JPH0647605B2 - 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. As examples of this, lentinan from Shiitake mushrooms and schizophyllan from Suehirotake mushrooms are known. All of these have β-1,3-glucan as the main chain,
It is said that glucose is branched by -1,6-bond.

It is said that pachyman 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 is represented by the formula (1), (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 represented by the formula, and a formula (II), (In the formula, Glu and the numbers have the same meanings as described above, and m is 0.
A second repeating unit represented by the formula (III), 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 Of 0 to about 30 and a chemical modification showing a molecular weight value of about 100,000 to about 1,500,000 in a gel high performance liquid chromatography using an aqueous sodium chloride solution having a concentration of 0.1 mol / l as a mobile phase. It provides a polysaccharide.

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

(1) Molecular weight A gel permeation high performance liquid chromatography using a sodium chloride solution with a concentration of 0.1 mol / l as a mobile phase, and using G-6000PW manufactured by Toyo Soda Co., Ltd. as a column, the gel weight was 100,000 to 150 It elutes at the position of 10,000 retention times.

(2) Elemental analysis value C: 4.0% to 45.0% H: 5.3% to 6.4% N: 1.0% to 4.6%.

(3) Sulfuric Acid Decomposition Completely hydrolyzing the chemically modified polysaccharide with 2N-sulfuric acid at 80 ° C. for 18 hours and analyzing by gas chromatography revealed that
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 Fig. 1 shows the infrared absorption spectrum by the potassium bromide tablet method.

(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 separated and fixed. 2,3,4-tri-O-methyl glucose and 2,3,4,6
-Tetra-O-methyl glucose may be separated / fixed 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 is 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 represented by the 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 consisting of a third repeating unit represented by the formula (I)
Per 100 glucopyranosyl group units, the number of repeating units having a carbonyl group represented by formula (IV) is about 20 to about 85, and the number of third repeating units represented by formula (III) is 0. Polysaccharides of about to about 30 can be prepared by reacting with tris (hydroxymethyl) aminomethane to form a Schiff base and reducing it.

The aldehyde type β-1,3-glucan used as a starting material in this method (hereinafter referred to as the method of the present invention) can be prepared, for example, by the same method as disclosed in JP-A-55-25409, except that It can be obtained without reduction treatment and acid hydrolysis. In other words,
ularia auriculajudae) fruiting body is extracted with an alkaline aqueous solution, and a portion which is not dissolved in the alkaline aqueous solution (hereinafter referred to as an alkali-insoluble portion) is decomposed with periodate.

In the method of the present invention, the reaction of forming a Schiff base from an aldehyde type β-1,3-glucan and tris (hydroxymethyl) aminomethane is performed in an aqueous medium in the amount of 1 g of the former to about 0.001 to 0.5 of the latter. Mol, preferably 0.01 to
It can be carried out by adding 0.1 mol. The aldehyde type β-1,3-glucan at that time is well dispersed in the aqueous medium. The amount of the aqueous medium relative to the aldehyde type β-1,33-glucan is about 10 to about 1,000 by weight.
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 at least equivalent to the carbonyl group in the aldehyde β-1,3-glucan used as the starting material. The concentration of the reducing agent is not limited, but it may be about 0.001 molar concentration or more, for example, about 0.001 to about 0.1 molar. The reaction temperature is about 0 to about 80 ° C., preferably about 10 to about 50 ° C., and the reaction time is usually several hours to 5 days, preferably about 2 days.

By this reaction, 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 capacity of 250 ml, 0.1 mol of tris (hydroxymethyl) aminomethane was added thereto, and distilled water was added to bring the total amount to 100 ml. And Disperse the aldehyde β-1,3-glucan well with a disperser, then add pH with hydrochloric acid.
It was adjusted to 7, and stirred for 2 days with a magnetic stirrer.

