JP2911097B2 - Glycoprotein and appetite suppressant containing the same as active ingredient - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel glycoprotein isolated from the fruit body of Pleurotus ostreatus and an appetite suppressant containing the same as an active ingredient.

[0002]

2. Description of the Related Art Hitherto, it has been reported that a glycoprotein isolated from a fruit body of a mushroom has an antitumor effect (JP-A-58-118519, JP-A-58-12129).
7). On the other hand, tetrahydropyridine compounds, deoxy-
It has been reported that D-glycol derivatives, aminopolysaccharides such as chitin, and triglyceride derivatives have an appetite-suppressing effect (JP-A-57-159714, JP-A-60-81).
127, JP-A-62-123122, JP-A-3-220
123).

[0003]

However, there is no report on a glycoprotein isolated from the fruit body of Pleurotus ostreatus and its appetite-suppressing activity.

[0004]

Means for Solving the Problems Thus, the present inventors have
In view of the above-described circumstances, as a result of studying glycoproteins isolated from the fruit body of Pleurotus ostreatus and its appetite-suppressing action, when a prescribed treatment is applied to the fruiting body of Pleurotus ostreatus, a novel glycoprotein is isolated, It has been found that the protein has an excellent appetite suppressing effect.

That is, the present invention relates to a glycoprotein isolated from the fruit body of Pleurotus ostreatus having the following characteristics 1) to 6) and an appetite suppressant containing the glycoprotein as an active ingredient. 1) Number average molecular weight (gel filtration method, dextran conversion):
70000-90000 2) Isoelectric point: 8.2-9.2 3) Sugar content (phenol-sulfuric acid method): 2.2-3.0% by weight 4) N-terminal amino acid sequence: alanine-threonine-valine-lysine- Isoleucine-threonine-alanine-threonine-proline-arginine-glutamine-phenylalanine-glycine 5) Lectin activity: yes 6) Appetite-suppressing action: yes

The oyster mushroom (Pleurotus ostreatus) used as a raw material in the present invention is of the order Agaric
ales), a mushroom belonging to the family Tricholomataceae. In the present invention, the fruit body of oyster mushroom (umbrella and / or handle) is used, and the fruit body may be either an artificial cultivation or a natural product. However, it is particularly preferable to use one having a large umbrella and a thick handle.

[0007] The glycoprotein of the present invention can be obtained from the fruit body of Pleurotus ostreatus through the following extraction step, precipitation step and fractionation step.

[0008] In the extraction step, the fruit body of Pleurotus ostreatus, its crushed or crushed material, its dried product or its dried and crushed product is dissolved in water, physiological saline or a 20% by weight or less polar organic solvent. Extraction treatment is performed with an aqueous solution to obtain an extract. In this case, the polar organic solvent includes methyl alcohol, ethyl alcohol, acetone and the like. For example, 10 to 30 times the amount of water is added to the dried and crushed fruit body of Oyster mushroom, and the mixture is extracted at room temperature, preferably at 40 ° C. or lower, more preferably with stirring for about 10 to 30 hours, and filtered or Centrifuge to obtain an extract.

Since the target glycoprotein is contained in the extract obtained in the extraction step, the extract, its concentrated solution or its dried product can also be used as an active ingredient of the appetite suppressant.

In the precipitation step, the extract obtained in the extraction step is subjected to a precipitation treatment with ammonium sulfate or a polar organic solvent to obtain a precipitate. In this case, the polar organic solvent includes methyl alcohol, ethyl alcohol, acetone and the like. The extract may be concentrated under reduced pressure, preferably concentrated under reduced pressure at a temperature of 40 ° C. or lower, and subjected to a precipitation treatment. Ammonium sulfate is 60 to
When it is added in the range of 90% by weight saturation and the amount of the polar organic solvent is in the range of 1 to 3 times, the substance containing the objective glycoprotein precipitates. For example, ammonium sulfate is added to the extract obtained in the extraction step to a saturation of 90% by weight, and the mixture is allowed to stand at room temperature to precipitate a substance containing the desired glycoprotein, followed by filtration or centrifugation to obtain a precipitate. .

Since the target glycoprotein is contained in the precipitate obtained in the precipitation step, the precipitate or a dried product thereof can also be used as an active ingredient of the appetite suppressant.

In the fractionation step, the precipitate obtained in the precipitation step is subjected to a chromatographic fractionation treatment to obtain a fraction containing the desired glycoprotein. As will be described later in detail in the Examples, the chromatographic fractionation treatment is repeatedly performed using a combination of different carriers and a mobile phase, and the objective glycoprotein can be sequentially fractionated and isolated. The precipitate obtained in the step is dialyzed against water, and the remaining liquid (the inner liquid of the dialysis membrane) is distilled off under reduced pressure, preferably at a reduced pressure of 40 ° C. or lower.
After freeze-drying at the same temperature or lower, the freeze-dried product can be subjected to affinity chromatography to isolate a target glycoprotein.

