JPS61194033A - Glycoprotein coriolan, and production thereof - Google Patents

Abstract

NEW MATERIAL:A glycoprotein coriolan having the following characteristics. Elemental analysis, C 50.0-52.0%, H 7.2-8.2%, N 2.0-6.0%, the rest part is O; color reactions, shown in the table I; pH, 4.4 as a solution obtained by dissolving 0.1g of the compound in 100cc of water; molecular weight, >=2,000,000 (gel-filtration, etc.); solubility, soluble easily in water and insoluble in acetone, etc.; composition of the constituent sugars, shown in the Table II; composition of the constituent proteins, shown in the Table III; ratio of protein to sugar, 1:2-4; color, white (freeze-dried product); decomposition point, 223 deg.C (by scanning differential thermal balance). USE:Antitumor agent. PREPARATION:The cultured product, fruit body, or mycelia of Coriolus pubescens is extracted with an aqueous solvent at <=50 deg.C, preferably at 0-30 deg.C, and subjected to gel-filtration to obtain the objective glycoprotein coriolan having a molecular weight of >=2,000,000. It can be administered by intravenous injection, oral administration or rectal infusion, at a dose of preferably <=500mg daily for injection and <=5g daily for oral administration.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel glycoprotein Coriolan.
n) and its manufacturing method.

More specifically, the present invention relates to Coriolan, a glycoprotein with antitumor activity, and a method for producing the same.

In recent years, there have been many reports that substances showing antitumor effects can be obtained from Basidiomycetes (Japanese Patent Laid-Open No. 54-140711,
JP-A No. 55-42564), and there are also known products that have been commercialized and used for actual treatment.

In 1961, the anticarcinogenic properties of the fruiting bodies and culture filtrate of 638 species of higher fungi were systematically tested in Ehrlich's ascites carcinoma, and the anticarcinogenic properties were found in fungi of 10 species of the family Arunocarinae and 20 species of the family Shimetaceae, including C. versicolor. was reported to be included. [Nobuhiko Komatsu, Hironori Terayo, Research on anticancer substances produced by higher fungi, Ministry of Education Research Report Collection (1961, Cancer), 16
0°1962).

Based on these results, the present inventors conducted intensive research in search of anti-tumor substances regarding various closed classes.
It has been found that the fruiting body and mycelium of Coriolus pube+5csns, which is in the order Coriolus, family Coriolus, genus Coriolus, contains a glycoprotein with a molecular weight of 2,000,000 or more, and that this substance has an excellent antitumor effect.

That is, the t2. ooo, Coriolan, a glycoprotein with excellent antitumor and immunostimulatory properties, can be obtained by separating over 000 polymeric substances;
The present invention has been completed based on the result that the separation of this substance can be achieved more advantageously than by gel filtration.

The present invention is Coriolan, a glycoprotein with a molecular weight of 2,000,000 or more.

Further, the present invention is a method for cultivating P. chinensis and collecting Coriolan, a glycoprotein with a molecular weight of 2,000,000 or more, from the culture.

Furthermore, the present invention involves extracting a culture, fruiting body, or mycelium of Yakifutake with an aqueous solvent, treating the resulting extract with a gel filtration method, and collecting Coriolan, a glycoprotein with a molecular weight of 2,000,000 or more. This is the way to do it.

The fungi used as a raw material in the present invention are Cor.
Any strain may be used as long as it belongs to the genus iorug pubescens). Coriolua pubesce, which has been entrusted to the Institute of Fine Technology as an example of Yakifutake
ns(Fr,) Quel, FERM P-65
61 is effectively used.

The extract raw material of the present invention may be in the form of natural or artificially cultured fruiting bodies or mycelia, and if it is artificially cultured using a liquid medium, it may be present in the cultured mycelium and the liquid medium during culture. All, including those released or eluted, can be used as raw materials for extracts.

From an industrial point of view, industrial mycelium cultivation using a liquid medium is preferable since it allows stable extraction raw materials to be obtained regardless of the season.

