灰树花D组分荣获美国国家专利,编号US5,854,404

Antitumor substance extracted from grifola

An antitumor substance having high immunopotentiating activity, extracted and fractionated from mycelia or fruit bodies of Grifola with water can be obtained by adding alcohol to its extract at a final concentration of 20 to 60%, preferably at a final concentration of 20 to 50% by volume (low-concentration addition) to remove floating or adhering matter from it.

Primary Examiner: Huff; Sheela
Assistant Examiner: Reeves; Julie E.
Attorney, Agent or Firm: Davidson, Davidson & Kappel, LLC

What is claimed is:

1. A glucan/protein complex produced by the steps of:

(a) thermally extracting mycelia or fruit bodies of Grifola with water at a temperature of from 50.degree. C. to 135.degree. C.;

(b) adding alcohol to the resulting water-soluble extract at a final concentration of 20 to 60% by volume, allowing said extract to stand in a vessel at a temperature of 1.degree. to 25.degree. C., and removing floating matter on the liquid or in the liquid or matter adhered to the vessel wall; and

(c) addition alcohol to said extract at a final concentration of 80 to 99% by volume, allowing said extract to stand at 1.degree. to 25.degree. C., and recovering said glucan/protein complex in the form of precipitates.

2. A glucan/protein complex produced by the steps of:

(a) thermally extracting mycelia or fruit bodies of Grifola with water at a temperature of from 50.degree. C. to 135.degree. C.;

(b) adding alcohol to the resulting water-soluble extract at a final concentration of 20 to 50% by volume, allowing said extract to stand in a vessel at a temperature of 1.degree. to 5.degree. C., and removing floating matter on the liquid or in the liquid or matter adhered to the vessel wall; and

(c) adding alcohol to said extract at a final concentration of 80 to 90% by volume, allowing said extract to stand at 1.degree. to 5.degree. C., and recovering said glucan/protein complex in the form of precipitates.

3. A glucan/protein complex produced by the steps of:

(a) thermally extracting mycelia or fruit bodies of Grifola with water at a temperature of from 50.degree. C. to 135.degree. C.;

(b) adding alcohol to the resulting water-soluble extract at a final concentration of 20 to 60% by volume, allowing said extract to stand in a vessel at a temperature of 1.degree. to 25.degree. C., and removing floating matter on the liquid or in the liquid or matter adhered to the vessel wall; and

(c) concentrating said extract to form said glucan/protein complex in the form of precipitates, or concentrating said extract into dryness to form said glucan/protein complex.

4. A glucan/protein complex produced by the steps of:

(a) thermally extracting mycelia or fruit bodies of Grifola with water at a temperature of from 50.degree. C. to 135.degree. C.;

(b) adding alcohol to the resulting water-soluble extract at a final concentration of 20 to 50% by volume, allowing said extract to stand in a vessel at a temperature of 1.degree. to 5.degree. C., and removing floating matter on the liquid or in the liquid or matter adhered to the vessel wall; and

(c) concentrating the solution to form said glucan/protein complex in the form of precipitates, or concentrating said extract into dryness to form said glucan/protein complex.

5. The glucan/protein complex according to any one of claims 1, 2, 3 or 4, wherein the ratio of glucan to protein is in the range of from 80:20 to 99:1.

6. An antitumor agent having tumor-growth inhibition and cellular immunocompetent activity, which consists essentially of the glucan/protein complex of any one of claims 1, 2, 3 or 4 as an active ingredient.

7. The glucan/protein complex according to any one of claims 1, 2, 3 or 4, wherein Grifola is Grifola frondosa, Grifola albicans Imaz, Grifola umbellatus or Grifola gigantea.

8. The glucan/protein complex according to claim 2, wherein the ratio of glucan to protein is in the range of from 80:20 to 99:1.

9. The glucan/protein complex according to claim 3, wherein the ratio of glucan to protein is in the range of from 80:20 to 99:1.

10. The glucan/protein complex according to claim 4, wherein the ratio of glucan to protein is in the range of from 80:20 to 99:1.

