JPS6377901A - Novel polysaccharides - Google Patents

Abstract

PURPOSE:To obtain novel polysaccharides which comprises a low-molecular beta-1,3-glucan excellent in water solubility and can give an antitumoral agent which shows an excellent effect at a low dosage, by extracting volvaria with an aqueous alkali solution and decreasing the MW of the extract. CONSTITUTION:Fruit bodies or mycelia of volvaria are extracted with 5-100pts.wt., per pt.wt. their dry weight per extraction, an aqueous alkali solution (0.1-3-N concentration) at 30 deg.C or below in an inert gas atmosphere such as nitrogen, and quaternary acidic substances, etc., are precipitated from the extract by the addition of a quat. ammonium salt and filtered off. The supernatant liquid is irradiated with ultrasonic waves at a power of 100-1,000W and a frequency of 5-100kHz for 20-500hr, and this treatment is stopped when the molecular weight reaches several thousand to 100,000 when it is traced by means of gel filtration high-performance liquid chromatography in which dimethyl sulfoxide is used as the mobile phase. A polysaccharide comprising first repeating units of formula I, second repeating units of formula II and third repeating units of formula III (wherein Glu is a glucopyranosyl group and numerals are positions of bonding), wherein the number of said third repeating units present is 4-12 on the average, and the sum of said second and third repeating units is 16-33 is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to the field of industrial application.
vacea) and its anti-MS agent.

[Conventional technology]

+9 Extraction method using alkaline solution, lentinan, and schiffilan from only basidiomycete bacteria. β-1,3 glucan derived from Fukurotake, which has a unique structure with a lower branching frequency than the branched β-1,3 glucan obtained from scleroglucan and the fruiting body of auricular fungus, can be obtained, and this β-1, It is known that 5-glucan is effective as an antitumor agent (Japanese Patent Application Laid-Open Nos. 59-45301 and 1983).
9-55424).

[Problems to be solved by the invention 5] However, the β-1,5 glucan derived from Fukurotake is difficult to dissolve in water or physiological saline after being dried by freeze-drying or the like. Furthermore, even if the solution is in a solution state during production, when operations such as desalting and concentration are performed, precipitation still occurs over time, which poses a practical problem.

[Means for solving problems]

The present inventors conducted intensive research to solve this fatal drawback, and found that when the molecular weight of the β-1,3 glucan was lowered to a certain range, the solubility in water was surprisingly completely improved. At the same time, we discovered that the medicinal efficacy is almost the same as that of the above-mentioned β-t3 glucan, and moreover, the optimal dosage is unexpectedly lower, leading to the present invention.

Generally speaking, it is said that the lower the molecular weight of β-1,3 glucan, which has antitumor properties, the lower its medicinal efficacy, and for schizophyllan, the lower limit of its molecular weight is approximately 1
It is said to be 0,000. There is also a theory that the reason for the decrease in drug efficacy due to this decrease in molecular weight is that the triple helix structure of glucan is involved in the expression of drug efficacy, and that the decrease in molecular weight destroys this structure.

However, even if the molecular weight of the novel polysaccharide of the present invention is 100,000 or less, its medicinal efficacy does not decrease at all. Moreover, according to the polysaccharide of the present invention, the optimal dose is lowered, resulting in the provision of a practically more useful antitumor agent.

The novel polysaccharide of the present invention can be obtained by reducing the molecular weight of the β-1,3 glucan by a known method. For example, methods such as ultrasonic treatment, enzyme treatment with β-1°3 glucanase, and chemical treatment with acid or the like are used.

