JPH0645647B2 - Chemically modified polysaccharide and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a chemically modified polysaccharide and a method for producing the same.

Polysaccharides derived from basidiomycetes have attracted attention because they show antitumor properties. Known examples of this include lentinan from Shiitake mushrooms, and schizophyllan from Suehirotake mushrooms. All of these have β-1,3-glucan as the main chain,
It is said that glucose is branched by β-1,6-bond.

It is said that bakiman from Basidiomycetes, scleroglucan from microorganisms of the genus Sclerotium, and polysaccharides derived from jellyfish have the same structure.

However, even with polysaccharides having such a similar structure, there is a difference in their antitumor properties, and one of the causes is a branched sugar, that is, a side chain form (for example, side chain). It may be due to the difference in the number and length of chains.

All of these polysaccharides were obtained by a simple extraction and separation operation from the fungus that is the origin, or were derived from the thus obtained polysaccharide by a simple reaction such as oxidation or reduction. .

The present invention has the formula (I): (In the formula, Glu represents a glucopyranosyl group, and a number represents a bonding position), a first repeating unit having a β-1,3-glucopyranosyl group unit as a repeating unit, and a formula
(II), (In the formula, Glu and numbers have the same meanings as described above, and X is N.
A hydroxyaminocarbonyl group or a (2-hydroxyethyl) amino group, and m represents an integer of 0 to 2), or a second repeating unit represented by the formula (III), (In the formula, Glu and the numbers have the same meanings as described above, and n is 0.
To an integer of 2), the number of the second repeating unit is about 20 to about 85 per 100 glucopyranosyl group units of the formula (I), the third repeating unit. Is a chemically modified polysaccharide showing a molecular weight of about 100,000 to about 1,500,000 in gel ultra high performance liquid chromatography using an aqueous solution of sodium chloride having a concentration of 0.1 mol / mol as a mobile phase. .

The chemically modified polysaccharide of the present invention typically has the following physical properties,
It exhibits chemical properties.

(1) Molecular weight A gel permeation liquid chromatography using a sodium chloride solution with a concentration of 0.1 mol / mobile as a mobile phase, and using G-6000PW manufactured by Toyo Soda Co., Ltd. as a column, gel filtration was performed to obtain a molecular weight of 100,000 to 1,500,000. Elute at the retention time position.

(2) Elemental analysis value Gives a value that substantially matches the value expected from the chemical formula. That is, when X in the formula (II) is an N-hydroxyaminocarbonyl group, C: 32.5% to 43.2% H: 4.5% to 6.2% N: 1.9% to 8.6% is given to give (2-hydroxyethyl) In the case of an amino group, a value of C: 35.0% to 43.0%, H: 5.0% to 6.2%, N: 2.0% to 11.0% is given.

(3) Sulfuric acid decomposition The chemically modified polysaccharide was completely hydrolyzed with 2N-sulfuric acid at 80 ° C. for 18 hours, and analyzed by gas chromatography.
Glucose is found but glycerol is not.

(4) Hydrochloric acid decomposition No precipitation occurs even if the chemically modified polysaccharide is dissolved in a 1N-hydrochloric acid aqueous solution and boiled.

(5) Solubility Soluble in water and dimethyl sulfoxide, but insoluble in methanol, ethanol, acetone, and benzene.

(6) Infrared absorption spectrum Infrared absorption spectra obtained by the potassium bromide tablet method are shown in Fig. 1 (when X in the formula (II) is an N-hydroxyaminocarbonyl group) and Fig. 2 (also (2-hydroxyethyl)). (In the case of amino group).

(7) Methylation analysis When methylated sugar obtained by hydrolysis after methylation was induced to alditol acetate and analyzed by gas chromatography, 2,4-di-O-methyl glucose and
2,4,6-Tri-O-methyl glucose is isolated and identified. 2,3,4-tri-O-methyl glucose and 2,3,4,6
-Tetra-O-methyl glucose may be separated or identified, or may not be detected at all.

