JPH05117303A - Water-soluble polysaccharide originating from yamabushitake (fungus) and antitumor agent mainly containing the same - Google Patents

Abstract

PURPOSE:To obtain the subject neutral aqueous solution of polysaccharide which can be used as a major ingredient of an antitumor agent because of its high antitumor activity, by extraction of YAMABUSHITAKE, a fungus (Hericium erinaceum), or its treated product with an organic solvent, then subjecting the extract to ion-exchange treatment. CONSTITUTION:Fruit bodies of YAMABUSHITAKE are combined with an organic solvent such as ethanol, homogenized, allowed to stand one day to effect extraction. The extraction mixture is filtered with suction to remove the residue, the residue of organic solvent extraction is subjected to repeated filtration operations with physiological salt solution at 4 deg.C for 2 hours to filter off the residue, which is combined with an aqueous solvent such as hot water at 100 deg.C and extracted with boiling water under stirring for 3 hours. The extract is cooled down, centrifuged, the supernatant is combined ethanol to separate the precipitate. The extract is centrifuged after cooling, the supernatant is combined with ethanol to separate the precipitate, which is dialyzed in flowing water to collect the water-soluble fraction. Then, the fraction is subjected to anion-exchange chromatography to effect elution with water whereby the polysaccharide of high antitumor activity originating from YAMABUSHITAKE is obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a water-soluble polysaccharide derived from Hericium erinaceum, which is a mushroom of the mushroom family, and an antitumor agent mainly comprising the water-soluble polysaccharide.

[0002]

2. Description of the Related Art Conventionally, it has been reported that a water-soluble fraction derived from Pleurotus cornucopiae has antitumor activity (Abstracts of the 1991 Annual Meeting of the Japanese Society of Agricultural Chemistry, p. 364). However, since this water-soluble fraction is a crude product obtained as a hot water extract of Physarum edulis fruit bodies, a hot water extract of liquid culture mycelia, or an alcohol precipitate of the culture filtrate, it has a drawback that its antitumor activity is low.

[0003]

The problem to be solved by the present invention is that the previously reported water-soluble fraction derived from Pleurotus cornucopiae has a low antitumor activity.

[0004]

However, the present inventors have
As a result of investigating what contributes to the antitumor activity among the water-soluble fractions derived from Yamabushitake, a specific water-soluble polysaccharide obtained by purifying the water-soluble fraction, particularly the water-soluble polysaccharide It was found that the specific glycoprotein complex and the polysaccharide obtained by further purifying the protein contributed.

That is, according to the present invention, a neutral water-soluble polysaccharide obtained by extracting the organic solvent extraction residue of Physcomitrella communis or its processed product with an aqueous solvent and subjecting the extract to an ion exchange treatment, the water-soluble polysaccharide The present invention relates to a glycoprotein complex and a polysaccharide obtained by purifying by molecular weight fractionation or affinity chromatography, and an antitumor agent containing these as a main agent.

[0006] In the present invention, first, the Phellinus linteus or a treated product thereof is extracted with an organic solvent to obtain an extraction residue. The edible mushrooms to be extracted include fruit bodies, cultured mycelia, culture solutions, and the like, and processed products thereof include crushed products, ground products, concentrated solutions, dried products, and the like. The organic solvent used for extraction includes methanol, ethanol, acetone, diethyl ether, hexane, etc., but methanol, ethanol, acetone, etc. containing about 10 to 20% of water may be used.

Next, the organic solvent extraction residue is extracted with an aqueous solvent to obtain the extract. Prior to extraction with an aqueous solvent, it is also effective to extract the organic solvent extraction residue with physiological saline to remove the free protein fraction contained in the organic solvent extraction residue. The aqueous solvent used for extraction is a solvent mainly composed of water, and may be water containing about 10 to 20% of methanol, ethanol, acetone or the like, but hot water is preferable.

Finally, the aqueous solvent extract is subjected to ion exchange treatment to obtain a water-soluble polysaccharide. Prior to the ion exchange treatment, it is also effective to add, for example, ethanol to the aqueous solvent extract and to collect only the free polysaccharide fraction contained in the aqueous solvent extract. When the aqueous solvent extract is subjected to anion exchange chromatography, the neutral water-soluble polysaccharide obtained as the non-adsorbed portion is the water-soluble polysaccharide according to the present invention.

The neutral water-soluble polysaccharide thus obtained is
It exhibits higher antitumor activity than the water-soluble fraction contained in the aqueous solvent extract before the ion exchange treatment.

The neutral water-soluble polysaccharide is subjected to molecular weight fractionation by, for example, gel filtration to obtain a galactomannoglucoxylan protein complex and a mannogalactoglucoxylan protein complex. Subjecting the galactomannoglucoxylan protein complex to affinity chromatography,
A xylan protein complex and a galactomannoglucoxylan are obtained by separating into an adsorbing part and a non-adsorbing part.

[0012] These glycoprotein complexes are also compared with the water-soluble fraction contained in the aqueous solvent extract before the ion exchange treatment,
Although it shows high antitumor activity, the galactomannoglucoxylan separated by subjecting it to the affinity chromatography described above is particularly excellent.

The antitumor agent according to the present invention is mainly composed of the neutral water-soluble polysaccharide, glycoprotein complex and / or polysaccharide described above. The content of these in the preparation can be determined so as to be 10 to 1000 mg / kg for oral administration and 1 to 10 mg / kg for parenteral administration (both to mouse).

