JPS6147519B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing protein polysaccharides having antitumor activity. Specifically, the present invention relates to a method for producing a protein polysaccharide having an antitumor effect from a culture medium obtained by culturing cells of a new mushroom belonging to the genus Agaricus. Many methods for producing antitumor substances from basidiomycetes have been proposed in the past, and for example, the production of antitumor substances by extraction with an aqueous solvent from the fruiting bodies of basidiomycetes was proposed in Japanese Patent Publication No. 51-22042. It is stated in the No. The present inventors first discovered a new variety of mushroom belonging to the genus Agaricus, Agaricus agaricus.
We have proposed a method for producing protein polysaccharides with antitumor activity from the fruiting bodies of Heinem. As a result of collecting protein polysaccharides and intensive research on their pharmacological effects,
The present invention was completed based on the discovery that a substance having a stronger antitumor effect than the substance obtained from the fruiting body can be produced from a culture medium, which is advantageous for industrial production. The basidiomycete used in the present invention was collected outside the city of Pietade, São Paulo, Brazil, and was recently successfully cultivated for the first time in Japan.
This is a new, unregistered variety. The method for cultivating fruiting bodies using this basidiomycete is described in Japanese Patent Application No. 52-54638. next,
The mycological properties of Kawariharatake, a new variety of mushroom used in the present invention, are shown. (1) Fruiting body The shape of the fruiting body is bell-shaped, and the size of the cap is 5.
It is ~15cm long and has a unique scent. When the cap opens, spores are formed unevenly, and spore formation is delayed and it takes time for the plant to turn black. When the folds are observed under a microscope, the club bodies and side threads are weak. Compared to other known mushrooms of the same genus, the stalk is thicker and longer, and the spores turn black later. It is also characterized by its strong aroma and the delicious and sweet meat of the stalk. (2) Grows well on wort agar medium and potato glucose agar medium (PD medium). White hyphae grow on the PD medium, turning the medium brown. (3) Optimal growth conditions for mycelium PH = 6.0-6.5 28℃ (4) Range of mycelium growth PH = 4.0-8.0 10-33℃ (5) Temperature range for fruiting body development 20-30℃
The optimum temperature is 24-28℃ (6) A mycelial network is formed during fruiting body formation. (7) A large amount is generated when the compost is covered with ridges of soil during the formation of fruiting bodies. When comparing the above mycological properties with known mushrooms of the same genus, such as Agricus campestris Fr., they differ in that the stalks are thicker and the spores turn black more slowly, and they are also more fragrant and the flesh of the stalks is different. It is delicious and sweet, which makes it unique and unique. Based on these mycological properties, this basidiomycete was determined to be a new fungal species belonging to the genus Agaricus, and is considered to be a new species belonging to the genus Agaricus.
heterosistes Heinem.et Gooss). This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under accession number 4731. Furthermore, the present protein polysaccharide can also be produced using artificial or natural mutant strains of the basidiomycete. The strain used in the present invention may be cultured by either a solid culture method or a liquid culture method commonly used for culturing basidiomycetes, but the latter method is preferably used in terms of productivity. The culture medium may be any conventional medium formulation as long as it contains the various nutrients necessary for the growth of the bacteria. That is, any carbon source that can be assimilated, such as glucose, sucrose, maltose, or starch, can be used as the carbon source. Examples of nitrogen sources include ammonium sulfate, ammonium nitrate, sodium nitrate, urea, etc.; examples of natural complex nutritional sources include potato extract, carrot extract, malt extract, peptone, V-8 youth, koji extract, yeast extract, yeast powder, and onion. Examples of inorganic salts that can be used include extracts, corn stave liquor, etc., such as phosphoric acid, salts, magnesium salts, calcium salts, iron salts, potassium salts, and other inorganic salts. In addition, trace elements, vitamins, etc. necessary for growth may be added as appropriate. Cultivation is usually carried out under aerobic conditions, such as a shaking culture method or an aerated agitation culture method. The culture temperature is preferably 20 to 37°C, preferably around 30°C. Growth is good when the culture pH is 3.0 to 9.0, preferably 4.5 to 7.0. The number of days for culturing varies depending on the culture conditions, but it is sufficient as long as there is growth of mycelium, and usually 2 to 3 days.
A good time is when maximum mycelium is produced for 30 days.