11. An antitumor agent having tumor-growth inhibition and cellular immunocompetent activity, which consists essentially of the glucan/protein complex of claim 2 as an active ingredient.

12. An antitumor agent having tumor-growth inhibition and cellular immunocompetent activity, which consists essentially of the glucan/protein complex of claim 3 as an active ingredient.

13. An antitumor agent having tumor-growth inhibition and cellular immunocompetent activity, which consists essentially of the glucan/protein complex of claim 4 as an active ingredient.

14. An antitumor agent having tumor-growth inhibition and cellular immunocompetent activity, which consists essentially of the glucan/protein complex of claim 5 as an active ingredient.

15. The glucan/protein complex according to claim 2, wherein Grifola is Grifola frondosa, Grifola albicans Imaz, Grifola umbellatus or Grifola gigantea.

16. The glucan/protein complex according to claim 3, wherein Grifola is Grifola frondosa, Grifola albicans Imaz, Grifola umbellatus or Grifola gigantea.

17. The glucan/protein complex according to claim 4, wherein Grifola is Grifola frondosa, Grifola albicans Imaz, Grifola umbellatus or Grifola gigantea.

FIELD OF THE INVENTION

The present invention relates to an antitumor substance having high immunopotentiating activity, which was extracted and fractionated from mycelia or fruit bodies of a "Maitake" mushroom (Grifola).

BACKGROUND OF THE INVENTION

Polysaccharides consisting of .beta.-1,6-linked glucose main chain with .beta.-1,3-linked glucose branches or consisting of .beta.-1,3-linked glucose main chain with .beta.-1,6-linked glucose branches extracted from mycelia or fruit bodies of Grifola, is known to have anticancer activity (see Japanese Patent LOP Publication No. 210901/1984).

A process for producing an anticancer substance, which comprises a combination of the steps of extracting Grifola, Grifola gigantea (Tonbimai) or Laetiporus sulphureus (Masutake) with hot water, concentrating the extract under reduced pressure, precipitating the concentrate with an organic solvent, dialyzing the precipitates to remove low-molecular-weight substances, and extracting impurities with a lipophilic organic solvent to remove them from the Jialysate, is also known (see Japanese Patent Publication No. 16047/1968).

However, the prior processes described in Japanese Patent LOP Publication No. 210901/1984 and Japanese Patent Publication No. 16047/1968 are not necessarily appropriate for providing a larger amount of pharmaceutical preparations and health foods efficiently from limited resources because their purification steps are considerably complicated and the products contain substances inhibiting immunopotentiating activity.

SUMMARY OF THE INVENTION

Under these circumstances, the present inventors did extensive research on a method of extracting Grifola and on various extracts obtained in its process, and as a result, it was made possible to efficiently obtain an antitumor substance with superior immunopotentiating activity. The main feature is to enhance antitumor activity and an immunopotentiating activity by removing floating or adhering matter by adding alcohol at a final concentration of 20 to 60% by volume (low-concentration addition) to a water-soluble extract resulting from thermal extraction of mycelia or fruit bodies of Grifola with water.

That is, the present invention relates to a glucan/protein complex having immunopotentiating activity, which is prepared in the steps of:

(1) thermally extracting mycelia or fruit bodies of Grifola with water;

(2) adding alcohol to the resulting water-soluble extract at a final concentration of 20 to 60% by volume (low-concentration addition), allowing it to stand at a temperature of 1.degree. to 25.degree. C., and removing floating matter on the liquid or in the liquid or adhering matter to the vessel wall; and

(3) adding alcohol to the solution at a final concentration of 80 to 99% by volume (high-concentration addition), allowing it to stand at 1.degree. to 25.degree. C., and recovering the resulting precipitates, or after the step (2), concentrating the alcohol solution to form precipitates or concentrating it into dryness in a usual manner, as well as to an antitumor agent comprising the same as an active ingredient.

In the present invention, the "Maitake" mushroom (Grifola) may be Grifola frondosa, Grifola albicans Imaz., Grifola umbellatus, Grifola gigantea etc., and these can be used in fresh or dried form, if necessary cut into pieces, or in powder form.