The present invention firstly provides a first repeating unit represented by -→3) β-D-Glu (1□) obtained from Fukurotake, a second repeating unit represented by the formula, and The third repeating unit represented by the formula β-D-Glul ↓ β-n-01u: ↓ 11 → 5) β-D-axu6 (t −-→ (in each formula, Glu represents a glucopyranosyl group, represents a bonding position), and the average number of third repeating units is about 4 to about 12 per 100 of the sum of the numbers of first, second and third repeating units,
The sum of the numbers of the second and third repeating units is also about 16
This polysaccharide has a molecular weight of several thousand to about 100,000 in gel-based high-performance liquid chromatography using dimethyl sulfoxide as a mobile phase, and is suitable for methylation analysis. According to 1
46-tetra-0-methylglucose, 2.4.6-)
Di-0-methylglucose, '2. 4-tri-o-methylglucose and 2.4-di-0-methylglucose; 2. 1 4,6-chitler O-methylglucose
When the molar ratio is 2.4.6-) ly-0-methylglucose is about 2.7 to about 5.0.1-
Methylglucose from about 11 to about 11L55 and 2.
It consists of a novel polysaccharide that provides 4-di-Q-methylglucose in a proportion of about 0.7 to 1.3.

Second, the present invention provides, as an active ingredient, a first repeating unit essentially represented by the formula % (1) obtained from Fukurotake, the formula β-D-Glu.

↓ 1-→3) β-D-Glu' (the second expressed as 1-1→
It consists of a repeating unit of formula 4 and a third repeating unit represented by formula % (in each formula, Glu represents a glucopyranosyl group, and the number represents the bonding position),
1st. The average number of third repeating units is about 4 to about 12 per 100 of the sum of the numbers of second and third repeating units, and the sum of the numbers of second and third repeating units is also about 16. It is an anti-malignant drug containing a polysaccharide having a molecular weight of from several thousand to about 100,000 when measured by high-performance liquid chromatography using dimethyl sulfoxide as a mobile phase, and Methylation analysis revealed that this polysaccharide is 2,3,4,6-tetodiO-methylglucose, 2.4.6-)! J-〇-methylglucose, 2.44-tri-o-methylglucose and 2.4-di-0-methylglucose, 1.46
-Tetra-0-methylglucose is 1, the molar ratio is 2.4.6-)di-0-methylglucose #2.7 to about 5.0. To Otsu 4-trilomethylglucose approx. 0. 1 to about 0.35 and 2,4-di-O-methylglucose in a ratio of about 0.7 to 1.3.

The physical and chemical properties of the polysaccharide of the present invention are as follows.

(1) Solubility Soluble in water, aqueous alkaline solution and dimethyl sulfoxide, and substantially insoluble in methanol, ethanol, n-butanol, acetone, benzene, toluene, ethyl acetate, propylene glycol, pyridine, etc.

(2) Optical rotation of a 0.5N aqueous sodium hydroxide solution of the polysaccharide of the present invention (
15%), the specific optical rotation is [α]■≠−12°.

(3) Elemental analysis value When carefully and thoroughly dried, it gives a value close to the calculated value from 0nHznOn, and 0343-45% H3S, 6-&4N: below the limit of quantification. (4) Infrared absorption analysis Infrared absorption spectrum by KBr tablet method. Shown in Figure 1. Absorption in the 896 muscle is unique to the β-bond of the D-glucose radical.

(51015-NMR analysis δ value (ppm) s 1056.86.8.7/>9.
75j! , 742°7 five paper x Roy and beak weight on 61.4.

(6) Color reaction Anthrone reaction: @Ninhydrin reaction; Negative dish carbazole reaction; Negative (7) Molecular weight: eluted in the range of 1,000 to about 100,000 molecules in gel-based high performance liquid chromatography using dimethyl sulfoxide as the mobile phase. do.

(8) Reaction with cetylpyridinium chloride No precipitate is formed with cetylpyridinium chloride in the aqueous solution.

(9) Constituent Saccharide When the polysaccharide of the present invention is hydrolyzed in a 1N sulfuric acid aqueous solution at 100°C and then identified by paper chromatography, and after being converted into alditate acetate and identified by gas chromatography, only glucose is detected. .

By a1 bond mode methylation analysis, the molar ratio of 2.3,4,6-tetra-0-methylglucose 10
02,4.6-)di-0-methylglucose approx. 2.7
from about 052.44-)di-0-methylglucose
about α1 to about α35 and 2,4-di-0-methylglucose about 0.7 to about 1.3.

When enzymatically degraded using exo-type β-1,3 glucanase, glucose and gentiobiose are produced.Also, after sufficient oxidation with sodium metaperiodate and reduction with sodium borohydride, under mild conditions, e.g.
or IIL2 normal sulfuric acid, 80°C to 100°C, 1
When hydrolyzed for about 2 hours, linear β-1,3 glucan from which glucose groups corresponding to side chains have been removed is obtained.