Since the fractionated solution obtained in the fractionation step contains the objective glycoprotein, the fractionated solution, its concentrated solution or its dried product is used as the active ingredient of the appetite suppressant.

As described above, the glycoprotein of the present invention isolated through the extraction step, the precipitation step and the fractionation step has the following characteristics 1) to 6), and particularly has an isoelectric point of 8.2. ~
It has a basic property of 9.2 and has a lectin activity and an appetite-suppressing action. 1) Number average molecular weight (gel filtration method, dextran conversion):
70000-90000 2) Isoelectric point: 8.2-9.2 3) Sugar content (phenol-sulfuric acid method): 2.2-3.0% by weight 4) N-terminal amino acid sequence: alanine-threonine-valine-lysine- Isoleucine-threonine-alanine-threonine-proline-arginine-glutamine-phenylalanine-glycine 5) Lectin activity: yes 6) Appetite-suppressing action: yes

The glycoprotein of the present invention has a pH of 5.8-1.
In the region of 0.0, human erythrocyte agglutinating ability is exhibited, but lectin activity is lost due to specific binding to 2'-fucosyllactose, and lectin activity is rapidly lost in a wet state at a temperature of 65 ° C or more. Therefore, in the present invention, when all of the extraction step, precipitation step and fractionation step, and when the extract, precipitate or fraction obtained in each step are concentrated and / or dried, these treatments are carried out at 65 ° C. It is preferably carried out at the following temperature, more preferably at a temperature of 40 ° C. or less.

[0016]

【Example】

Test Category 1 (Separation of Glycoprotein) As a raw material, fruit bodies of oyster mushrooms cultivated from Iwate Prefecture and buried in soil were used. The fruit body having an average umbrella diameter of 4.5 cm was freeze-dried at 40 ° C. or lower and pulverized by a mixer. To 200 g of the pulverized product, add 4 liters of water at 4 ° C.
Extraction treatment was performed for 4 hours, followed by filtration to obtain an extract. The extract was concentrated under reduced pressure to 1/2 volume at 40 ° C., ammonium sulfate was added to the concentrate to 90% by weight saturation, the mixture was allowed to stand at room temperature to precipitate, and centrifuged to obtain a precipitate.

The precipitate was dialyzed in running water, and the remaining solution (the inner solution of the dialysis membrane) was freeze-dried at 40 ° C. or lower, and the freeze-dried product was subjected to the following chromatographic fractionation treatment. First, the lyophilized product was added to 500 ml of sodium acetate buffer (pH 5.
0) and supplied to CM Toyopearl (trade name, manufactured by Tosoh Corporation) which had been equilibrated with the same buffer solution in advance. After sufficient washing with the same buffer, the aqueous sodium chloride solution was linearly flowed out with a concentration gradient of 0 → 1 M, and a fraction A which flowed out with a 0.25 M aqueous sodium chloride solution was obtained.

Next, ammonium sulfate was added to the above fraction A so as to have a concentration of 1 M, and the solution was supplied to butyl toyopearl (trade name, manufactured by Tosoh Corporation) which had been equilibrated with a 1 M aqueous solution of ammonium sulfate. After washing sufficiently with the same ammonium sulfate aqueous solution, the ammonium sulfate aqueous solution was linearly flowed out at a concentration gradient of 1 → 0 M, and a fraction B which flowed out with a 0.25 M aqueous ammonium sulfate solution was obtained.

Further, the above fraction B was dialyzed against running water to obtain a desalted residual solution (the inner solution of the dialysis membrane).
Hydroxyapatite was equilibrated with 0 mM sodium phosphate buffer (pH 6.8). After sufficiently washing with the same buffer, the sodium phosphate buffer was flowed out linearly with a concentration gradient of 10 mM → 3 M, and fraction C that flowed out with the 0.17 M sodium phosphate buffer was obtained.

Finally, the above-mentioned fraction C was dialyzed against running water, and the remaining liquid (the inner solution of the dialysis membrane) was freeze-dried at 40 ° C. or lower. The freeze-dried product was added to 5 ml of PBS ｛10 m
M phosphate buffer (pH 7.4) +0.15 M aqueous sodium chloride solution 、, and then subjected to gel filtration on Toyopearl HW55F (trade name, manufactured by Tosoh Corporation) pre-equilibrated with the same PBS. Thus, 140 mg of the glycoprotein of the present invention having the above-mentioned properties was obtained. This glycoprotein (hereinafter, referred to as sample 3) was subjected to an appetite suppressing effect test.