The thermal extraction is carried out at 50.degree. to 135.degree. C. for 15 minutes to 3 hours. For rapid extraction, this treatment is carried out under pressure at 100.degree. C. or more, for example, at 2 atmospheric pressure at about 120.degree. C. in a pressure pot for 30 minutes to 1 hour or thereabout.

The water used is distilled water, purified water, ion-exchanged water, tap water etc. About 4 to 20 parts, preferably 4 to 10 parts by volume of water is used per part of dried Grifola by weight. If fresh Grifola is used, about 2 to 10 parts, preferably 2 to 5 parts by volume of water is used per part of Grifola by weight.

In the step (2), alcohol added to the extract can be methanol, ethanol etc. Alcohol is added to the extract at a final concentration of 20 to 60% by volume. Alcohol with a water content of 0 to 50% can be used. When left at a temperature of 1.degree. to 25.degree. C. for 1 to 20 hours after addition, there occurs floating matter on the liquid or in the liquid or adhering matter to the vessel wall and these are removed by filtration or with a pipette, net, etc.

Because removal of the floating and adhering matter brings about an enhancement in the antitumor activity and immunopotentiating activity of the extract, the step of removing said matter is extremely important. For this step, it is essential to add alcohol at a final concentration of 20 to 60% by volume, preferably at final concentration of 20 to 50%.

To the solution obtained in the step (2) is added alcohol at a final concentration of 80 to 99%, preferably at a final concentration of 80 to 90% by volume (high-concentration addition), and then it is allowed to sand at 1.degree. to 25.degree. C., preferably at 1.degree. to 5.degree. C. to precipitate the desired substance, or alternatively the solution obtained in the step (2) is concentrated under heating to form precipitates or concentrated into dryness under heating.

The properties of the resulting substance of the present invention are as follows:

Appearance: hygroscopic powder in shades of brown.

Solubility: dissolved in water, an alkaline solution and dimethyl sulfoxide.

Coloration reaction: positive in anthrone reaction and ninhydrin reaction.

Aqueous solution property: neutral to weakly acidic.

Molecular weight: distributed around 1,000,000.

Analysis of the substance obtained in the present invention indicated that its main components are glucan and protein. After purification by column chromatography, it was found that the major component of the antitumor substance having immunopotentiating activity obtained by the present invention is a glucan/protein complex where the glucan/protein ratio varies mainly in the range of 80:20 to 99:1 depending on the qualities of Grifola as the starting material, conditions for extraction and purification, etc.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

(1) Extraction Method

500 g of fruit bodies of dried Grifola frondosa were extracted with 5 L of distilled water at 120.degree. C. for 60 minutes, and to 950 ml of the resulting soluble fraction was added ethanol at a final concentration of 45% by volume. When this solution was allowed to stand at 4.degree. C. for 12 hours, viscous and dark brown matter was formed on the liquid, in the liquid or on the vessel wall. This substance was removed with a pipette. After addition of ethanol at a final concentration of at least 80% by volume, the solution was allowed to stand at a low temperature of 4.degree. C. to give 3 g precipitates in shades of dark brown to black. The resulting substance was positive in both anthrone reaction and ninhydrin reaction. After purification by column chromatography, this substance was found to be glucan/protein complex where the glucan/protein ration was 96:4.

As a result of its examination by gel filtration chromatography on a TSK gel GMPW.sub.XL column, it was found that its molecular weight is distributed around 1,000,000. When its glucan moiety was hydrolyzed and examined qualitatively for neutral glucan by high performance liquid chromatography, only glucose was detected.

The examination of its protein moiety by an automatic amino acid analyzer (only tryptophan was examined by high performance liquid chromatography) indicated that the protein is composed of glutamic acid, aspartic acid, alanine, leucine, lysine, glycine, isoleucine, serine, valine, proline, threonine, arginine, phenylalanine, tyrosine, histidine, tryptophan, methionine, crystine etc.

