JPH06183981A - Antiulcer agent containing muco polysaccharide extracellularly produced by chlorella bacterium - Google Patents

Abstract

PURPOSE:To obtain an antiulcer agent containing a mucopolysaccharide which is produced by extracellular secretion from a specific bacterial strain belonging to the genus Chlorella as an active ingredient, having strong antiulcer action on various ulcers, being non-toxic, capable of readily purifying and collecting in a large amount. CONSTITUTION:This antiulcer agent contains a muco polysaccharide extracellularly produced and secreted from Chlorella sp. K-4035 strain belonging to the genus Chlorella as active ingredient. This mucepolysaccharide has the following properties: Physical property: white powder; color reaction: positive to Molisch reaction, phenol sulfuric acid reaction, etc., and negative to ninhydrin reaction and iodine starch reaction, etc.; elementary analysis: C 38.14+ or -1.59%, H 6.28+ or -0.29%, N 1.38+ or -0.34; molecular weight: 800000-1200000; pH: 4.5-5.5; solubility: soluble in water, insoluble in alcohol, acetone, etc.; constitutional sugar: rhamnose, arabinose, mannose and uronic acid. Furthermore, the muco polysaccharide is obtained e.g. from the culture filtrate after culturing Chlorella sp. K-4035 strain in a liquid medium at 30 deg.C for 4 days.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an anti-mucopolysaccharide produced and secreted by a unicellular green alga of the genus Chlorella extracellularly (hereinafter sometimes referred to as "extracellular mucilage chlorella polysaccharide"). Regarding tumor agents.

[0002]

2. Description of the Related Art Mushrooms and bacteria are known as proteins or polysaccharides having antitumor activity. All of these have been found to exhibit antitumor effects by enhancing the defense function of the body against tumors.

On the other hand, with respect to green algae such as single-celled green algae of the genus Chlorella, the extraction / separation of antitumor glycoproteins having a molecular weight of 120,000 and composed of several types of sugars has been reported (JP-A-61- 69728 publication). This substance is
The antitumor effect is enhanced by the hydrolysis treatment with ze, and it exhibits a direct cell killing effect.

[0004]

However, since this substance is an intracellular substance, it contains various components, and since it is not secreted outside the cells, it is difficult to fractionate and purify it in large quantities. Therefore, the antitumor effect of this substance has not been fully utilized.

On the other hand, Chlorella sp. K-4035 (Chlorella
sp. K-4035) strain is known to produce and secrete a viscous polysaccharide outside the cell, although it is disclosed in JP-A-61-96992.
No antitumor effect has been previously recognized for this substance.

The present invention provides a mucopolysaccharide having an antitumor effect, which is produced and secreted by a unicellular green alga of the genus Chlorella extracellularly, and therefore can be easily purified and collected in a large amount. The present invention aims to provide an antitumor agent containing

[0007]

[Means for Solving the Problems] As a result of earnest research, the present inventors have surprisingly found that the above-mentioned chlorella sp. The inventors have found that they have a strong antitumor effect, and completed the present invention.

That is, the antitumor agent of the present invention is produced outside the microbial cell of Chlorella sp. K-4035 strain belonging to the genus Chlorella.
It is characterized by containing a secreted viscous polysaccharide as an active ingredient. The antitumor agent according to the present invention will be described below. The physicochemical properties of the viscous polysaccharide produced and secreted by the Chlorella sp. K-4035 strain outside the cell are as follows. 1. Property: White powder 2. Color reaction: Maurish reaction + Phenol-sulfuric acid reaction + Ferric chloride reaction + Anthuron-sulfuric acid reaction + Carbazole-sulfuric acid reaction + Elson-Morgan reaction-Ninhydrin reaction-Iodine starch reaction-3. UV absorption spectrum: no peculiar absorption 4. Infrared absorption spectrum: As shown in FIG. Elemental analysis: C: 38.14 ± 1.59% H: 6.28 ± 0.29% N: 1.38 ± 0.34% 6. Molecular weight: 800 to 1.2 million 7. Specific optical rotation: α = + 0.031 ± 0.06 ° (0.2%, 20 ° C.) 8. Melting point: unclear, carbonized at about 220 ° C 9. Viscosity: η = about 1500 cp (1%, 25 ° C.) 10. pH: 4.5-5.5 (1%) 11. Solubility: Soluble in water, insoluble in alcohol, acetone, ether 12. Constituent sugar: including rhamnose, arabinose, mannose, uronic acid

The taxonomic properties of the chlorella sp. K-4035 strain producing this chlorella extracellular mucopolysaccharide and the method for producing the extracellular mucopolysaccharide are described in the above-mentioned JP-A-61-96992.
It is described in detail in the publication.

