JPH0248161B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to β-D-glucan extracted from the pine-like mycelial mass of a new strain of maitake, Glyfuora frondotsa bal tocatiana. In recent years, since the anticancer effects of certain polysaccharides have been known, interest in polysaccharides has increased in this field, and various studies have been conducted and the results have been published. In particular, there has been much research into polysaccharides derived from basidiomycete mushrooms, and for example, protein polysaccharides obtained from the extracellular culture of Corsicolor versicolor have already been put to practical use. A similar method for obtaining anticancer polysaccharides from extracellular culture products for mushroom strains belonging to Maitake fungi was proposed in Japanese Patent Publication No. 52-44386; requires many and complicated means for separation and purification. This invention does not use a cultured medium as a raw material as mentioned above, and the edible part of a new strain of maitake, Grifola frondotsa bal tocatiana, whose use has not been considered at all, after culturing the fruiting body. It was discovered that the pine-like mycelial mass that remained after the collection of the edible hyphae contained a substance with high antitumor activity, and based on this knowledge, intensive research revealed that this substance was β 1 → 3) A structure in which the main chain is a bonded glucan, and for every three β-D-glucose residues, there is a branch of one glucose residue that is β-(1 → 6) bonded to the carbon at the C-6 position. It was confirmed that this is a β-D-glucan having a repeating unit structure, leading to the present invention. This will be explained in detail below. What is the pine-like mycelial mass of maitake used in this invention? “Primary Colored Japanese Fungi Illustrated Encyclopedia” co-authored by Mutsuya Imazeki and Tsuneo Motoki
A new strain of the genus Maitake, Glyfuora frondotsa, which belongs to the family Salmonaceae, according to (Nursery Company)
Maitake mushrooms are artificially cultivated in a medium prepared by adding various nutrients to maitake, mainly consisting of sawdust, and the mycelium that remains after harvesting the edible cap is formed by a pine-shaped accumulation. In the case of bottle cultivation or bag cultivation, this corresponds to the base part of the fully heated maitake, which is about 2 to 3 cm in length. This was discarded as an edible portion, and the weight ratio is 8 to 10 to 100 edible portions (bulk portions). In addition, when a high-density maitake seed is transplanted into a mountain forest or other soil and cultivated, the growth process is slightly different and the form of ripening is also different, but in this case as well, the inedible parts will become inedible according to the above procedure. A pine-like mycelial mass may be used. The β-D-glucan of the present invention can be obtained by extracting the raw material, the pine-like mycelial mass, with hot water, low temperature or high temperature alkaline solution, and performing low temperature alkali extraction on the hot water extraction residue.
Furthermore, by subjecting this extraction residue to high-temperature alkali extraction, the target substance can be efficiently extracted. After removing the edible part from artificially cultivated maitake mushrooms, foreign matter such as the attached medium is removed from the remaining pine-like mycelial mass, and the cleaned pine-like mycelial mass is finely chopped in the case of fresh products, or finely chopped in the case of dried products. is destroyed.
The finely chopped or crushed pine-like mycelial mass is extracted with hot water in a pressure vessel such as an autoclave. Extraction is carried out in several batches using 50 to 100 times the amount of water as the pine-like mycelial mass, at a temperature of 120 to 125°C, and for 50 to 100 minutes per batch. Conventionally known extraction of mushroom components requires degreasing prior to extraction, but the inventors' experience has shown that degreasing with an organic solvent increases the removal load during subsequent purification, which is undesirable. Since there is no problem even without degreasing, it is preferable to omit degreasing in this invention. The extracted product is separated into an extraction residue and a hot water extract (F-1) by a conventional separation means such as centrifugation or filtration. The extraction residue is then subjected to low-temperature extraction with a 5-10% dilute alkaline solution. As the alkali, caustic soda and caustic potash are used, with caustic soda being preferred for subsequent treatment. 10-15 of extraction residue
Divide the amount of the alkaline solution into several times and use 3
Extract at ~5°C for 20-30 hours/time. The extracted product is separated into an extraction residue and an extract (F-2) in the same manner as described above. Regarding the low-temperature alkali extraction residue, use an alkaline solution of approximately the same level as above and heat it at 50 to 70℃ for 20 minutes.