(2) Antitumor Test

The substance obtained in (1) above (referred to hereinafter as "Substance A") and a dried substance obtained in the same manner as in (1) above except that the step of adding alcohol at low concentration (final concentration of 20 to 60% by volume) for removing floating matter on the liquid or in the liquid or adhering matter to the vessel wall was not carried out (referred to hereinafter as "Substance B") were dissolved respectively in physiological saline. Each solution was administered intraperitioneally into C3H mice with transplanted MM-46 carcinoma 10 times at a dosage of 0.1 mg/kg to examine its effect on tumor growth inhibition. The results are shown in Table

TABLE 1 ______________________________________ ##STR1## ______________________________________ (15 mice per group, .asterisk-pseud. ttest: there was a significant difference of 5% or less).

The tumor growth inhibition (%) was determined according to the following formula:

Tumor Growth Inhibition (%)=[1-(average tumor weight (g) in treatment group/average tumor weight (g) in control group)].times.100

the group given Substance A indicated a significantly stronger inhibitory effect on tumor growth than that of the group given Substance B. Five days after each test substance was given, macrophages and killer T cells were collected from the control group (given physiological saline only), the group given Substance A and the group given Substance B, and the activity of the cellular immunocompetent cells was determined in terms of uptake of 'H-thymidine. The results are shown in Table

TABLE 2 ______________________________________ Cellular Immunocompetent Cell Activity (.sup.3 H-Thymidine Uptake Ratio) Macrophages Killer T Cells ______________________________________ Control group 100.0 100.0 (given physiological saline) Group given substance A 203.5 284.5 Group given substance B 157.2 233.7 ______________________________________

It was found from the above results that Substance A exhibits stronger antitumor activity and immunopotentiating activity than those of Substance B.

EFFECT OF THE INVENTION

The above results indicate that immunopotentiating activity and tumor growth inhibitory activity are enhanced by removing the floating matter on the liquid or in the liquid or the adhering matter to the vessel wall occurring by adding alcohol at a final concentration of 20 to 60%, preferably at a final concentration of 20 to 50% by volume to the hot water extract from Grifola.

Hence, the feature of the present invention lies not in simply extracting polymeric .beta.-glucan, but in effectively providing a glucan/protein complex having high immunopotentiating activity from limited resources by a simple method.

The substance obtained according to the present invention is of low toxicity and high safety and can be orally administered as health foods and pharmaceutical preparations, especially antitumor agent, in the form of tablets, capsules, liquid, syrup etc.

Antitumor substance extracted from grifola

用水提取自Grifola的菌丝体或子实体并分级分离得到的具有高免疫增强活性的抗肿瘤物质，可以通过向其提取物中添加终浓度为20％至60％，优选终浓度为20％至50％的酒精来获得。体积（低浓度添加）以除去其中的漂浮物或粘附物。

主考官： 哈夫；Sheela

助理考官： 里夫斯；朱莉E.

律师，代理或事务所： Davidson，Davidson＆Kappel，LLC

要求：

通过以下步骤制备1.一种葡聚糖/蛋白质复合物：

（a）用在水从50.degree的温度下热提取奇果菌的菌丝体或子实体。摄氏135度 C。;

（b）以20至60体积％的最终浓度将醇添加到所得的水溶性提取物中，使所述提取物在1℃的温度下放置在容器中。到25.degree C.并除去液体上或液体或粘附在容器壁上的物质中的漂浮物；和

（c）加入到醇所述提取物在以体积计80〜99％的终浓度，使所述提取物站在1.degree。到25.degree C.，并以沉淀物的形式回收所述葡聚糖/蛋白质复合物。

2.一种葡聚糖/蛋白质复合物，其通过以下步骤产生：

（a）在50°C的温度下用水热提取Grifola的菌丝体或子实体。摄氏135度 C。;

（b）以20至50体积％的终浓度将醇加入到所得的水溶性提取物中，使所述提取物在1℃的温度下放置在容器中。至5度 C.并除去液体上或液体或粘附在容器壁上的物质中的漂浮物；和