The antitumor agent of the present invention can be prepared in the form of oral administration agents or parenteral administration agents such as intravenous infusion agents and intraperitoneal injection agents. Specific examples include the form of an aqueous solution having an isotonic solution such as physiological saline as a base material.

Further, the antitumor agent of the present invention comprises, in addition to the above-mentioned chlorella extracellular viscous polysaccharide, an additive usually used in pharmaceuticals, that is, a pharmaceutically acceptable buffering agent, surfactant, Preservatives and the like can be added.

[0012]

EXAMPLES The present invention will be described in detail below with reference to examples. Example 1 Chlorella sp. K-4035 strain was inoculated into 1.3 liters of a medium having the composition shown in Table 1 below, and aerobically cultured at 30 ° C and pH 7 for 4 days. Table 1 −−−−−−−−−−−−−−−−−−−−−−−−−−− Culture land composition -------- glucose 40 g / l urea 2 g / l magnesium sulfate 1 g / liter of monopotassium phosphate 1.5 g / l vitamin B ₁₂ 8 [mu] g / l iron-EDTA 30 mg / l Arnon A ₅ solution 0.6 ml / liter ---------------------------------

After diluting the obtained culture solution with water twice,
The cells were removed by centrifugation and further filtered using Standard Supercell (Jones Manville). To the filtrate, 1% of cetablon was added to precipitate the polysaccharide, which was washed with water, alcohol and acetone in order to remove excess setablon.

Next, when it was adsorbed on a Dowex 1-X4 (Cl type) column and eluted with a NaCl concentration gradient, the peak portion eluted near 0.8 M was taken and ethanol precipitation was repeated 3 times, It was dialyzed for one day in cold water.
Then, by freeze-drying, about 5 g of a polysaccharide was obtained. Example 2

Using the 8-week-old female BALB / c normal mouse, the toxicity test of the polysaccharide obtained in Example 1 was conducted. The mice were divided into 3 groups of 10 mice each, and 1 group was used as a control group. The polysaccharide was dissolved in physiological saline at 5 mg / ml, and 0.2 ml per animal was administered once every two days.
At this time, one group was intraperitoneally injected, and the other one group was subcutaneously injected. The change in average body weight of each group after the start of administration was measured and used as an index of toxicity. The results are shown in Table 2 below. Table 2 ------------------------------------ Mean body weight (g) Administration method -------- −−−−−−− 7 days after administration 14 days after administration −−−−−−−−−−−−−−−−−−−−−−−−−−−− Non-administration (subject) 20.5 21.7 23.6 Intraperitoneal injection 20.3 21.4 23.1 Subcutaneous injection 20.5 21.9 23.5 ----------------------- −−−−−−−−

As is clear from Table 2, there is no difference in the average body weight between the mice to which the chlorella extracellular mucopolysaccharide according to the present invention was administered and the mice of the control group, and there was no difference in growth. No abnormality was found. Therefore, it is clear that the chlorella extracellular mucilage polysaccharide according to the present invention has no toxicity. Example 3

Using 8-week-old female ICR mice, the antitumor effect of the antitumor agent using the polysaccharide obtained in Example 1 above was tested. Sarcoma 180 was added at 1 × 10 ⁶ to all mice tested. Cells were injected intraperitoneally. These mice were divided into 6 groups of 10 mice each, one of which was used as a control group, and the other 5 groups were each administered with a polysaccharide by intratumor injection, intravenous injection, intraperitoneal injection, subcutaneous injection and oral administration. , The survival days after tumor transplantation were measured. Polysaccharides were dissolved in physiological saline to 1 mg / ml and
0.2 ml each was administered 10 times in total, once a day from the day after the tumor implantation. The results are shown in Table 3 below. Table 3 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Administration method Mean survival days (days) Life extension effect ^* (%) −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Not administered (control) 14.5 0.0 Intratumoral injection 31. 2 115.2 Intravenous injection 24.0 65.5 Intraperitoneal injection 26.5 82.8 Subcutaneous injection 19.8 36.6 Oral administration 18.1 24.8 −−−−−−−−−−−−− −−−−−−−−−−−−−−−−−−− * Life-prolonging effect = (mean survival days of test group−mean survival days of control group) × 100 / mean survival days of control group

