily soluble in water, insoluble in methnol, ethanol, chloroform, ether, acetone, ethyl acetate, and n-hexane. Color of substance: white. Saccharide content: 85-90wt% calculated as glucose measured by phenol-sulfuric acid method.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel antitumor substances as well as Rhizopus oryzae isolated and fixed by the present inventor.
oryzae) M-24 strain.

BACKGROUND ART Brewed products are produced by fermenting various grains by using filamentous fungi, namely Rhizopus oryzae. However, when this bacterium is cultured in a conventional medium consisting of ammonium sulfate, (magnesium llitate, monobasic potassium phosphate, potassium carbonate, calcium carbonate, glucose, and agar), its propagation property is relatively weak; It was lacking in saccharification power and production of organic acids.

Therefore, the present inventor tl' carried out extensive research to improve the enzyme activity of the soybean, cultivated the bacterium using powdered slate extract as a basic culture medium, and collected the spores in sterile sand. As a result of repeated pure cultivation in 1~1, a fixed strain with strong enzymatic activity was obtained, and surprisingly, when grains were fermented with this strain, it was found to be an excellent antitumor substance. The present invention has been completed based on the discovery that this can be produced.

Therefore, the present invention provides a novel antitumor substance. More importantly, the present invention provides a method for producing a novel anti-Pan cancer substance using a new bacterial strain belonging to Rhizopus oryzae.

The bacterial strain fixed by the present inventor and used in the present invention has the following autologous properties.

(1) Morphological characteristics Colonies were grown on potato dextrose agar medium at 30%
At ℃, it turns pale white and then turns gray. The vegetative hyphae are well developed, grow quickly, and form branches up to 23 μm in width. #3 When attached to the substrate, the #3 branches form a branched temporary support. Spore paste stalks can also grow directly from hyphae, but a typical stalk is called IJ! It is generated from the part where temporary mounting occurs due to adhesion to the surface. Usually one erect, sometimes two erect. Unbranched, initially colorless and then turning brown. Middle school 7.8~
14 μm 4- The sporangia are spherical to hematocyst-shaped, initially colorless, and turn black when mature. It has abophysis with a diameter of 62-150 pm at the bottom of the spore. The columnar axis is subspherical to oval, width 34-1
It is 10 μm. The ridge that releases the spores collapses into the shape of a closed cup. Spores olive-gray, 6.6-9, 3 p.
m x 4.6 to 6.3 μm, with a slightly angular subspherical to oval shape and a rounded edge pattern on the surface.

tW membranous spores are formed in large numbers in vegetative hyphae and aerial hyphae, are early-growing, and are spherical, oval, or cylindrical in shape. No zygospores are observed.

(ID Physiological properties q) Growth range No growth at temperature = 5°C. Poor at 10℃.

Grows at 15-40℃. Optimum temperature 30℃0 fl)I:
s~7 (2) NaCA salt tolerance 1%, good growth. 3% poor. 5% did not grow.

■ v-p test knee ■ Fermentable sugars D-glucose (-Fl, D-fructose), D-
i rough)-su(→, sucrose (4), maltose (
→, starch (-F), raffinose H1 inulin (→
, Glycerin (T) ■ Use of sodium citrate 20 ■ Use of sodium citrate: + ■ Use of nitrogen sources Ammonium sulfate (→, Sodium glutamate (for),
Peptone (→, sodium nitrate (c), sodium nitrite ←) (!3) Acid production (relaxation) Regarding more than 20 monological properties, ■・■・1tlns (1950) and Zy
As a result of an investigation with reference to Cha@t at (1969), the present strain was found to be Rhizopus oryzae, but its enzymatic activity was higher than that of known bacteria, so it was identified as a new strain, and Rhizopus oryzae was identified as a new strain.・Rhlzopum oryzae
Went atPrlnaen -Ge@rl1gm
) M-24 (hereinafter referred to as [Rhizopus oryzae M-24
FbnMp-7195 and was designated as FbnMp-7195 by the Institute of Microbiology, Agency of Industrial Science and Technology.
Deposited as.

The antitumor substance of the present invention is Rhizopus oryzae M-2.
It is produced by fermenting grains using 4 and collecting water-soluble components in the fermentation liquid.

