JPS60155203A - Chemically modified polysaccharide and its production - Google Patents

Abstract

NEW MATERIAL:A chemically modified polysaccharide having a main chain comprising beta-1,3-glucopyranosyl group of formula I (wherein Glu is glucopyranosyl and the numbers are positions of bonding) as repeating unit and a second repeating unit of formula II (wherein m is 0-2) and/or a third repeating unit of formula III (wherein n is 0-2) bonded to the main chain. USE:Antineoplastic agents. PREPARATION:A Schiff base obtained by reacting an aldehyde-form beta-1,3-glucan having a main chain comprising beta-1,3-glucopyranosyl groups of formula I as repeating unit and a carbonyl group-containing repeating unit of formula IV and/or a repeating unit of formula III bonded to the main chain with hydroxyurea is reduced.

Description

[Detailed description of the invention] It is something.

Polysaccharides derived from basidiomycetes have attracted attention for their antitumor properties. Known examples of this include lentinan from shiitake mushrooms and schizophyllan from suehitake mushrooms. All of these are β-1.

It is said that the main chain is 3-glucan, and glucose is branched by one β-1,6 bond.

It is said that Bakiman from the basidiomycete, scleroglucan from microorganisms of the genus Sclerotium, and polysaccharides derived from wood ear fungus have similar structures.

However, even among polysaccharides with similar structures, there are differences in their antitumor properties, and one of the reasons for this is the presence of branched sugars, that is, the form of side chains (e.g., This may be due to differences in the number and length of chains.

All of these polysaccharides were obtained by simple extraction and separation operations from their fungi, or were derived from the polysaccharides obtained in this way through simple reactions such as oxidation and reduction. .

The present invention provides a β-1,3-glucopyranosyl group unit represented by the formula (I), 1 [3) β-D-Glu (1÷→ (in the formula, Glu represents a glucopyranosyl group, and the numbers indicate the bonding positions). The main chain as a repeating unit and the formula (It) bonded to this main chain, (wherein Glu and numbers represent the same meanings as above, and group, m represents an integer from 0 to 2), or this second repeating unit and formula (1) (in the formula, Glu and numbers represent the same meanings as above, n indicates an integer of 2 or 2)
The present invention provides a chemically modified polysaccharide consisting of a third repeating unit represented by:

The chemically modified polysaccharide of the present invention preferably has about 20 to about 85 second repeating units represented by formula (If) per 100 glucopyranosyl group units in the main chain, and the number of second repeating units represented by formula (If) is preferably about 20 to about 85.
It is a polysaccharide in which the number of third repeating units represented by I) is about 0 to about 30.

The chemically modified polysaccharide of the present invention typically has the following physical properties.
Indicates chemical properties.

111 When performing gel f filtration using a gel r superperformance liquid chromatography using a sodium chloride solution with a molecular concentration of 0.1 mol/l as a mobile phase and using Toyo 1st G-6000PW as a column, the molecule II - Elutes at a retention time of 100,000 to 1,500,000.

(2) Elemental analysis value Gives a value that substantially matches the value expected from the chemical formula. That is, when X in formula (n) is an N-hydroxyamide carbonyl group, C: 32.5% to 43.2% H: 4.5% to 6.2% N: 1.9% to 8.6 %, and in the case of (2-hydroxyethyl)amino group, give values of C: 35.0% to 4580% H: 50% to 6.2% N: 2.0% to 110%. give.

(3) Sulfuric acid decomposition Chemically modified polysaccharides were completely hydrolyzed with 2N sulfuric acid at 80°C for 18 hours and analyzed by gas chromatography.
Glucose is present, but glycerol is not.

(4) Hydrochloric acid decomposition Even when a chemically modified polysaccharide is dissolved in a 1N aqueous hydrochloric acid solution and boiled, no precipitate is produced.

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

(6) Infrared absorption spectra The infrared absorption spectra obtained by the potassium bromide tablet method are shown in Figure 1 (when X in formula (II) is an N-hydroxyamide carbonyl group) and Figure 2 (also for (2-hydroxyethyl)).
In the case of amino group).

(7) Methylation analysis The methylated sugar obtained by post-mortem hydrolysis of methyl is induced into alditol acetate and analyzed by gas chromatography. Tri0-methylglucose is isolated and identified. 2,5.4-)di-0-methylglucose and 2.3.4.6-tetra-0-methylglucose may be separated and identified, or may not be recognized.

Although the chemically modified polysaccharide of the present invention has very strong antitumor properties, its toxicity to mammals is extremely low, and its acute toxicity to mice is LDso value of 1500 μ/Y or more.

