JPH08165302A - D-glucan protein complex, method for separating the same, liver function improving agent and appetite enhancer containing the D-glucan protein complex as an active ingredient, and food and drink containing the D-glucan protein complex - Google Patents

Abstract

(57) [Summary] [Object] The present invention provides a novel D-glucan protein complex which exhibits excellent liver function-improving action and appetite-stimulating action even by oral administration, a method for separating the same, and a D-glucan protein complex. Liver function improver and appetite enhancer as active ingredients, and D
-Provides a food or drink containing a glucan protein complex. [Structure] The D-glucan protein complex of the present invention comprises a specific β-glucan protein complex.
(1 → 6): β- (1 → 3) -D-glucan protein complex.

Description

Detailed Description of the Invention

[0001]

The present invention relates to the genus Agaricu
s) mushroom, Agaricus blazei
), A specific D-glucan protein complex isolated from the fruiting body of the so-called Himematsutake mushroom, a method for separating the same, a liver function improving agent and an appetite enhancer containing the D-glucan protein complex as an active ingredient, and the D-glucan. The present invention relates to a food or drink containing a protein complex.

[0002]

2. Description of the Related Art Conventionally, it has been reported that substances separated from fruit bodies of various edible mushrooms have their own pharmacological effects. Also for Kawariharatake, it has been reported that the extract obtained by subjecting the fruiting body to hot water extraction has a liver function-improving effect and an appetite-increasing effect (JP-A-2-
124829, JP-A-6-239761). However,
In the above-mentioned conventional reports on Kawariharatake, the obtained extract is a so-called crude product, the body of the substance exerting the liver function improving effect and the appetite enhancing effect is unknown, and the degree of such effect is correspondingly low. There is a drawback that.

[0003]

The problem to be solved by the present invention is that, in a conventional report on agaricus, the obtained extract is a so-called crude product, which is a substance exhibiting liver function improving effect and appetite enhancing effect. The main point of this is unknown, and the degree of such effects is correspondingly low.

[0004]

The present invention, therefore, provides β- (1 → 6): β- which is separated from fruit bodies or mycelia of Kawariharatake, crushed products thereof, or dried products thereof.
(1 → 3) -D-glucan protein complex (hereinafter referred to simply as D-
Glucan protein complex), a method for separating the same, a liver function improving agent and an appetite enhancer containing the D-glucan protein complex as an active ingredient, and a food or drink containing the D-glucan protein complex.

The D-glucan protein complex of the present invention can be obtained by the following first step, second step and third step. 1st step: Kawariharatake fruiting bodies or mycelium, crushed products or dried products thereof are subjected to hot water extraction treatment,
Step of obtaining the extract Second step: Precipitation treatment of the extract with a polar organic solvent,
Step of obtaining the precipitate Third step: Anion exchange treatment is performed on the precipitate to obtain β- (1 → 6): β- (1 → 3) -D-glucan protein complex in the water eluate. Process of obtaining

In the first step, fruiting bodies or mycelia of Kawariharatake, crushed products thereof, or dried products thereof are subjected to hot water extraction treatment to obtain the extract. Usually, about 10 times amount of hot water is added to a dried and pulverized product of fruiting body or mycelium of Kawariharatake, and after hot water extraction treatment under boiling and stirring for several hours, filtration or centrifugation is performed to obtain an extract.

In the second step, the extract obtained in the first step is subjected to a precipitation treatment with a polar organic solvent to obtain the precipitate. As the polar organic solvent, methyl alcohol, ethyl alcohol, acetone and the like can be used, but due to their properties,
Preference is given to using ethyl alcohol. Usually, ethyl alcohol is added to the extract obtained in the first step so as to have a concentration of 80% by weight or more, followed by precipitation treatment, followed by filtration or centrifugation to obtain a precipitate. The obtained precipitate is preferably dialyzed, for example, in running water.

In the third step, the precipitate obtained in the second step is subjected to anion exchange treatment to obtain the D-glucan protein complex of the present invention in the water eluate. For the anion exchange treatment, various anion exchange resins, anion exchange membranes and the like can be used, but DEAE (diethylaminoethyl) cellulose is preferably used in view of the degree of purification. Usually, the precipitate obtained in the second step is treated with DEAE cellulose (C
l ^-) subjected to a column packed with eluting with water, after obtaining the aqueous eluate is non-adsorbed fraction, the water eluate was lyophilized, the D- glucan protein complex of the present invention obtain.