In the present invention, it is desirable to use a weakly acidic to weakly alkaline, preferably approximately neutral, aqueous solvent to extract the culture, fruiting body, or mycelium of Yakifutake, and specifically, it is -4 to 10, preferably ...Water (purified water, etc.) or an aqueous solution of 6 to 8 is desirable. An alkaline aqueous solution such as an aqueous sodium hydroxide solution is not preferred because it induces changes in the active ingredients contained in Yakifutake and reduces the medicinal efficacy of the final extract. Particularly, pre-alkaline aqueous solutions (IN to 2N) are not preferred. In the present invention, it is preferable to perform extraction using a physiological salt solution.

Physiological saline solution is a mixed solution of salts that is used as a medium to maintain the normal function of excised organs and tissues over a long period of time. It is also called a balanced salt solution because the ion antagonism is properly balanced. A typical example of this is physiological saline.

Regarding the extraction temperature, care should be taken because if the extraction temperature is higher than 50°C, the extraction of the active ingredients will be insufficient, and if it exceeds 100°C, the activity of the active ingredients will decrease due to heat.

Therefore, extraction is preferably carried out under mild conditions of 50°C or lower, more preferably 0 to 30°C, most preferably 3 to 27°C, particularly VC 3 to 7°C. Extraction can be accelerated by grinding or the like.

After the extraction is completed and the water-soluble polymer substance is recovered as described above, it is treated by gel filtration to obtain an extract.

This extract is preferably subjected to solid-liquid separation by a conventional method such as centrifugation prior to gel filtration. The extract obtained by extraction as described above is subjected to a gel filtration method to separate substances having a molecular weight of 2,000,000 or more that are dissolved in the extract. In gel A4, 5ephacryl
-S-200 (manufactured by Pharmacia) is preferably used.

Purification of the above-mentioned extract according to the present invention is preferably carried out under as mild conditions as possible and at a low temperature. Unlike ultrafiltration and reverse osmosis, gel filtration can be performed under normal pressure.
Meets this requirement. In addition, gel DV filtration is performed at 0 to 60°C.
Preferably it is carried out at 3 to 27°C, more preferably at 6 to 7°C. If this temperature is too high, Coriolan may be denatured.

The gel filtration method in the present invention has a molecular weight of 2,000,000
If the above fractions are separated, the glycoprotein Coriolan can be isolated as is, which is extremely effective. On the other hand, the limit a, II! The purpose of the filtration method and reverse sulfur pressure is to remove low molecular weight substances;
In contrast to the gel filtration method, which passes through the pores of a rane membrane, gel filtration is possible at normal pressure, and the process is much more physiological and simple.

In the present invention, molecular weight 2.000,
The glycoprotein Coriolan is obtained as a white powder by dialyzing more than 1,000 ml of the glycoprotein into water and freeze-drying it. The elemental analysis value of this white powder is typically C,50,0
~52.0,)(,7,2~8.2%,N,2.0~6
, 0chi residue is 0, and this substance is water-soluble,
The aqueous solution is white and transparent. In addition, when the molecular weight of this substance was measured using a molecular sieve, it was found that 5epharose C
Since it is eluted at the void volume position of L 2 B, the molecular weight is 2.000,000 or more.

Next, the physicochemical properties of the glycoprotein Coriolan of the present invention will be shown.

+11 Elemental analysis Elemental analysis of this material was conducted using PerkinElmer model 240C, and the results showed that carbon was 50.0 to 52.0%, hydrogen was 72 to 8.2%, and nitrogen was 2.0 to 6.0%. , and the remainder was oxygen.

(2) Color Reaction Table 1 Table 1 shows the results of various color reaction tests for K obtained by dissolving this substance in water.

The results in the table above indicate that the substance is a tang protein.

(3) Dissolve 0.12 of this substance in 100CH of water, Cor, n
When the value was measured using ing pHmeter 140, a value of 4.4 was obtained.

(4) Molecular weight of this substance is 0.15 M NaC4NllH2PO4/N
a, HPO4 to ρttZ2 A made 0, D 2 To
As a result of measuring the molecular weight of this substance by ultracentrifugation, the sedimentation coefficient of 82° was 100 or more. The measurement was performed using Beckman Prep, UV 5ca.
ner & L 8 Centrifuge was solid-phased.

