JPS62209091A - Antitumor active polysaccharide - Google Patents

Abstract

NEW MATERIAL:Antibiotic grifolan-N having the following physical and chemical properties. Elemental analysis (%): C 38.9-40.1, H 5.8-6.0, N <= determination limit. Molecular weight (gel filtration method): 1X10<5>-1X10<7> distribution. Melting point: 230 deg.C (decomposition). Specific rotatory power: [alpha]D=29.2+ or -0.5 (C=0.1, H2O). Solubility: readily soluble in water, alkali and dimethyl sulfoxide, insoluble in ethyl alcohol, ether, etc. Color reaction: positive in Molish reaction and negative in iodine reaction, etc. Appearance: white cotton state, etc. USE:An anti-cancer drug. PREPARATION:For example, a mycelium of Grifola frondosa var tokachiana (FERM P-4979) is subjected to contact reaction in a liquid medium containing one or more of low-molecular saccharide (e.g. glucose, etc.), high-molecular saccharide (e.g. starch, etc.), etc., at acidic pH at 20-30 deg.C for 2-8 days.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) This invention relates to an anti-tumor active polysaccharide obtained using Maitake mushrooms.

(Prior art) Many anti-tumor active substances derived from mushrooms have been known, including many for maitake, for example,
4386, a method for extracting anticancer substances from a cultured medium obtained by culturing maitake in a natural or synthetic nutrient liquid medium, and JP-A-6Q-58925, an antitumor active substance extracted from maitake fruiting bodies, etc. There is.

(Problems to be solved by the invention) - In the above-mentioned prior art, for example, Japanese Patent Publication No. 52-44386
Since the method disclosed in the publication uses a cultured medium as a raw material, it has the disadvantage that it requires a lot of complicated time and effort to separate and purify various organic and inorganic components mixed in the extract.
The method disclosed in Japanese Patent Publication No. 0-5892 uses fruiting bodies as raw materials, and therefore has the disadvantage that there is no problem in securing sufficient raw materials.

(Means for solving the problem) As mentioned above, in the conventional technology, separation of the target object,
There was still room for improvement due to the complexity of purification and problems remaining in the supply of raw materials.

In view of the above, as a result of intensive research, the inventors of the present invention published the patent publication No. 59-18995, which was invented by some of the inventors.
When we further investigated the polysaccharide obtained by contacting maitake mycelium with a carbohydrate solution, we unexpectedly found that it had antitumor activity, and based on this knowledge, we extracted neutral components from the polysaccharide obtained above. When fractionated, both fractions had high anti-J! l! It was confirmed that there was tumor activity, and as a result of physical and chemical analysis, as detailed below,
He recognized that it was a 1→3 glucan and completed this invention.

(a) Elemental analysis value C38.9-40.1%, H5.8
~6.0% N quantification limit (+) Molecular weight (gel filtration method) lxlO5 ~ lx107
Distributed to
．． 1.1120) (e) Infrared absorption spectrum (KBr
Method) As shown in Figure 1 (F) 13C-NMR spectrum (DMSO-d, middle) As shown in Figure 2 (G) Solubility in solvents Water, alkali, dimethyl sulfoxide ([) MSO)
Easily soluble in organic solvents such as ethyl alcohol, methyl alcohol, ether, and acetone.

(H) Color reactions The Molisch reaction, the Anthrone sulfuric acid reaction, and the phenol sulfuric acid reaction all give a positive result, and the iodine reaction, the ninhydrin reaction, and the Biillet reaction all give a negative result.

(S) Basic, acidic, neutral Aqueous solution is neutral (S) Color and shape of substance White, flocculent (Action) Maitake mycelia used in this invention are commonly known as Shiromaitake and Kuromaitake. Maitake mycelium is obtained by culturing Maitake mushrooms in a liquid or solid culture medium, and these Maitake mushrooms are those that have been isolated from natural Maitake mushrooms and have retained their characteristics through pure culture or subculture, or strains that are stored in various preservation institutions. Any strain that maintains its characteristics in the same way can be used, for example, as a preserved strain, Grifola frondos
a) IFO4911゜IFo7040, etc., which are obtained from nature, such as Grifola frondosa pal Tocatiana (Grifola frondosa pal Tocatiana)
var tokachiana) (Fiber Engineering Laboratory No. 497
No. 9), among which Grifola frondosa pal Tocatiana is preferred.