Hot alkaline extraction is performed for ~30 hours, and the extract is separated into an extraction residue and an extract (F-3) in the same manner as above. Hot water extract (F-1), cold alkaline extract (F-2) and hot alkaline extract (F-1) obtained above.
For each of 3), low molecular weight components and salts are removed by dialysis, the dialyzed solution is concentrated, and then precipitated with ethyl alcohol. In this case, the alkaline extract is neutralized with an acid such as acetic acid or diluted hydrochloric acid and then dialyzed. The precipitate obtained by alcohol fractionation of the above extracts F-1, F-2 and F-3 is a crude fraction containing the glucan of the present invention with high purity, and these crude fractions are then separated under boiling conditions. If necessary, it can be dissolved in water using ultrasonic irradiation, and if the dissolution is insufficient, it can be dissolved in alkali and subjected to dialysis after neutralization. The liquid is adsorbed through a column chromatography packed in a gel agent, and the aqueous eluate fraction eluted with water is collected. As a gel agent, DEAE-type Cephadex, Cepharose, bio-gel or cellulose can be used, but DEAE adjusted to HCO ₃ type can be used.
-Sephadex-A25 is preferred. Among the water elution fractions derived from the extracts F-1, F-2, and F-3, the water elution fractions derived from F-2 and F-3 were subjected to dialysis treatment, and ethyl alcohol was added to the concentrated solution. Almost 100% pure glucan can be obtained by simply producing a precipitate, but the water eluted fraction derived from F-1 that involves heat treatment contains unnecessary glucan (α-type) other than the desired glucan. Since there is a risk of contamination, enzyme treatment with α-amylase is performed to remove α.
-F-
2. Glucan similar to the alcohol precipitate derived from F-3 can be obtained. The glucan thus obtained has the following physicochemical properties. (1) Elemental analysis values, etc. C: 41.0-44.2%, H: 6.9-7.3%, N: below the limit of quantification, no halogen or sulfur detected. Total sugar (as glucose (molecular weight 162)) by phenol sulfuric acid method is 93-95%, Lowry-
Although the protein content is determined to be less than 0.5% by the Folin method, this value is so small that it cannot be removed by ordinary purification methods, and is not essential for characterizing the β-glucan of the present invention. (2) Molecular weight 0.2M-NaOH equilibrium Sepharose CL-2B and
The molecular weight is recognized to be in the range of 500,000 to 1,500,000 based on gel overdistribution using a CL-4B (Pharmacia Japan) column. (3) Melting point: Decomposes thermally at approximately 230℃. (4) Specific rotation [α] ²⁰ _D = +5 to +10 (solvent H ₂ O, concentration C =
0.1) (5) Solubility Easily soluble in alkalis, water, and dimethyl sulfoxide, slightly soluble in acids, and insoluble in ethyl alcohol, methyl alcohol, ether, acetone, and other organic solvents. (6) PH of aqueous solution PH of 1% aqueous solution 6-7 (7) Type of constituent sugar Add 1 ml of 1M trifluoroacetic acid (CF ₃ COOH) to the substance of this invention and hydrolyze at 100°C for 5 hours. Then, the product was reduced with sodium borohydride (NaBH ₄ ) by a conventional method, acetylated with pyridine-acetic anhydride to convert it into an alditol acetate derivative, and purified with N ₂ by gas chromatography (Shimadzu GC-6A). gas
Feed at 60ml/min, from 170℃ to 250℃ at 4℃/min
As a result of raising the temperature to ℃ and analyzing it, only glucose was clearly detected without detecting fucose, xylose, mannose, or galactose, which indicates that glucose is the only constituent sugar. (8) Binding mode of constituent sugars Hakomori method (S. Hakomori, J.Biochem, 55,
205 (1964), the methylation analysis method
90% of the substance of this invention is completely methylated with methyl iodide in a sealed tube.
After decomposition with formic acid (120°C, 10 hours) and removing residual formic acid by evaporation, the residue was hydrolyzed with 1M trifluoroacetic acid (100°C, 5 hours) and evaporated to dryness. - Partially O-methylated-alditols produced by reduction of methylated sugars with _NaBH4 (room temperature, 2 hours) to give the corresponding alditols, followed by acetylation (with pyridine-acetic anhydride) in a conventional manner. Acetate was analyzed by gas chromatography (GLC) and gas chromatography-mass spectrometry (GLC-MS). In addition, gas chromatography (GLC) and gas chromatography-mass spectrometry (GLC)
−MS) are both 0.3%OV−275+0.4%XF−
A 1150/Gas chromQ column of 0.3 x 200 cm was used, and the temperature was raised from 120°C to 190°C at 2°C/min. The analysis results were converted to 1.5-di-O-acetyl-2,
Each O-
The molar ratio of alditol acetate to methylated sugar is shown in Table 1 (molar ratio was determined from the area of the chromatogram).