（c）在按体积计的80％至90％的最终浓度加入乙醇到所述提取物，使所述提取物站在1.degree。至5度 C.，并以沉淀物的形式回收所述葡聚糖/蛋白质复合物。

3.通过以下步骤产生的葡聚糖/蛋白质复合物：

（a）在50.degree温度下用水热提取Grifola的菌丝体或子实体。摄氏135度 C。;

（b）以20至60体积％的最终浓度将醇添加到所得的水溶性提取物中，使所述提取物在1℃的温度下放置在容器中。到25.degree C.并除去液体上或液体或粘附在容器壁上的物质中的漂浮物；和

（c）浓缩所述提取物以形成所述的析出物的形式葡聚糖/蛋白质复合物，或浓缩所述提取物至干燥以形成所述葡聚糖/蛋白质复合物。

4.通过以下步骤产生的葡聚糖/蛋白质复合物：

（a）在50.degree温度下用水热提取Grifola的菌丝体或子实体。摄氏135度 C。;

（b）以20至50体积％的终浓度将醇加入到所得的水溶性提取物中，使所述提取物在1℃的温度下放置在容器中。至5度 C.并除去液体上或液体或粘附在容器壁上的物质中的漂浮物；和

（c）浓缩该溶液以形成所述析出物的形式葡聚糖/蛋白质复合物，或浓缩所述提取物至干燥以形成所述葡聚糖/蛋白质复合物。

5.根据权利要求1、2、3或4中任一项的葡聚糖/蛋白质复合物，其中葡聚糖与蛋白质的比率在80:20至99：1的范围内。

6.具有肿瘤生长抑制和细胞免疫活性的抗肿瘤剂，其基本上由权利要求1、2、3或4中任一项的葡聚糖/蛋白质复合物作为活性成分组成。

7.根据权利要求1、2、3或4中任一项所述的葡聚糖/蛋白质复合物，其中，灰树花为灰树花，白花灰树Imaz，伞形花草或Grifola gigantea。

8.根据权利要求2的葡聚糖/蛋白质复合物，其中葡聚糖与蛋白质的比例为80:20至99：1。

9.根据权利要求3的葡聚糖/蛋白质复合物，其中葡聚糖与蛋白质的比例为80:20至99：1。

10.根据权利要求4的葡聚糖/蛋白质复合物，其中葡聚糖与蛋白质的比率为80:20至99：1。

11.具有肿瘤生长抑制作用和细胞免疫活性的抗肿瘤剂，其基本上由权利要求2的葡聚糖/蛋白质复合物作为活性成分组成。

12.具有肿瘤生长抑制和细胞免疫活性的抗肿瘤剂，其基本上由权利要求3的葡聚糖/蛋白质复合物作为活性成分组成。

13.一种具有肿瘤生长抑制作用和细胞免疫活性的抗肿瘤剂，其基本上由权利要求4的葡聚糖/蛋白质复合物作为活性成分组成。

14.具有肿瘤生长抑制和细胞免疫活性的抗肿瘤剂，其基本上由权利要求5的葡聚糖/蛋白质复合物作为活性成分组成。

15.根据权利要求2所述的葡聚糖/蛋白质复合物，其中，灰树花是灰树花，白灰树Imaz，灰树花或Grifola gigantea。

16.根据权利要求3所述的葡聚糖/蛋白质复合物，其中，灰树花是灰树花，白灰树Imaz，灰树花或Grifola gigantea。

17.根据权利要求4所述的葡聚糖/蛋白质复合物，其中，灰树花是灰树花，白灰树Imaz，伞形花草或Grifola gigantea。

具有高免疫增强活性的抗肿瘤物质技术领域本 发明涉及具有高免疫增强活性的抗肿瘤物质，其是从“舞茸”蘑菇（Grifola）的菌丝体或子实体中提取并分级分离的。
发明背景
由β-1，6-连接的葡萄糖主链和β-1，3-连接的葡萄糖支链组成的多糖或由β-1，3-连接的葡萄糖主链和β的葡萄糖组成。从Grifola的菌丝体或子实体中提取的-1，6-连接的葡萄糖分支已知具有抗癌活性（参见日本专利LOP公开号210901/1984）。