After that, the pH was adjusted to 6.5 with hydrochloric acid, 1.16 g of sodium cyanoborohydride was added, and the mixture was further reacted for 2 days while stirring. After the reaction was completed, the reaction solution containing the suspension was centrifuged and filtered, and the water-soluble fraction was dialyzed against running water. The dialysis tube contents solution was freeze-dried to obtain 160 mg of the desired chemically modified polysaccharide. (Yield 16%) The analysis results of the obtained chemically modified polysaccharide were as follows.

Molecular weight A gel permeation high performance liquid chromatography using a 0.1 mol / l sodium chloride aqueous solution as a mobile phase, and using G-6000PW manufactured by Toyo Soda Kogyo Co., Ltd. as a column, the gel permeation yielded 210,000. It was eluted at the retention time position.

Elemental analysis value C: 44.0% H: 5.8% N: 2.4% Infrared absorption spectrum An infrared absorption spectrum by the potassium bromide tablet method is shown in FIG.

Methylation analysis From the result of methylation analysis, the number of repeating units represented by formula (II) (per 100 formulas (I)) 82.5 The number of repeating units represented by formula (III) (formula (I) 100 0) Example 2 Using the aldehyde-type polysaccharide obtained in Raw Material Preparation Example 2, Example 1
Chemical modification was performed in the same manner as above to obtain a chemically modified polysaccharide.
(Yield 12%) Molecular weight Elution was carried out at the position of retention time of molecular weight 950,000.

Elemental analysis value C: 45.0% H: 5.9% N: 2.2% Methylation analysis The number of repeating units represented by formula (II) from the result of methylation analysis (per 100 formulas (I)) 74 Number of repeating units represented by formula (III) (per 100 formula (I)) 8.5 Example 3 Example 3 Effect of chemically modified polysaccharide of the present invention on Sarcoma 180 solid tumor in ICR mouse group Was tested. 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 transplantation of the cancer cells, the drug was intraperitoneally administered in an amount of 0.1 ml once a day for 10 days from the next day. The test group was used with the chemically modified polysaccharide of the present invention (obtained in Example 1) at a dose of 5 mg / Kg · day, and the control group was administered with physiological saline only. 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. After centrifugation, the alkali-extracted fraction was looked into 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) (Wherein Glu and the numbers have the same meanings as described above) and a first repeating unit having a β-1,3-glucopyranosyl group unit as a repeating unit 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 0 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 it for 20.
5 g of aldehyde type polysaccharide was obtained. (Yield 82%) This aldehyde-type polysaccharide is a β-substantially consisting of a repeating unit represented by the formula (I) and a repeating unit represented by the formula (IV).
It was 1,3-glucan. The value of m in formula (IV) is about an average value.
It was 0.1. The number of repeating units of the formula (IV) per 100 repeating units of the formula (I) was 82.5. The nitrogen content of this aldehyde-type polysaccharide was below the limit of quantification.

Raw Material Preparation Example 2 Raw material preparation was carried out 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 in 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. The number of repeating units represented by formula (III) was 8.5 and the number of repeating units represented by formula (IV) was 74 per 100 repeating units represented by formula (I).

[Brief description of drawings]

FIG. 1 is a diagram showing an infrared absorption spectrum 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 the numbers have the same meanings as described above, and m is 0.
A second repeating unit represented by the formula (III), 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 a sodium chloride aqueous solution having a concentration of 0.1 mol / l 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.
An aldehyde-β-1,3-glucan consisting of a third repeating unit represented by the formula:
(I) per 100 glucopyranosyl group units, the number of repeating units having a carbonyl group represented by the formula (IV) is about 20 to about 85, the number of the third repeating unit represented by the formula (III) Of the formula (I), wherein 0 to about 30 polysaccharides are reacted with tris (hydroxymethyl) aminomethane to form a Schiff base, which is reduced, (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), (Wherein Glu, m and numbers have the same meanings as above), 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 0 to about 30, and the 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 showing 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.