[Table] In Table 1, 2,3,4,6-tetra O-Me
2,4,6-tri-O-Me-D-G when the alditol acetate of -D-G is 1.0
The molar ratio of alditol acetate of 2,4-di-O-Me-D-G is 1.9 to 2.0 and 0.9 to
1.1 and other 2,3,4-tri-O-
Me-D-G, 2,3,6-tri-O-Me-D
-G and 3,4,6-tri-O-Me-D-G
of alditol acetate are both 0
~Since it is a trace, the binding mode of glucose known from (7) above is

[Formula] That is, (1 → 3) for every 3 bonded glucose residues (1 → 6) at the C-6 position of one of the glucoses in the main chain
It is recognized that the mode of attachment is with branches of the glucose units attached. (9) Smith degradation products and their methylation Smith degradation (Biochemistry Course 4 Chemistry of Carbohydrates (Part 2)
(edited by the Japanese Biochemical Society, P479-495, 1982), the substance of this invention was oxidized with sodium metaperiodate (NaIO ₄ ) and then reduced with sodium borohydride (NaBH ₄ ) to give a polysaccharide polyalcohol, (i)0.5 ~ Hydrolyzed with 1.0N-sulfuric acid (completely
As a result of (Smith decomposition), glucose and glycerin were detected as products, and (ii) the 0.1N-sulfuric acid hydrolyzate (relaxed Smith decomposition) was dialyzed, and glycerol was obtained from the external dialysis fluid, and only glucose was obtained from the hydrolysis of the internal fluid. were detected respectively. From the results (i) and (ii), it is known that the glucan has a structure having a (1→3) bond and a branch point at C-6. Furthermore, the polysaccharide polyalcohol and the dialysate solution of (ii) above are treated in the same manner as in (8) above to convert them into the corresponding alditol acetate derivatives, and GLC and
Table 2 was obtained as a result of GLC-MS analysis, and from this result, it was recognized that the structure was such that one glucose branched from the main chain glucan with (1→3) bonds at regular intervals.

[Table] (10) β-glucanase degradation products The substance of this invention contains exo-type β-(1→3)-D
- Glucanase (derived from Basidiomvcetes sp, manufactured by Sigma) was applied at PH4.8, 37°C for 24 hours,
Deactivate the residual enzyme and transfer the reaction solution to Bio-GelP-2
As a result of chromatographic separation using a (polyacrylamide gel) column, gentibiose (6-O
-(β-D-glucopyranosyl)-D-glucopyranose) and glucose were detected, and no other substances were detected. Therefore, (1→
3) Glucan is β-type D-glucose, and C-
It is known that the branch at the 6th position is one β-type D-glucose. (11) ¹³ C-Nuclear Magnetic Resonance (NMR) Spectral Analysis The substance of this invention was dissolved in deuterated dimethyl sulfoxide (DMSO-d ₆ ) as a deuterated solvent, and 60
The resulting spectrum, measured at 0.degree. C., is shown in FIG. The spectra were operated in Fourier transform NMR mode under conditions of complete decoupling of hydrogen nuclei. In Figure 1, the signal S ₁ consisting of three peaks observed in the δ value region of 86 ppm is β (1 → 3)
three D-glucopyranosyl residues A,
B,C are assigned to the carbon at the C-3 position, and 68.2ppm
The signal S ₂ observed in is attributed to the hydroxymethyl carbon at the C-6 position, which contains a β (1→6) bond. Therefore, from the two characteristic signals S ₁ and S ₂ that appeared in Figure 1, β
- (1→6) for every 3 bonded D-glucose residues 1 (1→6) bonded D-glucose residue
The structure of β-glucan, which has several branches, can be known. (12) Infrared absorption spectrum analysis Using a JASCO IRA-1 type spectrometer, the substance of this invention was mixed with KBr (powder) for infrared measurement,
Fig. 2 shows a spectrum diagram obtained by tableting and measuring by a conventional method. In FIG. 2, the absorption (P) observed at a wave number of 878 cm ^-1 is characteristic of β-glycosidic bonds, and it is known that the substance of the present invention is a glucan composed of glucose having β-glycosidic bonds. (13) Color reaction Color reaction name Reaction color Judgment Moritsch reaction Purple-red positive Anthrone sulfuric acid reaction Green positive Phenol sulfuric acid reaction Brown positive Ninhydrin reaction No color development Negative As the ninhydrin reaction is negative, it is composed of sugars that do not contain protein. It is known to be a polysaccharide. From the above analysis results of various physical and chemical properties, the constituent sugar of the β-D-glucan of the present invention is β-D-glucose, and its binding mode is β (1 → 3) bond D-
β at carbon C-6 for every 3 glucose residues
(1→6) A structure with one branched glucose residue bonded, i.e.,