Even in fractionation by gel filtration using 5epharose CL-2B, the molecule t2. o o o, o o
o or more.

(5) Solubility This substance is highly soluble in water and insoluble in acetone, ethanol, methanol and ether.

(6) Hitachi285 uses infrared absorption to absorb infrared rays.
Grating Infrared Spectro
When measured using a photometer, the results were as shown in FIG.

Absorption is 3375 CrrL", 2920 cm-'
.

2860m-', 1745(X-',
1 660cm-'.

1 520~1 550cm-', 1 470
crIL″′(.

1375crn→ , 1 250cttt-'
, 1070 cm'.

(7) The ultraviolet absorption of this substance is KontronUvi.
When measured using KON 810, the results were as shown in FIG.

i87 NMR (Proton Nuclear Magnetic Resonance) absorption spectrum, S+) Ansha $l! The NMR absorption spectrum of this substance was measured using a model XL300. This substance 2~
Dissolve 3q in 0.4 CCK of heavy water and conduct NMR at 600 MHz.
The results of measuring the absorption coefficient are shown in FIGS. 3 and 4. In addition, Figure 3 shows the result of melting and measuring at 50℃.
The figure shows the results of measurement after melting at 100°C.

(9) Types of constituent sugars Silica gel plate (Merck art, No. 5
55320X20cIL), and the types of constituent sugars were investigated by thin layer chromatography.

The sample was hydrolyzed with 2N-H, 80, for 4 hours in a boiling water bath, neutralized and filtered with barium carbonate. After reducing the P solution by 4 times,
Silica gel spray treated with phosphoric acid) (Mer
ekart, No, 5553 20X20cIrL)
Spot K and develop using developing solvent A for about 7 hours.
As a result of coloring with the coloring agent B, mannose, crucose,
The presence of caractose and fucose was confirmed.

A A mixture of isopropyl alcohol, acetone, and 0.1M lactic acid in a ratio of 4:4:2.

B Aniline 4d% diphenylamine 49. Acetone 2
00-1) A mixture of I, Po, 80chi30-.

(Composition ratio of 11 constituent sugars William N1eder Meier method (Ana
l, Biochem.

40.465-475 1971), the composition ratio of the constituent sugars of this substance was determined, and the results shown in Table 2 were obtained with glucose as 1.

Table 2 Composition ratio of constituent proteins The protein portion of this substance was hydrolyzed with 6N hydrochloric acid and subjected to amino acid analysis, and the results shown in Table 3 were obtained. Amino acid composition was expressed as a percentage of the total number of amino acid residues.

Note that cysteine and tryptophan were not quantified.

Table 3 q.Protein and sugar ratio The protein to sugar ratio of this substance is 1:2-4.

u31 Color of substance The lyophilized product of this substance is white in color.

14 Decomposition point The decomposition point of this substance is TG-DSC standard type CN8089A1.
As a result of measurement using a differential scanning calorimeter (manufactured by Rigaku Denki Co., Ltd.), it was decomposed at 223°C.

It has been concluded that the glycoprotein Coriolan of the present invention has excellent medicinal effects that were not found in conventional anticancer substances extracted from higher fungi. That is, it not only shows a remarkable antitumor effect against marcoma-180 solid tumors, but also
It was also highly effective against eama-180 ascites-type tumors when administered alone, and approximately 50 tumors were completely cured. Also in v
It has been proven that the itro system has a strong immune system activation effect. That is, as a result of adding the glycoprotein Coriolan to a mouse tile lymphocyte suspension and examining the linnosocyte blasting reaction, it was found that the ability to induce blasting was approximately 19 times that of the control group. There are no side effects due to intraperitoneal administration, and it is effective for the lungs, heart, liver, tiles,
Bone marrow, stomach, etc. were examined histologically, but no significant changes were found.

The glycoprotein Coriolan of the present invention can be administered intravenously, orally,
Rectal administration is possible, and in the case of injection, the dose is preferably about 500 m/day or less, and in the case of oral administration, the dose is preferably about 5 f/day or less.

