JPH0696601B2 - Novel polysaccharide substance LK-100, production method thereof and antitumor agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Use) The present invention relates to a novel polysaccharide substance LK-100 and an antitumor agent containing the same as an active ingredient.

(Background of the Invention and Prior Art) Conventionally, many physiological activities of polysaccharide substances have been studied mainly on mushroom substances, but recently, several antitumor actions have been reported for polysaccharide substances produced by lactic acid bacteria. (Japanese Patent Publication (A) Sho 58-204001, Agric. Biol. Che
m.47 (7) 1623-1625.1983). The inventor discovered that a certain type of lactobacillus produced a polysaccharide substance and succeeded in isolating it while conducting research on the dietary effect of fermented milk / lactic acid bacterium beverage. It was confirmed that the present polysaccharide substance LK-100 is a novel polysaccharide substance that has not been heretofore known, and when its physiological action was investigated, it was found to have remarkable antitumor activity, and the present invention was completed. Is.

(Object of the Invention) An object of the present invention is to separate and collect a novel polysaccharide substance LK-100 from a culture of a microorganism belonging to the genus Lactobacillus.

Another object of the present invention is to provide an antitumor agent containing the novel polysaccharide substance LK-100 as an active ingredient.

(Structure of the Invention) <Microorganism> The microorganism used in the present invention is a novel polysaccharide substance LK-10.
It has 0 productivity and is a bacterium belonging to the genus Lactobacillus. As an example, Lactobacillus sp. Glico1569
Has the above-mentioned characteristics, and can advantageously produce the polysaccharide substance LK-100 of the present invention, and can be effectively used in the present invention. The Lactobacillus species Glico 15
69 (hereinafter referred to as "1569 strain") has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, and the deposit number is Micromachine Research Institute of Microbiology No. 8887 (FERM P-8887).

Strain 1569 has the following mycological properties. In addition, the testing and classification of mycological properties are carried out by the Berjays Manual of Determinative Bacteriology 8th Edition (Berg
ey's Manual of Determinative Bacteriology 8th E
d,) and Mitsuoka's method (clinical examination, 18,1163,1974).

(1) Morphological properties On an MRS agar plate under carbon dioxide substitution under anaerobic conditions,
Form wet colonies. Gram stain positive rod-shaped bacteria,
No motility or ability to sporulate.

(2) Physiological properties Catalase production-Acid production from milk-Lactic acid DL Growth at 15 ° C + Growth at 45 ° C-Ammonia production from arginine + Gas production from glucose + Gas production from gluconic acid + Sugar of assimilating amygdalin - arabinose + Hirobiosu - esculin - fructose + galactose + glucose + gluconate + lactose + maltose + mannitol - mannose - melezitose - melibiose + raffinose - rhamnose - ribose + salicin - sorbitol - sucrose - trehalose - xylose - When the mycological properties of the above microorganisms were searched according to the classification method of the above-mentioned literature, it was a lactobacillus belonging to the genus Lactobacillus.

<Culture condition> The polysaccharide substance LK-100 can be separated and collected from the liquid fraction and the bacterial cell fraction of the culture after culturing the LK-100-producing bacterium. The culture medium may be any medium as long as it is a medium for lactic acid bacteria, but a medium made of a dairy raw material containing skim milk, whey and the like is preferable. As culturing conditions, anaerobic culturing under carbon dioxide substitution is advantageous, but aerobic static culturing is also sufficient. The culture temperature is preferably 15 to 35 ° C. The pH of the medium is preferably weak acidity of 6.0 to 6.5. The culturing period varies depending on various conditions such as aerobic culturing or anaerobic culturing, the composition of the medium and the culturing temperature, but generally it may be about 3 to 5 days, and after the culturing, the desired polysaccharide substance LK-100 is It accumulates in the culture.

<Separation and Purification> The LK-100 produced and accumulated by the culture is separated and purified in the next treatment step.