The chemically modified polysaccharide of the present invention has a very strong antitumor property, but its toxicity to mammals is extremely low, and its acute toxicity to mice has an LD ₅₀ value of 1,500 mg / kg or more.

The chemically modified polysaccharide of the present invention is dissolved or dispersed in a physiologically acceptable base material like a known antitumor polysaccharide, and then subcutaneously,
It can be administered by intramuscular, intravenous injection or other conventional methods. Dosage is about 0.1 per kg body weight
To about 100 mg, preferably about 1 to about 20 mg
It is a degree.

The chemically modified polysaccharide of the present invention has the formula (I), (In the formula, Glu represents a glucopyranosyl group, and a number represents a bonding position), a first repeating unit having a β-1,3-glucopyranosyl group unit as a repeating unit, and a formula
(IV), (In the formula, Glu and the numbers have the same meanings as described above, and m is 0.
A repeating unit having a carbonyl group represented by the formula (III) or a repeating unit having the carbonyl group and the formula (III), (In the formula, Glu and the numbers have the same meanings as described above, and n is 0.
Or an aldehyde-β-1,3-glucan composed of a third repeating unit represented by the formula (I) per 100 glucopyranosyl group units of the formula (I).
V), wherein the number of repeating units having a carbonyl group is about 20 to about 85, and the number of the third repeating unit of formula (III) is 0 to about 30, It can be produced by reacting with urea or 2-hydrazinoethanol to form a Schiff base, and reducing the Schiff base.

In this method (hereinafter referred to as the method of the present invention), the repeating unit having a carbonyl group represented by the formula (IV) of the aldehyde type β-1,3-glucan as a starting material is represented by the formula (II) according to the following reaction formula. Converted to the represented repeat unit.

Aldehi type β-used as a starting material in the method of the present invention
1,3-Glucan can be obtained, for example, by the same method as disclosed in JP-A-55-25409, but without the final reduction treatment and acid hydrolysis. That is, the fungus, Auricularia auriculajudae
A portion of the fruiting body that is extracted with an alkaline aqueous solution and is not dissolved in the alkaline aqueous solution (hereinafter referred to as the alkali-insoluble portion)
Can be prepared by decomposing the compound with periodate.

In the method of the present invention, the reaction of forming a Schiff base from an aldehyde β-1,3-glucan and hydroxyurea or 2-hydrazinoethanol is carried out in an aqueous medium in the amount of 1 g of the former to about 0.001 to about 0.5 mol of the latter, preferably about 1 mol of the latter. Is 0.01 or
This can be done by adding 0.1 mol. The aldehyde β-1,3-glucan at that time is well dispersed in the aqueous medium. The amount of the aqueous medium relative to the aldehyde β-1,3-glucan is about 10 to about 1.000 in weight ratio.
The amount is double, preferably about 30 to 300 times.
The liquidity during the reaction has a pH of about 5 to about 10, preferably about 6
Or about 8 An acid such as hydrochloric acid for adjusting the liquidity,
Alternatively, an alkali, such as sodium hydroxide, can be used.

The reaction is usually carried out at a temperature of about 5 to about 80 ° C., preferably about 1
Perform at 0 to about 50 ° C. Reaction time is usually about 10
Time to about 5 days, preferably about 2 days.

After forming the Schiff base in this way, reduction is carried out. As the reducing agent, a strong reducing agent such as sodium borohydride or sodium cyanoborohydride is used. Particularly, sodium cyanoborohydride is preferable. The amount of the reducing agent is the aldehyde type β-1,3-used as the starting material.
It is at least equivalent to the carbonyl group in the glucan. The concentration of the reducing agent is not limited, but about 0.001 molar concentration or more,
For example, about 0.001 to about 0.1 mol can be exemplified. The reaction temperature is about 0 to about 80 ° C., preferably about 1
0 to about 50 ° C, the reaction time is usually several hours to 5
It is about one day, preferably about two days.