Similar to known antitumor polysaccharides, the chemically modified polysaccharide of the present invention can be dissolved or dispersed in a physiologically acceptable base and administered subcutaneously.
It can be administered by intramuscular or intravenous injection or other conventional methods. Dosage is approximately 0 per kg of body weight
．． 1 to about 100 to about 100, preferably about 1 to about 2
It is about 0rv.

The chemically modified polysaccharide of the present invention is represented by the formula (1), -[3) β-D-Glu (1÷→ (in the formula, Glu represents a glucopyranosyl group, and the numbers indicate the bonding positions). A main chain having glucopyranosyl group units as repeating units and K bonded to this main chain, represented by the formula (N) (in the formula, Glu and numbers have the same meanings as above, and m represents an integer from 0 to 2) a repeating unit having a carbonyl group, or a repeating unit having this carbonyl group and the formula (m), (in the formula Gl
u and numbers have the same meanings as above, and n is
It can be produced by reacting an aldehyde-type β-1,3-glutoyl compound consisting of repeating units represented by (integer number of ) with hydroxyurea or hydrazinoethanol to form a thick base, and then reducing this. can.

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 which is the starting material is converted into a repeating unit having a carbonyl group represented by the formula (II) according to the following reaction formula. converted to the represented repeating unit.

It can be prepared by decomposing the alkali-insoluble part (hereinafter referred to as the alkali-insoluble part) with periodate.

In the method of the present invention, the reaction of aldehyde-type β-1,3-glucan and hydroxyurea or 2-hydrazinoethanol to form a thick base is carried out in an aqueous medium for 1 g of the former to about 0.001 to about 0.00 g of the latter. 5 moles, preferably 0
．． This can be done by adding 0.01 to 0.1 mol. In this case, the aldehyde type β-1°3-glucan is well dispersed in an aqueous medium.

The amount of the aqueous medium relative to aldehyde type β-1,3-glucan is about 10 to about 1.000 times, preferably about 30 to 300 times, by weight. The liquid property during the reaction is DH about 5 to about 10, preferably about 6 to about 8.

Acids, such as hydrochloric acid, or alkalis, such as sodium hydroxide, can be used to adjust the liquid properties.

The reaction is usually carried out at a temperature of about 5 to about 80°C, preferably about 1
The temperature is about 0 to about 50°C.

The reaction time is usually about 10 hours to about 5 days, preferably about 2 days.

After forming a Schiff base in this way, reduction is performed. As the reducing agent, a strong reducing agent such as sodium borohydride or sodium cyanoborohydride is used. Particularly preferred is sodium cyanoborohydride. The amount of the reducing agent is at least equivalent to the carbonyl group in the aldehyde-type β-1,6-glucan used as the starting material. Although the concentration of the reducing agent is not limited, it is about 0001 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 10 to about 50°C, and the reaction time is usually about several hours to about 5 days, preferably about 2 days.

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

The present invention will be explained in more detail below with reference to Examples.

Example 1 1 g of aldehyde type β-1,3-glucan obtained in Raw Material Preparation Example 1 was placed in a 250 ml flask, 0.1 mole of hydroxyurea was added thereto, distilled water was added, and the total amount was 1 ml.
It was set as l. After the aldehyde type β-1,3-glucan was well dispersed with a disperser, the pH was adjusted to 7 with hydrochloric acid.
The mixture was stirred with a magnetic stirrer for 2 days.

Thereafter, the pH was adjusted to 6.5 with hydrochloric acid, and then 116 g of sodium cyanoborohydride was added, and the reaction was continued for another 2 days with stirring. After the reaction was completed, the reaction solution containing the suspended matter was centrifuged and filtered, and the water-soluble fraction was subjected to running dialysis with tap water. The contents of the dialysis tube were freeze-dried to obtain the desired chemically modified polysaccharide 72m9. (Yield: Z2%λ) The analysis results of the obtained chemically modified polysaccharide were as follows.

When performing gel filtration using a gel filtration high performance liquid chromatography using an aqueous sodium chloride solution with a molecular weight concentration of 01 mol/) as a mobile phase and using Toyo Soda Kogyo's G-(SOOOPW) as a column, it was found that It eluted at the time position.