This substance was methylated by 2.4.6-)di-0-
Methylglucose and trace amounts of 2,3,4,6-tetra-
Give 0-methylglucose. This linear β-1,
When 3-glucan is enzymatically degraded with exo-type β-1,3-glucanase, only glucose is produced. Therefore, the main chain of this polysaccharide consists essentially only of β-1,3-linked glucose groups.

From these facts, this new polysaccharide is clearly a different substance from the β-1,5 glucan disclosed in Japanese Patent Application No. 57-157058 in terms of water solubility and molecular weight.

The novel polysaccharide of the present invention is a basidiomycete (
Volvariella Volvacea) It can be easily prepared by extracting the mushroom (Volvariella Volvacea) with an alkaline aqueous solution and then reducing the molecular weight.

The mushroom used as a raw material may be either its fruiting body or mycelium. These may be in a raw state or a dried product.

Shredding raw materials before extraction is effective for increasing extraction efficiency.

As the alkaline aqueous solution used for extraction, alkaline hydroxides such as sodium hydroxide and potassium hydroxide can be used. In particular, IJium hydroxide is convenient.

The concentration of the alkali used is not particularly limited, but it is preferably used within the range of about α1N to about 3N. The amount of alkaline aqueous solution used for extraction is usually about 5 to about 100 times the dry weight of the raw material per extraction. If this amount is too small, the extraction efficiency will be poor, and if it is too large, post-processing will be difficult.

This extraction step is preferably carried out under an atmosphere of an inert gas such as nitrogen in order to prevent polysaccharide decomposition or denaturation such as peroxidation. Furthermore, the reaction may be carried out in the presence of a reducing agent such as sodium borohydride.

There are no particular limitations on the extraction temperature, but it is preferably about 30°C or lower. This is because polysaccharides having even more branches are extracted when extracted at higher humidity, for example, at about 60 to 80° C., which is the temperature condition of normal hot alkaline extraction.

The extraction operation may be repeated.

Fukurotake, which is a raw material for the novel polysaccharide of the present invention, contains many components within its bacterial cells in order to maintain its own life. Therefore, if the polysaccharide is directly extracted with an alkaline aqueous solution, various impurities will be included in the target polysaccharide. Although it is possible to prepare the desired polysaccharide from this extract, it requires complicated steps. Since the polysaccharides of the present invention cannot be extracted with solvents such as water and alcohol, these impurities are removed using water, hot water, a buffered aqueous solution, alcohol, or a combination thereof prior to the step of extraction with an alkaline aqueous solution. It is preferable to remove it.

In addition, when performing alkaline aqueous solution extraction by such an operation,
Mannogalactan, a protein that becomes a miscellaneous substance.

Gly;-Dan-like polysaccharides and the like can be removed in advance.

The alkaline aqueous solution extracted in step 5 was prepared using hydrochloric acid,
Neutralize with acid such as acetic acid. Normally in this state the polysaccharide is dissolved in the salt solution. Next, a quaternary ammonium salt such as cetylpyridinium chloride is added to this solution to precipitate contaminating acidic substances as insoluble precipitates. This precipitation is
Remove by normal separation means such as filtration or centrifugation. The supernatant liquid obtained in this step is dialyzed as it is or with a concentrated liquid or water, and the dialysate is dried and then subjected to the next step of reducing the molecular weight.

As mentioned above, known methods such as ultrasonic treatment, enzyme treatment, and chemical treatment with acids can be used to reduce the molecular weight, but the method itself is simple and post-treatment is easy. It is usually convenient to use an ultrasonic treatment method because of this and because polysaccharides of arbitrary molecular weights can be easily prepared.

It is sufficient that the ultrasonic generator used has an output of 100 to 1000 W and a frequency of 5 to 100 KH2. Usually, one with an output of 100 to 100 kHz and a frequency of 10 to 20 KHz is used.