By these reactions, the water-soluble polysaccharide of the present invention is eluted into the aqueous medium. The polysaccharide of the present invention eluted in an aqueous medium can be isolated by a conventional method such as dialysis or freeze-drying after removing the insoluble fraction by a conventional method such as centrifugation.

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

Example 1 1 g of the aldehyde type β-1,3-glucan obtained in Preparation Example 1 of a raw material was placed in a flask having a volume of 250 ml, to which 0.1 mol of hydroxyurea was added and distilled water was added to make a total amount of 100 ml. The aldehyde β-1,3-glucan was well dispersed with a disperser, adjusted to pH 7 with hydrochloric acid, and stirred with a magnetic stirrer for 2 days.

After that, adjust the pH to 6.5 with hydrochloric acid, add 1.16 g of sodium cyanoborohydride, and add 2 more while stirring.
Reacted for days. After the completion of the reaction, the reaction liquid containing the suspension was centrifuged and filtered, and the water-soluble fraction was dialyzed against running water. The content of the dialysis tube was freeze-dried to obtain 72 mg of the desired chemically modified polysaccharide. (Yield 7.2%). The analysis results of the obtained chemically modified polysaccharide were as follows.

The retention time of the molecular weight of 210,000 was obtained by gel permeation using G-6000PW manufactured by Toyo Soda Industry Co., Ltd. Was eluted at the position.

Elemental analysis value C: 36.7% H: 5.1% N: 4.2% Infrared absorption spectrum The infrared absorption spectrum by the potassium bromide tablet method is shown in FIG.

Methylation analysis From the results of methylation analysis, the number of repeating units represented by the formula (II) (per 100 formulas (I)) 82.5 The number of repeating units represented by the formula (III) (per 100 formulas (I)) ) 0 Example 2 Example 1 using the aldehyde-type polysaccharide obtained in Raw Material Preparation Example 2
Chemical modification was performed in the same manner as above to obtain a chemically modified polysaccharide.
(Yield 5.1%). The results of the analysis conducted in the same manner as in Example 1 are as follows.

Molecular weight It eluted at the retention time position of molecular weight 950,000.

Elemental analysis value C: 39.1% H: 5.2% N: 3.5% Methylation analysis Number of repeating units represented by the formula (II) based on the result of the methylation analysis (per 100 formulas (I)) 74 formulas (III) Number of repeating units represented by (per 100 formula (I)) 8.5 Example 3 Aldehyde β- 1,3. -Glucan was chemically modified to obtain 83 mg of the desired chemically modified polysaccharide. (Yield 8.3%).
The results of the analysis conducted in the same manner as in Example 1 are as follows.

Molecular weight It eluted at the retention time position of molecular weight 210,000.

Elemental analysis value C: 38.3% H: 5.4% N: 4.6% Infrared absorption spectrum An infrared absorption spectrum by the potassium bromide tablet method is shown in FIG.

Methylation analysis From the results of methylation analysis, the number of repeating units represented by the formula (II) (per 100 formulas (I)) 82.5 The number of repeating units represented by the formula (III) (per 100 formulas (I)) ) 0 Example 4 Using the aldehyde-type polysaccharide obtained in Raw Material Preparation Example 2, Example 3
Chemical modification was performed in the same manner as above to obtain a chemically modified polysaccharide.
(Yield 5.2%). The results of the analysis conducted in the same manner as in Example 1 are as follows.

Molecular weight It eluted at the retention time position of molecular weight 950,000.