[0013]

[Examples] To 13 kg of Pleurotus cornucopiae fruiting body, 30 liters of 99% ethanol was added, homogenized, allowed to stand for 1 day, and suction filtered to obtain the residue. The operation of adding physiological saline to the residue and extracting at 4 ° C. for 2 hours was repeated 3 times to obtain 741 g of the residue.

To 741 g of the residue obtained above, 3 liters of hot water at 100 ° C. was added and stirred for 3 hours while boiling to obtain an extract thereof. After cooling the extract, 5 times the amount of 99% ethanol was added to the supernatant obtained by centrifugation, and the mixture was further centrifuged to obtain a precipitate. The precipitate was dialyzed in running water and freeze-dried to obtain 21.6 g of a water-soluble fraction (F0).

6.1 g of the water-soluble fraction (F0) obtained above was subjected to anion exchange chromatography (diethylaminoethyl-cellulose column) and eluted with water to give a neutral water-soluble fraction as its non-adsorbed portion. A polysaccharide (F1) was obtained.

Neutral water-soluble polysaccharide (F1) obtained above
1.6 g was subjected to gel filtration (Toyopearl HW-65F, manufactured by Tosoh Corporation) and eluted with a 0.3 molar aqueous sodium hydroxide solution to give 633 mg of galactomannoglucoxylan protein complex (F1-a) and manno. 403 mg of the galactoglucoxylan protein complex (F1-b) was obtained.

262 mg of the galactomannoglucoxylan protein complex (F1-a) obtained above was subjected to affinity chromatography using ConA (carrier: Toyopearl, manufactured by Tosoh Corporation) to obtain pH 7.0 0.1. By elution with a molar phosphate buffer, 89 mg of xylan protein complex (F1-a-α) was used as the adsorbing portion, and galactomannanoglucoxylan (F1-a) was used as the non-adsorbing portion.
-Β) 166 mg was obtained.

The water-soluble fraction (F0) thus obtained, the neutral water-soluble polysaccharide (F1), the galactomannoglucoxylan protein complex (F1-a), the mannogalactoglucoxylan protein complex (F1-) b), xylan protein complex (F1
-A-α), and galactomannoglucoxylan (F1
The composition of -a-β) is summarized in Table 1.

[0019]

[Table 1]

Note) In Table 1, sugar content: colorimetric assay (phenol-sulfuric acid method), protein content: colorimetric assay (Lori-improved method), sugar composition: gas chromatography method,
Glc: glucose, Xyl: xylose, Man:
Mannose, Gal: Galactose

Sarcoma 180 tumor cells (6 × 10 ⁶ ) were collected from 7 female mice of the JCR / SLC system as a set, and collected from these mice 7 days after transplantation and counted.
Were transplanted subcutaneously on the back. Then, 1 day after the transplantation, the above F0-F1-a-β dissolved in physiological saline was
It was intraperitoneally administered to give 0 mg / kg mouse (F0 to F1-a-β conversion). The administration frequency was once a day for 10 days. 28 days after transplantation, the tumor size (cm ³ ) was measured,
The tumor suppression rate (%) was calculated as compared with the control group (untreated group). The survival rate (%) was calculated in comparison with the control group (100), and the complete tumor elimination rate (animal / animal) was also determined. At the end of the test, the mortality rate (animal / animal) 61 days after transplantation was determined. The results are summarized in Table 2.

[0022]

[Table 2]

[0023]

As is apparent from the above, the present invention described above has an effect that the antitumor activity is higher than that of the water-soluble fraction derived from Yamabushitake mushroom which has been reported so far.

Front Page Continuation (72) Inventor Taku Mizuno 5-5 9 Seinan-cho, Fujieda City, Shizuoka Prefecture (72) Inventor Hitoshi Ito 2-3-3 Shiroyama, Tsu City, Mie Prefecture (72) Inventor Keishin Mori Nasu Tochigi Prefecture 2-10-10 Mishima, Nishinasuno-cho, Gunma 4 Mutual Residence No. 103 (72) Hideki Sakamoto 47 47 Iguchi, Nishinasuno-machi, Nasu-gun, Tochigi Prefecture (72) Inventor Chiharu Suzuki 3800 211 Otani, Shizuoka-shi, Shizuoka Prefecture

Claims (6)

[Claims] 1. A neutral water-soluble polysaccharide obtained by extracting an organic solvent extraction residue of Yamabushitake mushroom or a treated product thereof with an aqueous solvent, and subjecting the extract solution to an ion exchange treatment. 2. The water-soluble polysaccharide according to claim 1, which is a galactomannoglucoxylan protein complex obtained by molecular weight fractionation. 3. The water-soluble polysaccharide according to claim 2, which is a xylan protein complex obtained as an adsorption part by further subjecting it to affinity chromatography. 4. The water-soluble polysaccharide according to claim 2, which is galactomannoglucoxylan obtained as a non-adsorbed portion by further subjecting it to affinity chromatography. 5. The water-soluble polysaccharide according to claim 1, which is a mannogalactoglucoxylan protein complex obtained by further fractionating the molecular weight. 6. An antitumor agent comprising the water-soluble polysaccharide according to claim 1, 2, 3, 4 or 5 as a main ingredient.