The ultrasonic irradiation time requires 20 to 500 hours when the above-mentioned normal ultrasonic generator is used. If the irradiation time is too short, the molecular weight will not be reduced enough and the polysaccharide will not dissolve completely in water, and if the irradiation time is too long, the molecules will
Considering the irradiation time, it is not small and is a waste of time. In reality, the ultrasonic irradiation time is determined by tracking the ultrasonic treatment using high-speed liquid chromatography.
When the temperature is within the range of 10,000, the treatment is finished and the object of the present invention is obtained.

For ultrasonication, the above supernatant liquid may be used as it is, or
Once dried as described above, it may be redissolved and used. When redissolving, the alkali-extracted polysaccharide does not dissolve in water, so it is first dissolved in an alkaline aqueous solution, then neutralized with an acid such as acetic acid, and subjected to ultrasonication. There is nothing wrong with dissolving this polysaccharide in dimethyl sulfoxide, which is a solvent, and subjecting it to ultrasonication.

The concentration of the solution used for ultrasonication is approximately 0.1% to 1%. If the concentration is too low, the efficiency will be poor, and conversely, if the concentration is too high, the viscosity of the solution will become extremely high, making ultrasonic treatment practically impossible.

After irradiating ultrasound for a predetermined period of time, the target product is recovered from the solution, but the treated solution contains metal pieces from the ultrasonic crusher, oligosaccharides with extremely low molecular weights, and salts that have no medicinal properties. After removing the precipitate using normal methods such as centrifugation,
Remove salts and oligosaccharides with extremely low molecular weight using ultrafiltration or dialysis tubing. The solution thus obtained is dried to obtain the desired polysaccharide. The drying method in this case is
Appropriate drying means such as vacuum drying, freeze drying, spray drying, etc. can be used.

When using dimethyl sulfoxide, after centrifugation,
It is convenient to obtain the substance of the invention by diluting with water, dialysis and drying.

The antitumor agent of the invention may contain the polysaccharide thus obtained in a conventional formulation.

For example, it can be prepared by dissolving it in physiological saline containing an auxiliary agent if necessary.

[Effect]

The antitumor agent of the present invention can be easily administered, for example, intravenously, like immunostimulants such as lentinan and schizophyllan. It can also be used in combination with other antitumor agents.

〔Example〕

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

Example 1 [Alkali Extraction] 500 g of freeze-dried Fukurotake fruiting body was soaked overnight in 6 t of 11M sodium phosphate buffer solution containing 0.9% sodium chloride and having a pH of 7.0, and then ground with a mixer and a homogenizer. Furthermore, the same phosphate buffer 6
The mixture was incubated for 24 hours and centrifuged. The obtained precipitate was dispersed in 12 t of water and placed in an autoclave for 120 t.
Heated at ℃ for 30 minutes. After cooling, it was centrifuged to obtain a precipitate. This hot water extraction process was repeated once to almost completely remove the water-soluble fraction.

The water-insoluble fraction obtained in this way is purified by borohydride).
It was dispersed in 15 t of a 1N aqueous sodium hydroxide solution in which 5 g of IJum was dissolved. The mixture was stirred at 25° C. for 4 hours under a nitrogen stream and then centrifuged. This precipitate was subjected to repeated extraction operations using a 1N aqueous sodium hydroxide solution. Rain sodium hydroxide extracts were combined and neutralized with concentrated hydrochloric acid to adjust to pH &5.

The extract thus obtained was added to an aqueous cetylpyridinium chloride solution (
10%) was added dropwise with stirring until no new precipitate was formed, followed by centrifugation at 10,000 G for 30 minutes to remove the formed precipitate. An equal volume of methanol was added to this supernatant and stirred to precipitate the polysaccharide. 2.5 tons of distilled water was added to this precipitate, dispersed with a homogenizer, and then dialyzed in running water for one week. The dialyzed fluid was freeze-dried to obtain alkaline extract 659 from the fruiting body of Fukurotake.

[Ultrasonic treatment]

After dissolving the previously prepared alkaline extract 29 in 400 cc of 1N aqueous sodium hydroxide solution, the solution was adjusted to pH 7 with acetic acid.
, OK adjusted. Add BRANSON's Mode to this solution.
1185 ultrasonic continuous treatment equipment for a total of 3α5 hours, 1
Ultrasonic waves of 50 W and 20 KHz were irradiated.

