JPH03291232A - Antitumor agent and immunoactivator - Google Patents

Abstract

PURPOSE:To obtain an antitumor agent and immunoactivator having excellent antitumor activity and immunoactivity free from toxicity, comprising a polysaccharide prepared by culturing Lactobacillus sp.KPB-167 as an active ingredient. CONSTITUTION:An antitumor agent and immunoactivator having excellent antitumor activity and immunoactivity from toxicity, comprising a polysaccharide prepared by culturing Lactobacillus sp.KPB-167 (FERM P-9886), separating and collecting the culture solution thereof as an active ingredient, capable of safely administered to human because of extremely low toxicity. The polysaccharide has the following physical and chemical properties of (i) elemental analysis value: C: 39%, H: 7%, N: lower than detection value, (ii) a polymer substance having molecular weight of 1,000,000-5,000,000, (iii) color reaction: positive in Chromotrope-sulfuric acid reaction and negative in aniline hydrochloric acid reaction, naphthoresorcinol reaction, carbazole sulfuric acid reaction and Elson- Morgan reaction.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a non-toxic and safe polysaccharide with excellent anti-tumor activity and immunostimulatory activity, which contains a polysaccharide obtained by culturing Lactobacillus sp, KPB-167 as an active ingredient. Regarding anti-tumor and immunostimulant.

[Conventional technology]

Traditional fermented milk kefir from the Caucasus region of the Soviet Union is produced using a starter called kefir grain. These kefir grains have a yellowish-white elastic cauliflower-like border, and various lactic acid bacteria and yeast calyx coexist. Kefir granules are formed by conjugating lactic acid bacteria, yeast, etc. with poly111 (kefiran) produced by a type of capsular polysaccharide-producing lactic acid bacteria as a support. It has been disclosed that the polysaccharide extracted from this kefir grain has antitumor activity (Japanese Patent Application Laid-Open No. 128207/1983, Japanese Patent Publication No. 56-4
No. 09.

Special Publication No. 56-410). However, since kefir grains are composed of various microorganisms, it is difficult to culture and manage them, and the capsular polysaccharide-producing bacteria that produce the desired polysaccharide have not been isolated in a pure manner. Therefore, it is unclear what microbiological classification the bacteria belong to, and it is difficult to confirm that the bacteria is a substance with antitumor activity.
Stable production of this polysaccharide is difficult.

The present inventors previously isolated a capsular polysaccharide producing bacterium, Lactobacillus sp, KPB-167, from kefir grains, and discovered a method for producing polysaccharides produced by this microorganism, patent application No. 63-1.
The application was filed as No. 75637.

[Problem to be solved by the invention]

It is widely desired to provide a polysaccharide-like substance extracted from kefir granules that has consistently stable productivity, high quality, and activities such as antitumor activity.

The present invention provides an antitumor and immunostimulant that meets this need.

[Means to solve the problem]

In order to solve the above-mentioned problems, the present inventors cultivated Lactobacillus sp, KPB-167 (Feikoken Bacteria Serial No. 9886), which was isolated from kefir grains that the present inventors had previously invented, and cultivated it. We conducted research on the antitumor activity and immunostimulatory activity of the polysaccharide isolated and collected from the fluid, and found that this culture had extremely high antitumor and immunostimulatory activity.We also confirmed the polysaccharide that is the main component. The invention has been completed.

The present invention is directed to an antitumor and immunostimulant agent containing a polysaccharide isolated and collected from the culture solution obtained by culturing Lactobacillus sp, KPB-167 (KPB-167 (KPB-167), and the main component of the same component. It was discovered that the component is a polysaccharide with the following physical and chemical properties.

■ Elemental analysis value C: 39%, H: 7%, N: Below detection limit ■Molecular liter: Approximately 1 million to 5 million polymer substances■ Color reaction 12 Chromotrope-sulfuric acid reaction 10 il, aniline-hydrochloric acid reaction mountain, naphtho Resorcinol reaction lv, carbazole-sulfuric acid reaction V, Elson-Morgan reaction ■ Solubility in solvents Soluble in water, insoluble in methanol, ethanol, aptane, and ether.

■Basic, acidic, neutral Aqueous solutions of this substance are neutral.

■ Types and composition ratio of constituent sugars Glucose:galactose=1:1 ■Exterior appearance Tasteless and odorless white powder ■ Ultraviolet absorption spectrum Does not exhibit characteristic absorption.

■ Infrared absorption spectrum It shows characteristic absorption as shown in Figure 1.

Lactobacillus sp, K which produces the active ingredient of the present invention
PB-167 is a type of long bacillus belonging to the genus Lactobacillus that is isolated from kefir grains, and its mycological properties are as follows.

