JPH1180206A - Glucomannan isolated from Himematsutake culture and antitumor agent containing the same as active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antitumor agent by separating a glucomannan having a mannose chain of β-1, 2 bonds as its primary chain from an Agaricus blazei mushroom culture. SOLUTION: A mecelia is grown by inoculating an Agaricus blazei mushroom spawn to a liquid culture medium for basidiomycetes and shake-culturing it in an aerobic condition at 30 deg.C for 14 days. The mecelia is obtained by centrifugalizing the culture system. A extractive liquid is obtained by adding water 10 times as much to the mecelia and carrying out a hot water extraction under boiling for 2 hr. After the extractive liquid is vacuum-concentrated to 1/10, a supernatant liquid is obtained by centrifugation. The equal amount of 99% ethylalcohol is added to the supernatant liquid to precipitate polysaccharides. The precipitate is obtained by centrifugation and freeze-dried. The freeze-dried material is subjected to further treatments such as salt-out, separation, elution and the like to give a glucomannan having a mannose chain of β-1, 2 bonds as its primary chain of the formula (wherein Glc is a glucose residual group; and Man is a mannose residual group). This glucomannan exhibits a strong antitumor action.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to the genus Agaricus (Ag
aricus mushroom, Himematsutake (Agar)
icus blazei), a glucomannan isolated from a culture of Kawariharatake, and an antitumor agent containing the same as an active ingredient.

[0002]

2. Description of the Related Art It has been reported that a water-soluble component isolated from a mushroom culture has various pharmacological actions. As for Himematsutake, it has been reported that a water-soluble acidic polysaccharide component, a neutral polysaccharide component and a protein polysaccharide component separated from the fruit body have an antitumor effect (Japanese Patent Laid-Open No. 1-6).
7194, JP-A-1-67195, JP-A-2-7863
0) Further, it has been reported that a water-soluble complex component separated from the fruiting body has a liver function improving effect, an anticancer effect and an immunological function lowering improving effect (JP-A-2-124829,
JP-A-6-128164, JP-A-7-258107),
Furthermore, it has been reported that the water-soluble polysaccharide component separated from the cultured mycelium and the culture filtrate has an antitumor effect (JP-A-55-108292, JP-A-55-10829).
3). However, in these conventional reports, among a wide variety of water-soluble components isolated from the culture of Himematsutake,
It is not clear what the body exhibits pharmacological action, especially the body which exhibits antitumor effect.

[0003]

The problem to be solved by the present invention is to elucidate which of the various water-soluble components isolated from the culture of Himematsutake has a strong antitumor effect, It is in providing a more effective antitumor agent.

[0004]

Means for Solving the Problems Thus, the present inventors have
As a result of research to solve the above problems, it was found that, among various kinds of water-soluble components isolated from a culture of Himematsutake, a glucomannan having a specific structure has a strong antitumor effect.

That is, the present invention provides a glucomannan having a β-1,2 linked mannose chain as a main chain, which is isolated from a culture of Himematsutake and a repetition represented by the following formula 1, which is isolated from a culture of Himematsutake: The present invention relates to a glucomannan composed of units and an antitumor agent containing these glucomannans as an active ingredient.

[0006]

(Equation 1)

In the formula 1, Glc: glucose residue Man: mannose residue

[0008] The Himematsutake culture used in the present invention is a fruiting body, a cultured mycelium or a culture filtrate of Himematsutake.
These can be obtained by a solid culture method or a liquid culture method usually used for culture of basidiomycetes, but for convenience of operation, are preferably obtained by a liquid culture method. For example, carbon sources such as glucose, saccharose, maltose, ammonium sulfate, ammonium nitrate, nitrogen sources such as sodium nitrate, malt extract, yeast extract, natural complex nutrients such as corn staplica, phosphates, magnesium salts, potassium salts and the like A normal sterilized liquid medium adjusted to about pH 6.0 composed of inorganic salts and other trace elements is inoculated with a seed of the genus Himematsutake at a temperature of about 30 ° C. for about 10 to 25 days while preventing aerobic contamination with various bacteria. When shaking culture or aeration and stirring culture under the conditions, mycelium grows.At this stage, a culture mycelium of Himematsutake and a culture filtrate can be obtained by centrifuging or filtering the culture system. Some shock is applied to the culture system at the stage of growth to form fruiting bodies, and aerobically until the desired size is reached. Can be obtained fruiting bodies himematsutake by continuing the cultivation under.

