JPS6361002A - Novel polysaccharide substance lk-100, production thereof and antitumor agent - Google Patents

Abstract

PURPOSE:To obtain a novel polysaccharide substance having specific physical and chemical properties, extremely low toxicity and excellent antitumor activity, by culturing a microbial strain belonging to Lactobacillus genus. CONSTITUTION:A microbial strain belonging to Lactobacillus genus and capable of producing polysaccharide substance LK-100 (e.g. Lactobacillus sp. Glico 1569, FERM P-8887, etc.) is cultured to obtain the objective polysaccharide substance LK-100 having the following physical and chemical properties: Elemental analysis, C=38.46%, H=6.35%, N=0.04%, O=55.15%; specific rotation, [alpha]<25>D=+72.5 deg. (C=0.5%); melting point, discoloration takes place from about 230 deg.C, begins to melt at about 260 deg.C and decomposes at about 280 deg.C to form dark brown material; solubility, soluble in water and insoluble in methanol, ethanol, acetone and ether; liquid nature, pH of 0.5% aqueous solution is about 5.0; color of the substance, freeze-dried product is white fibrous substance; composition of constituent sugar, glucose:galactose=1:0.8; infrared absorption spectrum (KBr method), shown in the figure.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) 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) In the past, much research has been done on the physiological effects of polysaccharide substances, focusing on the polysaccharide substances of mushrooms, but recently there have been several reports of antitumor effects of polysaccharide substances produced by lactic acid bacteria. (Unpublished Patent Publication (A) 1982-204001, ^gr
ic, Bio1. chem, 47(7) 1623-1
625, 1983), while conducting research on the dietary effects of fermentation holes and lactic acid bacteria beverages, the inventor discovered that a type of lactic acid bacterium produced a polysaccharide substance, and succeeded in isolating it. It was confirmed that this polysaccharide substance LK-100 is a novel polysaccharide substance that had not been previously known, and when its physiological effects were investigated, it was found that it has remarkable antitumor activity, and the present invention was completed. It is.

(Object of the Invention) The 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) <Microorganisms> The microorganisms used in the present invention are novel polysaccharide substances LK
It has a production capacity of -100 and is a bacterial species belonging to the genus Lactobacillus. As an example, Lactobacillus sp. glyco1569 (Lactobacillus sp.
Illus sp, G11co1569) has the above characteristics, advantageously produces the polysaccharide substance LK-100 of the present invention, and can be effectively used in the present invention.

The Lactobacillus sp. glyco1569 (
r1569 strain (hereinafter referred to as "strain r1569") has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and its accession number is FERM P-8887.

Strain 1569 has the following mycological properties. For testing and classification of mycological properties, please refer to the PURGE AIDS Manual.
Of Determinative Bacteriology 8th Edition (
Bergey's Manual Determi
native Bacterto+ogy 8th E
d) and methods of trafficking (clinical examination, 18.1163.1)
974).

(1) Morphological Properties Wet colonies are formed on MrtS agar plates under anaerobic conditions with carbon dioxide replacement. Rod-shaped rods that are positive for Durham staining and have no motile or spore-forming ability.

(2) Physiological properties Gas production from glucose Production of gas from decagluconic acid Assimilation of decasaccharide Amygdalin - Arabinose
+ Semibiose
− Aesculin − Fructose + Galactose
+glucose
+ Gluconic acid Deca lactose + Maltose
+Mannitol
− Mannose − Fructose − Melichiose
+ Raffinose - Rhamnose - Ribose
+Salinn
-Sorbitol -Eucrose -Trehalose
-xylose
- When the mycological properties of the above-mentioned microorganisms are searched according to the classification method of the above-mentioned literature, the genus Lactobacillus (Lactobacillus
lactic acid I? It was quiet.