Now, to explain the polysaccharide of this invention using Grifola frondosa pal Tocatiana, the mycelium obtained by culturing a subculture of this strain in advance in a liquid or solid medium is collected by appropriate means. After rinsing with water if necessary, it is preferably dispersed and suspended in sterile water, and this is mixed with arabinose, xylose, fructose, glucose, mannose, galactose, maltose, sucrose, melibiose, lactose, raffinose, One or more of low molecular weight sugars such as inositol and mannitol, high molecular weight sugars such as starch, dextrin, and gum arabic, glycosides or their hydrolysates, refined molasses, and purified mucus during the sugar refining process. Add the carbohydrate solution containing the carbohydrate to a solution whose pH has been adjusted to an acidic range with an organic acid such as citric acid or lactic acid, and heat at 20 to 30°C while stirring.
When the contact reaction is carried out for 2 to 8 days, the mycelium acts enzymatically on the carbohydrates and produces a large amount of polysaccharides in the solution.

After the above reaction is completed, the mycelium and the supernatant are separated, and the separated supernatant is concentrated or precipitated by adding a sugar-insoluble solvent such as methanol, alcohol such as ethanol, or acetone without concentrating. is generated. At this time, ethyl alcohol is usually used. After forming a precipitate with such a solvent multiple times as necessary, the precipitate is separated and dried to obtain an almost white flocculent material.

, dissolve the white flocculent obtained above in urea solution or water,
DEAE regenerated into HCO,-type-Sephadex A-
25 to separate the neutral fraction.

A white cotton-like polysaccharide of the present invention is obtained by adding a solvent such as alcohol as described above to form a precipitate. In this case, when dissolved in a urea solution, the solubility is good, but dialysis is required to remove urea from the neutral fraction. - When dissolved in water, it has the advantage of not requiring dialysis of the neutral fraction, but it takes a little longer to dissolve than in a urea solution. Therefore, whether to use a urea solution or water to dissolve the substance should be selected depending on the circumstances.

do it.

As mentioned above, the polysaccharide of this invention can be produced by simply selectively extracting the neutral fraction from the material obtained by contacting maitake mycelium with a carbohydrate solution. is not necessary, and a sufficiently pure polysaccharide can be obtained by performing dialysis as necessary for isolation and purification of the neutral fraction.

The polysaccharide obtained above has β(1 → 3) bonded glucose residues for every 3 β(1 → 3) bonded glucose residues, according to the physical and chemical analysis results shown below.
→6) β- with one branched glucose residue attached
It is known that this polysaccharide is a glucan, and this polysaccharide is called Grifola.
Name it n-N.

This Grifolan-N exhibits high antitumor activity as explained in the Examples below.

Physical and chemical analysis (1) Elemental analysis values, etc. C: 3g, 9-40.1%, H: 5.8-6.0%
, N: Below the quantification limit, halogen and sulfur are not quantified.

(2) Molecular weight 0.2 mol NaOH/8 mol urea equilibrium Sepharose CL
-4B (Pharmacia Japan) column gel filtration chromatography revealed a molecular weight distribution range of 1X1
0' to lX10't'.

(3) Melting point It decomposes thermally at about 230°C.

(4) [α] at a concentration of 0.1 g/100 mQ in water at a specific optical rotation of 20°C
.

shows 29.2±0.5.

(5) Solubility: Easily soluble in water, alkali, and dimethyl sulfoxide (DMSO), but insoluble in organic solvents such as ethyl alcohol, methyl alcohol, ether, and acetone.

(6) Basic, acidic, and neutral aqueous solutions A 1% aqueous solution exhibits a neutral range (pH 6.3 to 6.5).

(7) Types of constituent sugars Hydrolyzate with trifluoroacetic acid (CF, C00H) was reduced to sodium borohydride (Na[304), and the alditol acetate derivative thereof was analyzed by gas chromatography. As a result, only glucose was found. is clearly detected, and other sugars such as fucose, xylose, mannose, and galactose are not detected.

(8) Binding mode of constituent sugars The results of methylation analysis using the Hakomori method are 2, 3, 4°6-
Chitramethyl-0-glycidol: 2,4.6-
Trimethyl-〇-D-glycidol: 2,4-dimethyl-〇-D-glycidol in a ratio of 1.0:1.7:1.0, 3.4.6-trimethyl-〇-D-glycidol, 2
,6-dimethyl-〇-〇-glycidol is a trace, so for every three 1→3 bonded D-glucose residues, 1→
It is known that glucan having 6-linked D-glucose branches is used as a repeating unit.