[0009] The glucomannan of the present invention is a water-soluble polysaccharide isolated from the culture of Himematsutake as described above.
As will be described in detail later, the glucomannan of the present invention can be isolated from the fruit body or cultured mycelium of Himematsutake as follows. First, the dried fruit body or cultured mycelium of Himematsutake is extracted with hot water to obtain an extract. The extract is concentrated, and the concentrate is subjected to alcohol precipitation to obtain a precipitate. The aqueous solution of the precipitate is deproteinized by salting out to obtain a non-protein fraction. Next, this non-protein fraction is fractionated by ion exchange chromatography by a stepwise method to obtain a water-eluting fraction, a 0.5 M aqueous sodium chloride solution eluting fraction and a 1.0 M aqueous sodium chloride eluting fraction. The major polysaccharides in these eluted fractions were completely methylated by the Hakomori method, completely hydrolyzed, alditolated and acetylated, analyzed by GC / MS, and combined with ¹³ C-NMR.
According to the analysis, glucomannan having a number average molecular weight of 100,000 to 2.5 million (GPC method, pullulan conversion, the same applies hereinafter) having a β-1,2 bond mannose chain as a main chain is contained.

The fraction eluted with the aqueous solution of 0.5 M sodium chloride is repeatedly purified by a gradient method by ion exchange chromatography and gel chromatography to obtain a substantially single peak purified fraction. The purified fraction was analyzed by GC / MS in the same manner as described above, and also analyzed by ¹³ C-NMR. As a result, it was found that glucomannan having an average molecular weight of 2083000 composed of the repeating unit represented by Formula 1 was contained. ing.

When the glucomannan of the present invention is separated from the culture filtrate of Himematsutake, the culture filtrate is concentrated, and the concentrated liquid is precipitated with alcohol to obtain a precipitate. Thereafter, this precipitate is treated in the same manner as described above to obtain glucomannan.

[0012] Glucomannan thus isolated from the culture of Himematsutake has a remarkable antitumor activity against ICR / Slc mice transplanted with Sarcoma 180 solid tumor.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention include the following 1) to 3). 1) Inoculating a liquid medium for basidiomycetes with a seed of Himematsutake and shaking the culture at 30 ° C for 14 days under aerobic conditions to grow mycelium. The culture is centrifuged to obtain mycelium.
A 10-fold amount of purified water is added to the mycelium and extracted with hot water for 2 hours under boiling to obtain an extract. The extract is concentrated under reduced pressure to 1/10 and centrifuged to obtain a supernatant. Equivalent volume of 99 in the supernatant
% Ethyl alcohol is added to precipitate the polysaccharide, centrifuged to obtain a precipitate, and freeze-dried. The freeze-dried product is salted out with ammonium sulfate, and fractionated into a protein fraction and a non-protein fraction. The non-protein fraction was subjected to DEAE-Sepha
Fractionate byose CL-6B gel chromatography. Elution was performed by distilled water, 0.5% by the stepwise method.
This is performed using an aqueous M sodium chloride solution, a 1.0 M aqueous sodium chloride solution, and a 2.0 M aqueous sodium chloride solution.
The glucomannan of the present invention is contained in the fraction eluted with distilled water, the fraction eluted with a 0.5 M aqueous sodium chloride solution, and the fraction with a 1.0 M aqueous sodium chloride solution. A glucomannan having a β-1,2 linked mannose chain as a main chain, which is isolated from a cultured mycelium of Himematsutake.