Grains used as fermentation raw materials in the method of the present invention include:
Examples include white rice, rice, side grain, barley, millet, millet, millet, barley, corn, and koriyan. These grains are soaked in water to absorb enough water, then boiled.
This is inoculated with Rhizopus oryzae-M-24 and fermentation is carried out. Fermentation is preferably carried out for 3 to 5 days by raising the temperature daily so that the temperature is 30°C at the beginning and about 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.

8- Thus obtained fl-ru anti-ll! J8 tumorous substance contains the following physical properties.

■ Elemental analysis value (approx.): C4o5'6, N7%, N1%
.

■Molecular cap: 5,000-100,000゜■Melting point: Does not show a clear melting point, but decomposition is observed above 220℃1.C) Specific optical rotation: [α)D59.9° (10% water).

■ Ultraviolet t1iIm yield spectrum: 257.263 n
It has a maximum absorption near m (Fig. 1).

Ant) Infrared absorption spectrum (KBr): 1650
．． It has an overland peak at 1400.1050 crn-' (Fig. 2).

■ Solubility in solvents: Easily soluble in water. Insoluble in methanol, ethanol, chloroform, ether, acetone, ethyl acetate and n-hexane.

■ Color reactions: Pieuret reaction, xanthoprotein reaction, Millon reaction, Anthrone-Kaya.

Acid reaction, phenol-sulfuric acid reaction, and cysteine mimic acid reaction were positive.

■ Distinguish between basic, acidic, and neutral ampholytic (!9 Color of substance: white.

O-sugar content The sugar content in terms of glucose measured by the phenol sulfuric acid method is about 85-90%.

0 Protein content: The protein content in terms of bovine serum albumin measured by the Folin-Rousseau 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 Msth-All! was intraperitoneally administered to 6-week-old BALB/e mice with an average size of ff12Of. i! After one week, the LHL tumor cerebellum cells were extracted together with water.
105 of these cells were subcutaneously transplanted into the groin region of another 6-week-old BALB/CC mouse, and the powder preparation obtained in Example 2 was dissolved in distilled water for injection. Times 1 (
1%! /fish for 7 consecutive days! 1￥ Administered directly into the tumor. Five weeks after tumor implantation, the diameter of solid lung cancer or solid tumor was excised and compared with a control group in which physiological saline was administered instead of the heavy-mi sample. The obtained results were expressed using the following formula.

Total number of mice: 11 - Table 1 Test Example 2 Mice that were completely cured by administration of the antitumor substance of the present invention no longer formed engraftable tumors even if they were re-inoculated with new fcK tumor cells. can not see.

M@th-Alli tumor cells obtained in the same manner as described in Test Example 1 were administered to mice (6 weeks after inoculation) that had completely healed by administering the substance of the present invention to M@th-Alli tumor PIA tumors. First, the tumor was inoculated subcutaneously in the groin on the opposite side where the tumor had been formed and healed, and the reproductive tumor formation was examined. These results are shown in Table 2.

Table 2 Test Example 3 Sarcoma 180 lung tumor cells were intraperitoneally inoculated into 6-week-old mice (ICR strain, female) with an average body weight of 22 f, and the tumor cells grown in bales for 1 week were removed together with ascites. Cell 4
10' mice were subcutaneously transplanted into the groin of another 6-week-old mouse (ICR female), and the powder preparation obtained in Example 2 was dissolved in distilled water for injection, and from 24 hours after transplantation, once a day. times 1 f/
'p was orally administered for 15 consecutive days. Five weeks after tumor implantation, the diameter of the solid tumor or the weight of the solid tumor was compared with that of a control group in which physiological saline was administered as a substitute for 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 inhibited by oral administration of the powder preparation at a single dose of tf, , 'Ky.

Table 3 Results of acute toxicity tests using mice and rats, L
D and Q strokes were more than 7'icy when administered by mouth, and the intracavity injection force was more than 29/'-9.

As is clear from the above test results, the antitumor substance JJt of the present invention has low toxicity and can be used to treat various tumors, especially solid til! Contains excellent suppressive action against tumors.