In the case of tablets, granules, powders, capsules, etc. for oral administration, the composition may contain such things as binders, encapsulating agents, excipients, lubricants, disintegrants, and wetting agents. In the case of an oral liquid preparation, it may be an oral solution, a shaken mixture, a suspension, an emulsion, or a syrup.
It may also be in the form of a dry product that is redissolved before use. Furthermore, such liquid preparations may contain additives and preservatives commonly used therein.

Compositions for injection may contain additives such as stabilizers, buffers, preservatives, tonicity agents, and the like, and are presented in unit-dose ampoules or multi-dose containers. The compositions may also be in the form of aqueous solutions, suspensions, solutions, emulsions in oily or aqueous vehicles, wherein the active ingredient is contained in a suitable vehicle, e.g. a pyrogen, before use. It may be in the form of a powder that does not need to be redissolved in sterile water.

Next, examples and test examples of the present invention will be shown.

Example 1 Physiological saline was added to fruiting body 2502 of Yakifutake mushroom,
Grind at 3 to 7°C, centrifuge the supernatant at 1200Xf for 20 minutes, concentrate the supernatant 250 to 50 by freeze-drying, and then 5ephacryl-8-20.
Gel filtration was performed using 0 (manufactured by Pharmacia), and the molecular weight was 2.0.
00,000 or more fractions were separated, and the sugar content was 6.6
The N9 glycoprotein Coriolan was obtained.

Example 2゜Malt agar medium (2 t malt extract, 1 t -1 nibton,
20 t of glucose, 20 t of agar, 1,000 d of purified water) was sterilized at 121°C for 15 minutes, and Coriolus pubesena (Fr) was added to it.
Qujl), inoculated with FERM P-6561, 25
℃ for 7 days, and the obtained Yakifutake mycelium was grown at 50°C.
〇-Malt medium in Erlenmeyer flask (malt extract 20t,
The mixture ratio was adjusted to 1f Bopton, 20f glucose, 1.000m purified water, and 1 white fungus was inoculated into J200d.
Cultivate at 25° C. for 7 days while stirring with a rotary shaker at a speed of 15 rpm.

The obtained Yakifutake liquid culture mycelium was mixed with 10 liters of malt medium in a jar fermenter at a rate of 50 (ld) [L, under aeration of 5 t/min, at 200 r.
Culture at 25° C. for 7 days under stirring conditions of pm. The culture solution was collected by centrifugation (5,000 rpm for 10 minutes), and after washing three times with purified water, 120f of ret-shaped mycelia (
Wet weight).

69Of the mycelia obtained in this way were collected, 690cc of physiological saline was added, and using DYNO-MILL,
Crush the bacterial cells to a diameter of 5μ or less, and pour the liquid into 12
The supernatant 65011It was obtained by centrifugation at 00xr for 20 minutes.

Remove 90sd of the 650ml supernatant and add 8ephacry.
Gel filtration was performed using l-8-200 (manufactured by Pharmacia) to obtain a polymer fraction with a molecular weight of 2,000,000 or more, 5QO1.
I got d.

The sugar concentration in the high molecular fraction 300d was determined by the Anthrone sulfate method.
As a result of measuring the protein concentration using the Lowry method, the sugar concentration was 155 μf/ld, and the tan and odor concentrations were 38.
It became - at 9μ.

The production amount of the obtained glycoprotein Coriolan was 57 M9/l per culture solution as sperm production, and 94 M9/l per culture solution as protein production.

Further, the production amount of the glycoprotein Coriolan per bacterial cell iF (wet weight) was 0-A8q/f as a volume, and Q as a tanx, 12q/l.

Test Example (a) 6 x 10'' Sarcoma-180 ascites tumor cells were implanted subcutaneously in the lumbar region of a mouse, and the glycoprotein Coriolan obtained in Example 2 was added daily to 20P/9 for 10 days from the day after the implantation, and intraperitoneally administered. Five weeks after tumor implantation, the tumor was removed, and the tumor growth inhibition rate was calculated from the average weight of the substance-administered group and the average weight of the control group.

As a result, the inhibition rate by intraperitoneal administration of sarcoma-180 to tumors in mice was 90ts.