(9) Smith Decomposition Products Glucose and glycerin were detected as products by complete Smith decomposition, and glycerin was detected from the external dialysis fluid of the relaxed Smith decomposition product, and only glucose was detected from the hydrolysis fluid of the internal fluid.1 → It is known that it is a glucan with a structure having 3 bonds and a branch at C-6.

(10) Infrared absorption spectrum The results measured by the KBr method using a JASCOIRA-1 type point-to-point meter are shown in Figure 1, and an absorption (P) characteristic of β-glycosidic bond orientation is observed at a wave number of 894 cm-1. From this, it is recognized that it is a β-glycoside bond structure.

(11)” C-NMR spectrum dissolved in deuterated dimethyl sulfoxide (DMSO-d, ) and measured by JEOL-FX200 spectrometer.
The results measured at 0°C are shown in Figure 2, with a δ value of 67 ppm.
Signal S engineering including the assignment of C-6 carbon included in the β (l → 6) bond in the region and β (l → 3) in the δ value 8 Sppm region
A signal S2 attributed to the C-3 carbon included in the bond and a signal attributed to the C-1 carbon of the β-bond in the δ value range of 1103 pp are characteristically recognized, and furthermore, the signal S2 shows three peaks. Therefore, a β-glucan structure is known that has three β(1→3) bonds and one β(1→6) bond.

(12) The color reactions Morish reaction, Anthrone sulfuric acid reaction, and phenol sulfuric acid reaction all showed positive results, and the iodine reaction showed negative α(
1→4) It does not show the presence of bound glucan, and the ninhydrin reaction and Biuret reaction are both negative, so it does not show the presence of protein or peptide.

As mentioned above, the antitumor active polysaccharide Grifo of the present invention
Since lan-N exhibits high antitumor activity, it is useful medicinally and can be used for intraperitoneal administration, tumor administration, intravenous administration, and can also be used for oral administration, and is available in various forms. It has anticancer drug uses.

This will be explained in more detail below with reference to Examples.

(Example) Example 1 Pre-subcultured Grifola frondosa pal Tocatiana
varTokachiana)
Cut 2 sections of 5 m + s x 5 mm from the medium surface containing No. 9).
Take a piece and inoculate it onto an autoclaved slope of 4% malt extract, 5% sawdust extract, 5% wheat extract, 25% agar, pis, s, and culture at 25°C for 3 weeks to use as a primary inoculum. . 5mm x 5mm from the above - next inoculum
Take out 3 sections and mix them with 2% sucrose (as sugar content).

Inoculated into 50 Peng Q autoclaved medium containing 0.4% polypeptone and adjusted to pH 5.5 with 10% lactic acid.
A secondary inoculum is obtained by statically culturing at 5°C for 3 days. This secondary inoculum to 5rtrQ was added to an autoclaved medium containing 2% pullose, 2% cane molasses (as sugar content), 0.6% polypeptone, 3 drops of soybean oil, and adjusted to pH 4.5 with 10% lactic acid. The mixture was inoculated into two 500-capacity Sakaro flasks each containing 100 tR, and cultured with shaking at 25°C for 2 weeks to use as a tertiary inoculum. The total amount of the inoculum obtained above was autoclaved containing 2% glucose, 12% sucrose (as 11i amount), 0.6% polypeptone, and 0.1% soybean oil, and adjusted to pH 4.5 with 10% lactic acid. Completed medium 6
Pour Q into a 1012 capacity jar fermenter, aeration rate 0.5VVM, temperature 25℃, stirring number 25Or.
, P, and Im for 6 days.

The contents obtained in the jar fermentor culture described above were centrifuged (30 QOr, p, m, 5 minutes) to collect bacteria, and after washing with water and centrifuging twice, the total amount of mycelia obtained was collected. A 10Q capacity jar fermenter containing 6Q of carbohydrate solution with 5% glucose, 10, s% citric acid, and pH 4.0 was charged into a jar fermenter with aeration rate of 0.5 VVM, 28°C, and stirring number 250.
After reacting for 2 days at R, P, and M, the contents were centrifuged to separate the mycelia and supernatant, and the supernatant was diluted with ethanol at a concentration of 40
Ethanol was added to make a precipitate by volume%, and the precipitate was centrifuged and freeze-dried.

6.5 g of dry matter was obtained. Using the reacted mycelium remaining after the centrifugation, a second reaction was carried out using the same carbohydrate as above to obtain 5.8 g of a dried product in the same manner.