一种生产抗癌物质的方法，该方法包括以下步骤的组合：用热水提取Grifola，Grifola gigantea（Tonbimai）或Laetiporus sulfureus（Masutake），在减压下浓缩提取物，用有机溶剂沉淀浓缩物，透析还已知有沉淀物可除去低分子量物质，并用亲脂性有机溶剂提取杂质以将其从加里氏产物中除去（参见日本专利公开号16047/1968）。
然而，日本专利LOP公开号210901/1984和日本专利公开号16047/1968中描述的现有方法不一定适合于从有限的资源有效地提供大量的药物制剂和保健食品，因为它们的纯化步骤相当多。复杂且产品中含有抑制免疫增强活性的物质。
发明内容
在这种情况下，本发明人对提取灰树花的方法和在其过程中获得的各种提取物进行了广泛的研究，结果，有可能有效地获得具有优异的免疫增强活性的抗肿瘤物质。主要特征是通过将终浓度为20％至60％（体积）的乙醇（低浓度添加）添加到由菌丝体热提取产生的水溶性提取物中，从而通过除去漂浮或粘附物质来增强抗肿瘤活性和免疫增强活性。或水的Grifola子实体。
即，本发明涉及具有免疫增强活性的葡聚糖/蛋白质复合物，其通过以下步骤制备：
（1）用水热提取灰树花的菌丝体或子实体；
（2）以20至60体积％的终浓度将醇加入到所得水溶性提取物中（低浓度添加），使其在1℃的温度下静置。到25.degree C.除去液体上或液体中的漂浮物或粘附在容器壁上的物质；和
（3）加入到醇在80至99％的终浓度的溶液体积（高浓度添加），使其静置在1.degree。到25.degree 并回收所得的沉淀物，或在步骤（2）之后，以常规方式浓缩醇溶液以形成沉淀物或将其浓缩至干，以及包含其作为活性成分的抗肿瘤剂。
在本发明中，“舞茸”蘑菇（Grifola）可以是Grifola frondosa，Grifola albicans Imaz。，Grifola umbellatus，Grifola gigantea等，它们可以以新鲜或干燥的形式使用，如果需要切成小块，或者以粉末形式。
热萃取在50°C下进行。到135度 持续15分钟到3小时。为了快速提取，该处理在100°C的压力下进行。或更高，例如，在2个大气压下约120.degree。C.在压力锅中放置30分钟至1小时左右。
所用的水是蒸馏水，纯净水，离子交换水，自来水等。每份干燥的Grifola按重量计使用约4至20份，优选4至10份体积的水。如果使用新鲜的Grifola，则每重量份Grifola使用约2至10重量份，优选2至5重量份的水。
在步骤（2）中，添加到提取物中的醇可以是甲醇，乙醇等。将醇以20至60体积％的最终浓度添加到提取物中。可以使用水含量为0至50％的酒精。当放置在1度的温度下。到25.degree 添加后的1到20个小时内，在液体上或液体中会出现漂浮物，或在容器壁上的粘附物会通过过滤或用移液器，网等除去。
因为除去漂浮物和附着物使提取物的抗肿瘤活性和免疫增强活性增强，所以除去所述物质的步骤非常重要。对于该步骤，必须以20至60体积％的最终浓度，优选以20至50％的最终浓度添加醇。
向步骤（2）中获得的溶液中，以终浓度为80至99％，优选以终体积为80至90体积％的醇（高浓度加入）加入醇，然后使其在70℃下进行砂磨。 1.度 到25.degree C.，最好在1度。至5度 C.沉淀所需的物质，或者将步骤（2）中获得的溶液加热浓缩以形成沉淀或加热浓缩至干。
本发明所得物质的性质如下：
外观：吸湿性粉末，呈褐色阴影。
溶解度：溶于水，碱性溶液和二甲基亚砜。
显色反应：蒽酮反应和茚三酮反应呈阳性。
水溶液性质：中性至弱酸性。
分子量：分布在1,000,000左右。
对在本发明中获得的物质的分析表明，其主要成分是葡聚糖和蛋白质。通过柱色谱法纯化后，发现通过本发明获得的具有免疫增强活性的抗肿瘤物质的主要成分是葡聚糖/蛋白质复合物，其中葡聚糖/蛋白质比率主要在80:20至99：1的范围内变化。取决于作为原料的Grifola的质量，提取和纯化的条件等。