From the culture of the bacteria producing the polysaccharide substance LK-100, the liquid fraction and the bacterial cell fraction can be obtained by centrifugation.

An organic solvent such as ethyl alcohol or acetone is added to the liquid section to obtain a precipitate. On the other hand, the cell division is subjected to hot water extraction, and then centrifuged to obtain an extraction supernatant, and an organic solvent is added to this supernatant to obtain a precipitate. The precipitate thus obtained from the liquid fraction and the bacterial cell fraction is redissolved in hot water, and an organic solvent such as ethyl alcohol or acetone is added to reprecipitate the same.

The reprecipitated precipitation substance is dissolved in ion-exchanged water (or distilled water may be used), and then the column is filled with an anion-exchange resin and loaded. Further, ion-exchanged water is lengthened to the column, the passing-through section which is not adsorbed by the anion-exchange resin is collected, and this is permeable to ion-exchanged water using a folding membrane (manufactured by Union Carbide). The desired purified polysaccharide substance is obtained by freeze-drying the inside solution of dialysis under reduced pressure.
LK-100.

The polysaccharide substance LK-100 thus obtained is a novel substance having the following physicochemical properties.

<Physicochemical properties of LK-100> Elemental breakdown value C: 38.46% H: 6.35% N: 0.04% O: 55.15% Molecular weight was measured by gel filtration using Sepharose 2B (Pharmacia Hane Chemicals). Column size 1.5
× 95 cm: Flow rate 0.3 ml / min: Fraction 1.5 ml: Load sample 3 mg: This substance is fractionated into the gel particle external volume (Void Volume) under the condition of 0.1 mol sodium chloride solution as a developing agent.

Specific irradiance [α ▲] ²⁵ _D ▼ = + 72.5 ° (C = 0.5%) Melting point This substance begins to change color from around 230 ℃, begins to dissolve at around 260 ℃, and decomposes to blackish brown at around 280 ℃.

 Red external absorption spectrum (KBr method) As shown in Fig. 1.

Solubility in solvent Soluble in water, insoluble in methanol, ethanol, acetone, ether.

 Color reaction i. Maurish reaction + ii. Anthrone reaction + iii. Cysteine-sulfuric acid reaction-iv. Aniline-hydrochloric acid reaction-v. Carbazole-sulfuric acid reaction- vi. Elson-Morgan reaction- vii. Ninhydrin reaction- basic, The pH of a 0.5% aqueous solution of the acidic or neutral substance is around 5.0.

 Material color The lyophilizate of this material is a white fibrous material.

Types and composition ratios of constituent sugars To 3 mg of sample, 1 ml of 0.5N sulfuric acid was added and hydrolyzed at 100 ° C for 14 hours, neutralized with barium carbonate and filtered, and the sugar contained in the filtrate was converted to alditol acetate. After changing, it was analyzed by gas chromatography.

Conditions for gas chromatography Column: 3% Silar 10C on Uniport B (100-200 mesh) Glass column (0.3 × 200 cm) Column temperature: 200 ℃ → 220 ℃ (2 ℃ / min) Carrier gas: Nitrogen, 50ml / min Results Glucose and galactose were detected, and the ratio was glucose: galactose = 1: 0.8.

The formulation of the anticancer agent of the present invention includes, for example, injections,
Examples thereof include drops, tablets, capsules, granules, powders and suppositories. The preparation of these preparations can be performed according to a usual method. In addition, supplements usually used in the technical field, for example, stabilizers may be appropriately blended or coated.

Examples of the administration method of the anticancer agent of the present invention include intravenous injection, subcutaneous injection, oral administration and rectal administration. Although the dose of the drug of the present invention varies depending on the age, body weight, and degree of disease of the patient, it is usually preferable to administer 0.1 to 15 g of LK-100 per day to an adult. These doses are administered at once or divided into appropriate times. In addition, LK-100 may be added to various foods, especially fermented milk, lactic acid bacteria beverages, medical foods or pharmaceuticals.