Anti-II of the present invention! 1! To administer the reticulum to humans or animals, conventional methods can be employed, such as oral administration, subcutaneous, intramuscular, intravenous or [11! It can be administered by injection, intrarectally, externally, or intravenously.

The dosage varies depending on the type of tumor, age, symptoms, etc., but is approximately 0.2 to 2,000 Mg/Ky/IE, especially 3 to 5
o.

It is preferable to administer the drug in 1 to 6 divided doses per day, 15-q/day to 9/day. 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 (1) 60 tons of soybeans are soaked in water for 12 hours to absorb water, 1 ton of drained water is added, and the mixture is boiled for 1 hour.

Calcium chloride 7f'1 is added to this to coagulate it, insoluble protein is removed, and water 1z is added to solution F to make a mucus solution.

On the other hand, 2009 potatoes were soaked in 0.196 Shodong water for 1 hour, washed with water, peeled, and water LTF was added thereto and boiled for 1 hour. This is filtered, and water is added to the P solution to make 1 t, which is used as B solution.

Mix the above Mu and B solution in a ratio of 6:4 and make 16- Use as basic culture solution.

(11) Slate 50f is crushed (70 to 80 mesh or less), placed in basic culture solution 500, and heated on a boiling water bath for 30 minutes. The slate extract obtained in this manner is added to a used book culture solution at a concentration of 5 to 10% to obtain a culture solution.

(-) Sand containing almost no organic matter is thoroughly washed with distilled water and roasted for 30 minutes in a dry heat sterilizer at 160°C.

(1v) Put the culture solution into a Petri dish, inoculate it with filamentous fungi (Rhizopus genus) isolated from Chinese Ishikiko, and inoculate it with 25
Cultivate at ~30°C for 5 to 10 days, select those that have sufficiently proliferated mycelia and spores, place them in 50f of sand obtained in step (iii), and stir thoroughly to mix in a sterile box. This was placed in a Petri dish and stored in a freezer at -4°C to 10°C for 5 days.The spores were separated and inoculated into a test tube medium (M@y@r's modified artificial medium) with one platinum loop. 5 in a thermostat at 25-30℃
Stored for ~10 days. This cold and constant temperature storage was repeated 10 times to obtain the fixed M-24 strain.

Example 2 Polished rice I Ky was immersed in well-washed drained water for 12 hours to absorb sufficient water, and then boiled. 400 sterilized water for this white rice
- and stir well to mix, and inoculate this with the 7' (M-24 strain) fixed in Example 1.
Fermentation was carried out at 40°C for 24 hours and then at 40°C for 24 hours. 1 to 1.5 t' (f) of water was added to this fermentation liquid, heated to 45°C, and filtered after 24 hours. It dried in ~80 seconds to obtain a white powder anti-tumor substance tube.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the antitumor substance, and Figure 2 shows the infrared absorption spectrum of the same substance. Applicant Akira Shinozaki

Claims (1)

[Claims] 1. An antitumor substance having the following physical properties: (1) Elemental analysis values (approximately): C40%, H7%, NIX. ■Molecular weight: 5,000-100,000゜■Melting point: Does not show a clear melting point, but decomposition is observed above 220°C. ■ Specific optical rotation: [α) D59.9° (10% water). ■ Ultraviolet absorption spectrum: Maximum absorption near 257.26 nm. ■ Infrared absorption spectrum (KBr): 1650
. 1400. Has an upper narrow peak at 1050 on-1. ■ Solubility in solvents: Easily soluble in water. Insoluble in methanol, ethanol, chloroform, ether, acetone, ethyl acetate and n-hexane. Mon) Color reaction: Piuret reaction, xanthoprotein reaction, Myron reaction, Anthrone-sulfuric acid reaction, phenol-sulfuric acid reaction, and cysteine-sulfuric acid reaction are positive. ■ Distinction between basic, acidic, and neutral: amphoteric [phase] Color of substance: white. 0 Sugar content: The sugar content in terms of dulcose measured by the phenol-sulfuric acid method is about 85-90%. [Phase] Protein content: The protein content measured by the Farin-Lowry method is approximately 1% in terms of bovine serum albumin. 2. A method for producing an antitumor substance, which comprises fermenting grains using Rhizopus oryzae M-24 strain and collecting water-soluble components from the fermentation.