Elemental analysis value C: 40.5% H: 5.8% N: 3.1% Methylation analysis Number of repeating units represented by formula (II) from the result of methylation analysis (per 100 formulas (I)) 74 formulas (III) The number of repeating units represented by (per 100 formula (I)) 8.5 Example 5 The effect of the chemically modified polysaccharide of the present invention on Sarcoma 180 solid tumor was tested in a group of ICR mice. For each ICR mouse, 6 × 10 ⁶ Sarcoma 180 ascites tumor cells were subcutaneously inoculated into the groin. The experimental group consisted of 6 animals.
After the cancer cell transplantation, 0.1 ml of the drug was intraperitoneally administered once a day for 10 days from the next day. In the test group, the chemically modified polysaccharide of the present invention (obtained in Example 1) was used at a dose of 5 mg / kg · day, and in the control group, physiological saline alone was administered. The tumor was extracted 35 days after the tumor transplantation and the weight was measured. The tumor suppression rate of each group was calculated by the following formula.

Here, C: average tumor weight of control group T: average tumor weight of test group The results are shown in Table 1.

The aldehyde type β-1,3-glucan used as a raw material in the present invention was prepared as follows.

Raw Material Preparation Example 1 Preparation of Alkali-Insoluble Part 504 g of commercially available dried chrysanthemum jellyfish was sufficiently pulverized with 1% sodium chloride aqueous solution 6 by a household mixer,
It was left standing overnight and immersed. Then, 1% aqueous sodium chloride solution 3 was added, and the mixture was further heated at 60 ° C. for 6 hours with stirring to sufficiently swell the fungus. To further miniaturize the fungus, crush it with a homogenizer and then
Hot water extraction was carried out in an autoclave at 20 ° C. for 20 minutes, followed by centrifugation to remove the hot water extraction fraction. The residual fraction was subjected to hot water extraction operation again in the same manner.

To the residual fraction thus obtained, water 9 and 324 g of sodium hydroxide were added (at this time, the total volume became 12), and alkali extraction was carried out at 60 ° C. for 4 hours under a nitrogen atmosphere. Centrifugation was performed to remove the alkali-extracted fraction to obtain an alkali-extracted residue. Water is added to this alkali extraction residue.
And 156 g of sodium hydroxide are added (at this time, the total volume is 9
Became. ), And alkali extraction operation was performed again in the same manner.

Water 10 was added to the alkali extraction residue, washing, centrifugation and resuspension were repeated until the pH of the suspension reached about 9. Dilute hydrochloric acid was added to the suspension to adjust the pH to 7.

Next, water 5 was added to this suspension and treated with a homogenizer to further subdivide the alkali extraction residue. Water was further added to the suspension and freeze-dried to obtain 146 g of an alkali-insoluble portion (yield 29%). This is essentially of formula (I) The first repeating unit having a β-1,3-glucopyranosyl group unit represented by the formula (Glu and the numbers have the same meanings as described above) and the formula (III) (Wherein Glu and the numbers have the same meanings as described above), and the repeating unit 1 of the formula (I)
It was a polysaccharide having about 82.5 repeating units of the formula (III) per 00. The value of n was about 0.1 on average.

Preparation of aldehyde-type polysaccharide In a narrow-mouth brown bottle with a content of 5, 25 g of alkali-insoluble part
Then, distilled water 5 was added, the alkali-insoluble portion was well dispersed with a magnetic stirrer, and then degassed using an aspirator. Then sodium metaperiodate 66
After g was added and dissolved, the mixture was reacted with stirring at room temperature for 7 days. After the reaction was completed, washing with water and centrifugation were repeated 3 times to remove the generated formic acid and the residual sodium metaperiodate. Add water to the solid phase and freeze-dry to 20.5
g of aldehyde-type polysaccharide was obtained (yield: 82%). This aldehyde-type polysaccharide is composed of β-1,3 consisting essentially of the repeating unit represented by the formula (I) and the repeating unit represented by the formula (IV).
-It was a glucan. The value of m in formula (IV) is about 0.1 on average.
Met. Further, the repeating unit represented by the formula (I) is 100
The number of repeating units represented by formula (IV) per unit is 82.5.
It was an individual. The nitrogen content of this aldehyde-type polysaccharide was below the limit of quantification.