<Culture Conditions> The polysaccharide substance LK-100 can be separated and collected from the liquid compartment and bacterial cell compartment of the culture after culturing the LK-100 producing bacteria. As the culture medium, any medium may be used as long as it is a medium for lactic acid bacteria, but a medium based on milky raw materials containing skim milk, whey, etc. is preferable. As for the culture conditions, anaerobic culture under carbon dioxide replacement is advantageous, but aerobic static culture is also sufficient. Culture temperature is 15~
35°C is preferred.

Further, 1) II of the medium is preferably weakly acidic, with a value of 6.0 to 6.5. The culture period depends on whether it is aerobic culture or aerobic culture, the composition of the medium,
It varies depending on various conditions such as culture temperature, but generally 3~
It takes about 5 days, and after the completion of the culture, the desired polysaccharide substance LK-1
00 accumulates in culture.

(Separation and Purification) LK-100 produced and accumulated through culture is separated and purified in the next treatment step.

(2) Production of polysaccharide substance LK-100 From an idle culture, a liquid fraction and a bacterial 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 bacterial cell fraction is extracted with hot water, then centrifuged to obtain an extraction supernatant, and an organic solvent is added to this supernatant to obtain a precipitate. The precipitates thus obtained from the liquid section and the bacterial cell section are redissolved in hot water, and similarly an organic solvent such as ethyl alcohol or acetone is added to re-precipitate.

■ After dissolving the reprecipitated precipitate in ion-exchanged water (distilled water is also acceptable), pressurize and load it into a column filled with anion-exchange resin. Further, ion-exchanged water is passed through the column, and the passed-through portion that is not adsorbed by the anion-exchange resin is collected, and this is dialyzed against ion-exchanged water using a dialysis membrane (manufactured by Union Carbide). The desired purified polysaccharide substance is obtained by freeze-drying the dialysis fluid under reduced pressure.
It is LK-100.

The thus obtained polysaccharide substance LK-100 is a new substance having the following physical and chemical properties.

<Physical and chemical properties of LK-100> ■ Elemental analysis value C 38.46% H: 6.35% N: 0.04% 0: 55.15% ■ Molecular weight Gel using Sepharose 2B (manufactured by Farman Fine Chemicals) Measured by filtration method. Column size 1
5X95cm: Flow rate 0.3mC/a+in: Fracunion 1.5mN + sample 3mg loaded: Developer 0.1
Due to the conditions of molar sodium chloride solution, the substance is fractionated into the gel particle Void Volume.

0 Specific light intensity [α-kun -725° (C = 0.5%) ■ Melting point This substance starts discoloring around 230°C, and has a temperature of 260°
It begins to dissolve at around 280°C and decomposes into a blackish brown color at around 280°C.

(2) Infrared absorption spectrum (KBr method) As shown in Figure 1.

■ Solubility in solvents: Insoluble in water, methanol, ethanol, acetone, and ether.

■ Color reaction 1, Morinyu reaction 111
Anthrone reaction Mono... Nostaino □ Sulfuric acid reaction -1v Aniline □ Hydrochloric acid reaction -■ Carbazole-Sulfuric acid reaction
-vj Elson-Morgan reaction
-~11 Ninhydrin reaction
-■ I)+1 of a 0.5% aqueous solution of basic, acidic, and neutral substances is around 50.

■ Color of substance The lyophilized product of this substance is white and fibrous.

[Co., Ltd.] Types and composition ratios of constituent sugars 1 mc of 0.5N sulfuric acid was added to 3 mg of the sample, and the mixture was hydrolyzed at 100°C for 14 hours, neutralized with barium carbonate, filtered, and the +8. After converting the sugar contained in & into alditol acetate form, it was analyzed by gas chromatography.

Gas chromatography conditions column = 3% 5ilar IOcon U
niport B (100-200 meters) Glass column (0.3x200cm) Column temperature: 200℃→220℃ (2℃/ml) Carrier gas/nitrogen, 50mC/min Result Glucose and galactose were detected, and the ratio was It was galactose-10,8.

Formulation forms of the anticancer agent of the present invention include, for example, injections,
Examples include drops, tablets, capsules, granules, powders, and suppositories. Preparation of these formulations can be carried out according to conventional methods. In addition, adjuvants commonly used in the technical field, such as stabilizers, may be appropriately blended, or a film may be applied.