2) The fraction eluted with the 0.5M aqueous sodium chloride solution of 1) was further subjected to DEAE-S by a gradient method.
Purify repeatedly by epharose CL-6B gel chromatography to obtain a substantially single peak purified fraction. The purified fraction contains the glucomannan of the present invention composed of the repeating unit represented by the formula 1. A glucomannan composed of the repeating unit represented by the formula 1, which is separated from the cultured mycelium of Himematsutake.

3) An antitumor agent comprising the glucomannan of 1) or 2) as an active ingredient.

[0016]

Example: 20 g of glucose, 15 g of yeast extract, 10 g of potassium hydrogen phosphate, magnesium sulfate heptahydrate 2
g, calcium chloride 0.1 g and calcium carbonate 0.4
g in 1 liter of water in a pH 5.5 liquid medium,
A seed fungus of Himematsutake was inoculated and cultured with shaking at 30 ° C. for 14 days under aerobic conditions to grow mycelium. The culture was centrifuged to obtain mycelium. A 10-fold amount of purified water was added to the mycelium, and hot water extraction was performed at 100 ° C. for 2 hours while stirring and boiling to obtain an extract. The extract was concentrated under reduced pressure to 1/10,
After cooling, the mixture was centrifuged to obtain a supernatant. The polysaccharide is precipitated by adding an equal volume of 99% ethyl alcohol to the supernatant,
After obtaining a precipitate by centrifugation, it was freeze-dried. The aqueous solution of the lyophilized product was salted out with ammonium sulfate and fractionated into a protein fraction and a non-protein fraction. The recovery rate of each fraction with respect to the lyophilized product was 18.0% and 66.4%.

Sarcoma 180 ascites carcinoma subcultured in the abdominal cavity of an ICR / Slc mouse was implanted subcutaneously in the axillary region, and 24 hours after the implantation, the protein fraction and the non-protein fraction were replaced with a mouse body weight of 1 kg. The dose was intraperitoneally administered at a rate of 10 mg per dose. The frequency of administration was once a day for 10 days.
Four weeks after transplantation, the diameter of the tumor was measured and compared with the control group to calculate the tumor suppression rate. Five weeks after transplantation, the complete tumor disappearance rate and mortality rate were examined. The results are shown in Table 1.

[0018]

[Table 1]

In Table 1, tumor suppression rate: (1-T / C) × 100 where T is the tumor volume of the administration group and C is the tumor volume of the control group. Tumor complete disappearance rate: The number of mice whose tumor completely disappeared relative to the number of mice subjected to the test was indicated. Mortality: expressed as the number of dead mice relative to the number of mice subjected to the test. These are the same below.

The non-protein fraction having a strong antitumor activity was identified as DEAE-Sepharose CL-6B.
Fractionation was performed by gel chromatography. Elution was performed by a stepwise method using distilled water, a 0.5 M aqueous sodium chloride solution, a 1.0 M aqueous sodium chloride solution, and a 2.0 M aqueous sodium chloride solution. The recovery rate of each eluted fraction relative to the non-protein fraction was 12.5%, 70.8%, 3.8
%, 2.8% and 0.7%.

Sarcoma 180 ascites carcinoma subcultured in the abdominal cavity of an ICR / Slc mouse was implanted subcutaneously in the axillary region. From 24 hours after the implantation, each of the above eluted fractions was added at a rate of 10 mg / kg of mouse body weight. Administered intraperitoneally. The frequency of administration was once a day for 10 days. Three weeks after transplantation, the tumor volume was determined and compared with the control group to calculate the tumor suppression rate. Five weeks after the transplantation, the complete tumor disappearance rate was examined. The results are shown in Table 2.