For aerated agitation culture, aeration volume is 0.1 to 10VVM, stirring speed
It is best to do this in the range of 30 to 800 rpm. After completion of the culture, the culture solution is centrifuged or filtered to remove mycelia and obtain a culture solution. The obtained culture solution contains active ingredients and can be used as is.
Although it exhibits antitumor properties, the desired protein polysaccharide can be collected as a precipitate either as is or by adding a water-soluble organic solvent or various salts after appropriate concentration. For example, by adding a water-soluble organic solvent such as alcohol or acetone, or various salts such as ammonium sulfate or calcium chloride, a daily substance is precipitated, filtered or centrifuged, washed with acetone or ether, and dried to form a powder. Obtainable. Before producing a precipitate from the culture solution, low-molecular substances can be removed by dialysis, ultrafiltration, gel filtration, etc., or decolorization and deionization can be carried out by activated carbon, ion exchange treatment, etc., as necessary. Further, the obtained precipitate may also be redissolved in water or the like and subjected to the above treatment, if necessary. Then, the operation of reprecipitating with an aqueous solvent such as alcohol can be repeated. The substance thus obtained is a pale brown to grayish white amorphous powder, and its 1% aqueous solution has a pH of
Shows 6.0. When color reactions were performed on this substance, anthrone-sulfuric acid reaction, phenol-
The sulfuric acid reaction, xanthoprotein reaction, and pholinthiocalt reaction were all positive, and the substance of the present invention is considered to be a protein polysaccharide based on these reactions. Examples of other physicochemical properties of the substance of the present invention are as follows. (1) Average molecular weight: 10 ⁵ to 10 ⁷ , measured by gel filtration method using Sepharose 4B. Dextran was used as a standard substance for molecular weight. (2) Specific optical rotation (for dialysis sample) [α] ²⁰ _D = +63° (2.0% weight/volume aqueous solution) (3) Elemental analysis N: 1.6%, C: 39.3%, H: 6.5%, O:
52.6%N content varies slightly depending on the degree of purification. (4) Constituent sugar Add sulfuric acid to 1N to a 1% aqueous solution of this substance, perform hydrolysis at 100℃ in a sealed tube for 4 hours, neutralize with barium carbonate, and use the supernatant as a primary silica gel. Original thin layer chromatography was performed. The developing solvent was n-propyl alcohol: ethyl acetate: water (7:1:2), and detection was performed by color development using diphenylamine-aniline. As a result, two types of spots with Rf = 0.34 (galactose) and 0.45 (mannose) were detected. Furthermore, the supernatant liquid obtained earlier was dried, trimethylsilylated, and then analyzed by gas chromatography. As a result, mannose was detected as the main component.
Glucose, galactose, and ribose were confirmed as trace components. (5) Amino acids Dissolve this substance in redistilled hydrochloric acid, freeze and seal on a dry ice bath under reduced pressure, and then hydrolyze at 110°C for 22 hours. This was dried, dissolved in a buffer solution with a pH of 2.2, and analyzed using an amino acid analyzer. As a result, aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, γ-aminobutyric acid, histidine,
Ornithine, lysine, and arginine were detected. (6) Solubility Soluble in water, 1N caustic soda, 1N aqueous ammonia, 1N hydrochloric acid, 1N sulfuric acid and formic acid. Insoluble in alcohol, acetone, ethyl acetate, ether, benzene, acetic acid, dimethyl sulfoxide, dimethyl formamide, acetonitrile and dioxane. (7) Ultraviolet absorption spectrum (see Figure 1) Measurement results for a 0.05% aqueous solution of this substance
It has absorption near 200nm and 250nm. (8) Infrared absorption spectrum (see Figure 2) 3400 (S), 2930 (M), 1640 (S), 1540
(W), 1400 (W), 1050 (S), 900 (W), 800
(W), exhibits characteristic absorption (wavenumber cm ^-1 ) of 570 (W). (Based on the KBr method.) (9) Viscosity (for dialysis samples) As a result of measuring a 2.0 (weight/volume)% aqueous solution at 20°C using a BL type viscometer (manufactured by Tokyo Keiki), it was 3.8 cps. (10) Taste and odor This substance and its aqueous solution are tasteless and odorless. (11) Decomposition point Does not show a fixed decomposition point or melting point. Carbonizes on ignition. According to various literatures, for example, Lentinus
The antitumor polysaccharide lentinan derived from edodes (Berg) Sing. has β(1→3) as its main chain, and β(1→
6), is a glucan with β(1→3) branching, and antitumor polysaccharide SPG (Schizophyllan) derived from Schizophyllum commue also has β(1→3) branches.