Hereinafter, the present invention will be described in more detail with reference to Examples, Formulation Examples and Test Examples.

Example <Example of separation and purification of LK-100> Whey medium Whey 100 ml (composition) Peptone 1.0 g Meat extract 1.0 g Yeast extract 0.5 g Tween 80 0.1 ml Dipotassium phosphate 0.2 g Sodium acetate 0.5 g Ammonium citrate 0.2 g Sulfuric acid Magnesium 0.02g Manganese sulphate 0.005g pH 6.4g Whey medium (composition) 10 with polysaccharide LK-100 producing bacteria 156
Nine strains [Microtechnology Research Institute No. 8887, (FERM P-8887)] were inoculated and statically cultivated at 32 ° C for 4 days under carbon dioxide. After the culture was completed, the pH was adjusted to 6.0 and the temperature was adjusted to 90 ° C. And heat-treated for 30 minutes. Polysaccharide substance LK-10 attached to bacterial cells by this heat treatment
It was possible to extract 0 into the culture compartment.

After the heat treatment, centrifugation (10,000 rpm, 15 minutes) gave 9580 ml of culture supernatant. The supernatant was desalted and concentrated with an ultrafiltration membrane having a molecular weight of 1,000,000 (manufactured by Millipore) to obtain 300 ml of a concentrated liquid. Use ethyl alcohol as the final concentration in this concentrate.
It was added so as to be 45% (v / v). By this operation, a precipitated substance became visible, and this precipitated substance was collected by centrifugation (10,000 rpm, 5 minutes) to obtain 1.18 g of the precipitated substance.

The precipitated substance was dissolved in ion-exchanged water by heating, and the insoluble substance was removed by centrifugation (10,000 rpm, 15 minutes). By repeating this operation three times, 1.12 g of crude purified polysaccharide substance was obtained. The crude product obtained here was heated and dissolved in ion-exchanged water, and the solution was poured into a column filled with Cl--type loaded diethylaminoethyl sephadex (DEAE-Sephadex). Ion-exchanged water was flowed to collect the flow-through fraction containing non-adsorbent substances in DEAE-Sephadex.

Concentrate this and use a foldable membrane to deionize water.
It was transposed for 3 days at ℃. After the folding, the inner solution of the folding was freeze-dried under reduced pressure to obtain 0.52 g of a freeze-dried preparation of the white fibrous polysaccharide substance LK-100. The yield was 52 mg / 1,000 ml of culture solution.

Formulation Example 1 (Injection / Drip) Add 5 g of powdered glucose to contain 500 mg of LK-100, aseptically distribute to a vial, seal, seal with inert gas such as nitrogen and helium, and store in a cool dark place. did. Before use, 100 ml of sterile physiological saline was added to give an intravenous injection.

Formulation Example 2 (tablets) [A] LK-100 25 g Lactose 300 g Hydroxypropyl cellulose 1.8 g Magnesium stearate 6.0 g [B] Cellulose acetate phthalate 18 g Hydroxypropyl methylcellulose phthalate 18 g [A] , Well mixed. It was directly pressed or kneaded further, and then granulated through a screen of an extrusion type granulator, and a sufficiently well dried product was pressed to produce tablets.

Next, the formed tablet was coated with the well-dissolved base [B] to obtain 500 enteric tablets.

Formulation Example 3 (Granule) [A] LK-100 50 g Lactose 500 g [B] Hydroxypropyl cellulose 20 g Ethanol 250 g [A] components are mixed, mixed well, and placed in a vacuum kneader to be deaerated. Next, after substituting with nitrogen gas, the component [B] is added and thoroughly mixed. This was granulated through a screen of an extrusion type granulator to obtain 510 g of granules.

Test Example 1 Using 6-week-old ddY male mice, a group of 7 mice was tested. Sarcoma 180, which was previously transplanted and subcultured every 7 days in ddY female mice, was collected 6 days after transplantation into mouse ascites and sterilized with physiological saline to a concentration of 2.5 × 10 ⁷ cells / ml. Then, 0.2 ml of the diluted solution was subcutaneously injected between the scapulae of the mouse.