After ultrasonication, the mixture was centrifuged to remove metal pieces and the like, and then dialyzed for one week using a dialysis tape with a cut-off molecule of f15000 to remove salts, oligosaccharides, and the like.

The dialysis fluid was freeze-dried to obtain polysaccharide 1.267 (yield 63%) of the present invention.

The physical and chemical properties of this polysaccharide were as follows.

(1) Elemental analysis values os454% H;&4 N: Below the limit of quantification (2) Solubility It quickly dissolved in neutral water at room temperature.

(3) Specific rotation (4) Molecular weight Toyo Soda Kogyo ■ using dimethyl sulfoxide as a mobile phase
It was eluted at a molecular weight of 45,000 by gel filtration high performance liquid chromatography using a column manufactured by G-6000FW. As the standard substance for molecular weight determination at this time, standard dextran manufactured by Pharmacia was used.

(5) Infrared absorption spectrum Figure 1 shows the 7-Lier transform infrared absorption spectrum obtained by the KKBr tablet method.

+61015 NMR analysis 1 Color reaction and reaction with cetylpyridinium chloride The physical and chemical properties were the same as described above.

(7) Constituent sugars and binding mode The obtained polysaccharide was methylated, further hydrolyzed, and then induced to alditol acetate, which was analyzed by gas chromatography. The obtained methyl Hokucho ratio is 2, to 4.6-tetra-0-methylglucose 1.
002, 4.6-) Li-Methylglucose
2.852.3.4-)di-0-methylglucose
0.242.4-di-0-methylglucose
1.02 separately, this polysaccharide is exo-type β-1,
It was enzymatically digested with 3 glucanases. Glucose and gentiobiose were obtained.

Furthermore, this polysaccharide was sufficiently oxidized with sodium metaperiodate and reduced with sodium hydride.

When this was further hydrolyzed in α1 normal sulfuric acid at 100° C. for 2 hours, a linear glucan from which glucose groups corresponding to side chains were removed was obtained.

When we conducted methylation analysis in the same way, we found that 2, 4
．． 6-tri-o-methylglucose and trace amounts of 2,44
．． 6-Chitler O-methylglucose was given. In addition, enzyme analysis using exo-type β-1,3 glucanase revealed that
Only glucose was produced.

Therefore, the obtained polysaccharide consists of repeating units of formula 11 formula 2 and formula 3, and the ratio of the numbers is 6 repeating units of formula 3 per 100 repeating units in total, 6 repeating units of formula 2 The sum of the repeating units of and the repeating units of formula 3 was 26.

Example 2 The objective product of the present invention t 209 (yield 60%) was produced in the same manner as in Example 1 except that the ultrasonic irradiation time was 120 hours.
I got it.

According to the method of Example 1, the molecular weight of this polysaccharide is 2-7
It eluted at the 10,000 position. It also dissolved rapidly in neutral water at room temperature.

Other physical and chemical properties of this polysaccharide are shown in Example 1
It was almost the same as the substance of

Example 3 The effect of the polysaccharide of the present invention on Sarcoma 180 solid tumor was tested using IOR mice weighing approximately 259 kg.

Six million ascitic Sarcoma 180 cells were inoculated subcutaneously from the groin to the back. The experimental group is 1 group 6
I took it as a fish. The drug was administered intraperitoneally once a day for 10 days starting the day after tumor cell inoculation. Physiological saline containing α5% carboxymethyl cellulose was used as the control group, and the test group was prepared by dissolving the polysaccharide of the present invention in the saline at a dosage of 2 kg/day. Using. Tumors were excised 35 days after tumor implantation and their weights were measured. The layer temperature suppression rate for each group was calculated using the following formula.

Tumor suppression rate Shin) = Lnie x 100 to C: Average tumor weight of control group T: average tumor weight of test group The results are shown in Table 1.