3) It is a glucan based on bonds. On the other hand, the present protein polysaccharide is mainly mannan-based and is clearly different from the anti-tumor polysaccharides that have been published so far. The protein polysaccharide of the present invention having the above-mentioned physicochemical properties has a remarkable antitumor effect (see reference examples below), and also has an immune-enhancing effect, an interferon-inducing effect, and a vascular function-improving effect (for example, an anti-tumor effect). Hyperlipidemic effect), liver function improving effect, etc. are also observed.
It is an extremely useful substance. The present invention will be specifically described below with reference to Examples. Example 1 Glucose 4g, yeast extract 4g, malt extract
A PH5.5 medium consisting of 10g of water and 1 part of water was heated at 120°C.
After sterilization for 20 minutes, the agar medium was inoculated with the cultivated Kawariharatake mycelium, which was then statically cultured at 30°C for 30 days with occasional stirring. 5 g of main culture medium 2.0 with a pH of 5.0 consisting of 20 g of glucose, 5 g of Ebios powder, 20 ppm of antifoaming agent Rheocon 1705W (product name manufactured by Lion Oil Co., Ltd.), and 1 part of water.
It was placed in a Sakaguchi flask and sterilized at 120°C for 20 minutes. This was inoculated with 200 ml of the above precultured medium, and cultured at a culture temperature of 30°C using a reciprocating shaker. After culturing for 30 days, the culture was stopped, the mycelium was removed by centrifugation, and the resulting liquid was purified using a rotary evaporator for 6 days.
The mixture was concentrated to one-fold, an equal amount of ethanol was added thereto, and the mixture was left standing at 4°C for 1 day to form a precipitate. The precipitate was collected by centrifugation, washed with acetone and then with ether, and after drying, 875 mg of light brown powder was obtained per medium. Example 2 Glucose 20g, yeast extract 5g and pure water 1
A culture medium adjusted to pH 5.0 consisting of was placed in an Erlenmeyer flask and sterilized at 120°C for 20 minutes. This was inoculated with Kawariharatake mycelium cultured on an agar medium (slant) and cultured stationary at 30°C for 3 weeks. A main culture medium was prepared by dissolving 50 g of glucose, 10 g of Ebios powder, and 20 ppm of the antifoaming agent Rheocon in 1 part pure water and adjusting the pH to 5.0. This medium 7.0 was placed in a 10 volume jar fermenter and sterilized at 120°C for 20 minutes. Inoculate this with 500 ml of the above pre-cultured medium,
Culture temperature: 30℃, aeration volume: 0.5vvM, stirring speed: 200~
Culture was performed at 300 rpm. After 10 days of culture, the culture was stopped and the mycelium was separated, followed by further centrifugation at 10,000 rpm for 15 minutes using a centrifuge to remove the mycelium. Concentrate the obtained liquid 6.0 to 500 ml using a rotary evaporator and add 500 ml of ethanol.
ml was added and left at 4°C for one day and night to form a precipitate. Next, the precipitate obtained by solid-liquid separation was mixed with acetone,
After washing with ether and drying, 7.5 g of light brown powder was obtained. This was dissolved in pure water and dialyzed against pure water using a dialysis tube (Union Carbide Visking tube). The resulting precipitate was removed by centrifugation, and the supernatant was lyophilized to yield 6.0 g of white. A powder was obtained. Reference Example 1 Effect on Sarcoma 180 Tumor 24 hours after tumor inoculation, the pale brown protein polysaccharide (undialyzed) obtained in Example 2 was dissolved in physiological saline, and 11 mice per group (Swiss albino mice) were dissolved. )
The prescribed amount was administered intraperitoneally once daily for 10 days.
The size of the sarcoma was measured on the 14th, 21st, 28th, and 35th after inoculation, and the inhibition rate was calculated from the results. The results are shown in Table 1. 【table】

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of the protein polysaccharide obtained according to the present invention, and FIG. 2 shows its infrared absorption spectrum.

Claims (1)

[Claims] 1. A method for producing a protein polysaccharide, which comprises culturing Kawariharatake belonging to the genus Agaricus and collecting the protein polysaccharide from the resulting culture solution.