具体实施方式
（1）提取方法
用5 L蒸馏水在120°C提取500 g的Grifola frondosa干果。C.反应60分钟，并向950ml所得的可溶性级分中加入乙醇，其终浓度为45体积％。当将该溶液放置在4度时。约12小时，在液体，液体中或在容器壁上形成粘稠的暗褐色物质。用移液管除去该物质。加入最终浓度至少为80％（体积）的乙醇后，使溶液在4℃的低温下静置。C.产生3 g暗褐色至黑色阴影的沉淀物。所得物质在蒽酮反应和茚三酮反应中均为阳性。通过柱色谱法纯化后，
通过在TSK凝胶GMPW XL柱上进行凝胶过滤色谱法检查的结果，发现其分子量分布在1,000,000左右。当其葡聚糖部分被水解并通过高效液相色谱定性检查中性葡聚糖时，仅检测到葡萄糖。
用自动氨基酸分析仪检查其蛋白质部分（通过高效液相色谱法仅检查色氨酸）表明该蛋白质由谷氨酸，天冬氨酸，丙氨酸，亮氨酸，赖氨酸，甘氨酸，异亮氨酸，丝氨酸，缬氨酸，脯氨酸，苏氨酸，精氨酸，苯丙氨酸，酪氨酸，组氨酸，色氨酸，蛋氨酸，透明质酸等。
（2）抗肿瘤试验
在上述（1）中获得的物质（以下称为“物质A”）和以与上述（1）中相同的方式获得的干燥物质，不同之处在于以低浓度（最终浓度为20至60）添加醇的步骤。未进行用于除去液体上或液体中的漂浮物或将其附着在血管壁上的漂浮物（以下称为“物质B”）分别溶解在生理盐水中的处理。将每种溶液以0.1 mg / kg的剂量腹膜内施予移植有MM-46癌的C3H小鼠10次，以检查其对肿瘤生长抑制的作用。结果示于表中
表1 ________________________________________（每组15只小鼠，.asterisk-pseud.ttest：有5％或更少的显着性差异）。
根据下式确定
肿瘤生长抑制率（％）： 肿瘤生长抑制率（％）＝ [1-（治疗组平均肿瘤重量（g）/对照组平均肿瘤重量（g））]×。 100
给予A物质的组对肿瘤生长的抑制作用明显强于给予B物质的组。给予每种测试物质5天后，从对照组（仅给予生理盐水）中收集巨噬细胞和杀伤性T细胞，给予物质A的组和给予物质B的组，并根据对'H-胸苷的摄取来确定细胞免疫活性细胞的活性。结果示于表中
表2 ______________________________________蜂窝免疫活性细胞活性（.sup.3 H-胸苷摄取率）巨噬细胞杀伤T细胞______________________________________对照组100.0 100.0（给予生理盐水）组给定的物质A 203.5 284.5组给定的物质B 157.2 233.7 ______________________________________
它是从所找到的上面的结果，物质A显示出更强的抗肿瘤活性和免疫增强活性比那些物质B.
本发明的效果
以上结果表明，通过加入终浓度为20％至60％的酒精，最好是在乙醇中加入20％至60％的酒精，可以去除液体上或液体中的漂浮物或血管壁上的粘附物，从而增强免疫增强活性和抑制肿瘤生长。从Grifola提取的热水的最终浓度为20至50％（体积）。
因此，本发明的特征不在于简单地提取聚合的β-葡聚糖，而是在于通过简单的方法有效地从有限的资源中提供具有高免疫增强活性的葡聚糖/蛋白质复合物。
根据本发明获得的物质具有低毒性和高安全性，并且可以作为健康食品和药物制剂，特别是抗肿瘤剂，以片剂，胶囊剂，液体剂，糖浆剂等形式口服给药。

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