Sample Average tumor weight Abscess inhibition rate Product of Example 1 021g 9 & 1% Product of Example 2 α
11 9aO control 5.39
Example 4 The dosage of the polysaccharide of the present invention was adjusted to α08. cL4, 2.0°10 and 50"S' / PC9/ (Tested in the same manner as in Example 5, except that it was 1 & 7. As a comparative example, the alkaline extract of Example 1 (insoluble in water, high molecular weight (1) which has not been sonicated (referred to as glucan A)
was tested. The results are shown in Table 2.

Table 2 Dosage and tumor inhibition rate Dosage Product of Example 1 Comparative example (Glucan A)
α08'9/9/day 99.0%
49% [lL4 99.6
9&? 2
9 & 1 99.610
64.9 9&
? 50 47
64.0 (The number indicates the tumor suppression rate) [Effects of the invention] The polysaccharide of the present invention has excellent medicinal efficacy and is soluble in water, so it can be used as a tumor suppressant and has a low It can be used in various dosages, further increasing its practical value.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an infrared absorption spectrum of the novel polysaccharide of the present invention.

Claims (8)

[Claims] (1) Essentially the formula → 3) β-D-Glu (1- The first repeating unit represented by the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ The second repeating unit and the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼ Consists of a third repeating unit represented by (in each formula, Glu represents a glucopyranosyl group, and the number represents the bonding position),
The average number of third repeating units is about 4 to about 12 per 100 total numbers of first, second, and third repeating units, and the sum of the numbers of second and third repeating units is about 4 to about 12. A polysaccharide which also has about 16 to about 33 molecules and gives a molecular weight of several thousand to about 100,000 when analyzed by high-performance liquid chromatography using dimethyl sulfoxide as a mobile phase. (2) The polysaccharide according to claim 1, which is a polysaccharide derived from Fukurotake. (3) Methylation analysis revealed that 2,3,4,6-tetra-
o-methylglucose, 2,4,6-tri-o-methylglucose, 2,3,4-tri-o-methylglucose and 2,4-di-o-methylglucose, 2,3,4
, 6-tetra-o-methylglucose is 1, the molar ratio is about 2,4,6-tri-o-methylglucose.
7 to about 5.0, 2,3,4-tri-o-methylglucose about 0.1 to about 0.35 and 2,4-di-o-
3. A polysaccharide according to claim 1 or 2, which is provided in a proportion of about 0.7 to 1.3 methyl glucose. (4) It is soluble in water, aqueous alkaline solution, and dimethyl sulfoxide, and substantially insoluble in methanol, ethanol, n-butanol, acetone, propylene glycol, benzene, toluene, and ethyl acetate. The polysaccharide according to any one of Items 1 to 3. (5) As an active ingredient, the first repeating unit is essentially represented by the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ The second repeating unit is represented by the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ and a third repeating unit represented by the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In each formula, Glu represents a glucopyranosyl group, and the number represents the bonding position),
The number of fifth repeating units is about 4 to about 12 on average per 100 total numbers of first, second and third repeating units, and the sum of the numbers of second and third repeating units is about 4 to about 12. An antitumor agent containing a polysaccharide having about 16 to about 33 polysaccharides and giving a molecular weight of several thousand to about 100,000 when measured by high-performance liquid chromatography using dimethyl sulfoxide as a mobile phase. (6) The antitumor agent according to claim 5, wherein the polysaccharide is a polysaccharide derived from Fukurotake. (7) Polysaccharides were found to be 2, 3, 4, 6 by methylation analysis.
-tetra-o-methylglucose, 2,4,6-tri-
o-methylglucose, 2,3,4,-tri-o-methylglucose and 2,4-di-o-methylglucose, 2,3,4,6-tetra-o-methylglucose in 1
2,4,6-tri-o-methylglucose in a molar ratio of about 2.7 to about 5.0, 2,3,4-tri-o-
- methylglucose from about 0.1 to about 0.35 and 2,
7. The antitumor agent according to claim 5 or 6, wherein the antitumor agent is given at a ratio of about 0.7 to 1.3 of 4-di-o-methylglucose. (8) The polysaccharide is soluble in water, alkaline aqueous solution and dimethyl sulfoxide, and is soluble in methanol, ethanol, n-butanol, acetone, propylene glycol, benzene,
The antitumor agent according to any one of claims 5 to 7, which is substantially insoluble in toluene and ethyl acetate.