A, Bacterial morphology (+) Cell size: 1-1,5x15-60 (
μm) (2) Shape: Rod-shaped (3) Motility: None 4) Spore presence: None (5) Durham staining: Positive Distilled water 00mJ2 B, Growth status in medium Milk whey agar medium Morphology of colonies when bacteria were smeared on the surface and cultured anaerobically at 30°C for one week (gas bag method or glove box method).Medium composition. Agar pI (00mfi 1g 0.5g o, os g O,5g 0.1g mfi 1g 5.5 *Nemotomoto 10% skim milk was adjusted to pH 4.6 with lactic acid, and after boiling for 30 minutes,
Filtrate after filtering out the precipitate.

Trade name of polyoxyethylene sorbitan monooleate Composition: MgSO4・7H204g MoSO4・4H200, Is g FeSO4・7H200, 18g NaCl 0.1g Colony properties Shape 2 circles Shape Color tone: White Size: 2-4 m Transparency: Transparent ridges : Convex circular shape
Hardness: viscous periphery Edge: smooth Structure: homogeneous, dewdrop-like surface bi-smooth C9 Physiological properties ■ Growth temperature = 30℃ (optimal), 20-35℃ (growth range) ■ Growth p) 1: 5.5-6 (optimal), 5-7.5 (growth range) ■ Attitude towards oxygen: Aerobic anaerobic, grows even under aerobic conditions, but grows better in the presence of CO2.

■ Pigment production I &: None ■ Catalase: Negative ■ Optical rotation of lactic acid produced from sugar: D (L) type ■ Production of acid and gas from sugar: D (-) - Arabinose D (+) - Xylose α - L-rhamnose D-ribose glucose D-mannose D(-)-fructose D-galactose sucrose maltose cellobiose slurry - strehalose melbiose raffinose meletitose D-mannitol D-sorbitol ester Rin salicin amicdalic acid + Gas production To use this bacterium to produce the active ingredient of the present invention, for example, after culturing in milk whey medium or modified MRS medium (shown in Table 1), the supernatant fraction of the culture solution and the bacterium It can be carried out by conventional separation and collection methods from body surface fractions.

Examples of milk whey medium and modified MR5 medium compositions are shown in Table 1, but are not limited thereto. In addition, the culture conditions include a temperature of 25 to 35°C, a pl (4,5 to 6,0
It is preferable to inoculate the bacteria, replace the gas phase with CO2 gas, and then culture under 1M&gas conditions. Usually 4~
When cultured for 6 days, polysaccharide, which is the active ingredient of the present invention, accumulates in the culture solution.

(Margins below) Table 1 (Medium composition) Note: The above milk whey is made by adjusting 10% skim milk powder to pH 4.6 with hydrochloric acid, boiling it for 30 minutes, filtering it, and adding Na.
This is a filtrate obtained by adjusting the pH to 6.8 with OH and boiling and filtering for an additional 30 minutes.

Lactobacillus sp, KP obtained as above
To separate and purify the active ingredient of the present invention from the culture solution of B-167, the culture solution is heated at 10.00 rpm.

Centrifuge for IO minutes and separate into culture supernatant fraction and bacterial cell surface fraction.

The same amount of an organic solvent (methanol, ethanol, acetone, etc.), preferably cold ethanol, is added to the supernatant fraction, and after standing in ice water for 15 minutes, the precipitated polysaccharide is collected by centrifugation. Dissolve this in an appropriate amount of distilled water, add ethanol again to reprecipitate the polysaccharide, and collect. The polysaccharide is purified by repeating this operation several times.

The polysaccharide in the bacterial cell fraction was obtained by centrifuging the culture solution, heat-extracting the bacterial cells in hot water at 95°C for 30 minutes, and centrifuging again to remove the supernatant in which the polysaccharides were dissolved. obtain. Next, the same amount of an organic solvent such as cold ethanol is added, and the polysaccharide is purified by reprecipitation several times in the same manner as for purifying the polysaccharide from the supernatant liquid described above.

Lactobacillus sp, KP obtained as above
The polysaccharide in the culture solution of B-167 is separated and purified and is a pure polysaccharide.

This polysaccharide has the following physical and chemical properties.

■ Elemental analysis values C: 39%, H: 7%, N: below the detection limit ■ Molecular weight Asahipak G5-710 (manufactured by Asahi Kasei ■) column was used, distilled water was used as the developing solvent, and the flow rate was 1.
By gel filtration analysis at 0 mfi/mn, approximately 100
It was ~5 million.