The thus obtained D-glucan protein complex of the present invention has the following characteristic values. 1) Average molecular weight; The average molecular weight of dextran conversion by gel filtration method is 30,000 to 50,000. 2) Sugar content; the sugar content in terms of glucose by the phenol-sulfuric acid method is 60.2 ± 10.9% by weight. 3) Sugar composition: Hydrolyzed with sulfuric acid under heating, the produced constituent sugars were induced to alditol acetate, and then subjected to gas chromatography, and then galactose of 5.2 ± 2.5 relative to 100 parts by weight of glucose. By weight, mannose is contained in the proportion of 4.1 ± 1.5 parts by weight, and in addition to these, trace amounts of fucose, xylose, arabinose, and ribose are accompanied. 4) β- (1 → 6) -D-glucan and β- (1 → 3)-
Ratio with D-glucan: In the infrared absorption spectrum by the KBr method (FIG. 1), absorption showing a β-glucoside bond was observed in the vicinity of 890-910 cm ⁻¹ , and it was confirmed to be β-D-glucan. The ¹³ C-NMR spectrum (Fig. 2) δ (PPM) shows that H- 1 -β- (1,
3) H-1 in bands 1, 4.49-4.51 showing binding
Bands 2, 3.90-3. Showing -β- (1,6) bond.
Band 3 showing H-6-β- (1,3) bond at 92,
Band 4 showing an H-6-β- (1,6) bond at 3.82-3.88 and H-2-β- at 3.25-3.27.
Band 5 showing a (1,3); (1,6) bond was observed, and from these results, both β- (1 → 6) -D-glucan and β- (1 → 3) -D-glucan were observed. It was confirmed to be a protein complex containing. Furthermore, converted from the ratio of the height and area of each band, β- (1 → 6) -D-glucan 1
Β- (1 → 3) -D-glucan 20 per 100 parts by weight
It was found that the content was ± 5 parts by weight. 5) Protein content: The protein content in terms of bovine serum albumin by the Lowry method is 35.8 ± 5.6% by weight. 6) Ash content: 2 to 5% by weight. 7) Others; the color is white to grayish white. Solubility is
It is soluble in water, DMSO (dimethyl sulfoxide) and dilute alcohol, but insoluble in organic solvents such as methyl alcohol, ethyl alcohol, acetone and diethyl ether. pH shows around 6.5. The specific rotation of sodium at the D line at 24 ° C. is around +41.0.

The D-glucan protein complex of the present invention is effective as a liver function-improving agent and has the feature that it does not show side effects even after long-term administration. Further, the D-glucan protein complex of the present invention is effective as an appetite enhancer, and has the feature that it enhances the movement of the stomach and especially the large intestine itself with little influence on the movement of the small intestine. The D-glucan protein complex of the present invention exerts a desired liver function-improving effect and an appetite-increasing effect both by oral administration and parenteral administration. In both experiments using rats,
The effective dose is usually 10 to 1000 mg / orally.
The range is 1 to 10 mg / kg for parenteral administration (administered intraperitoneally).

The most convenient method of using the D-glucan protein complex of the present invention as an orally-administered agent having an appetite-stimulating action is to provide the D-glucan protein complex as a food or drink, which is as follows. There are various methods. 1) Method of using D-glucan protein complex itself of the present invention 2) Method of using D-glucan protein complex of the present invention dissolved in water 3) Sugar to D-glucan protein complex of the present invention , A method of mixing and using acids, salts and flavors 4) The D-glucan protein complex of the present invention is a food such as a baked product, a fermented product, a paste product, a dairy product, an oil and fat product, a seasoning, a confectionery, or the like. Method of adding and using in the manufacturing process of beverages such as coffee, cocoa, tea, fruit juice, vegetable juice, fermented drinks, soft drinks, etc.

The D-glucan protein complex of the present invention is extremely stable, and is stable for at least 3 years when kept at a constant temperature, and its activity is not decreased even after sterilization at 120 ° C. for 20 minutes. Therefore, it is very easy to handle, and it is very convenient when preparing tablets, powders, injectable solutions and the like, and when it is contained in foods and drinks.