Examples of methods for administering the anticancer agent of the present invention include intravenous injection, subcutaneous injection, oral administration, and rectal administration.

The dosage of the drug of the present invention varies depending on the patient's age, body weight, level of physical fitness, and administration method, but it is usually preferable to administer 0.1 to 15 g of LK-100 per day for adults. These doses are administered at once or divided into appropriate doses.

Furthermore, LK-100 may be added to various foods, especially fermenters, lactic acid bacteria drinks, medical foods, or pharmaceuticals.

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

Example <Separation and purification example of LK-100> Whey medium Whey 100m
(Composition) Peptone 1
．． 0g meat extract 1.0g yeast extract 0.5g Tween 80
0.1 mρ dipotassium phosphate
02g Sodium acetate 0.5g Ammonium citrate 0.2g Magnenium sulfate 002g Manganese oxide
0.005g pH6,4g whey medium (composition') loQ, polysaccharide substance LK-10
0-producing bacteria 1569 strains [Feikoken Kanyori No. 8887, (F
Seed EflM P 8887) 3 and culture it statically at 32°C for 40 minutes under carbon dioxide, and after culturing, adjust the pH to 6.
．． The sample was heated to 0.0C and heat-treated at 90'C for 30 minutes. The polysaccharide substance LK-1 attached to the bacterial cells during this heat treatment
oo could be extracted into the culture fluid compartment.

After heat treatment, centrifugation (io, ooorpm, 15 minutes)
9580 mQ of culture supernatant was obtained. This supernatant liquid was desalted and concentrated using an ultrafiltration membrane with a molecular weight of 1 million (manufactured by Millibore) to obtain 300 mQ of a concentrated liquid. Etyl alcohol was added to this concentrate to give a final concentration of 45% (V/V). As a result of this purification, a precipitated material was observed, and this precipitated material was collected by centrifugation 1 (10,000 rpm, 5 minutes) to obtain 18 g of precipitated material.

This precipitated material was heated and dissolved in ion-exchanged water, and insoluble materials were removed by centrifugation (to, 000 rpm, 15 minutes). By repeating this operation three times, 1.12 g of crudely purified polysaccharide material was obtained. The crude product obtained here was heated and dissolved in ion-exchanged water, and diethyl and
Aminoethyl Sephadex (DEAE-S
A column packed with ephadex) was pressurized. Ion-exchanged water was passed through and the flow-through fraction containing non-adsorbable material on DEAE-Sephadex was collected.

Concentrate this and use a dialysis membrane against ion-exchanged water to
Dialysis was performed at 0°C for 3 days. After dialysis, the dialyzed fluid was freeze-dried under reduced pressure to obtain 0.52 g of a freeze-dried sample of white fibrous polysaccharide substance LK-100.

The yield was 52 mg/1000 culture fluids.

Formulation Example 1 (Injection/Drop) 5 g of powdered glucose was added to contain 500 mg of LK-100, and the vials were aseptically distributed, sealed, filled with inert gas such as nitrogen and helium, and stored in a cool, dark place. . Before use, sterile physiological saline IQO+nQ was added to prepare an intravenous injection.

Formulation example 2 (tablet) [Ako LK-10025g Lactose 300g Hydroquinopropyl cellulose
1.8g magnesium stearate
6.0g [B] Cellulose acetate phthalate
18g of hydroxypropyl methylcellulose phthalate, 18g of each ingredient were taken and mixed well. The mixture was either directly pressed or kneaded well, then formed into granules through the screen of an extrusion type granulator, and once thoroughly dried, the mixture was pressed to produce tablets.

Next, the mixture was well dissolved into molded tablets and coated with the base of B1 to obtain 500 enteric-coated tablets.

Formulation example 3 (granules) [A] LK-10050g Lactose 500g [B] Hydroxypropyl cellulose
20g ethanol
Take 250g of each ingredient [Ai], mix well, put in a vacuum kneader, and degas. Next, after substituting with nitrogen gas, add the [+3] ingredient and mix thoroughly.