■ Color reaction 1, chromotrope-sulfuric acid reaction 11 ii, aniline-hydrochloric acid reaction mountain, naphtresorcinol reaction lv, carbazole-sulfuric acid reaction V, Elson-Morgan reaction ■ Solubility in solvents Soluble in water, methanol, ethanol, acetone, Insoluble in ether.

■Basic, acidic, neutral Aqueous solutions of this substance are neutral.

■ Types and composition ratio of constituent sugars After hydrolyzing polysaccharides with 2N-trioleoacetic acid at 100°C for 3 hours, the decomposed solution was evaporated to dryness in a vacuum concentrator, dissolved in distilled water, and analyzed by liquid chromatography. I did it. The column used is 5UGARSP-+010C
[manufactured by Showa Denko], the developing solvent was distilled water, flow rate 0.7 mQ.
/min at a temperature of 80°C. As a result, it was found that it was composed only of glucose and galactose, and the composition ratio was glucose:galactose=1.1.

■Exterior appearance Tasteless and odorless white powder.

■ Ultraviolet absorption spectrum Does not exhibit characteristic absorption.

■　Infrared absorption spectrum It shows characteristic absorption as shown in Figure 1.

The physiological activities, particularly the antitumor activity and immunostimulatory activity of the polysaccharide isolated and purified by culturing the Lactobacillus sp, KPB-167 described above, are as follows.

(1) Antitumor activity A test was conducted using 8 ICR mice (male) aged 5 weeks and weighing approximately 30 g in - group. Sarcoma 180, which has been transplanted and subcultured in ICR mice (male) for 10 days in advance.
(Sarcoma [10) cells were diluted with sterile physiological saline to 1 x 10 cells/1 rill, and then transplanted subcutaneously into the right thigh of a mouse, and from the next day, the cells of the present invention dissolved in physiological saline were Active ingredient polysaccharide 0.
25- was continuously administered intraperitoneally for 7 days. Controls received only physiological saline.

After transplanting Sarcoma 180, the major and minor axes of the tumor were measured every day using calipers to determine the tumor suppression rate 300 days after tumor implantation.

The tumor inhibition rate was calculated using the following formula.

o-S Tumor suppression rate (%) = x short axis) The results are shown in Table 2.

As shown in Table 2, the polysaccharide that is the active ingredient of the present invention significantly suppresses tumors.

(2) Immunostimulatory effect Using ICR mice (male), 6 weeks old and weighing approximately 30 g, 6 mice in the - group were taken, and the polysaccharide, which is the active ingredient of the present invention, was administered to induce sheep red blood cells in the mouse pancreas. We investigated the increase or decrease in the number of antibody-producing cells. Sheep red blood cells 4x109 as antigen
Inject 0.25 mfi of Ce1ls/mfi into the mouse peritoneal cavity.
The administration port was designated as Day O. Based on Day O, the three days before the reference date, Day-3, -2, -1,
Polysaccharide solution 0.25+nfi is administered intraperitoneally to mice.

"Pre-administration group", Day+1 after the reference date. +2. +3
"Post-administration group" administered on Day-3 before the reference date
, 71 and later +1. 3 of the “before and after administration group” administered at +3
divided into groups. As a control, Day-3, -1°+1. +
3, physiological saline was administered at 0.25 mA to form a "control group". In addition, the concentration of all polysaccharide solutions is 40■/kg/
d ay.

All mice were dissected on the 6th day after administration of sheep red blood cells.
! ta was extracted, and the number of anti-sheep erythrocyte antibody-producing cells in the pancreas was measured by the Cunningham method. The results are shown in Table 3.

(Margin below) As shown in Table 3 and above, the polysaccharide which is the active ingredient of the present invention significantly increases the number of anti-sheep erythrocyte antibody producing cells per n* cell compared to the control group.

From this result, it is clear that the active ingredient of the present invention has an excellent immunostimulatory effect.

(3) Acute Toxicity Using ICR mice (male) aged 5 to 6 weeks old and weighing approximately 30 g, the polysaccharide of the present invention was administered intraperitoneally and orally once to each test group of 4 mice. Acute toxicity tests were conducted.

After administration, the mice in each test group were observed for general symptoms and mortality for 3 weeks.

As a result, the maximum concentration in the test group was 100 μ/cm after intraperitoneal administration.
No abnormalities were observed in the test group mice at the maximum concentration of 1500 μg/kg mice after oral administration. Therefore, it can be concluded that the polysaccharide which is the active ingredient of the present invention is an extremely safe substance.

The antitumor agent and immunostimulant of the present invention vary depending on the symptoms, age, etc., but in the case of an antitumor agent, the amount is 1 mg to 100 mg.
/kg/daL for immunostimulants: 1 mg −100
mg/kg/day for oral preparations such as tablets, capsules,
Parenteral preparations such as liquid preparations, such as injection preparations, are prepared using common formulation auxiliaries and conventional preparation methods.