[0013]

【Example】

Test Category 1 (Separation of D-glucan protein complex) Fruit bodies of Kawariharatake are crushed, dried and then crushed, and 10 liters of hot water is added to 1000 g of the crushed matter,
Hot water extraction treatment was carried out for 3 hours while gently stirring under boiling conditions. After the hot water extraction treatment, centrifugation was performed to obtain the separated liquid. A 5-fold amount of 99% ethyl alcohol was added to the separated liquid to perform a precipitation treatment, followed by centrifugation to obtain a precipitate. The precipitate was dialyzed in running water, freeze-dried,
40 g of a crude product was obtained (hereinafter, this crude product is referred to as sample R).

[0014] Samples R, DEAE cellulose (Cl ^-)
Was subjected to an anion exchange treatment. The water eluate obtained as the non-adsorbed fraction was lyophilized to obtain 30 g of the D-glucan protein complex of the present invention (hereinafter, this D-glucan protein complex is referred to as sample P). When the sample P was subjected to a concentration gradient elution with a saline solution by a gel filtration method using a Toyopearl HW-65F column, it was divided into three types of D-glucan protein complexes. In the test and the appetite enhancement test, it had the same effect as the sample P. When the sample P was analyzed, it had the above-mentioned characteristic values.

Test Category 2 (Liver Function Improvement Test) In each experimental group, 5 Wistar rats were used (all male), and the following experimental groups 1 to 4 were conducted. Experimental group 1: Olive oil was intraperitoneally administered at a rate of 5 ml / kg. Experimental group 2; carbon tetrachloride, which is a substance that induces toxic liver damage, was dissolved in olive and the solution was used as carbon tetrachloride for 0.25
It was administered intraperitoneally at a rate of ml / kg. Experimental group 3; Sample R was orally administered at a rate of 100 mg / kg for 7 days, and then carbon tetrachloride was intraperitoneally administered on the 8th day in the same manner as in Experimental group 2, and the sample R was further administered for 3 days. 100 ml
It was orally administered at a rate of / kg. Experimental group 4; Sample P was orally administered at a rate of 50 mg / kg for 7 days, and then carbon tetrachloride was intraperitoneally administered on the 8th day in the same manner as in Experimental group 2, and the sample P was further administered for 3 days. 50 mg / kg
Orally.

Regarding the Wistar rats of each experimental group,
24 hours after the end of administration, serum GOT (glutamic acid-oxaloacetate transaminase) and GPT (glutamic acid-pyruvate transaminase) were measured,
In addition, TG (triglyceride) in the extracted liver was measured, and the results are shown in Table 1 as mean value ± standard error. GOT and GPT are based on Boehringer Ma
nnheim) colorimetric method using GOT and GPT color tests, and expressed in Karmenn units / ml. Also, TG is van Handell
), Etc., and indicated as wet weight (mg / g).

[0017]

[Table 1]

When the measurement results were verified, GOT, GP
Experimental group 3 and experimental group 4 for both T and TG
Was significant at 0.05% of the experimental group 2, and experimental group 4 was significant at 0.05% of the experimental group 3.

Test Category 3 (Appetite Enhancement Test) In each experimental group, 10 ICR mice were used (5 males).
The following test groups 5 to 7 were carried out. Experimental group 5: After being preliminarily bred for 2 weeks with MF (trade name, Oriental Yeast Co., Ltd.), which is a standard breeding sample powder, the temperature was 23
Under an environment of ± 2 ° C. and a relative humidity of 55 ± 5%, tap water was freely ingested while MF was continuously ingested. Experiment group 6: After preliminary breeding in the same manner as in Experiment group 5, under the same environment as in Experiment group 5, while freely ingesting tap water,
MF containing 10% by weight of sample R was freely taken. Experimental group 7: After preliminarily breeding in the same manner as in experimental group 5, under the same environment as in experimental group 5, while freely ingesting tap water,
MF containing 10% by weight of sample P was freely taken.

The ICR mice of each experimental group were bred for a total of 10 weeks including the preliminary breeding, and the weight value (g) and the weekly feed intake (g) were measured every week after the preliminary breeding.
The average value of these was calculated. The results are shown in Tables 2 and 3.

[0021]

[Table 2]

[0022]

[Table 3]

At the same time, p of fresh urine collected from ICR mice of each experimental group after 5, 7, and 9 weeks was measured.
H, protein, glucose, ketone body, occult blood reaction, urobilinogen and bilirubin were measured, and Na, K, C were also measured.
Although a and P were measured, no significant difference was observed between the experimental groups. After 10 weeks, blood was collected from ICR mice of each experimental group, and then all the animals were dissected to measure the wet weights of various organs of each individual, and the histopathological examination was also performed. No abnormality was found in any of them.