Furthermore, a third reaction was carried out using the same carbohydrates as above using the reacted mycelium remaining in the second reaction (however, the number of days for the reaction was set to 3 days), and 5.0 g of dried product was obtained in the same manner. .

Thoroughly mix the dried products obtained in the first to third steps above.

Add 1 g of this to an 8 molar urea aqueous solution at approximately 2 mg/mQ.
DEAE-
A neutral fraction (passing through the urea solution eluate) was collected using a column packed with Cellphadex A-25200mR, dialyzed for 72 hours with a cellulose tube (manufactured by Shiraimatsu Kikai), and the internal solution was 1.5 times amount of ethanol is added to form a precipitate, which is lyophilized to form a white flocculent or fine fiber-like antitumor active polysaccharide Grifolan-
Obtained 6 g of NO. This product showed the above-mentioned physical and chemical properties.

Example 2 Antitumor activity of Grifolan-N obtained in Example 1 (Part 1) IRC-strain 6-week-old mice (male, weight 27-30
g) Sarcoma 180 tumor cells 5 in groups of 10 animals
X10' was inoculated subcutaneously in the groin area, and this was designated as day 0.
A test solution prepared by dissolving the aforementioned Grifolan-N in physiological saline in a test amount was administered via various routes five times on days 1.3, 5.7, and 9, and disassembled on the 35th day after inoculation of tumor cells. When the inhibition rate and degree of complete regression of IIIR tumor growth were investigated, as shown in Table 1, Grifolar+-H was found to have a high tumor growth inhibitory effect.

1) *P<0.05. Book IP (0,
01, heavy water * p<o, oot Example 3 Antitumor activity of Grifolin-N obtained in Example 1 (according to the procedure of Example 2, the inhibition rate was investigated when the administration port was changed. IRC - strain 6 week old mice (male, weight 2
7 to 30 g), 5 x 10' Sarcoma 180 tumor cells were inoculated subcutaneously in the groin area in groups of 10 animals,
Let this be day zero. Grifol dissolved in saline
A test amount of an-N was intraperitoneally administered on predetermined days before and after day zero, and on the 35th day counting from the day after day zero, the mice were dissected and the state of the tumor was investigated. The results are shown in Table 2, and a high suppressive effect was observed even when administered before the tumor inoculation site.

Such a deterrent effect due to pre-administration is due to the conventionally known polysaccharide substance obtained from the fruiting body of Maitake mushroom (Japanese Patent Application Laid-Open No. 60-58925
This is characteristic of Grifolan-N compared to the previous publication. The reason for this effect of pre-administration is unknown and awaits future research.

Table 2 Dosage Dosage schedule Tumor weight (g)
Deterrence rate μg/times x times (day)
Average ± SD (%) 100X5 -9. −
7. -5. -3. -1 0.67±1.62
86.2100X5 ÷1. +3. +5.
+7. +11 1.13±3.57 76.7
100x5 +11. +13. +15. +17.
+19 0.73+1.29 84.9 control
+1.43.45. +7. +9 4.84±3.78
- (Effect) According to the present invention, fractionation and purification of the polysaccharide are extremely easy, and the obtained polysaccharide has high antitumor activity, so it has extremely high utility value as a tumor suppressor. , and allows for cheap leftover production.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum of Grifolan-N of the present invention. Figure 2 shows 13C-N of Grifolan-N of this invention.
An MR spectrum is shown.

Claims (1)

[Claims] Antitumor active polysaccharide Grifo having the following physicochemical properties
lan-N. (a) Elemental analysis value C38.9-40.1%, H5.8
~6.0% N quantification limit (b) Molecular weight: 1 x 10^5 - 1 x 1 by gel filtration method
Distribution at 0^7 (c) Melting point Thermal decomposition at 230℃ (d) Specific optical rotation [α]_D 29.2±0.5 (C=0
．． 1, H_2O) (e) Infrared absorption spectrum (KBr method) As shown in Figure 1 (f) ^1^3C-NMR spectrum (DMSO-d_
6) As shown in Figure 2, (g) Solubility in solvents, Easily soluble in water, alkali, dimethyl sulfoxide, Insoluble in organic solvents such as ethyl alcohol, methyl alcohol, ether, acetone, etc. (H) Color reaction mode. The Lisch reaction, Anthrone sulfuric acid reaction, and phenol sulfuric acid reaction all show positive results, and the iodine reaction, ninhydrin reaction, and Buret reaction all show negative results. (i) Basic, acidic, neutral Aqueous solution is neutral (n) Color and shape of substance White, flocculent