The amino acid composition of the glycoprotein obtained above is shown in Table 1.

[Table 1]

Separately, in the same manner as described above, a crushed fruit body of Pleurotus ostreatus is extracted with water, and the extract is subjected to precipitation treatment with ammonium sulfate to obtain the precipitate. Lyophilized at a temperature of to obtain a lyophilized product. This freeze-dried product (hereinafter, referred to as sample 2) was subjected to a test for an appetite suppressing effect. Separately, in the same manner as described above, a crushed fruit body of Pleurotus ostreatus was extracted with water, and the extract was freeze-dried at a temperature of 40 ° C. or lower to obtain a freeze-dried product. This freeze-dried product (hereinafter, referred to as sample 1) was subjected to a test for an appetite suppressing effect.

Test Category 2 (Test for Appetite Suppressing Action) A basic feed and a test feed described in Table 2 were prepared. These diets were divided into four groups, and each group was allowed to freely feed five rats, and the total food consumption (g) and body weight change (g) three days after the free feeding were measured. 3 is shown.

[0024]

[Table 2]

[0025]

[Table 3]

In Table 3, the control group: a group that had free access to the basic feed Test group 1: a group that had free access to the test feed to which sample 1 was added Test group 2: a test feed to which the sample 2 was added Ingested group Test group 3: A group in which the test feed to which sample 3 was added was fed freely. Numerical values of total food intake and body weight change: mean ± SEM Suppression rate: ｛(average of total food intake in control group) Value-average value of total food consumption of each test group) / average value of total food consumption of control group /
× 100 *: significant at a risk rate of 0.1%

[0027]

As is clear, the glycoprotein of the present invention described above has an effect of exhibiting an excellent appetite suppressing effect.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Hideki Sakamoto 47-12 Iguchi, Nishinasuno-machi, Nasu-gun, Tochigi Prefecture JP-A-2-270899 (JP, A) JP-A-3-118328 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C07K 14/375 A61K 35/84 A61K 38/00 BIOSIS (DIALOG) CA (STN) REGISTRY (STN)

Claims (6)

(57) [Claims] 1. A glycoprotein having the following characteristics 1) to 6 ), which is isolated from the fruit body of Pleurotus ostreatus. 1) Number average molecular weight (gel filtration method, dextran conversion):
70000-90000 2) Isoelectric point: 8.2-9.2 3) Sugar content (phenol-sulfuric acid method): 2.2-3.0% by weight 4) N-terminal amino acid sequence: alanine-threonine-valine-lysine- Isoleucine-threonine-alanine-threonine-proline-arginine-glutamine-phenylalanine-glycine 5) Lectin activity: Yes 6) Appetite-suppressing action: Yes 2. An appetite suppressant comprising the glycoprotein according to claim 1 as an active ingredient. 3. An appetite suppressant comprising the glycoprotein-containing extract according to claim 1, a concentrated solution thereof or a dried product thereof as an active ingredient, which is obtained through the following extraction step. Extraction step: Extracting the fruit body of Pleurotus ostreatus, its crushed or crushed material, its dried product or its dried and crushed product with water, physiological saline, or an aqueous solution in which a polar organic solvent is dissolved within 20% by weight or less. And obtaining the extract 4. An appetite suppressant comprising the glycoprotein-containing precipitate or a dried product thereof as an active ingredient, which is obtained through the following extraction step and precipitation step. Extraction step: Extracting the fruit body of Pleurotus ostreatus, its crushed or crushed material, its dried product or its dried and crushed product with water, physiological saline, or an aqueous solution in which a polar organic solvent is dissolved within 20% by weight or less. Precipitation step: a step of precipitating the extract with ammonium sulfate or a polar organic solvent to obtain the precipitate 5. An appetite suppressant comprising, as an active ingredient, a fractionated solution containing the glycoprotein according to claim 1, which is obtained through the following extraction step, precipitation step and fractionation step, a concentrated solution thereof or a dried product thereof. . Extraction step: Extracting the fruit body of Pleurotus ostreatus, its crushed or crushed material, its dried product or its dried and crushed product with water, physiological saline, or an aqueous solution in which a polar organic solvent is dissolved within 20% by weight or less. And a step of obtaining the extract. A precipitation step: a step of precipitating the extract with ammonium sulfate or a polar organic solvent to obtain a precipitate. A fractionation step: a chromatographic fractionation of the precipitate, the glycoprotein according to claim 1. Obtaining a fractionation solution containing 6. The appetite suppressant according to claim 3, wherein all the processes including the respective steps are carried out at a temperature of 65 ° C. or lower.