As is clear from Table 3, the antitumor agent of the present invention was found to have a life-prolonging effect by any administration method, and the highest life-prolonging effect could be obtained especially in intratumoral injection. Example 4

Using 8-week-old female BALB / c mice,
The antitumor effect of the antitumor agent using the polysaccharide obtained in Example 1 above was tested. Meth A fibrosarcoma was administered to all mice tested in an amount of 3 × 10 ⁶ per mouse. Cells were inoculated subcutaneously. These mice were divided into 5 groups of 10 mice each, one of which was used as a control group, and the other 4 groups were respectively administered with polysaccharide by intratumor injection, intravenous injection, intraperitoneal injection and subcutaneous injection, and tumor transplantation was performed. Tumor size was measured 13 days later. The polysaccharide was dissolved in physiological saline at a concentration of 1 mg / ml, and this was administered at 0.2 ml per animal once every 2 days from 2 days after tumor inoculation. The results are shown in Table 4 below. Table 4 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Tumor tumor growth Complete regression Administration method Size ^* Suppression rate ^** Number of individuals (mm ² ) (%) (Animal) ---------------------------------------------------------------------------------------- 253 0.0 0 Intratumoral injection 42 83.4 6 Intravenous injection 93 63.2 3 Intraperitoneal injection 89 64.8 2 Subcutaneous injection 156 38.3 0 −−−−−−−−−−−−−−−−−−−−−−−− −−−−−−−− * Tumor size = long diameter (mm) × minor diameter (mm) of tumor after 13 days ** Tumor growth inhibition rate = (tumor size of control group−tumor of test group) Size) x 100 / (size of tumor in control group)

As is clear from Table 4, the antitumor agent of the present invention exhibited a tumor growth inhibitory effect by any administration method. In particular, intratumoral injection showed the highest suppressive effect, and the number of completely regressing individuals was also highest.

[0021]

As described above, the antitumor agent of the present invention is
It shows no toxicity and shows high antitumor effect against various tumors in various dosage forms.

The mucopolysaccharide, which is the active ingredient of the antitumor agent of the present invention, is easily produced and secreted in large quantities because Chlorella sp. K-4035 strain is produced and secreted outside the cells. It is possible to

[Brief description of drawings]

FIG. 1 is a diagram showing an infrared absorption spectrum of a viscous polysaccharide produced and secreted extracellularly by Chlorella sp. K-4035 strain, which is used as an active ingredient of the antitumor agent of the present invention.

Claims (2)

[Claims] 1. A chlorella sp. K-403 belonging to the genus Chlorella.
5 An antitumor agent containing, as an active ingredient, a viscous polysaccharide produced and secreted outside the bacterial cell by 5 strains. 2. The antitumor agent according to claim 1, wherein the viscous polysaccharide has the following physicochemical properties. 1. Property: White powder 2. Color reaction: Maurish reaction + Phenol-sulfuric acid reaction + Ferric chloride reaction + Anthuron-sulfuric acid reaction + Carbazole-sulfuric acid reaction + Elson-Morgan reaction-Ninhydrin reaction-Iodine starch reaction-3. UV absorption spectrum: no peculiar absorption 4. Infrared absorption spectrum: As shown in FIG. Elemental analysis: C: 38.14 ± 1.59% H: 6.28 ± 0.29% N: 1.38 ± 0.34% 6. Molecular weight: 800 to 1.2 million 7. Specific optical rotation: α = + 0.031 ± 0.06 ° (0.2%, 20 ° C.) 8. Melting point: unclear, carbonized at about 220 ° C 9. Viscosity: η = about 1500 cp (1%, 25 ° C.) 10. pH: 4.5-5.5 (1%) 11. Solubility: Soluble in water, insoluble in alcohol, acetone, ether 12. Constituent sugar: including rhamnose, arabinose, mannose, uronic acid