Separately, cancer-bearing mice, mice to which mitomycin C as an anticancer agent was administered at a rate of 0.5 mg / kg, and X
When an appetite enhancement test was carried out in the same manner as in the experimental groups 5 to 7 described above using a mouse that was irradiated with whole rays at 300 R, almost the same results as in the experimental groups 5 to 7 were obtained.

Separately, an acute toxicity test was carried out by oral administration of sample P, and LD50 for mice was 300.
> 50 mg / kg, LD50 for rats is 2500
The D-glucan protein complex of the present invention did not have a problem regarding toxicity, even if it was more than mg / kg and also from the result of subacute toxicity test on rat and the result of general pharmacological test on rabbit.

Test Category 4 (Production of food and drink) 100 g of sugar, 15 g of honey, 5 g of caramel, 0.75 g of ascorbic acid, 0.3 g of citric acid, 0.2 of lemon flavor
g and 10 g of sample P were prepared to produce a health drink.

2 kg of apple juice, 0.5 ascorbic acid
And 10 g of sample P were prepared to prepare apple juice.

[0028]

EFFECTS OF THE INVENTION As is apparent, the present invention described above exhibits excellent liver function-improving action and appetite-stimulating action, and since such action is sufficiently exhibited by oral administration, its specific use In that case, there is an effect that it is very simple.

[Brief description of drawings]

FIG. 1 is an infrared absorption spectrum diagram of the D-glucan protein complex of the present invention.

FIG. 2 ¹³ C-NM of D-glucan protein complex of the present invention
R spectrum diagram.

[Explanation of symbols]

 1-5 band

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C07K 1/18 8318-4H 1/30 8318-4H 14/375 8318-4H

Claims (9)

[Claims] 1. An agaricus (Agaricus blazei)
Β- (1 → 6): β- (1 → 3) -D- which has a characteristic value of 1) to 6) below, which is separated from the fruiting bodies or mycelia of C., crushed products or dried products thereof. Glucan protein complex. 1) Average molecular weight (gel filtration method, dextran conversion); 3
0000-50000 2) Sugar content (phenol-sulfuric acid method, glucose conversion); 6
0.2 ± 10.9 wt% 3) Sugar composition; galactose 5.2 ± 2.5 parts by weight, mannose 4.1 ± 1.
5 parts by weight and trace amounts of fucose, xylose, arabinose and ribose 4) β- (1 → 6) -D-glucan and β- (1 → 3)-
Ratio with D-glucan: β- (1 → 6) -D-glucan 100 parts by weight, β- (1 → 3) -D-glucan 20 ± 5 parts by weight 5) Protein content (Lowry method, cattle) (Serum albumin conversion);
35.8 ± 5.6% by weight 6) Ash content; 2-5% by weight 2. β- (1 → 6): β- according to claim 1.
A method for separating a (1 → 3) -D-glucan protein complex, which comprises the following first step, second step and third step. First step; fruiting body or mycelium of Agaricus blazei, crushed product thereof or dried product thereof to obtain an extract by hot water extraction. Second process; the extract is a polar organic solvent. Precipitation treatment with
Step of obtaining the precipitate Third step: Anion exchange treatment is performed on the precipitate to obtain β- (1 → 6): β- (1 → 3) -D-glucan protein complex in the water eluate. Process of obtaining 3. The separation method according to claim 2, wherein the precipitation treatment is carried out with ethyl alcohol. 4. The separation method according to claim 2 or 3, wherein anion exchange treatment is performed with DEAE cellulose. 5. β- (1 → 6): β- according to claim 1.
A liver function improving agent, which comprises a (1 → 3) -D-glucan protein complex as an active ingredient. 6. The liver function improving agent according to claim 5, which is an orally administered agent. 7. β- (1 → 6): β- according to claim 1.
An appetite enhancer comprising a (1 → 3) -D-glucan protein complex as an active ingredient. 8. The appetite enhancer according to claim 7, which is an orally administered drug. 9. β- (1 → 6): β- according to claim 1.
A food or drink comprising a (1 → 3) -D-glucan protein complex.