(10' blIX Sarcoma-180 ascites tumor cells were intraperitoneally transplanted into mice, and the glycoprotein Coriolan obtained in Example 2 was intraperitoneally administered daily on 2011P/9 for 14 days from the day after the transplant. Control group All died within 60 days. Those surviving 60 days after transplantation and without ascites were considered completely cured.

As a result, the complete cure rate by intraperitoneal administration (single administration) for Marcoma-180 ascites-type tumors was 50%.

[Brief explanation of drawings]

Figure 1 shows the infrared absorption of the glycoprotein Coriolan.
Figure 2 shows UV absorption filter, Figure 3 and Figure 4 show N.
It is a figure showing an MR absorption spectrum.

Claims (1)

[Claims] 1. Coriolan, a glycoprotein having the following physical and chemical properties. (1) Elemental analysis The results of elemental analysis of this material show that carbon is 50.0 to 52.0%;
7.2-8.2% hydrogen, 2.0-6.0% nitrogen, and the balance is oxygen. (2) Color Reaction Table 1 Table 1 shows the results of various color reaction tests on the substance dissolved in water. The results in the table above indicate that this substance is a glycoprotein. (3) pH value When 0.1 g of this substance was dissolved in 100 cc of water and the pH value was measured, a value of 4.4 was obtained. (4) Molecular weight This substance is 0.15M NaCl, NaH_2PO_4/N
Adjust the pH to 7.2 with a_2HPO_4 (0.02% N
aN_3 addition), the molecular weight of this substance was measured by ultracentrifugation, and the sedimentation coefficient S_2_0 was 100 or more, indicating that the molecular weight was 2,000,000 or more. Sepha
Even in fractionation by gel filtration using roseCL-2B, the molecular weight is 2,000,000 or more. (5) Solubility This substance is highly soluble in water and insoluble in acetone, ethanol, methanol and ether. (6) Infrared absorption spectrum The infrared absorption spectrum of this substance is shown in FIG. 3375cm^-^1, 2920cm^-^1, 286
0cm^-^1, 1745cm^-^1, 1660cm
^-^1, 1520-1550cm^-^1, 1470
cm^-^1, 1375cm^-^1, 1230cm^
The respective absorptions are shown near -^1 and 1070cm^-^1. (7) Ultraviolet absorption spectrum The ultraviolet absorption spectrum of this substance is shown in FIG. (8) NMR absorption spectrum Dissolve 2 to 3 mg of this substance in 0.4 cc of heavy water, and
The results of NMR absorption spectra measured at Hz are shown in FIGS. 3 and 4. Note that FIG. 5 shows the results of measurement after melting at 50°C, and FIG. 4 shows the results of measurement after melting at 100°C. (9) Types of constituent sugars This substance was hydrolyzed with 2NH_2SO_4, and the presence of mannose, glucose, galactose, and fucose was confirmed by thin layer chromatography. (10) Composition ratio of constituent sugars William Nider Meier method (Anal
．． Biochem. 40, 465-475, 1971), the composition ratio of the constituent sugars of this substance was determined, and the results shown in Table 2 were obtained with glucose as 1. Table 2 (11) Composition ratio of constituent proteins The protein part of this substance was hydrolyzed with 6N hydrochloric acid and subjected to amino acid analysis, and the results shown in Table 3 were obtained. The amino acid composition is based on the total number of amino acid residues. Expressed as a percentage. Note that cysteine and tryptophan were not quantified. Table 3 (12) Protein to sugar ratio The protein to sugar ratio of this substance is 1:2 to 4. (13) Color of the substance The lyophilized product of this substance is white. (14) Decomposition point The decomposition point of this substance was measured using a differential scanning calorimeter, and the result was that it decomposed at 223°C. 2. Cultivate Yakifutake and obtain a molecular weight of 2,000 from the culture.
A method for producing the glycoprotein Coriolan, which comprises collecting the glycoprotein Coriolan. 3. Extract the culture, fruiting body or mycelium of Yakifutake with an aqueous solvent, and treat the resulting extract by gel filtration,
A method for producing the glycoprotein Coriolan, which comprises collecting the glycoprotein Coriolan with a molecular weight of 2,000,000 or more.