Elemental analysis values C: 36.7% H: 5.1% N: 4.2% Infrared absorption spectrum The infrared absorption spectrum obtained 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 formula (II) (main chain 10
0 pieces) 82.5 pieces Number of repeating units represented by formula (III) (main chain 1
Example 2 Example 1 using the aldehyde polysaccharide obtained in Raw Material Preparation Example 2
Chemical modification was performed in the same manner as above to obtain a chemically modified polysaccharide. (
The results of analysis conducted in the same manner as in Example 1 were as follows.

molecular weight The molecular weight was eluted at a latency time of 950,000.

Elemental analysis value C: 391% H: 5.2% N: 5.5% Methylation analysis From the results of methylation analysis, the number of repeating units represented by formula (n) (main chain 100
per unit) 74 units The number of repeating units represented by the formula (n+) (main chain 10
Example 3 Chemistry of aldehyde type β-1,3,-glucan was carried out in the same manner as in Example 1 except that 0.1 mol of 2-hydrazinoethanol was used in place of 01 mol of hydroxysilver system. The desired chemically modified polysaccharide 83m9 was obtained by modification. (Yield 83%
) The results of the analysis conducted in the same manner as in Example 1 were as follows.

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

Elemental analysis values ('': 5B3% H: 54% N: 4.6% Infrared absorption spectrum The infrared absorption spectrum obtained by the potassium bromide tablet method is shown in Figure 2.

Methylation analysis From the results of methylation analysis, the number of repeating units (main chain 100
(per unit) 82.5 Number of repeating units represented by formula (1) (main chain 100
Example 4 Example 3 Using the aldehyde 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: 52%), and the results of analysis conducted in the same manner as in Example 1 were as follows.

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

Elemental analysis value C: 40.5% H: 58% N: 3.1% Methylation analysis From the results of methylation analysis, the number of repeating units represented by formula (If) (main chain 10
0 per unit) 74 Number of repeating units represented by formula (m) (main chain 100
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 x 10 6 Sarcoma 18o ascites cells were subcutaneously inoculated in the groin area. The experimental group consisted of 6 animals per group. Once a day for 10 days from the next day after cancer cell transplantation
The drug was intraperitoneally administered in olml. The test group included
5 m9 of the chemically modified polysaccharide of the present invention (obtained in Example 1)
The control group received only physiological saline. On day 35 after tumor implantation, the tumor was excised and its weight was measured. The tumor suppression rate of each group was calculated using the following formula.

where 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 wood ear mushrooms were thoroughly ground with 61 g of a 1% aqueous sodium chloride solution using a household mixer.
- Allowed to stand and soak day and night. Thereafter, 1% aqueous sodium chloride solution 31 was added, and the mixture was further heated at 60° C. for 6 hours with stirring to sufficiently swell the wood ear. In order to further refine the wood ear mushrooms, after crushing them with a homogenizer,
Hot water extraction was performed in an autoclave at ℃ for 20 minutes, and the hot water extracted fraction was removed by centrifugation. Regarding the residual fraction,
The charcoal water extraction operation was carried out in the same manner.

91 g of water and 324 g of sodium hydroxide were added to the residual fraction thus obtained (the total volume was 121 at this time), and alkaline extraction was performed at 60° C. for 4 hours under a nitrogen atmosphere. The alkaline extraction fraction was removed by centrifugation to obtain the alkaline extraction residue. Add 81 g of water and 156 g of sodium hydroxide to this alkaline extraction residue (at this time, the total volume is 91 g).
1), the same alkali extraction operation was performed again.

Water 101 was added to the residue of the alkali extract, and the operations of washing, centrifugation, and resuspension were repeated until the pH of the suspension became approximately 9. Dilute hydrochloric acid was added to the suspension to adjust the pH to 7.

Next, 51 liters of water was added to this suspension, which was then 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 alkali-insoluble portion (yield: 29%).

This substance is substantially illuminated by the formula (I) -03) β-D-Glu (1-3-+ (in the formula, Glu and numerals have the same meanings as above)
, 3-glucopyranosyl group unit as a repeating unit, and a repeating unit bonded to this main chain represented by formula (m) (in the formula, Glu and numbers have the same meanings as above). Repeat unit 1
The polysaccharide contained approximately 82.5 repeating units of formula (I) per 00 units. The average value of n was approximately 01.