Raw Material Preparation Example 2 A raw material was prepared in the same manner as in Preparation Example 1 except that the amount of sodium metaperiodate used in the preparation of the aldehyde-type polysaccharide of Raw Material Preparation Example 1 was changed to 13.2 g to obtain an aldehyde-type polysaccharide. This aldehyde-type polysaccharide is substantially β-1,3 consisting of a repeating unit represented by the formula (I), a repeating unit represented by the formula (III) and a repeating unit represented by the formula (IV).
-It was a glucan. The values of n in the formula (III) and m in the formula (IV) were both about 0.1 on average. In addition, the number of repeating units represented by the formula (III) was 8.5 and the number of repeating units represented by the formula (IV) was 74 per 100 repeating units represented by the formula (I).

[Brief description of drawings]

FIG. 1 and FIG. 2 are diagrams showing infrared absorption spectra of the chemically modified polysaccharide of the present invention.

Claims (3)

[Claims] 1. A formula (I), (In the formula, Glu represents a glucopyranosyl group, and a number represents a bonding position), a first repeating unit having a β-1,3-glucopyranosyl group unit as a repeating unit, and a formula
(II), (In the formula, Glu and numbers have the same meanings as described above, and X is N.
A hydroxyaminocarbonyl group or a (2-hydroxyethyl) amino group, m is an integer of 0 to 2), or a second repeating unit represented by the formula (III), (In the formula, Glu and the numbers have the same meanings as described above, and n is 0.
To an integer of 2), the number of the second repeating unit is about 20 to about 85 per 100 glucopyranosyl group units of the formula (I), the third repeating unit Is a chemically modified polysaccharide having a molecular weight of about 100,000 to about 1,500,000 in gel ultra high performance liquid chromatography using an aqueous sodium chloride solution having a concentration of 0.1 mol / mol as a mobile phase. 2. The formula (I), (In the formula, Glu represents a glucopyranosyl group, and a number represents a bonding position), a first repeating unit having a β-1,3-glucopyranosyl group unit as a repeating unit, and a formula
(IV), (In the formula, Glu and the numbers have the same meanings as described above, and m is 0.
A repeating unit having a carbonyl group represented by the formula (III) or a repeating unit having the carbonyl group and the formula (III), (In the formula, Glu and the numbers have the same meanings as described above, and n is 0.
Or an aldehyde-β-1,3-glucan composed of a third repeating unit represented by the formula (I) per 100 glucopyranosyl group units of the formula (I).
V) is a carbonyl group-containing repeating unit having a number of about 20 to about 85, and the third repeating unit of the formula (III) has a number of 0 to about 30. A formula characterized by reacting with urea or 2-hydrazinoethanol to form a Schiff base and reducing it.
(I), (Wherein Glu and the numbers have the same meanings as described above), a first repeating unit having a β-1,3-glucopyranosyl group unit as a repeating unit, and a formula (II), (In the formula, Glu, m and the numbers have the same meanings as described above, and X
Represents an N-hydroxyaminocarbonyl group or a (2-hydroxyethyl) amino group), or a second repeating unit represented by the formula (III), (In the formula, Glu, n and the numbers have the same meanings as described above), and a second repeating unit represented by the formula (II) per 100 glucopyranosyl group units of the formula (I). The number of repeating units is about 20 to about 85, the number of the third repeating unit represented by the formula (III) is about 0 to about 30, and an aqueous solution of sodium chloride having a concentration of 0.1 mol / l is used as a mobile phase. A method for producing a chemically modified polysaccharide having a molecular weight value of about 100,000 to about 1.5 million in gel ultra high performance liquid chromatography. 3. Aldehyde type -β- used as a starting material
The production method according to claim 2, wherein the 1,3-glucan is obtained by oxidizing the alkali-insoluble portion of the fruiting body of Pleurotus cornucopiae with periodate.