[Formula] is a repeating unit structure, and it is recognized that this unit is a β-D-glucan composed of a large number of linked units, and the β-glucan of this invention is as explained in the examples below. As a result of experiments using mice,
Dosage per dose: 20 to 30 g per mouse body weight
It has extremely high antitumor activity with a 93-97% growth inhibition rate for transplanted Sarcoma 180 solid sarcoma when administered in small doses in the 100 μg range, and no significant changes are observed even when temporarily administered in large doses. .
Because of its physiological activity, the β-D-glucan described above is also useful as a medicinal agent, and can be used as an anticancer drug in a variety of ways, including intraperitoneal administration, intratumoral administration, intravenous administration, and oral administration. It has the following.
This will be explained in more detail below using Examples. Example 1 Forty 1-volume wide-mouth polyethylene bottles were filled with a medium prepared from sawdust, soybean meal, soybean meal, and soil extract, and the maitake fungus strain Glyfuora frondotsa bal tocatiana (Feikoken The mature maitake mushrooms were inoculated with Mycobacterium no. 4979) and cultured for about 60 days. The mature maitake mushrooms were removed from the cultivation bottle, leaving about 3 cm of the base, and the upper part was collected as the edible part. This weight was approximately 5100g. A portion approximately 3 cm long at the base was collected and foreign substances such as attached medium were removed to obtain approximately 410 g of a pine-like mycelium mass. This was washed with water to the extent of rinsing, dried in the sun, and then ground in a small pulverizer (sample mill, rotation speed: 10,000 rpm) to form a powder. The moisture content of this powder was 5.2%. 65 g of this powder was extracted with hot water using 600 ml of water/time at 121°C for 60 minutes (using an autoclave). This operation was repeated 7 times, and the extraction residue and extract liquid (including washing liquid) were separated by centrifugation. Got 4.8. The entire amount of this extract was concentrated under reduced pressure to a volume of about 400 ml, and 400 ml of ethyl alcohol of 99% or higher was added thereto and gently stirred.
Precipitate the alcohol-insoluble fraction at 15,000 p.m.
The precipitate was collected by centrifugation for 10 minutes and dried with acetone/ether to obtain 9.73 g of powder. 3 g of this powder crude fraction was dissolved by heating under boiling using ultrasonic waves to a concentration of 5 mg solute/ml water, and after cooling, 600 ml of the solution was dissolved in DEAE-Sephadex A25 (HCO ^- type ₃ ) 50
Pass 300 mg of solute/time through a ml column and adsorb it with 200 mg of water.
ml/time to obtain 2000 ml of water elution fraction, which was dialyzed in demineralized water for 24 hours using a cellulose tube (manufactured by Shiraimatsu Kikai).The dialyzed solution was concentrated to about 100 ml, and the salt concentration, PH 0.1M Tris-HCl buffer PH6.9
(use 0.1% _NaN3 to prevent spoilage), add 3 mg of crystalline α-amylase (Sigma), and heat at 37°C.
The reaction solution was treated for 24 hours, heated to inactivate the enzyme, dialyzed in demineralized water for another 24 hours, concentrated the dialyzed solution under reduced pressure to a volume of 100 ml, and centrifuged at 15,000 rpm for 15 minutes. 150 ml of 99% or higher ethyl alcohol was added to the obtained supernatant, and the resulting precipitate was collected by centrifugation and thoroughly dried with acetone/ether to obtain 460 mg of an almost white powder. This powder exhibits the physicochemical properties summarized in Table 3 and is the β-D-glucan of the present invention. Example 2 The hot water extraction residue obtained in Example 1 contains 5% urea.
Add 10% caustic soda solution 600ml/time, 4℃, 20
The extraction operation was repeated three times under different conditions, and the extraction residue and extract liquid (including washing liquid) were separated by centrifugation.
I got it. After neutralizing this extract with concentrated acetic acid
8 in running water using visking cellulose tubes
Dialysis was carried out for several days, and the dialysis fluid was centrifuged at 15,000 r.p.m for 10 minutes. The supernatant obtained was concentrated to a volume of about 400 ml, and
The alcohol-insoluble fraction was precipitated by adding 400 ml of 99% or more ethyl alcohol, centrifuged at 15,000 rpm for 10 minutes, the precipitate was collected, and dried with acetone/ether to obtain 4.07 g of powder (crude fraction). Aqueous fraction 2000 obtained by treating 3 g of the above powder with DEAE-Sephadex A25 column in the same manner as in Example 1
ml in demineralized water using cellulose tubes.
The internal solution obtained by dialysis for 24 hours was concentrated under reduced pressure to a volume of 100 ml.
150 ml of 99% or more ethyl alcohol was added to the supernatant obtained by centrifugation, and the resulting precipitate was collected by centrifugation and sufficiently dried with acetone/ether to obtain 450 mg of an almost white powder. This powder exhibits the physicochemical properties summarized in Table 3 and is the β-D-glucan of the present invention. Example 3 Urea 5 was added to the low-temperature alkaline extraction residue obtained in Example 2.
Add 600 ml of 10% caustic soda solution containing 65°C.
Hot alkaline extraction was performed for 60 minutes to obtain 800 ml of extract (including washing liquid). This extract was cooled, neutralized with concentrated acetic acid, and treated in the same manner as in Example 2 to obtain 2.88 g of powder (crude fraction). The entire amount of this powder was treated in the same manner as in Example 2 to obtain 460 mg of almost white powder. This powder shows the physical and chemical properties summarized in Table 3,
It is the β-D-glucan of this invention.