Preparation of aldehyde type polysaccharide Contents: 25 g of alkali-insoluble portion in a narrow-mouthed brown bottle with a capacity of 5 mm.
After adding 51 liters of distilled water and thoroughly dispersing the alkali-insoluble portion using a magnetic stirrer, the mixture was degassed using an aspirator. Then sodium metaperiodate 66
After adding and dissolving 9, the mixture was allowed to react at room temperature for 7 days with stirring. After the reaction was completed, water washing and centrifugation were repeated three times to remove the generated formic acid and the remaining sodium metaperiodate. Add water to the solid phase and lyophilize for 20
．． 59 aldehyde type polysaccharides were obtained. (Yield: 82%) This aldehyde type polysaccharide consists essentially of a main chain represented by formula (1) and a repeating unit represented by formula (IV).
, 3-glucan. The average value of m in formula (IV) was approximately 01. Also, the main chain 10 represented by formula (1)
The number of repeating units represented by the formula (■) per 0 units was 82.5. The nitrogen content of this aldehyde type polysaccharide was below the limit of quantification.

Raw Material Preparation Example 2 Raw material IAI was produced in the same manner as in Raw Material Preparation Example 1 except that the amount of sodium metaperiodate used in the preparation of the aldehyde polysaccharide was changed to 13.29 to obtain an aldehyde polysaccharide. Ta. This aldehyde type polysaccharide consists essentially of a main chain represented by formula (1), a repeating unit represented by formula (II), and a repeating unit represented by formula Ov).
, 6-glucan.

The average values of m in formula (lit) and formula (IV) were approximately 01. Also, the main chain 10 represented by formula (I)
The number of repeating units represented by formula (m) was 8.5, and the number of repeating units represented by formula (N) was 74.

[Brief explanation of drawings]

FIG. 1 and FIG. 2 are diagrams showing infrared absorption spectra of the chemically modified polysaccharide of the present invention. Patent applicant: Toyo Soda Kogyo Co., Ltd. (%)

Claims (7)

[Claims] (1) Formula (I), i→3) β-1,3-glucopyranosyl group unit represented by β-D-Glu (1→→ (in the formula, Glu represents a glucopyranosyl group, and the numbers indicate the bonding positions) Formula (I[), -+3) β-D-Glu6(1→
→ A second compound represented by or this second repeating unit and a third repeating unit represented by the formula (El), (in the formula, Glu and numerals have the same meanings as above, and n represents an integer from 0 to 2) A chemically modified polysaccharide consisting of repeating units. (2) The number of second repeating units is about 20 to about 85 and the number of third repeating units is about 20 to about 30 per 100 glucopyranosyl group units in the main chain, claim 1. Chemically modified polysaccharides as described. (3) The claimed polysaccharide is a chemically modified polysaccharide having a molecular weight of about 100,000 to about 1,500,000 in gel f-superperformance liquid chromatography using an aqueous sodium chloride solution with a concentration of 0.1 mol/l as a mobile phase. Chemically modified polysaccharide according to FM item 1 or 2. (4) Formula (I) publication → 3) β-D-Glu (1 → → (in the formula, Glu represents a glucopyranosyl group, and the numbers indicate the bonding position) β-1,3-gelcopi-2-nosyl group unit A main chain having as a repeating unit and a carbonyl group bonded to this main chain represented by the formula or a repeating unit having this carbonyl group and a repeating unit represented by formula (1), (in the formula, Glu and numerals have the same meanings as above, and n represents an integer from 1 to 2). Type-: Formula (1) % Formula % (1 (Formula A main chain having repeating units of β-1,3-glucopyranosyl group represented by Glu and numbers have the same meanings as above), and a main chain with the formula (
n) a second repeating unit represented by (representing taste), or this second repeating unit and formula (11) (in which Glu, r
1. A method for producing a chemically modified polysaccharide, which comprises obtaining a chemically modified polysaccharide consisting of a third repeating unit represented by (l and numerals have the same meanings as above). (5) The number of repeating units having a carbonyl group represented by formula (■) is about 20 to about 85 per 100 glucopyranosyl group units in the main chain, and the number of repeating units represented by formula (1) is about 20 to about 85. Using 0 to about 30 aldehyde-type -β-1,3-glucan as a starting material, the number of second repeating sites represented by formula (I[) per 100 glucopyranosyl group units in the main chain is Approximately 20
5. The manufacturing method according to claim 4, wherein the chemically modified polysaccharide has a number of third repeating units represented by formula (11) ranging from about 85 to about 30. (6) A patent claim for obtaining a chemically modified polysaccharide having a molecular weight of about 100,000 to about 1.5 million in gel filtration high performance liquid chromatography using an aqueous sodium chloride solution with a concentration of 0.1 mol/l as a mobile phase. The manufacturing method according to item 4 or 5. (7) Aldehyde type-β-1 used as starting material,
3-Glue force y is obtained by oxidizing the alkali-insoluble portion of wood ear fruiting body with periodic acid, Claim 4
The manufacturing method described in any one of Items 6 to 6.