[0022]

[Table 2]

In Table 2, FA-1a: a fraction eluted with distilled water FA-1b: a fraction eluted with a 0.5 M aqueous sodium chloride solution FA-1c: a fraction eluted with a 1.0 M aqueous sodium chloride solution *: compared with the control group Significant difference at 0.05% risk

0.5M with particularly strong antitumor activity
The fraction eluted with the aqueous solution of sodium chloride was repeatedly purified by a gradient method using DEAE-Sepharose CL-6B gel chromatography to obtain a purified fraction having substantially a single peak. The average molecular weight of the purified fraction is 208
3000. The purified fraction was completely methylated by the Hakomori method, completely hydrolyzed, alditolated and acetylated, and subjected to GC / MS. As a result, four main peaks (No. 1 and No. 1) were observed on the gas chromatogram.
2, No. 4 and No. 4. 9) was detected. From the mass spectra described in Table 3 of these peaks and the relative retention times on the gas chromatogram described in Table 4, these peaks (alditol acetate of partially methylated sugar) were obtained.
Are 1-acetyl-2,3,4,6-tetramethyl-glucitol, 1,2-diacetyl-3,4,6
-Trimethyl-mannitol, 1,3-diacetyl-
2,4,6-trimethyl-glucitol and 1,2,6
-Triacetyl-3,4-dimethyl-mannitol.

[0025]

[Table 3]

[0026]

[Table 4]

In Tables 3 and 4, methylated saccharide: alditol acetate of partially methylated saccharide Relative retention time: In GC (gas chromatogram), peak no. Each peak No. 1 when the retention time (minute) of No. 1 is 1 Relative retention time Molar ratio: peak No. in GC (gas chromatogram) 1 when the area of No. 1 is set to 1. Area ratio of

From the results in Tables 3 and 4, the purified polysaccharide is composed of glucose and mannose,
Monosubstituted glucose, C-1 and C-3 substituted glucose, C
-1 and C-2 substituted mannose, C-1, C-2 and C-
It became clear that the 6-substituted mannose has a structural skeleton composed of approximately 1: 1: 1: 1. From this, the existence of the structural part shown in Formula 1 was analyzed.

In the formula (1), a 1,2-bonded mannose;
For the 1,6-linked glucose and the 1,3-linked glucose, the purified fraction was subjected to ¹³ C-N to confirm whether each binding mode was α or β.
Subjected to MR. Each signal area ratio of the ¹³ C-NMR spectrum appears at 2: 1: 1, and the signal at 105.0147 ppm is located at C-1 of β-1,2 linked mannose and at 1: 1.
The signal at 03.3682 ppm was assigned to C-1, β-1,3 linked glucose, and the signal at 101.0465 ppm was assigned to glucose C-1, β-1,6 linked from glucose to mannose. From these results, it was analyzed that the binding mode between mannoses, the binding mode between mannose and glucose, and the binding mode between glucoses represented by Formula 1 were all β bonds.

The ICR / Slc male mouse (5
Week) and in an acute toxicity test (one week observation) by intraperitoneal or oral administration to male Wistar rats, the glucomannan of the present invention was 1000 mg / kg and the mortality rate was 0/6. No statistically significant difference was observed between the control group, such as weight gain, urinalysis, blood test results, and relative organ weight ratio.

[0031]

As is clear from the above, the novel glucomannan of the present invention isolated from the culture of Himematsutake has a strong antitumor activity.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Hitoshi Ito 2-3-10 Shiroyama, Tsu City, Mie Prefecture 7-3-8 Sumiyoshi Yamate, Higashinada-ku, Kobe-204

Claims (6)

[Claims] 1. A β-isolated from a culture of Agaricus blazei
Glucomannan having a mannose chain having a 1,2 bond as a main chain. 2. A glucomannan composed of a repeating unit represented by the following formula 1, which is isolated from a culture of Himematsutake. (Equation 1) (In the formula 1, Glc: glucose residue Man: mannose residue) 3. The glucomannan according to claim 1, wherein the culture of Himematsutake is a fruit body of Himematsutake. 4. The glucomannan according to claim 1, wherein the culture of Himematsutake is a cultured mycelium of Himematsutake. 5. The glucomannan according to claim 1, wherein the culture of Himematsutake is a culture filtrate of Himematsutake. 6. An antitumor agent comprising the glucomannan according to claim 1, 2, 3, 4 or 5 as an active ingredient.