This was formed into granules through the screen of an extrusion type granulator to obtain 510 g of granules.

Test example! The test was conducted using 6-week-old male ddY mice in the − group. Sarcoma TGO (Sarcoma 180), which had been previously transplanted and passaged every 7 days in ddY female mice, was harvested 6 days after transplantation into the ascites of the mouse, and 2.5X 107c
It was diluted with sterile physiological saline to a concentration of el Is/mσ, and 0.2+nQ of the diluted solution was subcutaneously injected into the interscapular region of mice.

Starting from the day after transplantation, physiological saline was administered intraperitoneally to the control group, and the polysaccharide substance LK-100 obtained in the example dissolved in physiological saline was administered intraperitoneally to the test group once a day for 20 consecutive days. The dosage is 10
There were two levels: 0 mg/kg and 250 mg/kg. The day after the final administration of polysaccharide substance LK-100, the tumor was excised and its weight was measured. The tumor cell growth inhibition rate was determined according to the following formula.

(control group average tumor weight) The results are shown in Table-1.

Table-1 Test Example 2 ddY system 6i1! The test was conducted using 7 aged male mice in - group.

Sarco-v180 (Sarcoma 180), which had been previously transplanted and passaged every 7 days in ddY female mice, was harvested 6 days after transplantation into mouse ascites, and 2.5X 10'cell
It was diluted with sterile physiological saline to a concentration of ls/m12, and 0.2 ml of the diluted solution was injected subcutaneously between the shoulder blades of mice.

From the day after transplantation, the test group received 3.
Polysaccharide substance LK-10 dissolved in physiological saline at 0% 4 degrees
0.1 ml of 0.0 solution was forcibly administered directly into the stomach using a sonde. In the same manner, for the control group, 0.1 m of physiological saline was added.
12 were administered. The day after the final administration of 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 in Test Example 1.

The results are shown in Table-2.

Table 2 (Effects of the Invention) The polysaccharide substance LK-100 of the present invention exhibits excellent antitumor activity.

In addition, the polysaccharide ffLK-100 has extremely low toxicity (it provides an antitumor agent with no side effects for humans or animals).

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an infrared absorption spectrum of the polysaccharide substance LK-100 of the present invention. Patent applicant Glico Kyodo Dairy Co., Ltd. RIKEN

Claims (1)

[Claims] 1. Polysaccharide substance LK-100 (
1) Elemental analysis values C: 38.46% H: 6.35% N: 0.04% O: 55.15% (2) Cannot be measured because it is a molecular weight high molecular substance. (3) Specific luminous intensity [α] ＾2＾5_D=+72.5°(C=0.5%)(
4) Melting point This substance begins to discolor at around 230°C, begins to dissolve at around 260°C, and decomposes into a blackish brown color at around 280°C. (5) Infrared absorption spectrum (KBr method) As shown in FIG. (6) Solubility in solvents Soluble in water, insoluble in methanol, ethanol, acetone, and ether. (7) Color reaction i, Molish 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 - (8) The pH of 0.5% aqueous solutions of basic, acidic, and neutral Betsumoto substances is around 5.0. (9) Color of the substance The lyophilized substance is white and fibrous. (10) Types of constituent sugars and composition ratio glucose:galactose=1:0.8. 2. A method for producing polysaccharide substance LK-100, which comprises culturing polysaccharide substance LK-100-producing bacteria belonging to the genus Lactobacillus, and separating and collecting polysaccharide substance LK-100 from the culture. 3. The polysaccharide substance LK-100 producing bacterium belonging to the genus Lactobacillus is Lactobacillus sp. glyco156.
9 (Lactobacillus sp. Glico
1569) according to claim 2. 4. Polysaccharide substance LK-100 according to claim 1
An antitumor agent containing as an active ingredient. 5. The antitumor agent according to claim 4, which is in an oral administration form. 6. The antitumor agent according to claim 4, which is in a parenteral administration form.