【table】

[Table] Example 4 The glucan obtained in Example 2, which is one of the glucans of the present invention, was tested for toxicity and antitumor activity using mice as specimens. (1) Body weight change due to large dose ICR mouse, 6 weeks old (male, weight 27-30g)
was divided into an untreated group (6 mice), an intraperitoneal administration group (5 mice), an intravenous administration group (5 mice), and an oral administration group (5 mice).The oral dose was 6 mg/mouse, and the other doses were 1 mg/mouse. The product was administered to mice at a dose of 3 mg/mouse, and body weight changes were observed for 10 days after administration. As shown in Table 4, no significant changes were observed overall.

【table】

[Table] (2) Antitumor activity The glucan of this invention and the crude fraction obtained in the process were tested. ICR-strain 7-week-old mice (male, weight 27~
Sarcoma 180 solid sarcoma cells (5 x ¹⁰⁶ cells/mouse) were transplanted into the right inguinal region of the mouse, and the test amount was administered every day for 10 days from the next day on a once/day dosing schedule. The size of the sarcoma (maximum diameter x minimum diameter mm ² ) was measured every week, and the weight of the sarcoma removed on the 35th day was measured to investigate the growth inhibition rate and complete regression. These results are shown in FIG. 3 and Table 5.

【table】

【table】 [Brief explanation of drawings]

FIG. 1 shows the ¹³ C-nuclear magnetic resonance (NMR) spectrum of the substance of the present invention, and FIG. 2 shows the infrared absorption spectrum of the same substance. FIG. 3 is a graph illustrating an example of the antitumor activity of the same substance, and shows the progress over time of Sarcoma 180 transplanted into mice by administration of the same substance and its crude fraction. The meanings of (a) to (g) shown in FIG. 3 are as shown in the following table.

【table】

【table】

Claims (1)

[Scope of Claims] 1 β having the following physicochemical properties obtained by extraction of the pine-like mycelial mass of the cultured fruiting body of the Maitake strain Glyfuora frondotsa bal tocatiana.
β- characterized in that the main chain is a β-1,3 bond, and the repeating unit is a structure in which one β-1,6-linked glucose is branched for every three β-1,3-linked glucose residues. D-glucan. (a) Elemental analysis values C 41.0-44.2% H 6.9-7.3% N Below the quantification limit (b) Molecular weight (gel filtration method) 500,000-1,500,000 (c) Melting point Decomposes at approximately 230°C (d) Specific optical rotation [ α〕 ²⁰ _D = +5 to +10 (C = 0.1,
H ₂ O) (e) Infrared absorption spectrum (kBr tablet method) Shown in Figure 2. (f) ¹³ C-NMR spectrum shown in Figure 1. (g) Solubility in solvents Easily soluble in alkalis, water, and dimethyl sulfoxide, slightly soluble in acids, insoluble in organic solvents such as ethyl alcohol, methyl alcohol, ether, and acetone (h) Color reaction Moritsch reaction, Anthrone Both sulfuric acid reaction and phenol sulfuric acid reaction are positive, ninhydrin reaction is negative (li) Distinction between basic, acidic and neutral 1% aqueous solution is slightly acidic to neutral (PH6-7) (n) Color and shape of substance Almost white Powder body.