Next, examples of the present invention will be described.

〔Example〕

Example 1 Production of active ingredients Tryptone 10 g IQ, meat extract 10 g/Q, yeast extract 5 g IQ, lactose 20 g/Q, K
2HP0.2 g/Q, 2 ammonium citrate
gIQ, Tween 801gIQ, sodium acetate 5g
/ Q, vgso4・7I (200,511g/Q
, MnSO4・4H200,28g/Modified M of Q
Put 200 mfi of R5 medium into a screw cap neck, inoculate it with Lactobacillus sp, KPB-167 strain, replace the gas phase with CO2 gas, and then statically culture at 30°C for 4 days under anaerobic conditions. , was used as the preculture solution.

Main culture is tryptone 20g/Q, meat extract 20g
/Q, yeast extract 10g/Q, lactose 100gIQ
, K2HP0.2 gIQ, ammonium citrate 2gIQ, Tween 801g/Q, sodium acetate 5gIQ, MgSO
4-7H200, 58g/Q, MnSO4・4H2
00, 28g/Q and calcium chloride 0.555
Add 30 trr (l) of preculture solution to a culture flask containing 1.5 Q of MRS medium for producing kefiran at g/Q, and after replacing the gas phase with CO2 gas, heat at 30°C, pus,
Culture was performed under 0 control. During culture, p due to the production of lactic acid.
Since H was lowered, 5N caustic soda was added dropwise to control the pH, and stirring was performed using a magnetic stirrer.

After culturing for 6 days, the polysaccharides were recovered from the culture supernatant. 6 That is, the culture solution IQ was centrifuged at 10.00 rpm for 15 minutes and separated into a culture supernatant fraction and a bacterial cell fraction. Polysaccharides can be recovered from the culture supernatant fraction by adding the same amount (IQ) of cold ethanol (9
After adding 9.5%) and standing in ice water for 15 minutes, the mixture was centrifuged at 10.00 rpm for 15 minutes to obtain a polysaccharide in the precipitate. After dissolving the obtained polysaccharide in 500rnfl of distilled water, further 500mQ of cold ethanol (99,5
%) and allowed to stand in ice water to precipitate the polysaccharide. This ethanol precipitation operation was repeated three times to purify the polysaccharide, which was then freeze-dried to obtain 1.97 g of a white polysaccharide powder.

Example 2 Tablet (1) Polysaccharide obtained in Example 1 20g lactose
100g Crystalline cellulose 30g Potato starch 30g Magnesium stearate 2g Mix the ingredients (1) to (4) and separate them in advance (4
) is added as a glue, made into granules, and dried. Add (5) to this and mix to make 1 g 200 square tablets.

Example 3 Capsule (1) Polysaccharide obtained in Example 1 20g (2)
Lactose 100g (3) Crystalline cellulose 50g (4)
Talc 10g or more (1
) to (4) were mixed and filled into 200 square capsules.

〔Effect of the invention〕

The present invention is an antitumor and immunostimulant having excellent antitumor activity and immunostimulatory effect. Furthermore, it is a useful drug with extremely low toxicity and can be safely administered to humans.

[Brief explanation of drawings]

FIG. 1 shows an infrared absorption spectrum of polysaccharide, which is an active ingredient of the present invention. Dense Speech Loose I

Claims (1)

[Claims] 1. Lactobacillus sp. An anti-tumor and immunostimulant containing a polysaccharide isolated and collected from the culture solution obtained by culturing KPB-167 (KPB-167 (Feikoken Bacteria No. 9886)) as an active ingredient. 2. Antitumor and immunostimulant whose polysaccharide has the following physical and chemical properties. (1) Elemental analysis value C: 39%, H: 7%, N: below the detection limit (2) High molecular weight substance with a molecular weight of approximately 1 million to 5 million (3) Color reaction i, chromotrope-sulfuric acid reaction + ii, Aniline-hydrochloric acid reaction - iii, naphresorcinol reaction - iv, carbazole-sulfuric acid reaction - v, Elson-Morgan reaction - (4) Solubility in solvents Soluble in water, insoluble in methanol, ethanol, acetone, and ether. (5) Basic, acidic, and neutral Aqueous solutions of this substance are neutral. (6) Types of constituent sugars and composition ratio glucose:galactose = 1:1 (7) Appearance: Tasteless and odorless white powder (8) Ultraviolet absorption spectrum characteristics: Shows no absorption. (9) Infrared absorption spectrum Shows characteristic absorption as shown in Figure 1.