From the day after transplantation, once a day for 20 consecutive days, physiological saline was administered to the control group, and the polysaccharide substance LK-100 obtained in the example dissolved in physiological saline was intraperitoneally administered to the test group. The dose was in two stages of 100 mg / kg and 250 mg / kg. On the day after the final administration of the polysaccharide substance LK-100, the tumor was excised and its weight was measured. The tumor cell growth inhibition rate was calculated according to the following formula.

The results are shown in Table-1.

Test Example 2 Using 6-week-old ddY male mice, a group of 7 mice was tested. Sarcoma 180, which had been previously transplanted and subcultured every 7 days in ddY female mice, was collected 6 days after transplantation into mouse ascites and sterilized with physiological saline to a concentration of 2.5 × 10 ⁷ cells / ml. It was diluted with water and 0.2 ml of the diluted solution was subcutaneously injected between the scapulae of mice.

From the day after transplantation, once a day for 20 consecutive days, 3.0% in the test group
Polysaccharide substance LK-100 solution 0.1% dissolved in physiological saline
ml was forced into the stomach directly by a sonde. In the same manner, 0.1 ml of physiological saline was administered to the control group. On the day after the final administration of the polysaccharide substance LK-100, the tumor was excised and its weight was measured. The tumor cell growth inhibition rate was determined according to the formula of Test Example 1.

The results are shown in Table-2.

(Effect of the invention) The polysaccharide substance LK-100 of the present invention exhibits excellent antitumor activity.
Further, the polysaccharide substance LK-100 has extremely low toxicity and provides an antitumor agent having no side effect on humans and animals.

[Brief description of drawings]

FIG. 1 is a drawing showing an infrared absorption spectrum of the polysaccharide substance LK-100 of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kiyoshi Naganuma 967-1 Ogami-cho, Akishima-shi, Tokyo Akio Dormitory Examiner Kenji Hiromi

Claims (6)

[Claims] 1. A polysaccharide substance LK-10 having the following physicochemical properties:
0 Elemental analysis value C: 38.46% H: 6.35% N: 0.04% O: 55.15% Molecular weight Cannot be measured because of high molecular weight. Specific irradiance [α ▲] ²⁵ _D ▼ + 72.5 ° (C = 0.5%) Melting point This substance begins to change color around 230 ° C, begins to dissolve at around 260 ° C, and decomposes to blackish brown at around 280 ° C. Red external absorption spectrum (KBr) As shown in FIG. Solubility in solvent Soluble in water, insoluble in methanol, ethanol, acetone, ether. Color reaction i. Maurish reaction + ii. Anthrone reaction + iii. Cysteine-sulfur reaction-iv. Aniline-hydrochloric acid reaction-v. Carbazole-sulfuric acid reaction- vi. Elson-Morgan reaction- vii. Ninhydrin reaction- basic, The pH of a 0.5% aqueous solution of acidic and neutral substances is around 5.0. Material color The lyophilizate of this material is a white fibrous material. Type and composition ratio of constituent sugars Glucose: galactose = 1: 0.8. 2. A method for producing a polysaccharide substance LK-100, which comprises culturing a polysaccharide substance LK-100-producing bacterium belonging to the genus Lactobacillus and separating and collecting the polysaccharide substance LK-100 from the culture. Law. 3. A polysaccharide substance LK-10 belonging to the genus Lactobacillus.
0 producing bacteria is Lactobacillus sp.
The method according to claim 2, which is 9 (Lactobacillus sp. Glico 1569). 4. An antitumor agent comprising the polysaccharide substance LK-100 as an active ingredient. 5. The antitumor agent according to claim 4, which is an oral dosage form. 6. The antitumor agent according to claim 4, which is a parenteral dosage form.